Abstracts BIOMIN XV: 15th International Symposium on Biomineralization

9–13 September 2019 • Munich, Germany

1. Keynote lectures (K1 – K8) ................................................................................................................. 2

2. Talks (T1 – T89) ................................................................................................................................... 4

3. Posters (P1 – P107) ............................................................................................................................. 31

Design/Layout Layout: www.conventus.de

Editorial Deadline: 31 August 2019

1

K 1

On ion transport and concentration toward mineral formation

in sea urchin larvae Keren Kahi1, Neta Varsano1, Andrea Sorrentino2, Eva Pereiro2, Peter Rez3,

Steve Weiner1 and Lia Addadi*1

1Department of Structural Biology, Weizmann Institute of Science, Rehovot,

Israel 2ALBA Synchrotron Light Source, MISTRAL Beamline−Experiments Division, Barcelona, Spain 3Department of Physics, Arizona State University, Tempe, AZ, USA

During mineralized tissue formation, organisms are faced with a major

problem in ion transport and concentration. Suffice it to consider that marine

animals must concentrate calcium by >4 orders of magnitude relative to its concentration in sea water to deposit calcite or aragonite minerals. Sea urchin

larvae are good model organisms for studying ion processing during skeleton

formation, because of the extensive knowledge accumulated on the processes related to mineral deposition. Sea urchin larvae have endoskeletons

composed of two calcitic spicules, deposited by primary mesenchymal

spicule-forming cells (PMCs). PMCs take up seawater through endocytosis1 into a complex network of vacuoles. Within the PMCs, calcium

ions are translocated from the seawater vacuoles to various organelles and

vesicles where they accumulate, and subsequently precipitate as an amorphous calcium carbonate (ACC). The amorphous precipitates are finally

translocated to the spicule, where they crystallize.

We address the question of the form in which calcium ions are stored in

different locations in the cell, whether dissolved or solid and in which

structural phase. In order to locate and characterize calcium content in individual vesicles we performed cryo-soft X-ray microscopy (cryo-SXM)

on dispersed PMCs. The presence of concentrated calcium ions was detected

by imaging the cells in the energy range before and after the calcium L-absorption edges. We characterized the chemical environment of the calcium

ions using X-ray absorption spectroscopy. We observe hundreds of particles

containing Ca in each PMC. The particles are composed of different forms of highly disordered phases of calcium salts, presumably carbonate. We also

developed methods for quantitative evaluation of calcium ion concentrations.

We observed concentrations diluted relative to ACC (19M), but concentrated relative to sea water (10mM). The spectroscopic and analytical data thus

together indicate a transition through a series of amorphous calcium

carbonate phases in the low molar concentration range. These data shed light on the intracellular transport and concentration pathways of calcium ions in

PMCs. This may well be relevant to other organisms, and thus lead to a

deeper understanding of biogenic mineral formation. 1. N. Vidavsky, S. Addadi, A. Schertel, D. Ben-Ezra, M. Shpigel, L. Addadi,

S. Weiner, Calcium transport into the cells of the sea urchin larva in relation

to spicule formation. Proc. Natl. Acad. Sci. U.S.A, 113(45), 12637-12642,

2016. 2. E. Beniash, J. Aizenberg, L. Addadi, S. Weiner, Amorphous

calcium carbonate transforms into calcite during sea urchin larval spicule

growth. P. Roy. Soc. B-Biol. Sci., 264 (1380), 461-465, (1997).

K 2

Biomineralization in echinoderms: developmental mechanisms

and evolution. Charles Ettensohn*1 1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh,

PA USA

All adult echinoderms have a calcite-based endoskeleton. Embryonic and

larval patterns of skeletogenesis, however, vary greatly across the phylum,

revealing a rich history of evolutionary modifications to the developmental programs that underlie biomineral formation in this group. The formation of

the skeleton has been particularly well studied in embryos of euechinoid sea

urchins, which have served as a major experimental model for developmental biologists for more than a century. The cellular behaviors that underlie

skeleton formation in sea urchin embryos have been described in detail and

many gene products that play essential roles in biomineral formation have

been identified. In addition, a complex transcriptional network that underlies

skeletogenesis has recently been elucidated. This gene regulatory network

links the early specification of embryonic skeletogenic cells (primary mesenchyme cells, or PMCs) to their cellular behaviors and biomineral-

forming properties. The PMC gene network is proving to be a powerful tool

for understanding the genetic and molecular control of skeletogenesis in echinoderms and the evolution of biomineralization.

K 3

Getting to the roots of apatite-based biomineralization of dental

hard tissues: from Conodonts and Cichlids to related

bioinspired materials

Elena V. Sturm*1 (née Rosseeva) 1Physical Chemistry, Zukunftskolleg, University of Konstanz, Konstanz,

Germany

Chordates and especially vertebrates represent the most highly advanced and

complex group of organisms. The formation of their hierarchical apatite-organic based hard tissues is evolutionary optimized and exhibits high

structural complexity on various length scales and amazing mechanical performance. The major objective of our research is to explore the

biomineralization processes involved in the formation of dental (and dental-

like) apatite-based hard tissues of earliest and modern vertebrates. Specifically, in my presentation I will focus on the detailed characterization

of morphology-structure-composition-property relationships of hard tissues

of feeding apparatus of Conodonts and Cichlid Fishes. We elaborate and combine exciting evolutionary model systems with cutting-edge

spectroscopy, microscopy and diffraction techniques to analyze the

structural, chemical and morphogenetic basis of the dental hard tissue biomineralization process. Our new understanding of dentical and tooth

structure in Conodont and Cichlids could also advance strategies for

synthesizing bioinspired and biomimetic materials and deepen our knowledge of their morphogenesis process.

K 4

Coral Biomineralization: linking pieces of the puzzle

Sylvie Tambutté*1 1Department of Marine Biology, Centre Scientifique de Monaco, Monaco

Coral biomineralization is the process that leads to the formation of a calcium carbonate exoskeleton. As for other biominerals, two essential questions are:

What do we know about the control of coral biomineralization? How can we

link biological control with physico-chemical processes? I will show how studies performed at the Centre Scientifique de Monaco since the 90’s have

provided insight into the coral biomineralization process from the whole

organism to the gene by combining molecular and physiological approaches. I will present how data obtained from experiments conducted in different

coral compartments: the tissues, the extracellular calcifying medium and the

skeleton can be linked together and can help in deciphering where and how biological control occurs. More specifically, I will present 1) the

measurements of pH, carbonate and calcium in the extracellular medium that

we have obtained by developing in vivo approaches, and 2) the mechanisms involved in transepithelial ion transport that we have characterized by

molecular and physiological approaches. Finally, I will show how changes

in environmental parameters such as seawater pH influence biological control and can provide information on coral biomineralization.

K 5

Towards bone-on-a-chip: cell differentiation and extracellular

matrix organization Sana Ansari1,2,3, Esther Cramer1,2, Johanna Melke2,3, Keita Ito2,3, Sandra

Hofmann2,3, Nico Somemrdijk1,3, Anat Akiva*1,3 1Laboratory of Materials and Interface Chemistry and Center for Multiscale Electron Microscopy, Department of Chemical Engineering and Chemistry.

Eindhoven University of Technology, Eindhoven, The Netherlands. 2Institute for Complex Molecular Systems. Eindhoven University of Technology, Eindhoven, The Netherlands. 3Department of Biomedical Engineering. Eindhoven University of

Technology, Eindhoven, The Netherlands.

Introduction

The formation of bone involves a cascade of biological and chemical events that control the process of collagen mineralization in bone. In this process,

bone cells named osteoblasts form a 3D organized collagen matrix and

secrete several non-collagenous proteins (NCPs) that control the mineralization process. However, the precise mechanism by which these

NCPs control the mineralization of collagen fibrils is not yet clear. In an

attempt to get a direct observation on the time and place of the expression of these proteins, and to study their role in collagen mineralization, we use an

advanced in vitro model system.

Materials and methods. Here, human mesenchymal stromal cells (hMSCs) are seeded on 3D silk

scaffold and are exposed to osteogenic medium and to continuous

mechanical stimulation. Depending on the composition of the osteogenic medium and the specific mechanical load, we can control the differentiation

of the hMSCs to osteoblasts and sequentially into osteocytes – the last step

in osteoblast differentiation.

Results

Using a unique bioreactor, we monitor the cellular and extracellular

development of the system. The stem cells transformed from long and

2

elongated cells to round shaped cells, which are typical to osteoblasts. In this stage, the cells also expressed specific osteoblastic proteins. These proteins,

such as osteocalcin, osteopontin, and osteonectin where located intra and

extracellular, embedded in the collagen matrix. In a later time point, when the cells are embedded in the collagen matrix, another morphological change

occured, the transformation into osteocytes as judged by the formation of

long processes. The osteocyte formation was further supported by the expression of unique osteocyte marker proteins such as DMP-1 and

sclerostin.

Conclusion

In this study, we show for the first time that in vitro differentiation of hMSCs

into osteoblast and osteocytes is achievable. The system shows the main

characteristic features of bone, resembling the process of bone formation. This system will allow to obtain a better understanding of the role of specific

proteins on collagen mineralization, in a system that is close as possible to in

vivo bone.

K 6

Bioinspired magnetite nanoparticles as smart drug

nanocarriers and hyperthermia agents C. Jimenez-Lopez*1, A. Peigneux1, Y. Jabalera1, F. Oltolina2, G. Iglesias3, M. Prat2 1Universidad de Granada (España), Department of Microbiology,

Granada, Spain 2Università del Piemonte Orientale A. Avogadro, Dipartimento di Scienze

della Salute, Novara, Italy 3Universidad de Granada (España), Department of Applied Physics, Granada, Spain

Among the biologically controlled biomineralization process, few has raised so much expectations as the magnetosome formation by magnetotactic

bacteria. Magnetosomes are composed of a magnetite (or greigite) crystal

enveloped by a lipidic bilayer and they are crucial for those bacteria survival. Therefore, magnetotactic bacteria control magnetosome formation at the

genetic level, so the magnetite crystals have the necessary characteristics to

maximize their response to the magnetic field. Magnetosome research has raised interest in several areas, from Astrobiology to Nanotechnology, since

magnetosomes are the ideal magnetic nanoparticle that could be used in

many applications, mainly clinics, both in diagnosis and in therapeutics. However, the problem for their use is the extremelly low yields in which

magnetosomes are produced. In particular, the need for a directed targeted chemotherapy has become a

matter of growing interest in order to increase efficiency, to reduce the cost

of systemic treatments and, even more important, to reduce the undesirable secondary effects related to the systemic distribution of the chemotherapeutic

molecule and thus, its interaction with healthy cells. This is not only true and

crucial in tumor treatment, either by means of chemotherapy or immunotherapy, but also for local targetable diseases like local infections. In

this context, novel MamC-mediated biomimetic (magnetosome-like)

magnetic nanoparticles (BMNPs) are proposed as valuable carriers for targeted chemotherapy because of the size (36 ± 12 nm) and of surface

properties conferred by MamC coating. They are superparamagnetic at room

and body temperatures, have a large magnetic moment per particle, mediate hyperthermia, are cytocompatible, and, having a negative surface charge at

physiological pH, can be efficiently coupled with a variety of molecules and

antibodies directed against receptors overexpressed in target cells displaying coupling stability, while releasing DOXO at acidic pH. This release can be

enhanced by hyperthermia. As an example, the nanoassembly DOXO-mAb-

BMNPs has been characterized and demonstrated that it selectively recognizes the Met receptor (overexpressed in many cancers), binds

efficiently to Met+ tumor cells, and discharges DOXO within their nuclei

more efficiently than DOXO-BMNPs, exerting cytotoxicity. These data represent proof of concept for in vivo experiments in which the controlled

dual targeting (mAb-mediated and magnetic) approach and combined

(chemotherapy and hyperthermia) therapy are been studied. Peigneux, A., Oltolina, F., Colangelo, D., Iglesias, G. R., Delgado, A. V.,

Prat, M., Jimenez-Lopez, C. (2019) Functionalized Biomimetic Magnetic

Nanoparticles as Effective Nanocarriers for Targeted Chemotherapy. Particle and Particle Systems Characterization. DOI:10.1002/ppsc.201900057.

Nudelman, H,, Valverde-Tercedor, C., Kolusheva, S., Widdrat, M.,

Grimberg, N., Levi, H., Nelkenbaum, O., Davidov, G., Faivre, D., Jimenez-Lopez, C., Zarivach, R. (2016) Structure function studies of the magnetite-

biomineralizing magnetosome-associated protein MamC. Journal of

Structural Biology 194, 244-252.

K 7

The unique molecular physiologies of biomineralizing

phytoplankton: Animal, vegetable and mineral C. Brownlee*1 1Marine Biological Association, UK University of Southampton, UK

The marine eukaryotic phytoplankton account for around one quarter of

global productivity, roughly equivalent to the terrestrial rainforests. They also underpin much of global biomineralization in the form of biogenic

silica (diatoms) and calcite (coccolithophores). Despite the major

importance of these groups in the Earth’s largest ecosystems, their physiologies remain relatively poorly studied. Combined functional

genomics and single cell biophysics approaches are beginning to reveal

surprising novel features, including ion channels not previously found in eukaryotes. Their roles in excitability, signalling and cellular homeostasis

will be discussed. The discovery of novel silicon transporters is also

shedding new light on mechanisms of coccolithophore biomineralization. Together, these studies are providing new insights into the evolution of

major classes of eukaryotic ion channels and membrane transporters. The

rationale and approaches for studying single cell physiology in situ in the oceans will also be discussed.

K 8

Patterns in the evolutionary acquisitions of mineralized

skeletons

S. Porter*1, J. Moore1, L. A. Riedman1, R. Wood1, R. Rickaby1 1University of California at Santa Barbara, Earth Science, Santa Barbara, United States

Mineralized skeletons evolved many times within the eukaryotes, including

dozens of times within the Metazoa. These numerous independent acquisitions provide an opportunity to test hypotheses about the origin and

evolution of skeletons, including the factors controlling the initial choice of

mineralogy and the reasons why mineralized skeletons evolved in the first place. Earlier work in this area focused on the evolution of carbonate

skeletons by metazoans, showing that acquisitions of aragonitic skeletons

were clustered during times of aragonite seas (when the Mg/Ca ratio of seawater favored aragonite precipitation) and those of calcitic skeletons

during calcite seas. We have since expanded this dataset to include other

skeletal mineralogies and other eukaryotic clades, including both extinct and extant taxa, and have identified more than 80 independent acquisitions of

mineralized skeletons in eukaryotes, along with their time of first appearance,

their mineralogy, and, for the earliest animals, their microstructures and habitats at the time they first evolved.

Several interesting patterns have emerged from this preliminary dataset.

First, skeletal acquisitions among animals are clustered in time, with half appearing in the early Cambrian, and another 25% in the Ordovician through

Devonian periods. No acquisitions are recorded for the later Paleozoic. A

smaller cluster of acquisitions occursed in the mid-Triassic to Jurassic; nearly all of these later skeletons evolved within the cnidarians and annelids. In

contrast, skeletal acquisitions in non-metazoan eukaryotes are distributed throughout the Phanerozoic, with no obvious clustering in time. This

supports the view that ecological factors affecting only animals (e.g., the

appearance of carnivores), rather than physical factors affecting all marine organisms (e.g., increased Ca2+), were a primary driver of skeletal

biomineralization in animals. Second, acquisitions of phosphatic skeletons

are concentrated in the Neoproterozoic and Cambrian, perhaps reflecting higher levels of phosphate in the oceans at this time, though our count of

independent acquisitions may be inflated by post-mortem phosphatization of

organic skeletons., In contrast with calcareous and siliceous skeletons, which are distributed about equally among autotrophs and heterotrophs, phosphatic

skeletons are only known to occur in heterotrophs, perhaps reflecting the

higher cost for autotrophs to use a limiting nutrient to form a skeleton. Finally, the skeletal microstructures and habitats of the earliest carbonate

biomineralizers suggest phases of increasing biological control over

biomineralization, with the earliest carbonate skeletons forming disorganized microstructures and inhabiting only carbonate environments. These were

succeeded in the latest Ediacaran and early Cambrian by calcifying animals

that lived in both carbonate and siliciclastic environments and formed more organized, though still relatively simple, fibrous microstructures.

3

T 1 Nanostructure and growth of calcite crystals of the shell plates of the

barnacle Austromegabalanus psittacus (Crustacea, Cirripedia,

Balanidae)

A. Checa*1,2, E. Macías-Sánchez3, A. Sánchez-Navas4, A. Rodríguez-

Navarro4, N. A. Lagos5 1Universidad de Granada, Estratigrafía y Paleontología, Granada, Spain 2CSIC-Universidad de Granada, Instituto Andaluz de Ciencias de la Tierra,

Armilla, Spain 3Max Planck Institute of Colloids and Interfaces, Potsdam, Germany 4Universidad de Granada, Mineralogy and Petrology, Granada, Spain 5Universidad Santo Tomás, Centro de Investigación e Innovación para el Cambio Climático, Santiago, Chile

The calcareous plates enclosing the body of the barnacle Austromegabalanus

psittacus are mainly made by calcite crystals. The crystals are arranged into

bundles, within which all crystals are strictly co-oriented. The c-axes of crystals of different bundles are roughly perpendicular to the growth

surfaces. Observations of crystals appearing on the free growth surfaces

reveal changes in size (from 1 to 5 µm) and morphology (from irregular to euhedral rhombohedral). A remarkable feature of these crystals is their

coarse nanogranular ultrastructure. The {104} rhombohedral faces are

distinctly much smoother than other types of surfaces. In turn, the nanogranules making up the crystals are arranged into rod-like structures

(which we call lineations), which diverge at large angles from neat lines.

Examination of incomplete rhombohedra indicate that the lines from which the lineations diverge are at large angles to the {104} faces and clearly in

continuity with the rhombohedron edges. That is, they align in parallel to ˂-

441> directions of calcite. The complete sequence of growth from irregular granules to {104} rhombohedra can be reconstructed. The {104} faces begin

to appear at or close to their centers and spread towards the edges by the

progressive lateral addition of nanogranule lineations. The last rhombohedral elements completed are the edges. Rhombohedra are usually incomplete,

because the grains are quickly overgrown by other neighbors during plate

growth. TEM observations reveal that the nanostructure consists of crystalline domains, which are surrounded by amorphous areas. This is in

agreement with AFM observations, which show the existence of two phases

with different contrast. All the above observations give important insight into the growth mechanisms of these calcite biocrystals. We propose that during

the transformation of ACC into calcite, due to the force of crystallization,

organic molecules are expelled from the crystal lattice. Growth of the crystalline domains takes place preferentially along the directions, which are

the strongest periodic bond chains (PBCs) in calcite. Accordingly, organic

molecules are barely incorporated along these directions and become expelled sidewards. This implies that the formation of nanogranules and their

arrangement into lineations in barnacle calcite crystals is a

crystallographically-controlled phenomenon and cannot be explained by a particle-aggregation process, as previously shown for molluscan biogenic

aragonite.

T 2

Towards understanding larval bivalve calcification

mechanisms in a changing ocean F. Melzner*1, K. Ramesh2, M. Clark3, G. Nehrke4 1GEOMAR, Marine Ecology, Kiel, Germany 2University of Gothenburg, Gothenburg, Sweden 3British Antarctic Survey, Cambridge, United Kingdom 4AWI Bremerhaven, Bremerhaven, Germany

Marine larval bivalves (mussels, oysters) rapidly secrete first shells within

less than one day, thereby precipitating a calcium carbonate mass that corresponds to their own body mass. This remarkable synthesis effort

requires massive transfer of inorganic carbon and calcium, as well as proton

removal from the site of calcification. These transport mechanisms are unknown so far. Similarly, it is not clear, whether amorphous precursor

phases are formed intracellularly, or whether carbonate formation is located

in dedicated extracellular spaces at the organism-mineral interface. A better mechanistic characterization of shell formation processes is important, as it

allows us to understand the high vulnerability of larvae to ongoing ocean

acidification. We use mussel larvae (Mytilus) as model species and utilize a variety of

approaches, ranging from in vivo confocal microscopy, in vivo

confocal Raman microscopy, microelectrode recordings to transcriptomic approaches to learn more about early shell mineralization and how it is

impacted by ocean acidification.

Using in vivo calcein pulse-chase and confocal microscopy, we can show that (1) it is unlikely that biomineral precursors are formed in large vesicles in

cells. This finding is supported by in vivo confocal Raman microscopical analysis of first secreted shell components at the trochophore stage, which

(2) exclusively indicates presence of aragonite in the shell. While it is

possible that very small intracellular carbonate particles are produced intracellularly, it is equally possible that mineral formation is an extracellular

process. We therefore characterized the carbonate chemistry environment at the shell-tissue interface using minute microsensors for pH, calcium and

carbonate and find (3) that veliger larvae can elevate calcium carbonate

saturation state of the calcification space, which could be instrumental in increasing rates of carbonate precipitation. However, larvae (4) are not able

to maintain this regulatory effort when seawater pH decreases. This finding

could explain reduced calcification and dissolution rates when larvae are exposed to simulated ocean acidification. In order to identify ion transport

proteins that support calcification substrate transport and pH regulation, we

use substrate limitation assays in combination with transcriptomics and can (5) show that SLC4 and 26 family member candidate genes are probably vital

for bicarbonate transport and pH regulation.

References: Ramesh et al. 2017 Nature Communications 8:1709

Ramesh et al. 2018 Journal of the Royal Society Interface 15: 20170723

Ramesh et al. 2019 Ecology & Evolution (in press)

T 3

Terebratulide brachiopod shell biomineralization by mantle

epithelial cells M. Simonet Roda*1, A. Ziegler2, E. Griesshaber1, X. Yin1, U. Rupp2, D. Henkel3, V. Häussermann4,5, J. Laudien6, U. Brand7, A. Einsenhauer3, W.

W. Schmahl1 1Ludwig Maximilians Universität München, Department of Earth and Environmental Sciences, Munich, Germany 2University of Ulm, Central Facility for Electron Microscopy, Ulm,

Germany 3GEOMAR Helmhöoltz Centre for Ocean Research, Marine

Biogeochemistry/Marine Systems, Kiel, Germany 4Pontificia Universidad Católica de Valparaíso, Facultad de Recursos Naturales, Escuela de Ciencias del Mar, Valparaíso, Chile 5Huinay Scientific Field Station, Puerto Montt, Chile 6Alfred-Wegener-Institut Helmholtz-Zentrum fur Polar- und Meeresforschung, Bremerhaven, Germany 7Brock University, Department of Earth Sciences, Ontario, Canada

The shell of modern rhynchonellide and terebratulide brachiopods is a hybrid

composite where an extracellular biopolymer matrix is reinforced by calcite.

Both material components are secreted by outer mantle epithelium (OME) cells.

In order to understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with

TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium

(OME) cells of juvenile and live Magellania venosa shells. Imaging was carried out on embedded/etched, chemically fixed/decalcified and high-

pressure frozen/freeze-substituted shell pieces taken from the commissure,

central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound

organelles (Simonet Roda et al. 2019a, Simonet Roda et al. 2019b).

At the commissure, the OME is multi-cell layered, while in central shell regions it is single-cell layered. OME cells form at the commissure oblique

extensions that, in cross-section, are round below the primary layer and flat

underneath the fibres. At central shell regions, OME cells are considerably thinner in comparison to cells at the commissure.

When actively secreting shell carbonate extrapallial space is lacking as OME

cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to

extracellular matrix membranes that line the proximal surface of fibres and

tonofilaments connect apical to basal hemidesmosomes. This stabilizes the contact of epithelium and the fibres and keeps the mantle in place. Individual

fibres are secreted by several cells at the same time. This requires

communication and tight cooperation of neighbouring OME cells for the coordinated secretion of organic membrane and mineral, explaining the high

abundance of gap junctions between cells.

There is not any observation in the cell ultrastructure in our study that can be taken as evidence for a vesicular transport based mineralization process. On

the contrary, the absent or very narrow (in the range of nanometers) space

between the epithelium and the growing fibres, together with the absence of significant differences in the volume fraction of vesicles between secreting

and non-secreting regions of the OME, as well as the extreme reduction in

cell thickness at sites of mineral secretion suggests, that in modern Magellania venosa (and likely in all Rhynchonellida and Terebratulida

forming the fibrous microstructure) mineral transport to the sites of

mineralization occurs via ion transport mechanisms through the cell membrane and not by transport of mineral by organelles such as vesicles.

Simonet Roda, M., Ziegler, A., Griesshaber, E., Yin, X., Rupp, U., Greiner,

M., Henkel, D., Häussermann, V., Eisenhauer, A., Laudien, J., Schmahl, W.W., Terebratulide brachiopod shell biomineralization by mantle epithelial

cells, Journal of Structural Biology,

https://doi.org/10.1016/j.jsb.2019.05.002 Simonet Roda, M., Griesshaber, E., Ziegler, A., Rupp, U., Yin, X., Henkel,

D., Häussermann, V., Laudien, J., Brand, U., Eisenhauer, A., Checa, A.G.,

4

Schmahl, W.W., 2019b. Calcite fibre formation in modern brachiopod shells. Sci. Rep. 9, 598.

T 4

How hepatopancreas cells of a terrestrial crustacean take up

and release calcium after ingestion of mineral from the old

shed cuticle U. Rupp*1, A. Ziegler1 1Ulm University, Central Facility For Electron Microscopy, Ulm, Germany

The hepatopancreas of isopods serves for food digestion, storage of lipids and carbohydrates, storage of essential metals as well as accumulation and

detoxification of xenobiotic metals. Metal is accumulated in lysosomal metal

containing granules in the two major cell types (S and B) of the hepatopancreas (Dallinger and Prosi, 1989). A recent µCT study on mineral

shifts in moulting Porcellio scaber has shown that mineral is present within

the hepatopancreas lumen, however, only when the animal has ingested the old shed cuticle (exuviae) after moult (Ziegler et al., 2017). This suggests

uptake of mineral from the ingested exuviae by the hepatopancreas and

recycling for mineralisation of the new cuticle. The aim of this study was i) to reveal if the cells of the hepatopancreas

contain mineral from the exuviae, ii) if the metal-containing granules

contribute to the uptake of calcium and its release for mineral recycling, and iii) to find pathways for mineral/calcium transport from the exuviae to the

hemolymph. We have therefore investigated the mineral distributions within

the cytosol and organelles of hepatopancreas S- and B-cells. We used P. scaber at the postmoult stage that have ingested their exuviae after

the moult, those that have not ingested their exuviae as a negative control, and animals at the intermoult stage, 9-10 days after exuviae ingestion that

have a fully mineralized cuticle.

We used STEM, TEM multi-image acquisition, EDX and EFTEM for the analysis of the ultrastructure and mineral distribution in high pressure frozen

and freeze substituted hepatopancreas tissue. Ultramicrotomy of resin

embedded samples was performed using a piezo-driven oscillating diamond knife and propane-1,3-diol as floatation medium for the sections to minimize

loss of mineral.

The cryofixed samples reveal numerous extracellular vesicles (exosomes) and many multivesicular bodies containing proexosomes in both cell

types. After high-pressure freezing and freeze-substitution, granules in

sections of S as well as B-cells contain well-preserved mineral deposits. In animals that have ingested the exuviae, we found a significantly higher

calcium concentration in the metal granules of S cells in comparison to

control animals. In B cells the calcium concentration in lysosomal granules was much lower and independent of moulting stage or exuviae ingestion. We

observe intracellular seams of mineral along the microvilli and the lateral

plasma membranes, consisting of phosphorus co-localizing with calcium and occasionally with calcium and zinc in S-cells and with iron or calcium in B-

cells. Small granules composed of calcium and phosphorous occur also

between cells and within the basal lamina. In intermoult animals, such mineral seams and granules are less abundant and absent in

control postmoult animals that have not ingested their cuticle.

The results indicate that calcium uptake from the ingested exuviae takes place across the apical plasma membrane of the cells and that

lysosomal metal granules in S-cells accumulate calcium for mineralization

of the new cuticle. Transport into the haemolymph occurs in the form of calcium phosphate via the basolateral membrane and the basal lamina. Co-

localization of zinc with calcium suggests similar pathways for the two

elements. Prosi F. and Dallinger R. 1988. Cell Biol. and Toxicol. 4: 81–96.

Ziegler A. Neues F. Janáček J. Beckmann F. Epple. M. 2017. Arthropod

Struct. Dev. 46: 63–76. Supported by the DFG ZI 368/11-1.

T 5

Identification of extracellular vesicles involved in the

biomineralization of the hen eggshell L. Stapane1, N. Le Roy1, J. Gautron*1 1French National Institute of Agricultural Research (INRA), Bird Biology and Poultry, Nouzilly, France

Question The eggshell is a critical barrier against mechanical stresses and microbial penetration. Its integrity is essential to maintain the hygienic quality of this

basic human food and to limit the number of downgraded eggs. In such a

context, we are looking for eggshell strength specific markers in order to optimize egg quality.

The eggshell is made of 95% mineral phase (calcium carbonate on calcite

form) and an organic matrix (3.5%) mostly containing proteins. Eggshell formation arises from an extra-cellular biomineralization process, which

takes place in a fluid that contains eggshell precursors and involves a

transient phase of amorphous calcium carbonate (ACC). This work aims at

exploring the presence and the role of extracellular vesicles to stabilize ACC and to address it to the mineralization site.

Results In a first approach, we used real time qRT-PCR to assess the expression of

vesicular target genes in several tissues. The results confirmed a high

expression of vesicular target genes (edil3, anxa1, anxa2, pdcd6ip) in oviduct portions where mineralization takes place. In this study, we have also

explored the role of EDIL3 and MFGE8 proteins in chicken shell at key

stages of shell mineralization, and confirmed they could bind Ca2+ and vesicles, thanks to an EGF-like calcium-binding domain and a F5/8C

phospholipid-binding domain. It was therefore suggested that both proteins

could be involved in the vesicular transport of calcium. In a second approach, electronic microscopy coupled with elementary analysis was used to observe

the uterine fluid collected during eggshell biomineralization. Data obtained

highlighted the presence of extracellular vesicles (~ 300 nm) containing calcium carbonate. Finally, Western Blot analysis confirmed the presence of

EDIL3, a key vesicular protein in the purified vesicle fraction.

Conclusions The results of this study showed for the first time the involvement of

extracellular vesicles in the transport of calcium carbonate for the

biomineralization of hen"s eggshell. We proposed a model of calcification using vesicles to stabilize ACC and explaining the fast deposition of the

crystalline calcite oriented layer in the shell. The proteins described in this

study will have to be explored as biological markers for a selection of chicken layers with improved mechanical properties.

T 6

STIM1 a calcium sensor promotes the assembly of an ECM

that contains extracellular vesicles and factors that modulate

mineralization A. George*1, Y. Chen1 1University of Illinois at Chicago, Oral Biology, Chicago, United States

Introduction

Biomineralization is a dynamic process in which living organisms deposit

mineral in the extracellular matrix. Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. Several

proteins work in coordination and contribute to this tightly regulated process.

STIM1 (Stromal interaction molecule 1) is one such protein that has been recently identified to function in bone and enamel mineralization. The

STIM1 protein is a calcium sensor localized to the membrane of the

endoplasmic cells and is well recognized for its physiological role in the endoplasmic reticulum. We have demonstrated earlier that DMP1

stimulation of preosteoblasts lead to calcium release from internal Ca2+

stores and this store depletion is sensed by the ER Ca 2+ sensor STIM1. Store-operated calcium entry is one of the major Ca 2+ influx mechanisms

following store depletion in the ER.

Objectives

To demonstrate a role for STIM1 in dentin matrix mineralization.

Materials and Methods

In order to understand the function of STIM1 during dentin mineralization, we overexpressed STIM1 in dental pulp stem cells (DPSCs) to generate p-

EF1α-STIM1 in which EF1a promoter drives the expression of STIM1. Cell

lines were characterized for the overexpression and knock down of STIM1. The transgenic cells along with the control were cultured for 7, 14 and 21

days in growth and differentiation media and the morphology of the cells and

topology of the matrix were examined by Field Emission Scanning Electron Microscopy. Further, we examined matrix mineralization by Alizarin red and

von Kossa staining. Gene expression analysis by RT-PCR was performed to

identify cell differentiation in the presence and absence of STIM1. Immunofluoresence was performed to confirm the localization of osteogenic

markers in the presence and absence of STIM1. Results: Transgenic cell

lines overexpressing and knockdown of STIM1 was successfully generated as assessed by fold changes in STIM1 mRNA expression. FESEM results

demonstrate that STIM1 overexpressing cells released large amount of

extracellular microvesicles and matrix mineralization. Interestingly, knockdown of STIM1 resulted in fewer microvesicles and less mineralized

matrix. Alizarin and von Kossa staining demonstrate the differentiation of

stem cells into odontogenic phenotype with STIM1 overexpression.

Conclusion

STIM1 is a crucial molecule in biomineralization as it influences release of

large amounts of extracellular vesicles and formation of mineralized matrix. This was impaired in the absence of STIM1. Downregulation of

differentiation markers suggest that STIM1 promotes differentiation

probably by mobilizing intracellular calcium ions. As Ca2+ functions as second messengers, therefore their role in cellular differentiation suggest that

STIM1 can promote intracellular Ca 2+ oscillations and thus provide a signal for activation of downstream and upstream effectors that promote

differentiation and matrix mineralization.

5

Acknowledgements

NIH-NIDCR DE 11657 and the Brodie Endowment Fund.

T 7

Intraperiostracal calcification in the Bivalvia- tuning in to the

aerials of Catillopecten E. Harper*1, G. Kamenev2, F. Varela- Feria3, J. Taylor4, E. Glover4, A.

Checa5

1University of Cambridge, Earth Sciences, Cambridge, United Kingdom 2Russian Academy of Science, 2. National Scientific Center of Marine

Biology, Vladivostok, Russian Federation 3University of Seville, Seville, Spain 4The Natural History Museum, Life Sciences, London, United Kingdom 5Uniiversity of Granada, 5. Department of Stratigraphy and Paleontology,

Granada, Spain

Most studies of molluscan biomineralization concentrate on deposition of

shell layers onto and below the periostracum. However, in the last decade there has been an increased appreciation of intraperiostracal calcification –

where biominerals are secreted within the periostracum - producing often

intricate and elaborate structures. Thus far most of this intraperiostracal calcification has been reported in the Bivalvia, where it is widespread in the

Anomalodesmata, Palaeoheterodonta, Pteriomorphia and Imparidentia. This

distribution hints that this is either a plesiomorphic character or that there is such a facility within the Bivalvia for such intraperiostracal calcification that

it has evolved polyphyletically in different situations.

In this study we present a detailed study of recently discovered intraperiostracal calcification in a clade in which it has not be observed

before. Members of the Propeamussiidae, the "glass scallops", are a denizens

of the deeper part of the oceans, where they produce fragile thin shells and pursue an actively carnivorous lifestyle. Amongst them, Catillopecten

natalyae from the abyssal plain (4,860-5,680 m depths) adjacent to the Kuril-

Kamchatka and Japan trenches (Pacific Ocean) produce an extraordinary ornament of intricate processes with multiple projections, looking like tiny

aerials and antennae. They are interesting because of their intricacy and the

fact that they are calcitic rather than the more typical aragonitic nature of intraperiostracal calcifications. Using a combination of SEM and EBSD we

show that that these intricate structures are produced entirely within the periostracum and must form in a way that allows growth in a progressively

outwards direction (i.e. entirely different from normal secretion whereby

growth occurs inwards from the surface of the shell). Each aerial is crystallographically continuous with the underlying prism. The stem of each

aerial is shown to be parallel to the c-axis of calcite and the typical three-fold

bifurcations of the antennae correspond with the three short diagonals of the calcite rhombohedra. There may be multiple aerials from a single prism in

the outer layer of the shell, and those of a single prisms show a remarkable

co-orientation, whilst those on adjacent prisms show a slightly different orientation. We will propose a model for the formation of these intricate

aerials and also to compare these with other forms of intraperiostracal

calcification in the Bivalvia. The function of these fascinating structures remains unknown!

We acknowledge Russian Foundation for Basic Research (grant no. 19-04-

00281-a) and CGL2017-85118-P (Spanish Ministry of Economy, Industry and Competitivity)

T 8

The dynamics of structural mesoscale dislocations in nacre M. Beliaev*1, I. Zlotnikov1 1Technische Universitaet Dresden, B CUBE - Center for Molecular Bioengineering, Dresden, Germany

Molluscan shells ultrastructures are a paradigm of complex hierarchical

biocomposite structures formed in the course of extracellular biomineralization. Morphogenesis of these ultrastructures is known to follow

thermodynamically driven self-assembly processes in accordance with the principles of classical materials science. Sheet nacre ultrastructure is a highly

regular and periodic architecture composed of thin organic membranes and

aragonitic layers, which are made of flat mesocrystalline platelets. Similar to classical crystal growth, nacre deposition is accompanied by incorporation of

two-dimensional defects, such as dislocations and twinning, that are integral

to its formation process. However, in contrast to a generic atomic-scale lattice, in sheet nacre, these defects occur on the mesoscale level.

Specifically, twinning in aragonite is known to be responsible for the shape

of nacre platelets and structural dislocations in the layered assembly are key to its morphogenesis. In the present work, we employ synchrotron-based

nanotomographic imaging combined with machine learning post-processing

techniques to visualize and understand the nature and the dynamics of mesoscale dislocations in the nacre of the bivalve Unio Pictorum in 3D. By

drawing an analogy to processes in classical materials science, we shed light

on the role of structural dislocations and their interaction in sheet nacre formation. This work is a step towards a deeper understanding of

fundamental thermodynamic and kinetic principles that drive the self-assembly of nacre.

T 9

Morphogenesis in the mollusc Atrina vexillum - an epitome for

ideal coarsening D. Zöllner*1, I. Zlotnikov1 1B CUBE - Center for Molecular Bioengineering, TU Dresden, Dresden, Germany

The microstructure of polycrystalline materials is closely linked to many

materials properties. This fact makes the phenomenon of the migration of individual grain boundaries between neighboring crystals and therewith the

coarsening of the whole grain structure a major focal point in classical

materials science. Out of the different growth modes observed in natural and synthetic materials—such as rocks, metals and alloys—the case of ideal

coarsening resp. grain growth is given special attention. Even though the

basic assumption for ideal coarsening—which is homogeneity of the physical properties of the boundaries—seems highly unrealistic, many analytical as

well as numerical investigations focus on this case due to the reduction in

problem complexity. As a result, the large variety of analytical models were rarely found to describe the microstructural evolution of experimentally

measured polycrystalline grain networks. In the present work, it is shown that

biomineralization of the prismatic architecture in the shell of the mollusc Atrina vexillum can be described qualitatively and quantitatively by

conventional thermodynamic, kinetic and topological considerations from

classical materials science. Hence, a biogenic polycrystalline material is presented as an epitome for ideal coarsening.

T 10

The role of residual stresses in biomineral morphogenesis

revealed by 3D dark-field x-ray microscopy V. Schoeppler*1, P. Cook2, I. Zlotnikov1 1TU Dresden, B CUBE - Center for Molecular Bioengineering, Dresden,

Germany 2University of Natural Resources and Life Sciences, Institute of Physics and Materials Science, Vienna, Austria

Residual internal stresses occur in numerous synthetic, geological and

biogenic crystals having desirable or undesirable effects on materials

performance. Specifically, a number of recent studies have demonstrated the significance of residual stresses in the mechanical functionality of a number

of biomineralized tissues. However, the role of these forces in biomineral

morphogenesis was never previously examined, mainly due to the lack of an appropriate characterization approach, which requires spatial and

crystallographic characterization of the mineral on several length scales.

Most of the state-of-the-art methods are either surface techniques yielding 2D information or allow only limited 3D analysis of very small sample

volumes.

In this work, we employed the recently developed technique—dark-field x-ray microscopy—to study the relationship between residual stresses and

crystallographic properties of biogenic calcite in the prismatic ultrastructure in the bivalves Pinna nobilis and Pinctada nigra in 3D. This method,

developed at the European Synchrotron Radiation Facility (ESRF), utilizes

magnifying refractive lenses to map the intensity profile of hard x-rays diffracted from crystalline materials with an angular resolution of 0.01° and

allows to analyze millimeter sized samples with a spatial resolution of 50 nm.

Whereas the prisms in P. nobilis have an almost perfect single crystalline character, the growing prisms in P. nigra gradually change their

crystallographic orientation and split into sub-prismatic domains. Due to the

high angular resolution of the method, we were able to obtain unprecedented detail on the mosaicity of prisms in the two organisms and to demonstrate a

correlation between internal lattice strains and local crystallographic

properties of biogenic calcite in 3D. By comparing the experimental data from the two species, we not only shed a new light on the relationship

between structure and texture during the formation of biomineralized tissues,

but also demonstrate the role of internal stresses in biomineral morphogenesis.

T 11

Stabilizing the shell as an inducible defense - morphological,

microstructural and chemical shell-modifications in the

freshwater snail Physella acuta in response to different crayfish

species H. Eck*1, A. Schenk1, V. Grün1, C. Laforsch1 1University of Bayreuth, Animal Ecology I, Bayreuth, Germany

Predator induced morphological defenses are a widespread phenomenon in

the phylum of mollusks. It has been previously demonstrated that mollusks respond to shell-crushing crustacean predators with changes in their shell,

6

including shell size, weight and thickness. These changes in shell morphology are often associated with an increased shell stability, i.e. shell

crush resistance. However, there is still very limited understanding of the

mechanism, by which such an adaptive increase in mechanical stability is achieved, e.g. microstructural or chemical modifications of the shells.

Moreover, it remains elusive whether there is a specificity of predator

kairomones (semiochemicals released by the predator that allows prey organisms to detect the predator), i.e. if mollusks can differentiate between

closely related crustacean predators and adjust the expression of inducible

morphological defenses accordingly. Therefore, we studied if freshwater snails express different inducible morphological defenses when exposed to

different crayfish species. Furthermore, the chemical and microstructural

modifications, leading to an increasing crush resistance in shells of predator-exposed snails were analyzed. In this study, we particularly focus on the

inducible morphological defenses in the acute bladder snail Physella acuta

in response to the marbled crayfish Procambarus virginalis and the signal crayfish Pacifastacus leniusculus. The snails were exposed to kairomones

from the two crayfish species until they reached maturity. Subsequently, the

morphology, weight and stability was compared across treatments. The shells were then analyzed for predator induced changes in microstructure and

chemical composition using a wide range of analytical methods including

scanning electron microscopy (SEM), Raman- and Fourier-transform infrared (FTIR) spectroscopy, powder x-ray diffraction (PXRD), small-angle

x-ray scattering (SAXS) and thermogravimetric analysis (TGA). Our results

demonstrate that P. acuta responds to both predators with the expression of inducible defenses. Compared to non-exposed individuals, crayfish exposed

snails characteristically show an increased shell size and weight, a narrower aperture of the shell and an increased crush resistance of the shell.

Interestingly, individuals from both predator treatments do not differ

significantly from each other. Regarding chemical modifications, shells of crayfish exposed snails exhibit higher volumes of carotenoids, while there

are no quantitative differences in the other components of the organic or

inorganic fraction. The microstructure of the shell did not show pronounced differences between non-induced and crayfish induced snails. Our study

demonstrates that P. acuta expresses inducible morphological defenses in

response to two different crayfish species. The observation that the expression of the defensive traits did not differ between the two crayfish

species provides first indications that there may exist a universal crayfish cue.

T 12

Mural mechanics and morphology of cephalopod shells -

adaptation or artifact? R. Lemanis*1, D. Stier1, I. Zlotnikov1 1Technische Universität Dresden, B CUBE – Center for Molecular Bioengineering, Dresden, Germany

Cephalopods have exploited their biomineralized skeleton over the course of hundreds of millions of years for the purposes of buoyancy, hydrodynamics,

and protection against predation. Over this time, they have explored a huge

disparity of ultrastructures and shell geometries that reflect constructional and ecological boundaries. Testing the biomechanics of these structures

allows us to begin to untangle the evolutionary history and ecology of these

animals. Here, we aim to test the potential function of mural modifications in coleoid cephalopods using comparative computational mechanics. The

spiral cephalopod phragmocone is a hollow tube divided into chambers by

mineralized walls; the attachment zone between these walls and the shell is the mural zone. The only extant coleoid with a spiral phragmocone, Spirula

spirula, is imaged using high resolution computed tomography and used as a

basis to construct a representative coleoid shell model. Mechanical data from nanoindentation from the Spirula shell are used to inform finite element

models to test how the presence of mural modifications affect stress and

strain distributions due to hydrostatic pressure. Indentation data reveals a significant difference between the moduli of the shell and septa, however not

one large enough to arrest cracks through the region. Furthermore, the septa

show a stiffness gradient whose origins are not currently understood. Shells with and without the mural flap show no differences in their response to the

applied pressure, while the rear, adapical flaps seem to redistribute peak

stress values away from the attachment site and onto the stiffer shell wall. We are able to show that mural modifications have only a minor effect on the

mechanics of the shell. The mural flap, a defining character of Decabrachia,

seems to have no apparent mechanical function. This result challenges prior hypotheses that proposed the mural flap as an adaptation to deeper water

depths that facilitated the ecological separation between Decabrachia and

belemnites.

T 13

In-Situ TEM of calcium carbonate mineralization in the presence

of L-aspartic acid M. Longuinho1, N. Peña2, D. Ihiawakrim2, M. Farina*3, A. Rossi4, O.

Ersen2 1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 2Institut de Physique et Chimie des Matériaux de Strasbourg, Strasbourg,

France 3Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 4Brazilian Center for Physics Research, Rio de Janeiro, Brazil., Rio de Janeiro, Brazil

Introduction

Biomimetic syntheses are widely performed in the development of new

materials for pharmaceutical purposes and tissue regeneration, besides seeking to reproduce in vitro biomineralization processes. A better

understanding on how mineralization occurs in biological environment

allows the synthesis of materials with special properties, which ensure the reliability of the production. Among the biominerals, calcium carbonates

(CaCO3) are the most abundant, being present in primitive to complex

organism like the spicules in calcareos sponges and otoliths in the inner ear of vertebrates. Therefore, CaCO3 minerals are of great interest for

fundamental studies as a model for nucleation, growth and crystallization of

biogenic minerals. A great challenge in the biomineralization field is the understanding of the initial stages of the process, since the formation of a

critical nucleus, what occurs in a short period of time and at the molecular

level. This makes difficult the direct observation of this phenomenon with routine analytical imaging techniques. To overcome this restriction, we used

in situ techniques that allowed the observation of crystallization in the

aqueous medium in real time, like morphological changes, crystallinity and structural dynamic. Specifically, in situ Transmission Electron Microscopy

(in situ-TEM) enables acquisition of images and videos at Angstrom

resolution, as chemical and structural information during the development of mineral phases.

Objectives

The present work aims to understand how organic molecules, specifically aminoacids, influence CaCO3 biomineralization. As described in the

literature, there are specific proteins directly related to the biomineralization

which are capable of influencing the structure and kinetic of CaCO3 crystallization due to polarity, electrophilicity and presence of sulfate or

phosphate groups.

Material & methods

The experiment was carried out by mixing CaCl2 and Na2CO3 solutions,

with and without L-aspartic acid, and then the mixture was dropped in a chip of the in situ sample holder (Protochips) and analyzed in a JEOL-2100F

TEM.

Results & conclusion

Preliminary results show a region more electron dense, probably with

accumulation of ions where crystalline nuclei are formed. However, most of

them undergoes dissolution, before reaching a critical size (70 to 120 nm) in the samples without addition of the amino acid. The dissolution may have

happened because of the higher energy of the system leading to instability or

because of the energy of the electron beam during the analysis. In the experiment containing L-aspartic acid, it was also possible to observe

crystals with calcite-like morphology and organic vesicular-like structures

containing crystalline material. The existence of nanocrystals inside vesicular-like structures suggests that the amino acid can accumulate ions

inside these structures, inducing the precipitation of crystals. Such

phenomena are described in micron and millimeter scale using polymers and polypeptides as polymer induced liquid phase (PILP), however, never

described for small molecules like aminoacids forming nanoscale crystals.

Further studies will be carried out to better understand of this mechanism.

T 14

Affimer-directed control over calcium carbonate polymorphs I. Sandei*1, T. Gaule 2, F. Meldrum 1 1University of Leeds , Chemistry , Leeds, United Kingdom 2University of Leeds, Biological Sciences , Leeds, United Kingdom

A key factor in biomineralization is the use of organic molecules to direct the

formation of inorganic materials including silicates and magnetite. Being able to emulate this strategy synthetically and use organic molecules to

control the formation of inorganic materials is therefore a long-standing goal

in materials synthesis and crystallisation studies. Significant efforts have therefore been made to identify molecules that can produce inorganic crystals

with well-defined size, structure and morphology. One approach that has

received particular attention is the screening of libraries of polypeptides to identify individual molecules that are active in directing mineralisation.

There, polypeptides are typically displayed on phage, and active individuals

are selected based on multiple bio-panning rounds to identify strongly-binding individuals. These are anticipated to have a strong effect on

crystallisation.

7

Here, we use a phage-display approach to identify proteins that can direct calcium carbonate formation. In contrast to short polypeptides, these have

well-defined conformations that can be expected to be important in

controlling crystallisation. A 1.3∙1010 library of Affimers was employed, where these comprise a small protein scaffold of 81 amino acids. They are

extremely stable and displays significant beta sheet structure together with

one or two nine amino acid variable regions that display two variable loops. These were displayed on a modified form of the M13 phage major coat

protein (pVIII), and multiple bio-panning rounds were conducted to identify

individuals that bound strongly to calcite and aragonite at different pH conditions.

From the initial phage library, 14 different proteins were selected and

purified. The ones that strongly bound to calcite are particularly rich in basic and nonpolar amino acids in both loops. At pH 8.5 there is a prevalence of

negative charges (as the alpha carboxylic groups are mainly deprotonated)

with a great presence of histidine (H) and arginine (R) residues in Loop 1. For aragonite, we found a significant number of nonpolar amino acids, with

fewer basic residues than calcite and the appearance of aspartate (D) and

glutamate (E) residues in one of the two loops. Moreover, tryptophan (W) is one of the main residues present, together with polar amino acids carrying -

OH groups in the side chain.

Experiments are currently underway to explore the function of these proteins in directing calcium carbonate formation in bulk solution at

different pH values, and in the presence of magnesium ions. The proteins

will also be tethered to surfaces in the form of a monolayer, and their influence on calcium carbonate precipitation determined. This will enable

us to investigate whether the proteins behave differently in solution and when located on a solid substrate.

T 15

Synthetic, prismatic-type CaCO3 films via seeded

mineralization Y. Jiang*1

1Xiamen University, Xiamen University, China

Introduction

Biominerals, by taking advantage of their hierarchical architecture, reconcile

multiple functions which are otherwise contradictory in their synthetic counterparts. From the synthetic point of view, the delivery of hierarchical

architecture can be ascribed to the active roles of (multiple) soft matter,

which turns the mineralization proceeding in organism into self-organization processes. The control of a self-organization process for the delivery of life-

feathered synthetic minerals nonetheless remains technically challenging in

biomimetic mineralization.

Objectives

The primary focus of our work is to design specific microenvironment based

on the organization of multiple types of soft matter for the delivery of prismatic-type minerals with distinct architecture and remarkable mechanical

performance.

"Patients & methods" or "Materials & methods" We introduce seeded mineralization for the delivery of prismatic-type

CaCO3 films.

Results

The method starts with the deposition of granular CaCO3–polyacrylic acid

hybrid films on polymer substrates like chitosan, silk fibroin, or polyvinyl

alcohol. Next, an overgrowth procedure leads to prismatic-type CaCO3 overlayers. Our study shows that the presence of different additives can have

a tremendous impact on their morphological outcomes, while the selection

of polymer substrates determines the polymorphic form of the granular seed layer. In our very recent work, we study the impact of hydrogels on the

structural information of the prismatic overlayers, though hydrogels

themselves are not involved in the delivery of biogenic prismatic minerals. This prismatic CaCO3 films can be used for drug delivery applications.

Conclusion

To summarize, the synergistic effect of multiple types of soft matter and the proper use of biomimetic mineralization methods can create favored

microenvironments for the delivery of prismatic-type CaCO3 films in self-

organization processes. These CaCO3 films exhibit comparable mechanical performance to their biogenic counterparts. The rational design of

biomimetic mineralization systems helps for the mechanistic understanding

of biomineralization, and meanwhile, provides a practical tool to design proper materials for biomedical applications.

T 16

Synthesis of pyrite nanoparticles using the matrix protein from

the scaly-foot, Chrysomallon squamiferum T. Yamashita1, H. Matsuda1, Y. Suzuki2, N. Ahsan3, Y. Okada3, M.

Suzuki*1 1The University of Tokyo, Departiment of Applied Biological Chemistry, Bunkyo-ku, Tokyo, Japan 2The University of Tokyo, Deapartment of Earth and Planetary Science,

Bunkyo-ku, Tokyo, Japan 3The University of Tokyo, Research Center for Advanced Science and Technology, Meguro-ku, Tokyo, Japan

Introduction

Chrysomallon squamiferum (scaly-foot) is a deep-sea snail which was

discovered near the hydrothermal vent of the Central Indian Ocean Ridge "Kairei Field". This snail has scaly structures mineralized with iron sulfide

nanoparticles (pyrite, FeS2) on its foot, and the shell is covered with an iron

sulfide layer on the calcium carbonate layer. Pyrite nanoparticles have superior photovoltaic properties to be applied to photovoltaic power

generation in the solar panel. However, the methods of its inexpensive and

green chemical synthesis have not been established. Therefore, new industrial applications using the formation mechanism of pyrite nanoparticles

in the scaly-foot are required. In this research, in order to identify the

substances that have key factors to synthesize the sulfide nanoparticles in vitro, we tried to extract the organic molecules that bind to iron in the scaly-

foot, because the iron-binding organic molecules may have a function to

prevent the aggregation of particles.

Results and Discussion

In this study, we analyzed the organic molecules extracted from the scaly

structure and the shell in the scaly foot and searched for substances related to the production of iron sulfide nanoparticles by interaction with iron.

Pyrite in the scaly structure was dissolved by reducing with zinc and trivalent

chromium under anaerobic conditions. We used HPLC post column chelator method to identify the iron binding organic molecules. This result revealed

that large amount of low molecular weight organic matters having iron

binding ability existed in the scaly structure. This low molecular weight organic molecule was purified using cation exchange resin and reverse

phase-HPLC. We tried to determine its chemical structure using mass

spectrometry and NMR. On the other hand, 1 M acetic acid was used to dissolve the calcium carbonate

layer of the outer shell. Then, the insoluble iron sulfide layer in acetic acid was extracted using SDS-DTT. A specific protein band was detected from

the iron sulfide layer. LC-MS/MS analyses revealed that this band showed

an amino sequence of heme protein. Heme is consisting of tetrapyrrole ring that can make the complex with iron in vivo.

The heme protein may have some roles for the formation of nano pyrite in

vitro. We try to use the heme protein to synthesize an iron sulfide nanoparticles using various conditions.

T 17

Oriented crystallization of single crystalline strontium sulfate

in marine Acantharea V. Merk*1,2, P. Smeets2, J. Walker2, D. Joester2 1Florida Atlantic University, Department of Chemistry & Biochemistry;

Department of Ocean & Mechanical Engineering, Boca Raton, United States 2Northwestern University, Materials Science and Engineering, Evanston, United States

Introduction

Simple organisms, such as sea urchins, calcerous sponges, coccolithophores, or magnetotactic bacteria, pursue highly sophisticated strategies for growing

single-crystalline skeletal units. Acantharia radiolarians present yet another

prime example of single crystal engineering in nature. The marine protists build intricate star-shaped endoskeletons from smoothly curved strontium

sulfate (SrSO4) spicules, whose long axes coincide with the crystallographic

a-axis. Although Acantharia occur in the ocean's zooplankton from tropical to polar zones, the underlying crystallization mechanism remains virtually

unexplored.

Objectives

In this paper, we anticipate addressing the question how crystal texture is

controlled in a biological environment. The poor stability of Acantharia in

culture renders time-resolved observations of the spiculogenesis impossible. To infer the crystallization mechanism of biogenic celestite, we analyzed the

atomic- and nanostructure as well as the distribution of intercalated

biomolecules on various length scales.

Materials and Methods

We investigated the atomic and nanostructure of Acantharian skeletons from

the Solomon Islands using a combination of X-ray diffraction, Raman spectroscopy, electron-optical imaging and synchrotron-based techniques.

The sample preparation for transmission electron microscopy (TEM)

8

included targeted focused-ion beam (FIB) lift-outs from Acantharian spicules, mostly along primary crystallographic axes. Synchrotron X-ray

fluorescence mapping and Sulfur K-edge XANES spectra were obtained at

sector 13-ID-E at the Advanced Photon Source (APS), Argonne National Laboratory. Scanning wide-angle X-ray scattering data were acquired at the

P03 endstation, PETRA III at DESY.

Results

Acantharia grow highly ordered SrSO4 single crystals, whose crystal

structure is almost indistinguishable from geological and synthetic

counterparts. As markers for intracrystalline organic matter, sulfur-bearing amino acids and di-sulfides were detected across the protist"s skeleton by

microbeam X-ray absorption spectroscopy (XANES) at the sulfur K-edge.

Highly anisotropic small-angle X-ray scattering signals correspond to nanoscale electron density variations parallel to the crystallographic a-axis,

which are consistent with buried interphases in the SrSO4 biomineral.

Similarly, scanning TEM Z-contrast imaging showed nanodomains with a lower atomic number around the spicule center, which do not seem to

correspond to light metals (e.g. Na, K, Ca, etc.). In the diffraction contrast

image from the spicule cross-section, we observed concentric layers reflective of fluctuating growth.

Conclusions

Taken together, our findings suggest that biomolecules interact with specific planes and subsequently become introduced into the growing crystal. An

occlusion of specific biomolecules may guide the crystallographic

orientation of primary seed crystals, indicating genetic regulation of the crystal growth process. Furthermore, Acantharia present an excellent source

of bio-inspiration for alkali earth sulfates with tailored mechanical, electronic, and optical properties.

T 18

Influence of stress on biogenic calcite growth J. Colombani*1, B. Zareeipolgardani2, A. Piednoir1 1Université Claude Bernard Lyon 1, Institut Lumière Matière,

Villeurbanne, France 2Université Grenoble Alpes, ISTerre, Grenoble, France

Calcite, the most widespread crystalline form of calcium carbonate, is ubiquitous both in nature and in the industry. In particular, its precipitation

by living organisms under the form of shells is the major CO2 sink inside the

ocean, and it is used in multiple industrial sectors (ordinary cement manufacturing, paper bleaching, toothpastes, etc.). Therefore the knowledge

of its mechanisms of growth, both organic and inorganic, are of foremost

importance, in the modelling of the carbon cycle, in the simulation of oil reservoirs, or as a promising new route for biomimetic materials engineering.

One parameter, although almost always present during calcite growth, has

not been considered in most studies. When calcite grows from nuclei, they

eventually enter into contact, and stresses develop at their interfaces,

particularly if the material grows in a confined environment, for example

inside a living cell. So far, we have no idea on how this stress modifies the growth kinetics, or the morphology of the grown surface. To address this

issue, we have used an atomic force microscope (AFM), both to imply a local stress on a growing calcite surface, and to image it. We have found that the

stress has a double influence: it slows down significantly the growth, and

induces a change of the type of growing phase. Calcite biomineralization proceeds in presence of organic materials, among which amino acids are

widespread. Therefore we have investigated the influence of pentaglycine on

the growth kinetics. This amino acid shows the striking ability to cancel the phase transition induced by the applied force.

T 19

pH regulation in calcifying primary mesenchyme cells of the

sea urchin larva M. Hu*1 1University of Kiel, Institute of Physiology, Kiel, Germany

Question The sea urchin embryo develops an elaborate calcitic endoskeleton that has

been used by biologists to study the mechanisms of biomineralization. Amorphous calcium carbonate is formed in intracellular compartments and

exocytosed into the syncycial cable formed by primary mesenchyme cells

(PMCs). For intracellular precipitation of CaCO3, PMCs require HCO3-/CO32- concentrating as well as proton export mechanisms to promote

calcification. These processes are of fundamental importance in biological

calcification, but remain relatively unexplored even for mammalian systems.

Material & Methods Larval cultures: For our experiments we used the purple sea urchin

(Stongylocentrotus purpuratus) and larval cultures were maintained at 15°C in natural seawater for further experiments.

Recalcification assay: Skeletons of early pluteus larvae (3dpf) were

dissolved in MES buffered natural seawater adjusted to pH 6.0 for 12 h. After transfer to natural seawater (pH 8.1) larvae re-calcified their skeletons within

four days.

Life cell imaging: The pH sensitive dye BCECF_AM and BCECF-FA 10KD dextran were used to monitor intracellular as well as vesicular pH,

respectively. For cellular pH recordings the ectoderm was removed and

ratiometric pH recordings were performed. Immunocytochemistry and westernblot analyses: Localization and

quantification of PMC specific acid-base transporters was performed using

species-specific custom made antibodies. RT-qPCR and whole mount in situ hybridization: Expression levels of PMC

specific acid-base transporters were measured by qPCR and normalized to

the housekeeping gene SpZ12. Whole mount in-situ hybridization was performed to localize transcripts in the sea urchin larva.

Microinjection of GFP constructs and Morpholino knock-down: Micro-

injections were performed to insert morpholinos and BAC-GFP constructs into the fertilized egg (one-cell stage).

Results Here we demonstrate the SLC4 HCO3- transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic

endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary

mesenchyme cells with peak expression during de novo formation of the skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects

accompanied by decreased calcification rates and skeleton deformations. Re-

calcification experiments demonstrated an increase in PMC pHi and substantial elevations in intracellular [HCO3-] during skeleton rebuild driven

by SpSLc4a10. Despite overall reductions in proton export capacities of

PMCs in this phase, large filopodial cells containing acidic vesicles connect to the PMC syncytium and locally elevate pH regulatory capacities. These

cells do not express ALX1 a specific marker for skeletogenic mesenchyme cells. The process of skeleton rebuild and vesicular acidification in PMC

associated cells is sensitive to the V-Type-ATPase inhibitor bafilomycin.

Conclusion Intracellular accumulation of bicarbonate is a fundamental mechanism of

calcification in PMCs of the sea urchin embryo to develop and rebuild the

calcitic endoskeleton. We propose that during this phase PMCs shift from a NHE-based proton export mechanism to a vesicular storage of protons

generated through extensive precipitation of CaCO3. Non-PMC cells

associated to the skeletogenic syncytium potentially play a critical role in storage and removal of protons during extreme calcification efforts. These

results highlight the importance to better understand pH regulatory processes

in calcifying systems including cell-cell interactions and acid-base transport on the cellular and sub-cellular level.

T 20

The formation of fenestrations in Eucidaris tribuloides

embryonic spicules J. Walker*1, B. Moreno1, D. Joester1 1Northwestern University, Materials Science and Engineering, Evanston, United States

Skeletal patterning in sea urchin embryos is an important model system for

investigating biomineralization of calcium carbonate. Harnessing a tightly controlled growth mechanism, the organism is able to direct the formation of

single crystals that have complex curved and branched morphologies with

precise crystallographic directions. These single crystals of calcite, known as spicules, begin growing from a seed rhombohedral calcite crystal. The

deposition of these spicules and patterning of the skeletal structure is

controlled at the local level by the action of primary mesenchyme cells (PMCs). They fuse to create a syncytium, or privileged space, in which the

mineral is deposited and grows. The pattern of the skeleton is species

specific, and PMCs have been shown to be able to autonomously direct skeletal design including the formation of features such as fenestrations.

These are regularly spaced holes within the single-crystalline central rod that

is normal to the spicule direction, a property that is thought to increase the stiffness-to-weight ratios and fracture resistance of the structure. Replication

of this ability to produce consistent periodic features at multiple length scales

is vitally important for the creation of synthetic materials with specific and tunable properties.

In this work we investigated the formation of fenestrations in the embryonic

spicules of Eucidaris tribuloides, a tropical sea urchin, and the effect their presence has on crystallographic and morphological features of the spicule.

Spicule formation in both embryos of E. tribuloides and S. purpuratus were

investigated. Embryos from each species were separately grown in culture. Spicules were extracted from the embryonic cultures and examined by SEM

at Northwestern University Atomic and Nanoscale Experimental Centre

(NUANCE). The identified features were then further investigated by synchrotron white beam X-ray diffraction mapping using a 250 nm beam

size, which was obtained at beamline 34-ID-E at the Advanced Photon

Source, Argonne National Laboratory. SEM imaging of the extracted spicules at different time points allowed a

detailed visualization of the morphology of the fenestrations as the spicule

develops. Investigation of an individual spicule using white beam X-ray diffraction mapping enabled us to correlate the fenestrations to changes of

direction of the lattice planes within the crystal. Using a beam size of 250

9

nm it was possible to investigate these microscale features and observe changes to the (001) plane relating to the location in the spicule. Although

there was a high homogeneity in plane direction as would be expected for a

single crystal, small changes (>0.1 degrees) in rotation were identified in regions surrounding a fenestration.

White beam X-ray diffraction mapping can be used to correlatively with

electron microscopy to demonstrate local changes in lattice planes in and around micron-sized features. It can efficiently provide detailed

crystallographic information over a larger scale than other techniques such

as TEM, which can narrow down the area of interest for investigation using higher resolution but more labour intensive techniques. We have used it to

investigate lattice plane changes related to the formation of fenestration in

the growing spicule of a sea urchin embryo.

T 21

Effects of seawater Mg2+/Ca2+ ratio and diet on the

biomineralization and growth of sea urchins - applicability of

echinoderms in paleoenvironmental reconstructions D. Kołbuk*1, P. Dubois2, S. Di Giglio2, S. M’Zoudi2, J. Stolarski1, P.

Gorzelak1 1Polish Academy of Sciences, Institute of Paleobiology, Warsaw, Poland 2Université Libre de Bruxelles, Faculté des Sciences, Brussels, Belgium

It has been suggested that the skeletal Mg/Ca ratio of well-preserved fossil

calcifying marine invertebrates, including echinoderms, was predominantly

affected by the changes of Mg2+/Ca2+ ratio in the ancient seawater, which has varied between ~1.0 to 5.2 throughout the Phanerozoic. However, it has been

also demonstrated that the chemical composition of Recent and fossil echinoderms can be modified by a number of other factors, such as

temperature, salinity, metabolic processes, diet, and diagenesis. In this work,

we addressed the problem of diet vs. seawater chemistry impact on skeletal Mg/Ca ratio, in experimental conditions. Two phylogenetically distant

echinoid species, Psammechinus miliaris and Prionocidaris baculosa, were

cultured in low Mg2+/Ca2+ (~2.5 and 1.5) artificial seawater, prepared by lowering the Mg2+ and increasing the Ca2+ concentrations. Before the sea

urchins were exposed to low Mg2+/Ca2+ conditions, they were tagged with

manganese in order to identify newly-formed skeleton areas under cathodoluminescence (CL) and calibrate their growth rates. After tagging,

the echinoids were incubated for 21 days in seawater with different

Mg2+/Ca2+ ratios (~5.2, ~2.5, ~1.5 [mol/mol]) and fed with diets containing different amounts of magnesium (~0.3 wt% or ~20 wt%). Mg/Ca ratios in

the newly formed skeleton in different types of echinoid ossicles (test plates,

spines, teeth, and demipyramids) were determined using electron microprobe. Mg/Ca ratios in all ossicle types of both species decreased

proportionally with decreasing Mg2+/Ca2+ ratio of seawater, in which they

were growing. However, echinoids fed with magnesium-enriched diet typically displayed higher skeletal Mg/Ca ratios. Growth rates in some

ossicles (test plates and demipyramids) significantly decreased with the

reduction of ambient Mg2+/Ca2+ ratio. These results underscore the importance of diet in modulating Mg/Ca ratio of echinoderm skeleton and

suggest that caution should be exercised when using the skeletal Mg/Ca ratio

of fossil echinoderms as a direct paleoseawater Mg2+/Ca2+ proxy. [This research was funded by the National Science Centre (NCN) grant no.

2016/23/B/ST10/00990].

T 22

Genetic control over biomineralization in calcareous sponges O. Voigt*1, B. Fradusco1 1LMU Munich, Department of Earth- and Environmental Sciences, München, Germany

The ability to produce biominerals has been a key innovation in animal

evolution and allows animals to generate as essential structures as shells,

teeth and skeletons. The genetic mechanisms underlying the biomineralization process and their evolution are only poorly understood. It

seems that biomineralization evolved serval times in different animal linages,

but still utilize similar general principles and genetic pre-adaptations. We study biomineralization in the class of calcareous sponges, which belong

to the oldest animal phylum. These sponges gained the ability to produce

calcite spicules of different shapes in contrast to other sponge classes, which have siliceous spicules. They provide a simple system of

biomineralization: Calcareous sponges produce numerous spicules in a short

time (hours to days), and each is formed by only a few (e.g. two to seven) specialized cells.

Therefore, we aimed to identify the genetic mechanisms that control the

biomineralization system in calcareous sponges to understand general principles of biomineralization.

We applied a combination of genomics, transcriptomics, proteomics and

RNA in situ hybridization to identify key biomineralization genes of calcareous sponges and to obtain information about the spatial and temporal

expression during the spicule formation process.

Key components of the genetic biomineralization toolkit were identified, including carbonic anhydrases, bicarbonate transporters and secreted spicule

matrix proteins. We observed changes in the spatial and temporal expression

of these genes during the spicule formation process. Some matrix proteins are only produced by sclerocytes that form spicules of a specific shape.

Our results highlight the essential role of genetic control over the

biomineralization process even in as simple structures as the calcite spicules of calcareous sponges. Comparative analyses with multiple species will help

to reveal the genetic key-innovations obtained by the last common ancestor

of these sponge class.

T 23

Crystallographic misorientation between spicules in the

skeleton of the calcareous sponge sycettusa hastifera L. Souza Coelho1, M. Klautau2, M. Farina3, A. Rossi*1 1Brazilian Center for Research in Physics, Rio de Janeiro, Brazil 2Federal University of Rio de Janeiro, Biology, Rio de Janeiro, Brazil 3Federal University of Rio de Janeiro, Biomedical Science, Rio de Janeiro, Brazil

Introduction

The skeleton of calcareous sponges (phylum Porifera, class Calcarea) is

formed by Mg-calcite spicules ranging from approximately 10 to 2000 μm in

length and usually composed of two, three or four (rarely five) conics rays joined at the base [1,2]. One individual spicule behaves as a monocrystal

(trigonal system) with only few degrees of misorientation (<1°) [1,3]. The

species Sycettusa hastifera belong to the subclass Calcaronea. In this subclass the unpaired actine of tri and tetractine spicules is elongated

approximately in the [211] direction. The crystallographic direction of the

actines are conserved through the evolution of the classes. In the class Calcinea, the actines are elongated in the {210} directions. The spicules

normally have a specific position in the body of the sponge (cormus) and are

used as a taxonomic character. S. hastifera has triactine and tetractine spicules in the cortical, subcortical, subatrial and atrial regions of the

choanosome of the sponge.

Objectives

In this work, the crystallographic misorientations between spicules in the

cortex, subcortical, subatrial and atrial regions were identified in different

transversal sections of the tubular structure of the sponge (base, middle and top of the sponge tube) and in different area of each section of the tube. The

aim of this study is to relate the misorientation between spicules with the process of growth of the sponge skeleton and the resistance proprieties of the

aquiferous system tubes.

Materials and Methods

Samples from S. hastifera were collected in Arraial do Cabo city, Rio de

Janeiro, Brazil. The sponges were dehydrated with increasing concentrations

of ethanol and infiltrated with epoxy resin. The sponge embedded in resin was cut transversally to the tube direction using a diamond saw. Transversal

sections of the sponge were polished with alumina 1μm and then coated with

a thin carbon layer. Electron Backscatter Diffraction (EBSD) were performed in a Scanning Electron Microscope Jeol 7100FT equipped with an Oxford

EBSD detector. Kikuchi line patterns were recorded using Aztec and

Channel 5 softwares. Each transversal section was divided into four areas (1, 2, 3 and 4) from where cortical, subcortical, subatrial and atrial spicules were

studied. Stereographic projections were generated to study misorientation

between spicules. Spicule types were identified using an inverted optical microscope.

Results

The subcortical spicules were the less oriented spicule type from S. hastifera. The absence of a preferential orientation may be related with the existence of

pseudotriactine spicules which present similar morphology but different

crystallographic orientation compared to a conventional triactine. All spicule types in the middle section of the sponge tube were more well oriented than

in the base of the sponge. This result is related with the process of growth of

the sponge tube. The transversal section of the sponge tube had an oval symmetry. The areas of the section superposed to the larger diameter

presented spicules more well oriented compared to the areas in the smaller

diameter of the tube. The same spicule type may have different orientation in different area.

Conclusion

The spicule orientation in the cormus of S. hastifera present more or less misorientation depending on the transversal section of the sponge tube (base,

middle or top), the spicule type and the area of the transversal section.

10

T 24

Paradoxical mechanical properties of sponge spicules J. Werckmann*1, E. Bouzi2, E. Brodu2, M. Longuinho1, A. Porto Careiro3,

D. Ihiawakrim4, A. Rossi1, M. Klautau5, M. Farina5 1Brazilian Center of Physic Researsh, COMAN, Rio de Janeiro, Brazil 2University, Metz, France 3University, Marseille, France 4CNRS, Strasbourg, France 5University Fédérale, Rio de Janeiro, Brazil

Our study focuses on calcareous sponges. Their three-dimensional structure

is ensured by the entanglement of spicules within their soft cylindrical body,

composed mainly of collagen. These spicules have the distinction of being biogenic single crystals consisting of magnesium calcite. One of their

paradoxical properties is the conchoïdal fracture caused by mechanical stress,

whereas mineral calcite has fractures determined by {104} cleavage planes. The purpose of our work is to highlight the factors that are behind this

property. Thus we have implemented several characterization techniques

associated to transmission electron microscopy. Specimens of the species Paraleucilla magna were immersed in a 2.5% sodium hypochlorite solution

to completely remove the adhered organic tissue. The spicules were rinsed

with distilled water followed by 100% ethanol then air dried. Thin spicule cross section were obtained by Focused Ion Beam (FIB). Briefly, the

spicules were dispersed on a silicon substrate and metalized with a 200 nm

gold layer for surface protection and electrical conductivity. To improve the

protection, a 1 µ Pt layer was in situ deposited. To avoid ion damages,

progressive thinning process was implemented. To complete the process, a

current of 30 pA at an energy of 5 keV was used to clean and reduce the amorphous damaged layer produced during the former steps. Titan

transmission electron microscope operating at 300keV was used. The

electron diffraction obtained on sections extracted on the same spicule, whatever their orientation, are interpreted as originating a single crystal. In

addition, the high resolution images of the external spicule-membrane

interface show the presence of nanocluster attached or close to the surface of the spicule. Their FFT can be interpreted as coming from calcite

nanocrystals. The dark field image obtained when the microscope operates

in Scanning Transmission Electron Microscopy (STEM) mode reveals the existence of significant porosity. Transmission Kikuchi diffraction (TKD)

was implemented in a scanning microscope working at 30keV for mapping

the mosaicity. Before observation a thin layer of platinum was deposited to enhance the electrical conductivity of the sample and to protect them to the

beam damages. Spatial resolution was de of 10nm (pixel size) and the angular

resolution reached about 0.01°. Thanks to this angular precision, the maps of disorientation obtained show that the domains of the mosaic are smaller than

or equal to 10 nm. Based on our experimental results and bibliography (1, 2)

we conclude that the growth of spicules is a discontinuous process that leads

to the formation of a porous structure and that it is controlled by proteins that

catalyze the formation of nanocrystals that are deposited almost epitaxially

on the spicule in formation, resulting in the formation of a mosaic of very low disorientation (0.01°), undetectable by electron diffraction. During

growth a small amount of protein is included by chance in the spicules. These three factors: porosity, mosaicity and inclusion of proteins increase the

resistance of calcareous spicules to the mechanical stress entailing the

conchoidal fracture. This ensures a better resistance of the sponges to the mechanical stresses due to the ebb and flow of the sea and on the other hand

a better resistance to the action of predators.

1) Rossi et al. Acta Biomaterialia (2014)

2) L. Freeman et al. Angewandte Chemie (2010)

T 25

Rice plant biomineralization- Ultrastructure of biosilica, and

defense protein N. Ozaki*1,2, R. Abiko1, K. Tsuji1, T. Ishida1, M. Suzuki3, F. Nudelman2 1Akita Prefectural University, Department of Biotechnology, Faculty of

Bio-resource Sciences, Akita, Japan 2The University of Edinburgh, School of Chemistry, Edinburgh, United Kingdom 3The University of Tokyo, Graduate School of Agricultural and Life Sciences , Tokyo, Japan

Biologically formed amorphous silica (biosilica) are widely found in

bacteria, diatoms, marine sponges, terrestrial higher plants, some of which have been well characterized. Macromolecules in biominerals are known to

control mineralization. Biosilicas in diatom and marine sponge, are formed

under mild condition and neutral pH using unique macromolecules, such as silaffin, glassin, and long chain polyamines. The typical example of silicon

accumulating higher plants, gramineous plants (rice plants), produces a large

amount of biosilica in their leaf blades and rice husks. Although biosilicas deposited in rice plants play various important roles, such as enhancing

mechanical strength, improving disease resistance and photosynthetic activity, information on the molecular mechanisms involved in the biosilica

formation is very limited. In this study, we investigated the microstructure of biosilicas from rice plants (Oryza sativa) by using scanning electron

microscopy (SEM) and Cryo-FIB SEM. In addition, we extracted organic

matrices from biosilicas and characterized by LC-MS/MS analysis. SEM analyses revealed that the silicas of rice plants are composed of nanoparticles

that are several dozen nm in diameter, similar to diatom silica and to siliceous

spicules of sponge. We extracted a 11 kDa protein from silica from the leaf blades and a 140 kDa protein was identified as the only organic component

contained in rice husk"s silica. Protein Database searches revealed that the

11 kDa proteins belong to plant defense proteins known as LTPs, and the 140 kDa protein has an amino acid sequence similar to glucanase. Both proteins

are cationic proteins present in abundance in higher plants and are known to

play important roles in resistance to abiotic and biotic environment stress. Given their cationic nature, we hypothesize that they are involved in

regulating silica formation in the rice leaves and husks. Therefore, we are

currently investigating their function in controlling silica biomineralization.

T 26

The biology of silica deposition in leaf silica cell - a first full

description of plant bio-mineralization process R. Elbaum*1, S. Kumar2 1Hebrew University of Jerusalem, Institute of Plant Sciences, Rehovot,

Israel 2Weizmann Institute of Science, Plant & environmental sciences, Rehovot, Israel

Biomineralization in animals is a very common strategy creating skeletal elements. In plants, however, body design is flexible, and each organ is self-

supported by cell walls. Plant minerals are typically utilized in defense

mechanisms. Silica is a very abundant mineral in plants that may constitute 10% of the dry weight of certain tissues. Most astonishingly, very little is

known about the control over silica formation in plants. We discovered the

mechanism for silica deposition in epidermal silica cells of sorghum (Sorghum bicolor) leaves. Silica cell are almost completely filled with silica

when mature. We showed that silicification is confined to elongating leaves,

in a well-defined active silicification zone (ASZ). The mineralization initiates in live cells. Silica starts to form at the cell"s periphery, adjacent to

the primary cell wall. In contrast to other biomineralization processes, the

mineral is not formed inside vesicles. Instead, the silica cell protoplasts synthesize and secretes a unique protein that accelerates silica precipitation.

The protein is packed in vesicles and is secreted to the extracellular space, which is saturated with silicic acid. The silicic acid precipitates into silica,

creating a secondary wall inward to the primary cell wall, and restricting the

protoplast"s space. The thickening silica wall fills up the cell volume within a few hours. Alongside this fast process, the silica cell goes through

programmed cell death, the cell contents are possibly evacuated to

neighboring cells. This is a first description of silica deposition in plants.

T 27

Imaging and analysis of internal silicon pools in diatoms A. Gal*1 1Weizmann Institute of Science, Plant and Environmental Sciences, Rehovot, Israel

Diatoms are abundant unicellular algae that cover themselves with a cell wall

made of silica. The silicification process is under strict biological control, to

the extent that the delicate nanoscale architecture and fine ornaments are species-specific. The polymerization of silica from its soluble building

blocks is thought to take place inside a specialized organelle that is

responsible for precipitating the mineral phase and controlling its morphology. Our understanding of the inorganic precipitation mechanism of

diatom silica is still rudimentary, as conventional imaging and analytical

tools are inadequate to resolve the native-state structural information and chemistry related to the silicification process. For example, it is unclear what

are the mechanisms of silicon uptake and concentration from the

environment, and what are the conditions inside the silicifying organelle that

give rise to the precise morphology.

We use a suite of cryo electron microscopy techniques in order to extract intracellular structural and chemical information with nanoscale resolution.

3D serial imaging of whole Thalassiosira pseudonana cells, using cryo

focused-ion-beam scanning electron microscopy (cryoFIB-SEM), and energy dispersive spectroscopy (EDS), shows that the cells maintain high

intracellular concentration of Si throughout the cell cycle. In addition,

scanning transmission electron tomography (STEM tomography), enables us to visualize in 3D the ultrastructure of the silica deposition vesicle along the

silicification process. In the species Chaetoceros tenuissimus we followed

the formation of long silica needles with cryo electron tomography (cryoET) and discovered a new mechanism for the deposition of silica. Overall, our

direct approach to study the formation of diatom silica in situ by means of

advanced microscopy tools is yielding detailed understanding of the cellular controls that shape the silicification process in diatoms.

11

T 28

Control of biosilica morphology and mechanical performance

by the conserved diatom gene Silicanin-1 S. Görlich1, D. Pawolski1, I. Zlotnikov1, N. Kröger*1 1TU Dresden, B CUBE, Dresden, Germany

Diatoms represent a large group of unicellular, eukaryotic microalgae that

are most well known for their ability to produce cell walls made of

nanopatterned porous biosilica. The species-specifically patterned cell walls are paradigms for biological mineral morphogenesis and the evolution of

lightweight materials with exceptional mechanical performance. The

formation of biomineral building blocks often takes place within specialized intracellular vesicles, which in diatoms are called Silica Deposition Vesicles

(SDV). Recently two families of SDV membrane proteins have been

identified that are well conserved throughout all diatoms and may therefore play a fundamental role in diatom cell wall formation. One promising

candidate is Silicanin-1 (Sin1), which was shown to have moderate silica-

formation activity in vitro that is enhanced by the addition of long-chain polyamines. It was therefore speculated that Sin1 may influence the assembly

of biosilica-forming biomolecules within the SDV lumen. Here we describe

the CRISPR-Cas9 mediated gene knockout of Sin1 in Thalassiosira pseudonana. Although the mutants grew normally, the knockout cell lines

exhibit a reduced biosilica content and showed pleiotropic effects on silica

morphogenesis, which drastically compromised the strength and stiffness of their cell walls. These results identify Sin1 as a key player in the development

of specific structural features of the T. pseudonana cell wall and as essential

for the biogenesis of mechanically robust diatom cell walls, thus providing an explanation for the conservation of this gene throughout the diatom realm.

T 29

Investigation of the hybrid nanocomposites of the chiton tooth

stylus to find inspiration for new material development L. Stegbauer*1, E. E. Alp2, I. Moudrakovski3, P. Smeets1, R. Free1, S.

Wallace1, M. Hersam1, D. Joester1 1Northwestern University, Materials Science and Engineering, Evanston, United States 2Argonne National Laboratory, Advanced Photon Source, Sector 3, Lemont,

United States 3Max Planck Institute for Solid State Research , Stuttgart, Germany

Introduction

Organisms possess unparalleled control over the structure and properties of

mineralized tissues such as teeth and bones, creating curved single crystals

as well as tough and lightweight self-repairing skeletal structures. Organic matrices play an integral role in the selective formation of metastable mineral

precursors and their transformation into the final biomineral. The chiton is a

model system for the extracellular, matrix-mediated iron oxide.[1] Its radula is a ribbon-like rasping tongue with many rows of extremely hard, wear-

resistant, and self-sharpening teeth designed to withstand the stresses of

grazing algae on rocks.[2] So far, there have been numerous studies on the cuspid heads of the tooth, which consists of one of the hardest and most wear-

resistant biogenic materials, a magnetite/chitin composite. The underlying base, also called stylus, has not been investigated.

Objectives

In this study we aim to establish a better understanding of the biominerals in the radula tooth stylus of the chiton Cryptochiton stelleri. Modern material

fabrication requires large amount of energy to synthesize materials for

semiconductors, medicine, or construction, often employing extensive top-down fabrication steps at high temperatures and pressures. Biominerals can

serve as inspiration for finding innovative methods and new materials and

may also allow to decrease the carbon footprint of these processes. Such knowledge could inform the synthesis of new nanocomposite materials with

outstanding properties.

Materials and Methods

A comprehensive characterization of the stylus was performed with a suite

of techniques that provide insights into structure and compositions at

multiple length scales, including synchrotron Mößbauer spectroscopy (SMS), solid-state nuclear magnetic resonance (ss-NMR), X-ray

spectroscopy and electron imaging. Mechanical properties such as hardness

and reduced modulus were tested by nanoindentation. Results: We find evidence that the cusp-adjacent stylus is mineralized by

santabarbaraite, an amorphous iron hydroxy phosphate that was not known

to occur as a biomineral. The mineral occurs in very small nanoparticles adjacent to α-chitin nanofibers. The stylus possesses outstanding mechanical

properties that are comparable to steels. We propose that the organism

establishes complex spatial gradients in terms of the degree of mineralization (10-25 wt%) and mechanical properties across the stylus. Based on our

insights, we formulated bio-inspired inks for additive manufacturing,

demonstrating that the mechanical properties can be tuned over a wide range also in in vitro.

Conclusion

The stylus can bridge the gap of the vastly different mechanical properties of

the hard head and the radula membrane by varying the amount of deposition

of the biomineral santabarabaraite. The discovered mineralized component, the stylus of the chiton, is a biological nanocomposite material that exhibits

interweaved and co-aligned nanosized chitin fibers with nanosized

amorphous iron phosphate particles in a string-of-pearls manner. The combination of iron phosphate and chitin has impressive mechanical

properties over a wide tunable range which surpass many man-made

materials that require energy intensive manufacturing such as light-metal alloys. Deliberate selection of synthesis conditions inspired by the results of

the biomineral provide us with an artificial composite material that closely

resembles the stylus. The fabricated bulk AFP/chitosan is biodegradable, non-toxic, and processed at room temperature.

[1] L. R. Brooker, J. A. Shaw in Advanced topics in biomineralization,

IntechOpen, 2012, pp. 65–84. [2] a) J. C. Weaver, Q. Wang, A. Miserez, A. Tantuccio, R. Stromberg, K.

N. Bozhilov, P. Maxwell, R. Nay, S. T. Heier, E. DiMasi et al., Materials

Today 2010, 13, 42; b) H. A. Lowenstam, S. Weiner, On biomineralization, Oxford University Press, New York, 1989.

T 30

Reinforcement and adaptation of the mantis shrimp spike-

How crustacean cuticle became a perfect harpoon? Y. Delaunois*1, D. Ruffoni2, P. Compère1,3 1ULiege, Biologie, écologie évolution, Liege, Belgium 2Uliege, aerospace and mechanic, Liege, Belgium 3Uliege, Center for Applied Research and Education in Microscopy (CAREM), Liège, Belgium

Introduction

In the field of bioinspired materials, the crustacean cuticle is an example of

natural organo-mineral biomaterial able to endure a variety of strong stresses thanks to the combination of a complex fibre organization, adaptations and

controlled mineral deposition (Romano, Fabritius, & Raabe, 2007, Acta

Biomateriala 3, 301-309). Stomatopoda is a crustacean order including two groups: smashing and spearing mantis shrimps. Hence, the first group is

already well known for the mechanical abilities of its smashing limbs or

"hammer" (Patek & Caldwell, 2005, Journal of Experimental biology 208, 3655-3664), this study rather focuses on spearing mantis shrimps. Their

spearing appendages bear long spikes that impale fish in a fraction of second and are therefore designed to penetrate at high speed, to avoid escape of the

prey but also to resist to bending forces during the capture.

Objective

The aim of this study is to determine the structural and compositional

adaptations in the spike cuticle of spearing mantis shrimps to endure the

intense stress occurring during attacks.

Materials and methods

Spikes from Lysiosquillina maculata specimens were cut in segments and

either 2.5%-glutaraldehyde- or ethanol-fixed for TEM observation (after OsO4–staining and resin embedding) and for µCTscan and SEM-observation

respectively. Ethanol-fixed samples were also resin-embedded, then polished

for block-face imaging by BSE-SEM and EDAX under low vacuum conditions (0.4 Torr) in a ESEM-FEG XL-30 fitted with Bruker 129eV SDD.

Results

First, the µCTscan highlighted spike external features as curvature and serrations along both edges linked by grooves on the sides. When compared

to the decapod exoskeleton (classic model) and to the regular stomatopod

cuticle (cephalotoracic shield), the spike cuticle exhibits important modifications in its internal fibre architecture and mineralization. These

main changes are the lack of inner epicuticle and its replacement by a hyper-

mineralized exocuticle containing fluorapatite, and the alternating helicoidal (twisted plywood) and nematic (longitudinal orientation) arrangements of

chitin-protein fibres in the deeper procuticle layers. Also present in the

smashing limbs, theses procuticle layers were previously named the outer helicoidal layer, the striated layer and the inner helicoidal layer because no

clear subdivision between classical decapod layers (exo- and endocuticle)

was identified. The limit between exo- and endocuticle was identified in the thin outer helicoidal layer. In terms of composition, the endocuticle including

the 3 fibrous arrangements was seen to be mainly CaCO3-calcified with high

phosphate (and some fluor) proportion. The phosphate content gradually decreases toward the epidermis while inversely the substitution rates by Mg

increases. Nanoindentation tests also revealed significant variation in the

reduced modulus between the layers.

Conclusion

During evolution, the spearing limbs of mantis shrimps acquired a

specialized exoskeleton strongly modified in shape, in internal fibre architecture and mineral composition. These changes suit the intense

mechanical constraints, especially anisotropic stresses, resulting from the

spike shape and movement for rapid penetration in preys. They are also thought to cope with cracks propagations.

12

Acknowledgments: The first authors is a fellow of the FNRS-FRIA (Belgium)

T 31

Acid-induced demineralisation of enamel as a function of time

and pH R. Harper*1, R. Shelton1, J. James1, E. Salvati2, C. Besnard2, A.

Korsunsky2, G. Landini1 1University of Birmingham, School of Dentistry, Birmingham, United Kingdom 2University of Oxford, Department of Engineering Science, Oxford, United Kingdom

Acid-induced enamel demineralisation affects many people either by

exposure to acidic diets, acidic gases/ particulates from pollution (dental erosion) or to dental plaque acids (dental caries) with the latter currently

being the most prevalent disease in humans. This study aimed to develop in

situ micro-CT and light microscopy methods to determine progression of enamel demineralisation and the dynamic relationship between acid pH and

mineral density to aid in the development of improved prevention, diagnosis

and restorative treatments. Intact human third molars extracted for therapeutic reasons with full ethical

approval (National Research Ethics Committee; NHS-REC reference

09.H0405.33/ Consortium Reference BCHCDent332.1531.TB) were longitudinally sectioned into 500µm thick slices. A flat tipped 300µm

diameter needle was clamped perpendicularly on the external enamel smooth

surface before coating the entire surface with nail varnish to generate an ~300µm diameter circular non-varnished area on the slice. Varnished slices

for micro-CT in situ imaging were placed in a radiolucent Kapton® holder

(5 x 4.5cm pieces glued at the edges) containing lactic acid (10%, 0.5mL, pH 2.2) before sealing. Four samples were radiographically time-lapse imaged

hourly for 85h using a SkyScan 1172 micro CT scanner. Greyscale (related

to mineral density) loss was quantified for both the advance of the demineralisation front and extent of mineral density reduction. Varnished

slices for light microscopy in situ imaging were glued to the base of a petri

dish, to which 5ml of lactic acid (10%, pH 2.2) was added before sealing. Three samples were then time-lapsed imaged (every 100 seconds) using a

Zeiss Primotech D/A POL light microscope using a 5x objective. In addition,

18 varnished slices were incubated at 37°C in lactic acid (0.5%, 0.5mL) for three weeks at either pH 3.6, 4.0, 4.4, 4.8 and 5.2 whilst deionised water was

used as a control and were tomographically imaged using micro CT with lesion size and mineral loss quantified using an hydroxyapatite phantom.

Hourly micro-CT time-lapse sequences showed the depth of enamel

demineralisation progressed with time from the surface towards the dentine following a power-law function, which was 21% faster than the lateral

demineralisation progression after the 85h exposure to lactic acid (10%, pH

2.2). The minimum greyscale remaining (related to the remaining mineral density) within the induced enamel lesion followed an exponential decay,

while the total greyscale loss with time was linear, which showed a constant

anisotropic mineral release within the enamel architecture. Polarised light microscopy time-lapse sequences showed that once the demineralisation

front reached the Hunter-Schreger bands in enamel, there was preferential

demineralisation along those bands. Mineral density loss was linear with increasing pH acidity between pH 5.2 and pH 4.0 when incubated over a 3-

week period exposed to lactic acid (0.5%). At pH 4.0, there was complete

mineral loss at the centre of the demineralised area after the 3-week period and the linear function intercepted the x-axis at pH 5.6, near the critical HA

pH.

The time dependent linear total greyscale loss, exponential minimum greyscale remaining, power-law demineralisation front progression,

preferential lateral demineralisation and linear relationship between pH and

mineral density loss showed the intrinsic enamel structure affected the progression of demineralisation.

T 32

Remineralization potential of the enamel protein amelotin and

nano-hydroxyapatite M. Neshatian*1, J. Holcroft1, A. Kishen1, B. Ganss1 1University of toronto, Dentistry, TORONTO, Canada

Background Dental caries, which according to world health organization (WHO) is the

most prevalent chronic disease worldwide, is caused by acid-mediated demineralization of dental tissues. Resin-based composites are the most

commonly used restoration system for dental caries lesions. The integrity of

the interface between the tooth and the adhesive used to bond composite resin to the tooth tissue is crucial to the longevity of the restoration. Currently used

resin bonded composites have an average life span of only 5.7 years.

Therefore, remineralization of demineralized dentin, especially the tooth/restoration interface, is of considerable interest in restorative dentistry.

It may improve bond stability and delay or prevent restoration failures.

Amelotin (AMTN) is an enamel protein, which is expressed specifically during the enamel maturation stage and was shown to have a direct

promoting effect on mineral growth and formation both in vivo and in vitro.

Since AMTN is secreted on a pre-existing mineral layer during amelogenesis, we have used nano-hydroxyapatite (HA) to mimic the native

environment in which AMTN exerts its mineral promoting properties.

Objectives To investigate the ability of the combination of AMTN and Nano-HA to

improve mineralization in simulated body fluid (SBF) and remineralization

of demineralized dentin collagen. Hypothesis: combination of AMTN and nano-HA improves mineral formation both in SBF and dentin collagen.

Method

SBF was incubated with AMTN, nano-HA, or both at 37°C for up to 21 days. Light scattering was used to record the formation and growth of mineral

particles.

Coronal dentinal sections of human extracted molars were fully demineralized with 0.5M Ethylenediaminetetraacetic acid (EDTA).

Demineralized specimens were treated with AMTN, nano-HA or both and

incubated in SBF at 37°C. Specimens were fixed and imaged with scanning electron microscopy (SEM) to visualize mineral formation Results: The

combination of AMTN and nano–HA promoted mineral formation in SBF

significantly compared to controls (P<0.05) when tested by ANOVA. Minerals were visually identified on the surface of dentin disks as well as

within the dentinal tubules on the samples treated with both AMTN and

nano-HA in as little as 2 hours. Conclusion: combination of AMTN and nano-HA can accelerate the mineralization of demineralized dentin

Significance The accelerated mineral formation mediated by AMTN and nano-HA can be

further refined to engineer a complex that may enhance the durability of

dental restoration by creating a homogenous mineral interface between restorative material and native tooth tissue.

T 33

The hidden structure of mouse and human enamel P. Gilbert*1 1Univeristy of Wisconsin - Madison, Physics, Madison, WI, United States

Introduction Enamel is the hardest and most resilient tissue in any animal"s body. The

morphology of human and mouse enamel is well established: it consists of space-filling[1], aligned, parallel, ~50 nm wide, microns-long nanocrystals,

bundled into 5-micron-wide rods, previously known as prisms. The

orientation and arrangement of enamel crystals, however, are poorly understood.

Question 1. Are crystal orientations in human and mouse enamel as

previously assumed based on morphology alone, from SEM

images?

2. Do the observed crystal mis-orientations play a functional role in toughening enamel?

Methods We use polarization-dependent imaging contrast (PIC) mapping[2,3], a

synchrotron method that took a decade to refine and optimize[4,5], so it can

finally be used to measure and display the crystal c-axis orientation of carbonate or apatite biominerals. In a PIC map color quantitatively displays

the c-axis unit-vector orientation in polar coordinates, including the in-plane

and off-plane angles, displayed as hue and brightness, respectively, both referred to the polarization plane, not the image plane. The sample is

mounted vertically, and the beam illuminates it from the right with 30°

grazing incidence. The polarization plane is the plane in which the polarization vector rotates, which is perpendicular to the beam direction, thus

the polarization plane intersects the sample surface plane at 60°, and only a

vertical c-axis (displayed as cyan with full brightness), lies in both the polarization and the image plane[5]. Radiation damage to enamel is

minimal[6]. Before PIC mapping, enamel samples are embedded, polished,

coated[7].

Results PIC maps of mouse enamel show that, within a rod, crystals are co-oriented

with one another but not with the long axis of the rod[8], in human enamel they are not co-oriented with either: the c-axes of adjacent crystals are most

frequently mis-oriented by 1°-30°, and their orientation gradually changes up

to 30°-90° within each rod[9,5]. Molecular dynamics simulations demonstrate that mis-orientation of adjacent crystals induces crack

deflection[5], and thus toughens enamel.

Conclusions The newly observed mis-orientations contribute to make human enamel last

a lifetime.

References 1. L Yang, CE Killian, M Kunz, N Tamura, PUPA Gilbert, RSC-Nanoscale

(2011), DOI: 10.1039/C0NR00697A

2. PUPA Gilbert, A Young, SN Coppersmith, Proc Natl Acad Sci USA (2011), DOI: 10.1073/pnas.1107917108

13

3. CE Killian, RA Metzler, YUT Gong, TH Churchill, IC Olson, V Trubetskoy, MB Christensen, JH Fournelle, F De Carlo, S Cohen, J

Mahamid, FH Wilt, A Scholl, A Young, A Doran, SN Coppersmith,

PUPA Gilbert, Adv Funct Mater (2011), DOI: 10.1002/adfm.201001546 4. RA Metzler, M Abrecht, RM Olabisi, D Ariosa, CJ Johnson, BH Frazer,

SN Coppersmith, PUPA Gilbert, Phys. Rev. Lett. (2007), DOI:

10.1103/PhysRevLett.98.268102 5. E Beniash, CA Stifler, C-Y Sun, GS Jung, Z Qin, MJ Buehler, PUPA

Gilbert, submitted (2018),

6. T Parasassi, O Sapora, AM Giusti, G De Stasio, G Ravagnan, In J Rad Biol (1991), DOI: 10.1080/09553009114550061

7. G De Stasio, BH Frazer, B Gilbert, KL Richter, JW Valley,

Ultramicroscopy (2003), 10.1016/S0304-3991(03)00088-3 8. CA Stifler, N Kølln Wittig, M Sassi, C-Y Sun, MA Marcus, H Birkedal,

E Beniash, KM Rosso, PUPA Gilbert, J Am Chem Soc (2018), DOI:

10.1021/jacs.8b05547 9. RT DeVol, C-Y Sun, MA Marcus, SN Coppersmith, SCB Myneni,

PUPA Gilbert, J Am Chem Soc (2015), DOI: 10.1021/jacs.5b07931

Funding sources NSF: DMR-1603192; DOE: DE-FG02-07ER15899; DOE: DE-AC02-

05CH11231.

T 34

Unique three-dimensional structure of a fish mandible bone

subjected to unusually high mechanical loads E. Raguin*1, K. Rechav2, V. Brumfeld2, R. Shahar3, S. Weiner1 1Weizmann Institute of Science, Structural Biology, Rehovot, Israel 2Weizmann Institute of Science, Chemical Research Support, Rehovot,

Israel 3Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Koret School of Veterinary Medicine, Rehovot, Israel

Introduction

Much of our understanding of bone structure is based on two-dimensional

techniques. However, these methods provide limited insight into the

structural complexity found in these tissues, especially at the nanometer length scale. Here, we explore the structure-mechanics relations in 2D and

3D of a bone that is neither cortical nor trabecular at different hierarchical

levels of organization. This bone is found in the jaws of a fish that uses its tooth-jaw complex to crush hard-shelled bivalve mollusks. The bone is thus

subjected to unusually high compressive loads during mastication. The tooth-jaw complex is composed mainly of flat molar-like teeth characterized by the

absence of roots.

Objectives

To understand the structure-function relationships of the bone at the interface

of the tooth and the mandible.

Materials & methods

Jaws of adult black drum fish (Pogonias cromis) from Texas were studied.

Reflected light microscopy and back-scattered electron (BSE) microscopy of

polished sections, as well as micro-computed tomography (micro-CT) were used to assess at lower resolution the structural organization and density of

the bone near the contact area in 2D and 3D. We then used focused ion beam

with scanning electron microscopy (FIB-SEM) and the serial surface view (SSV) method to examine both demineralized (treated) bone and also dried

mineralized bone (untreated) to characterize the high resolution structure.

Both methods provide structural insights and the comparison shows that the untreated bone provides key structural information using FIB SEM, with less

risk of artifacts.

Results

Micro-CT and 2D polished sections of the mandibular bone revealed a highly

porous structure, with porosities ranging from tens to hundreds of microns in

diameter. MicroCT also showed that the teeth are supported by a circular narrow boney rim. The bone at the bone-tooth interphase is defined by a

small contact surface that is relatively more mineralized than most of the

jawbone. The interphase structure is characterized by the presence of fewer pores of approximately 5 to 50 μm in diameter compared to the other

mandibular bone. The nanoscale level presents the most unexpected

organization in both treated and untreated bones: rods of ordered collagen fibrils with their long axes parallel to the load direction are embedded inside

a disordered matrix. Untreated bones reveal the presence of nano-tubules

with diameters of 40 to 60 nm, which are aligned with the mineralized collagen fibrils.

Conclusions

We show an unexpected structure of bone at the tooth-bone interphase that is subjected to high mechanical loads: the collagen fibril nano-tubules fabric

is a novel structural motif not identified to date in bone. While the orientation

of the mineralized collagen fibrils in the rods provides the greatest resistance to compression, the function of the tubules remains enigmatic.

T 35

High resolution mechanical, elemental and ultrastructural

characterizations during zebrafish caudal fin regeneration P. Y. Chen*1, Y. R. Shih1, F. Bohns1, Y. J. Chuang2 1National Tsing Hua University, Materials Science and Engineering,

Hsinchu, Taiwan 2National Tsing Hua University, Life Science, Hsinchu, Taiwan

Introduction

Mineralization process during zebrafish fin regeneration has been studied,

mainly focused on the gene control mechanism, signal pathways and the roles

of collagen. Crystallization of mineral from amorphous to well-aligned crystal was evaluated by advanced X-ray analyses and cryo-electron

microscopy techniques. However, the micro/nano-scale mechanical

properties of zebrafish fin during regeneration have not been comprehensively investigated. In this study, micro-/nano-mechanical

properties at high spatial resolution of zebrafish caudal fin were investigated

by nanoindentation and PeakForce Quantitative Nanomechanical Mapping (PF-QNM). Mineralization and ultrastructure were evaluated by SEM,

Raman microscopy and electron probe X-ray microanalysis (EPMA) at

different locations and stages during the regeneration process. By combining these techniques, the relationship between the degree of mineralization and

mechanical properties during zebrafish caudal fin regeneration was

elucidated.

Materials & Methods

Wildtype AB strain zebrafish were used for the experiment. Fish caudal fins

were amputated and examined after 7 days post amputation (dpa), 14dpa, 21dpa and 28dpa. Nanoindentation (TI 980 TriboIndenter) was carried out to

probe localized mechanical properties (hardness and reduced elastic

modulus). AFM(PF-QNM) (Dimension Icon, Bruker) was used to analyze the nano-scale mechanical properties of the fin and characterize

nanostructure simultaneously. SEM (SU8010, Hitachi) and EPMA (JXA-

8500F, JEOL) were performed to characterize micro-structural features and high precision elemental analysis (Ca and P). In addition, by combing Raman

spectrometer (HORIBA, iHR550) and laser confocal microscope (Olympus,

IX71), Raman signal mappings were generated to correlate degree of mineralization and corresponding locations.

Results Nanoindentation results showed that the elastic modulus and hardness gradually decreased from proximal to distal regions of regenerated ray.

Nanoindentation mapping of individual proximal ray revealed that elastic modulus at the central region reached ~15GPa and dropped to ~3GPa toward

the edges and similar trends were observed in middle and distal rays. EPMA

analyses showed the decrease of Ca and P concentration from proximal to distal regions of the single ray and the mineral concentration declined from

the center to the edge within an individual ray, in agreement with

nanoindentation results. Additionally, PF-QNM showed low DMT modulus at distal region and higher modulus at the central region of individual ray.

SEM and AFM both showed non-mineralized collagen fibrils at the edge of

the ray while granular mineralized collagen fibrils were discovered at the center. Raman microscopy mapping showed a decrease of mineral signal

from proximal to distal region of regenerated fins after 7, 14, 21, 28 dpa.

Conclusion

We have applied multi-scale approaches, including elemental analysis,

ultrastructural characterization and mechanical testing to establish a

comprehensive analyses platform for the regeneration of zebrafish caudal fin. High precision EPMA elemental analysis and nanoindentation results both

confirmed that the regeneration and mineralization processes of zebrafish fin

start from the center and gradually spread to the edge of individual ray. This mechanical/materials science approach can not only explore bio-

mineralization process, but also have the potential to be used for in vivo

studies in the future.

T 36

Misorientation and enhanced hardness in tooth enamel C. Stifler*1, C. Y. Sun1, E. Beniash2, P. Gilbert1 1University of Wisconsin-Madison, Physics, Madison, United States 2University of Pittsburg, Oral Biology, Pittsburg, United States

Introduction

Teeth are subjected to extreme, repetitive forces and wear on a daily basis. Human enamel endures forces up to 770 Newtons, hundreds of times per day,

and must remain functional for decades [1]. By comparison, great white

shark enameloid exerts 7400 N of force when biting, but they shed their teeth regularly [2]. The mechanical stress that the teeth undergo suggests that there

are structural features in enamel that prevent catastrophic failure. While the

morphology of enamel is known to be space-filling [3], aligned groups of microns long, nanometers wide apatite crystals (called rods in humans and

bundles in sharks), the orientation of the crystals remains poorly understood.

Question

Is there a relationship between the degree of crystal misorientation in tooth

enamel of diverse animals and the hardness and elastic modulus?

14

Methods

We used a method called polarization-dependent imaging contrast(PIC)

mapping [4,5] to reveal the crystal orientations within rods in human or

mouse enamel and in bundles in sharkand parrotfish enameloid. PIC-mapping is a synchrotron method developed about a decade ago and has since

been refined, so that it can quantitatively measure c-axis orientations in

carbonate and apatite biominerals [6,7]. To create PIC maps, a series of photoelectron emission microscopy (PEEM) images ranging in x-ray

polarization from parallel to perpendicular is acquired the calcium L-edge

[8]. The intensity versus polarization curve that exists at every pixel in the stack is fit to a cosine squared curve according to Malus' law and orientation

information is extracted from the fit parameters.The colors in PIC maps

correspond to the c-axis orientation relative to the polarization of the x-rays: hue is the in-plane angle and brightness is the out-of-plane angle.The enamel

samples are embedded, polished and coated before analysis, and the x-rays

leave minimal damage to the sample [9,10].

Results

Analysis of PIC maps from mouse enamel [8],human enamel [7], great white

shark enameloid, and parrotfish enameloid [11] indicates that c-axis orientations of adjacent crystals are slightly misoriented by a few degrees,

the median misorientation ranging from 1.5-4°. In the case of human enamel,

crystals within a single rod can be misoriented up to 30-90°, but this change is gradual, similar to what was observed in nacre [7,12]. The observed

misorientation is positively correlated with the hardness and elastic modulus,

with R=0.86 and R=0.75, respectively.

Conclusions

Increased crystal misorientation in enamel is related to, and possibly causes enhanced hardness.

References

1.S Varga, S Spalj, ML Varga, SA Milosevic, S Mestrovic, M Slaj, European Journal of Orthodontics, (2011), DOI: 10.1093/ejo/cjq097

2.S Wroe, DR Huber, M Lowry, C McHenry, K Moreno, P Clausen, TL

Ferrara, E Cunningham, MN Dean, AP Summers, J. Zoology (2008), DOI: 10.1111/j.1469-7998.2008.00494.x

3.L Yang, CE Killian, M Kunz, N Tamura, PUPA Gilbert, RSC-Nanoscale

(2011), DOI: 10.1039/C0NR00697A 4.PUPA Gilbert, A Young, SN Coppersmith, Proc Natl Acad Sci USA

(2011), DOI: 10.1073/pnas.1107917108

5.CE Killian, RA Metzler, YUT Gong, TH Churchill, IC Olson, V Trubetskoy, MB Christensen, JH Fournelle, F De Carlo, S Cohen, J

Mahamid, FH Wilt, A Scholl, A Young, A Doran, SN Coppersmith, PUPA

Gilbert, Adv Funct Mater (2011), DOI: 10.1002/adfm.201001546 6.RA Metzler, M Abrecht, RM Olabisi, D Ariosa, CJ Johnson, BH Frazer,

SN Coppersmith, PUPA Gilbert, Phys. Rev. Lett. (2007), DOI:

10.1103/PhysRevLett.98.268102 7.E Beniash, CA Stifler, C-Y Sun, GS Jung, Z Qin, MJ Buehler, PUPA

Gilbert, submitted (2018),

8.CA Stifler, N Kølln Wittig, M Sassi, C-Y Sun, MA Marcus, H Birkedal, E Beniash, KM Rosso, PUPA Gilbert, J Am Chem Soc (2018),

DOI:10.1021/jacs.8b05547

9.T Parasassi, O Sapora, AM Giusti, G De Stasio, G Ravagnan, In J Rad Biol (1991), DOI:10.1080/09553009114550061

10.G De Stasio, BH Frazer, B Gilbert, KL Richter, JW Valley,

Ultramicroscopy (2003), DOI:10.1016/S0304-3991(03)00088-3 11.M Marcus, S Amini, CA Stifler, CY Sun, N Tamura, HA Bechtel, DY

Parkinson, HS Barnard, XXX Zhang, JQ Isaiah Chua, A Miserez, PUPA.

Gilbert, ACS Nano (2017), DOI:10.1021/acsnano.7b05044 12.RT DeVol, C-Y Sun, MA Marcus, SN Coppersmith, SCB Myneni, PUPA

Gilbert, J Am Chem Soc (2015), DOI:10.1021/jacs.5b07931

Funding Sources NSF: DMR-1603192

DOE: DE-FG02-07ER15899

DOE: DE-AC02-05CH11231

T 37

Investigation of Mg2+ incorporation into deciduous enamel and

its effect on the mechanical properties V. Kovacs Kis*1, A. Sulyok1, M. Hegedűs2, I. Kovács2,3, Z. Kovács2 1Centre for Energy Research Hungarian Academy of Sciences, Thin Film

Physics Laboratory, Budapest, Hungary 2Eötvös University, Department of Materials Physics, Budapest, Hungary 3Research Centre for Astronomy and Earth Sciences, HAS, Budapest, Hungary

The substitution of bivalent cations exhibiting biological activity, such as

Mg2+, in apatite, the main mineral reservoir of calcium and phosphorus in

vertebrates, is an intriguing research topic in biomaterial synthesis and fundamental biochemistry as well. Mg2+ is known to inhibit the growth of

apatite crystalline nuclei in biological environment [1]. Recently in has been

revealed that a Mg-rich amorphous calcium phosphate phase exists between the apatite nanocrystals in human dental enamel [2]. At the same time, it has

been demonstrated that Mg2+ incorporation into permanent tooth enamel

introduces changes in crystal structure, hardness and whiteness of the enamel [3]. Deciduous dental enamel has some specific microstructural features

which are different from permanent tooth enamel, e.g. the aprismatic outer

layer, having a decisive role in mechanical response and also in corrosion resistance and caries evolution.

In this contribution we investigate structural and mechanical properties of

sound deciduous molar enamel as function of Mg2+ incorporation via ion-exchange reactions. Furthermore, to elucidate the Mg2+ interaction with Ca-

phosphate nanoparticles, the same Mg-exchange experiments were

performed on synthetic hydroxylapatite (HAP) and amorphous calcium phosphate (ACP) nanopowders.

Structural investigation is carried out using micro-X-ray diffraction (µXRD)

scanning and transmission electron microscopy methods (SEM, TEM). Mechanical testing was done using depth sensing nanoindentation. The ion

exchange experiments were monitored by X-ray photoelectron spectroscopy

(XPS) and energy dispersive spectroscopy (EDS) in the TEM. Untreated sound enamel exhibits strong correlation between Mg2+

concentration, microstructure and nanohardness. The average (Ca+Mg)/P

value is 1.6, close to stoichiometric apatites. According to XPS, ion-exchange experiments on sound enmel resulted an increased Mg2+

concentration on the enamel surface up to 6 at% which stabilized at ca. 3 at%

with (Ca+Mg)/P =2.55 after 30 min sputtering time, corresponding to ca. 50 nm depth. Synthetic HAP sample exhibited less increase of Mg2+ content,

from 0.17 at% up to 1.95 at% and (Ca+Mg)/P from 1.64 up to 1.94. In case

of the ACP nanopowder, the formation of a new, probably amorphous phase was observed with characteristic morphology and Ca:Mg:P =1 : 1 : 2 ratio.

In case of HAP nanopowder, the Mg2+ incorporation occurs through surface hydration of the individual nanoparticles, as indicated by the increase of

(Ca+Mg)/P ratio. ACP nanopowder proved to be highly reactive in Mg-rich

environment. Enamel is a compact ceramic material, with crystallite size and morphology similar to HAP nanopowder but much less free crystalline

surfaces for hydration. The very high amount of incorporated Mg2+ even at

50 nm from the surface and the (Ca+Mg)/P ratio is supposed to be related to the low crystallinity material, probably with organic residue between the

enamel forming apatite nanowires. This intercrystalline material can serve as

either chemical or physical channel for Mg2+ ions, by faster dissolution rate or the presence of nanofractures, respectively. The effect of Mg2+ content on

the nanohardess is also discussed.

[1] Ding, Pan et al. (2014) Cryst. Growth Des. 14, 763−769 [2] La Fontaine, Zavgorodniy et al. (2016) Sci. Adv. 2, e1601145

[3] Abdallah, Eimar, Basset et al. (2016) Acta Biomaterialia 37, 174–183.

T 40

Osteoblast behavior and mineralization onto substrates with

controlled topographies R. Silva dos Santos1, P. Rougerie2, K. Anselme3, M. Farina*2 1Federal University of Rio de Janeiro, Institute of Biophysics Carlos Chagas Filho, Rio de Janeiro, Brazil 2Federal University of Rio de Janeiro, Institute of Biomedical Sciences, Rio

de Janeiro, Brazil 3Université de Haute-Alsace, Mulhouse, France

Introduction Topographical patterns can affect cell adhesion, proliferation, migration,

differentiation and gene expression. Osteoblasts in vivo encounter large,

curved topographical patterns such as Howship"s lacunae walls. Studying whether and how curved topography affect osteoblast biology and bone

matrix production thus can help us to understand bone formation in vivo and

enhance osteo-integrative processes in vivo. Interestingly, some studies report that artificial topographical elements like pillars or roughness can

improve matrix mineralization in vitro. However, these topographies are

usually irregular and/or contain edges. Therefore, they are of limited use to understand the reaction of osteoblast to curved surfaces more akin to what

they encounter in vivo. The aim of this work is thus to evaluate the influence

of curved substrate topography on osteoblast arrangement and mineralization, and the possibility to harness it to reproduce in vitro matrix

organization patterns observed in vivo, as in parallel fibered bone or osteons.

Methods Polydimethylsiloxane (PDMS) scaffolds were produced presenting three

types of cell-scale topographies: either edge-containing or smoothly curved

anisotropic substrates (grooves and ridges) or smoothly curved isotropic substrate (egg-box). The smoothly curved surfaces (isotropic or anisotropic)

are expected to better mimic in vivo environments compared to more classic

edge-containing surfaces. The scaffolds were subsequently coated with fibronectin, and further used for seeding primary rat calvaria preosteoblasts

(F-OST cells) that were then induced to mineralization. Cells attachment and

proliferation, quantity, composition and organization of the bone-like extracellular matrix were evaluated. Mineralized deposits were assessed with

Alizarin and Von Kossa stainings and cell distribution and orientation were

observed by staining for the actin cytoskeleton and laser scanning confocal microscopy.

15

Results Osteoblast cells aligned with the scaffold axes in all anisotropic substrates

although it was more evident in the topography with straight edges.

Mineralization occurred after two weeks without induction factors and after a few days upon induction, apparently also following the orientation of the

ridges and grooves, and it was more intense on the scaffold with straight

edges. The mineral deposits initiated in concave regions in all topographies, showing an alignment of the matrix with the substrates, which was not

observed in the control (flat PDMS). After approximately 10 days the

deposits tended to occupy the whole substrate. From confocal imaging we were able to generate topographical maps and 3D

reconstruction of our surfaces and determined the osteoblast positioning

relative to the local curvature at different stages of their proliferation and maturation.

Conclusions Osteoblasts cultivated over anisotropic surfaces orientate along the topography axis, indicating a topography-induced cell guidance and

suggesting a possible anisotropy of the produced mineralized matrix. We

found also that biomineralization initiated at concave regions. Putting together, these findings can help in the design of topography inducing

scaffolds for different purposes in biomineralization and bioengineering, as

the in vitro production of bone like tissue.

T 41

Influence of mineral properties and organic matrix

composition and structure on bone mineral dissolution A. Rodriguez-Navarro*1, N. Dominguez-Gasca1, C. Benavides-Reyes1, E. Sanchez-Rodriguez1, M. Greiner2, W. W. Schmahl2 1Universidad de Granada, Granada, Spain 2Ludwig-Maximilians-Universität, Department of Earth and Enviromental Sciences, Munich, Germany

Bone mineral dissolution is a highly complex process due to large chemical and structural heterogeneity of bone tissue. Bone mineral dissolution is

facilitated by the small crystal size of apatite crystals and large amount of

ionic substitutions. It is also dependent on the associated collagen matrix in which apatite crystals are integrated and on other proteins absorbed on crystal

surfaces. To better understand this process, we have studied in detail how

bone mineral chemistry and structure changes during demineralization using different analytical techniques such as electron microscopy, 2D X-ray

diffraction and infrared spectroscopy. We show that bone mineral and organic matrix characteristics of different types of bone tissues (i.e., bovine

enamel, bovine and avian cortical bone, avian medullary bone) strongly

influence bone mineral solubility and dissolution behavior. For instance, medullary bone mineral which is poorly crystalline and its organic matrix is

non-collagenous, dissolves much more rapidly than any other type of bone.

It is also shown than during demineralization, there is a selective dissolution of poorly crystalline disorganized bone mineral rich in labile carbonate.The

information gathered in this work provides a more complete picture of how

bone mineral dissolution occurs which is needed to understand basic but highly important processes such bone formation and resorption during the

bone remodeling.

T 42

Coral skeleton formation ? Concentrations and temporal

dynamics of pH, [CO32-] and [Ca2

+] in the extracellular

calcifying medium of a live tropical coral D. S. Sevilgen*1, A. Venn1, V. Planas-Bielsa1, E. Tarnbutté1, D. Zoccola1,

S. Tarnbutté1 1Centre Scientifique de Monaco, Monaco, Monaco

To build their skeleton, stony corals precipitate calcium carbonate (CaCO3 in

the form of aragonite) within an extracellular calcifying medium (ECM) which is located between the skeleton and the coral tissue. Increases in

important calcification parameters such as pH, calcium concentration [Ca2+]

and dissolved inorganic carbon species (DIC, e.g. [CO32-]) in the ECM can

facilitate the formation of CaCO3. Indeed, elevation of these parameters

increases the aragonite saturation state (Ωarag), a determinant that describes

the favorability of CaCO3 to form from solution. The objectives of our study were to access the ECM of live coral directly

with microsensors and conduct real time in vivo measurements in the ECM

of the growing edge of Stylophora pistillata microcolonies. The aim was to evaluate if and to which extend pH, [CO3

2-] and [Ca2+] are elevated.

Furthermore, we aimed at resolving temporal dynamics of the different ion

concentrations. Microsensor measurements were facilitated by inverted brightfield- and confocal microscopy and images of the studied coral region

were taken frequently throughout the experiments.

Our data showed that all parameters within the ECM, as well as derived DIC concentrations and Ωarag, were significantly elevated above seawater values.

Furthermore, by combining different approaches, we were able to follow

temporal dynamics of pH, [CO32-] and [Ca2

+] in the ECM and observe interrelations in the different ion concentrations.

Our study provides the most comprehensive in vivo characterization of

directly measured ECM carbonate chemistry parameters in a single live coral species to date, and highlights the role of active transport mechanisms for the

biological control of ECM carbonate chemistry. Moreover, the novel insights

into the temporal dynamics of the different ion concentrations improve understanding of carbonate chemistry dynamics during CaCO3 formation in

the ECM.

T 43

Modelling coral calcification - isotope and element fluxes in a

tightly constrained system F. Böhm*1, I. Taubner1, A. Eisenhauer1, M. Bleich2 1Geomar, Kiel, Germany 2University of Kiel, Institute of Physiology, Kiel, Germany

Isotopes are useful tools for studying biomineralization, where mineral formation mechanisms, material sources and transport pathways are reflected

in the isotopic composition of biominerals. On the other hand, applications

of isotopes as environmental proxies recorded in biominerals can profit siginificantly from a better understanding of the processing of elements and

isotopes during biomineralization.

We develop a numerical model of elemental and isotopic fluxes in scleractinian corals. Based on two recently published coral boxmodels (1-3)

our model quantifies ionic and molecular fluxes between seawater and coral

compartments: oral/aboral epithelia, coelenteron, calcifying medium, skeleton. It includes carbonic and boric acid chemistry and isotopes, Ca2+ and

its isotopes, and O2. Trans- and para-cellular fluxes are implemented.

The ion permeability of the oral epithelium was tuned to 45Ca tracer data and Ussing chamber results (4, 5). Diffusive ion permeabilities of the aboral

epithelium and the skeleton are constrained by electro-physiological data

from Ussing chamber experiments on Stylophora pistillata colonies (6). The para- and trans-cellular model fluxes were adjusted to yield calcification rates

equivalent to an extension on the order of 1 cm/year.

We find that the aboral epithelium is extremely tight. Para-cellular fluxes (2) and diffusion through the skeleton are of minor importance. Trans-cellular

transport is the only significant source of ions for calcification.

While small molecules like CO2 and B(OH)3 diffuse through cells, trans-membrane transport is necessary for ions like HCO3

-, Ca2+, Sr2+ or B(OH)4-.

Constraints from isotopes are used to design possible transport pathways in the model: While B/Ca ratios observed in coral skeletons can be achieved

with trans-cellular boric acid diffusion, the resulting boron isotope

composition disagrees with known coral values. However, correct isotope ratios are achieved assuming co-transport of borate ions with HCO3

- through

the cell membranes. Consequently, boron isotopes in corals are influenced

by pH and by bicarbonate transport rates. (1) Hohn S., Merico A. (2012) Biogeosci. 9, 4441-4454. (2) Hohn S., Merico

A. (2015) Front. Earth Sci. 2, 37. (3) Nakamura T. et al. (2013) Coral Reefs

32, 779-794. (4) Furla P.et al. (2000) J. exp. Biol. 203, 3445-3457. (5) Tambutté" E. et al. (2012) Proc. Royal Soc. B, 279, 19-27. (6) Taubner et al.

(2017) Limnol. Oceanogr. Meth., 15, 753-765.

T 44

Ion transporter gene expression is linked to the thermal

sensitivity of coral calcification C. Bernardet1, E. Tambutté1, N. Techer1, S. Tambutté1, A. Venn*1 1Centre Scientifique de Monaco, Marine Biology Department, Monaco, Monaco

Reef coral biomineralization is sensitive to temperature, which is one reason why coral reef ecosystems are threatened by climate change, but the

mechanisms underlying the thermal sensitivity of corals are poorly

understood. Furthermore, light is also a key factor in modulating biomineralization rates but a mechanistic understanding of how light

enhances coral calcification is lacking. We carried out a controlled laboratory

study that characterized the thermal performance curve (TPC) of calcification in light and darkness in the widely-used model coral species

Stylophora pistillata. Using the TPC to target low and high temperatures at

which calcification rates were depressed, we used gene expression analysis to investigate the role of ion transport mechanisms in the coral"s thermal

response. We focused the study on genes with functions in the transport and

regulation of dissolved inorganic carbon, calcium and H+. Our findings reveal a high degree of coherence between physiological responses (e.g.

calcification and respiration) with distinct gene expression patterns to the

different temperatures, and also to day and night conditions. At high and low temperatures, a core gene expression pattern emerged, pointing to thermal

effects on dissolved inorganic carbon transport mechanisms linked to

reductions in calcification rate. At high temperature but not low temperature, light stimulated calcification and the response of a more functionally diverse

group of genes. Overall, our results highlight biological control via ion

16

transport mechanisms linked to the thermal sensitivity of calcification, which is a subject of growing interest to emerging fields of study that seek to

improve the resilience of corals to climate change.

T 45

DNP-enhanced NMR of metamorphosing young corals shows

that skeleton construction entails modulation of organic

material S. Nasser1, M. Neder2, B. Uluca3, U. Akbey3, H. Heise3, T. Mass2, G.

Goobes*1 1Bar Ilan University, Chemistry, Ramat Gan, Israel 2University of Haifa, Marine Biology, Haifa, Israel 3Juelich Research Institute, Institute of Complex Systems, Juelich, Germany

The ability of corals to maintain homeostasis and mineralize has been

compromised by ocean acidification and temperature rise causing reefs to

recede and even vanish. Therefore, corals have been serving lately as important proxies of environmental impact on marine life. Early in their life,

corals undergo transformation from a swimming organism (planula) to an

benthic immobile one (polyp), that lives in colonies and forms exoskeleton for protection. During this metamorphosis process, they are vulnerable

leaving them exposed to detrimental external changes. It is therefore very

important to carefully characterize the two developmental states – planula and polyp- and to be able to assist corals withstand ongoing hazardous

variations in their surroundings.

Recently, we have shown using 13C MAS NMR on whole 13C-labeled young Stylophora pistillata corals that mineralization starts before the coral

settles. We also found using 2D 13C DARR that Glu-rich proteins bind soft amorphous mineral in the planula and Asp-rich proteins bind aragonite

crystals in the polyp. Regulation of the two mineral states by the disparate

proteins suited well the needs of the coral changing from motile to sessile. Here, we expand investigations of the metamorphosis using DNP-enhanced

MAS NMR measurements, obtaining favorable signal enhancements of

about 16- to 48-fold. Using 2D 13C DQ-SQ and PDSD measurements on the intact corals, labeled either via 13C carbonate/glycine or via 13C6

glucose/glycine, we follow the changes in organic level production related to

the transformation and the onset of aragonite precipitation. We monitor contribution of symbiotic dynoflagellates via carbonate metabolism by using

the latter as a food source.

The carbon fingerprint changes observed relate to increased carbohydrate production and certain proteins which were not detected without

enhancement. These changes are indicative of the transitions entailing onset

of colonization and expedited mineralization effort initiated after settling on the bottom of the ocean.

T 46

How inorganic spherulites grow- learning from corals C. Y. Sun*1,2, L. Gránásy3, C. Stifler1, T. Zaquin4, R. Chopdekar5, N.

Tamura5, J. Weaver6, J. Zhang1, S. Goffredo7, G. Falini8, M. Marcus5, T.

Pusztai3, T. Mass4, P. Gilbert1,2,9 1University of Wisconsin - Madison, Physics, Madison, United States 2University of Wisconsin - Madison, Materials Science, Madison, United States 3Wigner Research Centre for Physics, Institute for Solid State Physics and

Optics, Budapest, Hungary 4University of Haifa, Marine Biology, Haifa, Israel 5Lawrence Berkeley National Laboratory, Advanced Light Source,

Berkeley, United States 6Harvard University, Wyss Institute for Biologically Inspired Engineering,

Cambridge, United States 7University of Bologna, Biological, Geological and Environmental Sciences, Bologna, Italy 8University of Bologna, Chemistry, Bologna, Italy 9University of Wisconsin - Madison, Chemistry, Geoscience, Madison, United States

Introduction Modern coral skeletons are commonly assumed to consist of aragonite

crystals that grow spherulitically, that is, acicular crystals radiating from

common centers and fill space [1]. Although the name "spherulite" comes directly from its shape, morphology alone can be misleading. In an earlier

study, we determined quantitatively and found that spherulitic aragonite

crystals, both synthetic and biogenic from coral skeletons, exhibit a 0-35º misorientation of c-axes across grain boundaries [2]. Previously coral

skeletons were only been identified as spherulitic from morphological

observations, now we set out to characterize the crystal orientation distributions in coral skeletons from various genera and species to reveal

their structural differences.

Objectives The aim is to fully characterize the structure and crystal orientations in

diverse coral skeletons and to reveal the mechanism of spherulitic growth in

general.

Materials and Methods

Twelve different coral species analyzed here include different clades,

different growth morphologies, and different geographic origins. Polarization-dependent Imaging Contrast (PIC) mapping was used to

measure quantitative crystal orientations. PIC mapping was done using the

Photoelectron Emission spectroMicroscopy (PEEM) at ALS, LBNL. PIC mapping utilizes X-ray linear dichroism, which, for coral, is maximum at the

O K-edge π* peak (534 eV), and is done by rotating the linear polarization

of the illuminating X-rays from horizontal to vertical in 5º steps, acquiring a PEEM image at each polarization, and then fitting the intensity variation

versus polarization angle to a cosine-square curve, from which the c-axis for

each pixel can be deduced [3-5]. Electron backscattered diffraction and X-ray microdiffraction were done for further grain boundary analyses.

Phase-field simulation models with an orientation field [6] were used to

support the proposed formation mechanism.

Results Quantitative PIC maps confirmed that all coral skeletons form aragonite

spherulites. In addition, we discovered that 4 out of the 12 species show a different growth form of aragonite with tiny (0.2-2 µm), randomly oriented,

equant crystals, we termed "sprinkles". With quantitative PIC mapping, we

confirm that sprinkles are not spherulites due to their much larger c-axis misorientation angles across grain boundaries. We propose that the randomly

oriented sprinkles are the initially nucleated crystals, which later coarsen into spherulites: crystals with radially oriented c-axes have more space to grow

and thus become larger, at the expense of smaller, randomly oriented

sprinkles. Phase-field simulations support our proposed mechanism: in all spherulites the first nucleated crystals are randomly oriented sprinkles, which

then coarsen and become only radially oriented crystals.

Conclusions With quantitative crystal orientation analysis, we discovered randomly

oriented sprinkles in coral skeletons. We propose that sprinkles are the early-

stage nucleated crystals in all spherulites, whether biogenic, synthetic, or geologic, and then radially oriented crystals coarsen at the expense of non-

radially oriented ones. Phase-field theory confirms that this is indeed the

case. [1] Yang 2011, DOI: 10.1039/C0NR00697A

[2] Sun 2017, DOI: 10.1021/acsnano.7b00127

[3] Metzler 2007, DOI: 10.1103/PhysRevLett.98.268102 [4] Gilbert 2011, DOI: 10.1073/pnas.1107917108

[5] Killian 2011, DOI: 10.1002/adfm.201001546

[6] Gránásy 2005, DOI: 10.1103/PhysRevE.72.011605

T 47

Anisotropic lattice distortions caused by photosymbiosis in

scleractinian corals I. Coronado*1, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland

The symbiosis between scleractinian corals (host) and unicellular zooxanthellae (dinoflagellate algae, Symbiodinium spp), is the key to the

success of modern reefs. Symbionts deliver to coral host nutrients, resulting

from photosynthesis, and promote the light-enhanced skeletal calcification. Coral-algae symbiosis has had an important role in the reef evolution and

allowed them to thrive in different environments through the geological

record. Unfortunately, the identification of symbiosis in the fossil record is a paramount challenge. Several criteria have been applied with the purpose to

differentiate Recent and fossil symbiotic and asymbiotic corals on the basis

of their skeletons due to the lack of fossilization of zooxanthellae. Morphological, bio-geochemical, and lately microstructural differences have

been applied in order to distinguish the presence or absence of

photosymbionts. High-resolution X-ray diffraction (HR-XRD) studies have shown that crystal

lattice of bio-aragonite, in the molluscs and coral skeletons, is anisotropically

distorted as compared with that of geological counterparts. It has been suggested that these distortions result from the intercalation of organic

molecules into the crystallographic lattice during the biomineralization

process. It was assumed by our hypothesis that distinct biogeochemical differences found between symbiotic and asymbiotic corals should also be

reflected in changes of lattice parameters of bio-aragonite of these two

groups. In order to test this, we sampled a large suite of coral skeletons (30 symbiotic and 24 asymbiotic taxa) representing different phylogenetic lines.

High-resolution X-ray powder diffraction measurements were carried out at

MCX beamline of the Elettra Synchrotron Radiation Facility. The structural parameters were refined by Rietveld analysis and the lattice distortions (as

Δd/drf), crystallite size and microstrain analyses were performed. To

correlate the lattice distortions data with information about inorganic and organic molecules trapped into the crystals we collected also X-ray powder

diffraction data of isochronously annealed and bleached in block at different

17

time treatments of skeletons of selected taxa of both ecological groups. We also performed thermogravimetric analyses under an N2 atmosphere and

mass-spectroscopy measurements (using ICP-MS).

A linear variation between the lattice parameters of symbiotic and asymbiotic corals was observed. Orthorhombic constant ratios clustered both ecological

groups, confirming our original hypothesis. In addition, significant

differences in the biogeochemical composition were found, showing a complex correlation with anisotropic distortions. The observed

crystallographic vital effect seems to be related to physiological changes

resulting from the interaction between symbiont and coral host but also by the subsequent ecological variations of the studied samples.

Acknowledgements: This work was supported by the National Science

Centre (Poland) grant 2017/25/B/ST10/02221. The authors acknowledge the Elettra Synchrotron Radiation Facility (Trieste, Italy) for provision of MCX

beamline and synchrotron facilities (proposal: 20170388).

T 48

In-vivo and in-situ micro-Raman spectroscopy and imaging

study of mineral formation in the primary polyps of

pocilloporoidea corals I. Pinkas*1, M. Neder2, P. P. Laissue3, A. Akiva4, D. Akkaynak5, M.

Albéric6, O. Späker6, Y. Politi6, T. Mass2 1Weizmann Institute of Science, Chemical Research Support, Rehovot, Israel 2University of Haifa, Department of Marine Biology, Haifa, Israel 3University of Essex, School of Biological Sciences, Colchester, United

Kingdom 4Eindhoven University of Technology, Chemical Engineering and Chemistry, Eindhoven, Netherlands 5Princeton University, Department of Ecology & Evolutionary Biology ,

Princeton, United States 6Max Planck Institute of Colloids and Interfaces, Biomaterials, Potsdam, Germany

Introduction In reef-building corals, the earliest stages of mineral formation are critical to

the construction of reefs and their survival potential. The short time window of coral settlement, when massive, rapid calcification occurs, provides a

unique opportunity to study a range of bio-carbonate morphologies and to

understand the transformation of mineral phases into the mature coral exoskeleton.

Objectives To this end, we study two members of the pocilloporoid stony corals, Stylophora pistillata and Pocillopora acuta, during the first few days

after settlement. Following our discovery that mineralization starts even

before the polyp settlement and that the deposition of minerals is controlled by specific proteins1, we show that the initial mineral phase after settlement

is nascent magnesium calcite (Mg-Calcite), with rod-like structures in P.

acuta, and with dumbbell-like structures in S. pistillata.

Materials and methods The project utilizes several techniques to uncover the mechanism of skeleton

building in the coral larvae. Among them, SEM, EDS, Confocal fluorescence microscopy, XRD, XRF, molecular biology, bioinformatics, and in-

vivo and in-situ micro-Raman spectroscopy and imaging. The organisms

were collected from the Red Sea over several nights in February-May 2016 and 2017 by covering the corals with nets following peak releases of larvae.

Results Our measurements reveal that there are basic growth structures with similar characteristics for the two coral species that consist of Mg-Calcite (rods and

dumbbells). We verify this by both micro-Raman spectroscopy and

XRD/XRF. Confocal microscopy reveals vesicles filled with materials consisting of divalent ions (Ca2+, Mg2+, Sr2+) within the coral cell layers.

These rod and dumbbell-shaped structures, which, most likely, form the

centers of calcification (CoC), grow and merge to build the basic skeletal units of the polyp, the basal plate and the septa, in a spherulitic growth pattern

of aragonite needle shaped crystals. As these initial shapes (dumbbells) have

also been observed in calcium carbonate precipitation by bacteria, we have verified that these do not exist in the coral samples by fluorescence imaging

with fluorophores sensitive to bacteria, and by RNA sequencing of the

samples, showing that no relevant bacterial community is associated with the dumbbell structures.

Conclusions Based on our data, we suggest that mineralization in these corals occurs in three phases: first, ion rich vesicles of amorphous calcium carbonate (ACC)

are formed intracellularly. Then, the vesicles are transferred to the

calcification site, forming rod/dumbbell-shaped structures composed of nascent Mg-Calcite. During the third phase, the Mg-Calcite outer surface is

used for the growth of needle-shaped aragonite crystallites in a spherulitic growth pattern forming the septa and basal plate of the coral. These processes

take place at each center of calcification, starting at different times so that

one can observe all of them in the same polyp. We suggest a reason behind this complex process2.

References 1 Akiva A, Neder M, Kahil K, Gavriel R, Pinkas I, Goobes G, and Mass T,

Minerals in a Pre-Settled Coral Stylophora pistillata Crystallize via Protein

and Ion Changes, Nature Communications, 9, 1880; DOI: 10.1038/s41467-018-04285-7 (2018)

2 Neder M, Laissue P P, Akiva A, Akkaynak D, Albéric M, Spaeker O, Politi

Y, Pinkas I*, and Mass T*, Mineral formation in the primary polyps of pocilloporoid corals, Acta Biomaterialia, DOI:10.1016/j.actbio.2019.07.016

(2019)

T 49

Coral biomineralization toolkit differs in octocorallian vs

hexacorallian P. Ganot*1, N. Le Roy1,2, M. Fritz3, T. Rausch3, D. Aurelle4, A.

Haguenauer4, M. Arenda5, V. Benes3, D. Allemand1, S. Tambutté1 1Centre Scientifique de Monaco, Monaco, Monaco 2INRA, Nouzilly, France 3EMBL, Heidelberg, Germany 4CNRS, Marseille, France 5KAUST, Thuwal, Saudi Arabia

Introduction

Corals are calcifying organisms represented in diverse taxa of Cnidaria,

including Hexacorallia (e.g. reef-building corals), and Octocorallia (e.g. precious red corals). Corallium rubrum, the Mediterranean precious red

coral, produces two types of biomineralized structures, the sclerites and the

axial skeleton. Both are made of high Mg calcite precipitates and an organic matrix (OM) which is secreted by the scleroblast cells and the calcifying

epithelium, respectively. It is noteworthy that the red coral axial skeleton is

the material for one of the oldest forms of jewelry known to man.

Objectives

Hexacorallia and Octocorallia diverged around 550-700 Mya, before the first

mineralized cnidarian fossil dating. How evolutionary conserved are the processes controlling biomineralization between the reef-building corals and

the precious red corals? Although calcification in hexacorallia has been the

subject of several investigations, little is known on octocorallian calcification.

Mat&met

We combined microdissection technics with omics approaches to shed light on the synthesis and composition of the red coral biominerals. Proteomic

analysis of the decalcified biominerals allowed us to identify over 100 different proteins composing the sclerites and axial skeleton OMs.

Transcriptomic analysis of the calcifying tissues enabled us to ascertain

whether the OM proteins were specific to the calcification process or partaken with other regular extracellular matrix processes.

Results

Our results show that 1/ many (but not all) OM proteins are shared between the sclerites and the axial skeleton; 2/ in comparison to the known reef-

building coral OM proteins, octocorals seem to have evolved different

strategies: a marked utilization of collagen fibers but also, many novel proteins; 3/ only half of the red coral OM proteins are specifically expressed

by the calcifying cells; 4/ many of calcifying specific proteins appear to be

co-opted from already existing extracellular matrix gene families.

Conclusion

Coral is a vernacular name: calcification in different cnidarian taxa, although

using an evolutionary conserved "tool kit" (e.g. Carbonic Anhydrases), appears to result from independent recruitments of extracellular matrix genes

involved in other processes.

T 50

Biomineralization in cephalopods- first proteomic data on

Spirula spirula and Argonauta hians M. OUDOT*1, E. FARA1, P. NEIGE1, F. MARIN1 1CNRS / Université de Bourgogne - Franche-Comté, DIJON, France

Cephalopods constitute a major class of molluscs, from which only a part of

the living representatives possesses a calcified shell. The macro-evolutionary history of this clade shows a tendency to shell reduction, internalization and

finally, complete loss, from "basal" forms (nautilus) to the most derived ones

(octopus). Although phylogenetic relationships between living shell-bearing cephalopods are rather well established, molecular mechanisms of shell

formation are still poorly understood. In particular, skeleton-associated

proteins, which are supposed to constitute the 'molecular toolbox' for shell formation, have been partly characterized, and only in two cephalopod

genera, the nautilus and the cuttlefish.

The objective of the present study is to sketch the outlines of the skeletal matrix in cephalopods, to check whether representatives of this class use

similar or totally different 'molecular toolboxes'. In addition, our study aims

at describing precisely the microstructural characteristics observed in the models. Our ongoing study is primarily delimited to two species, the Ram's

horn squid Spirula spirula and the paper nautilus Argonauta hians. Note that

18

in this latter case, the shell is a calcitic eggcase secreted only by females and constitutes a remarkable example of a derived character, unique to argonautid

clade.

To this end, we have extracted - after two cleaning procedures - the shell calcifying matrices of the two studied species and performed proteomic

characterization followed by in silico investigations. The saccharidic

signature of soluble fractions has also been investigated by ELLA tests using a large set of lectins. In the case of S. spirula, all our investigations rest upon

two geographically different batches: one from Canary Islands, one from

Brazil. In parallel, we have studied the shell microstructural features of S. spirula and of A. hians by conventional SEM observations on polished

sections.

The shell of S. spirula has a complex microstructural organization. In particular, SEM observations evidence a peculiar feature, also described

among Spirula"s ancestors and defined as "mural flap": at the junction of the

septa with the shell wall, the lamello-fibrillar structure of the septa is inserted between two sub-layers of the internal microstructure of the wall, in the form

of a bevel. From a biochemical viewpoint, the organic matrix of S. spirula is

essentially proteinaceous and saccharidic with macromolecules of discrete and non-discrete molecular weight. The soluble fraction reacts with WGA,

LEL, jacalin and concanavalin A lectins. Our proteomic investigations

identified several protein hits. However, most of them occur with one peptide only and do not match with already known mollusc skeletal proteins. This

strongly suggests that the shell matrix of the ram"s horn squid is quite

different from that of other shell-bearing molluscs studied so far. We come to a similar preliminary conclusion by analyzing the proteomic data obtained

from the shell matrix of Argonauta hians, also characterized by a majority of 'one-peptide' hits. Our study emphasizes the diversity of shell matrix

components in cephalopods, which may put into question a single molecular

model for describing shell formation in this mollusc class.

T 51

Tools for understanding proteins of unknown function in

biomineralization A. Skeffington*1, A. Ohara2, A. Milentyev3, C. Heintze2, S. Görlich2, N.

Poulsen2, A. Fischer1, M. Brzezinka1, M. Gorka1, A. Graf1, N. Kröger2, A. Scheffel1 1MPIMP, Organelle Biology and Biotechnology, Potsdam-Golm, Germany 2B CUBE Center for Molecular Bioengineering, Dresden, Germany 3MPI of Molecular Cell Biology and Genetics, Dresden, Germany

Proteins have been found to be intimately associated with biominerals from

a wide range of organisms. Many roles for these proteins have been

postulated, including the stabilisation of amorphous phases, generation of hydrogels and supramolecular templates, control of crystal nucleation and

the control of crystal morphology. When examining the results of a

proteomics experiment designed to identify proteins associated with a given biomineral, studies often focus on those proteins with similarity to proteins

or domains of known function, which have normally been characterised in

organisms that do not biomineralize. However a number of the proteins identified in biomineralization proteomic experiments are normally

annotated as proteins of unknown function and indeed we would expect

proteins important for a particular mode of biomineralization to be largely restricted to those taxa that engage in this process. Thus, it is clear that to

understand taxon specific biomineralization processes we need to understand

the proteins of as yet unknown function associated with biominerals. Molecular genetic tools for many biomineralizing organisms are in the early

stages of development, meaning that experimental investigations into the

roles of proteins of unknown function are highly laborious. Thus, computational tools are required to investigate the properties of the proteins

and thereby guide and focus experimental work. In this presentation I will

describe a range of user-friendly computational tools for the analysis of proteins of unknown function. This includes tools to examine the statistical

properties of sequences, including local and global biases in amino acid

composition, the degree of intrinsic disorder and sequence complexity. In addition, the tools can be applied to find overrepresented motifs and can be

used for the visualisation and exploration of these data. I will also describe a

new method for the identification of novel domains independent of multiple sequence alignment methods. To illustrate the utility of these tools, I will use

our own proteomic data, focussing on the silica associated proteins of

diatoms and the calcite associated proteins of coccolithophores. I will also demonstrate that new information can be found in previously published data

sets using these methods, focussing on data from molluscs and echinoderms.

T 52

Osseointegrative one implant coatings H. Cölfen*1, J. Knaus1, M. Gießl1, D. Schaffarzcyck2 1Universität Konstanz, Physikalische Chemie, Konstanz, Germany 2stimOS GmbH, Konstanz, Germany

Implants are important materials to replace bone. The today commonly used

materials are titanium and polyetheretherketone (PEEK). While titanium is

very hard, PEEK has mechanical properties very similar to bone which is an advantage. However, while titanium is at least partly accepted by the bone

building osteoblast cells, PEEK is completely bioinert. This leads to a bad or

missing connection of the implant to the surrounding bone tissue and revision surgeries are often necessary with a chance of 15 – 40 % depending on the

implant. This is painful for the patient and causes enormous costs.

We have therefore developed a strategy to covalently coat the implant with a bone mimetic layer. We use a coupling chemistry to covalently bind gelatin

or collagen to a PEEK surface or alternatively to titanium. Afterwards, this

layer is mineralized with calcium phosphate resulting in an only 50 – 100 nm thick bone mimetic surface layer. Comparative cell tests show that

Osteoblasts like the bone mimetic layer and secrete collagen as a first step to

building new bone already within the first 12 h. Animal tests with a sheep model show the superior performance of the osseointegrative bone implant

surface in comparison to the common implant materials.

We have extended this work to covalently bound polysaccharide-based coatings using the same approach to bind and mineralize Hyaluronic acid.

Also, fully synthetic bone implant surface modifications are presented.

T 53

Multiscale characterization of bone matrix changes in a pre-

metastatic mouse model of breast cancer C. Liu*1, A. E. Chiou2, I. Moreno1, T. Tang1, W. Wagermaier1, M. Dean1,

P. Fratzl1, C. Fischbach2 1Max Planck Institute of Colloids and Interfaces, Department of

Biomaterials, Potsdam, Germany 2Cornell University, Meinig School of Biomedical Engineering, Ithaca, United States

Introduction Metastatic breast cancer often spreads to bone, resulting in incurable

osteolytic lesions and poor clinical outcome. While bone degradation is a

hallmark of bone metastasis, recent studies indicate that early-stage bone metastasis may depend on tumor cell adhesion to osteogenic regions.

Increasing evidence suggests that primary tumors can prepare metastatic

niches via circulating tumor-derived factors, but it remains unclear whether this also occurs in breast cancer bone metastasis, and if changes in the bone

matrix play a role in this process.

Objectives To test the hypothesis that primary breast cancer can alter bone matrix across

various length scales prior to metastasis, we have applied multiscale analysis techniques to characterize the bones of a mouse model of pre-metastatic

breast cancer.

Methods To model the effects of circulating tumor-derived factors, 3-week female

nude mice (n=5) received daily intraperitoneal injections of either tumor cell

conditioned media (TCM), collected from the human breast cancer cell line MDA-MB-231, or blank media (control), for a period of 3 weeks. Because

the proximal tibia and distal femur are common sites of bone metastasis in

mice, the hindlimbs were harvested for analyses. For each mouse, one tibia was fixed in 70% ethanol and subjected to micro-computed tomography

(micro-CT), backscattered electron (BSE) imaging, confocal laser scanning

microscopy (CLSM), Raman microspectroscopy and laboratory-based small-angle X-ray scattering (SAXS). The contralateral tibia was fixed, decalcified,

and processed for histological analysis. Distal femurs were used to harvest

RNA from trabecular bone for sequencing (RNAseq).

Results Micro-CT analysis of trabecular bone in the proximal tibia of TCM-injected

mice revealed that bone volume fraction, trabecular thickness, and trabecular separation increased significantly relative to control mice, while trabecular

number decreased. BSE agreed with these changes, and further indicated that

the increase in trabecular bone area for TCM-injected mice was most prominent within 200 µm to the metaphyseal growth plate. SAXS analysis

within this region demonstrated that the thickness of mineral crystals does

not change, but their degree of orientation was slightly decreased in TCM-injected mice compared to control mice. Bone dynamic histomorphometry

showed the cortical bone in TCM-injected mice has higher mineral

apposition rate compared to the control. Raman data showed increased mineral-matrix and carbonate-mineral ratios in the endocortical bone of the

TCM group. Ongoing experiments examine differences in bone remodeling

cells via immunostaining, collagen organization via second harmonic generation imaging and gene expression profiles via RNAseq.

Conclusion Together, these results suggest that circulating tumor-derived factors modulate both trabecular and cortical bone structures from the micro- to

nano-scales in sites prone to breast cancer bone metastasis. Changes in the

bone microstructure, degree of mineral crystal orientation, mineral apposition rate and carbonate-mineral ratio suggest that bone remodeling and

osteogenic activity may increase due to tumor-derived factors, which may

provide a favorable metastatic niche in bone, even before their arrival at bone tissues.

19

T 54

Effect of mother-of-pearl feeding on bone loss due to

ovariectomy in the rat K. D. Nguyen1, N. Laroche1, A. Vanden Bossche 1, Y. Bertache1, M. T.

Linossier 1, M. Thomas 1, S. Peyroche 1, M. Normand 1, L. Vico 1, M.

Rousseau*1,2 1U1059 Inserm - Sainbiose (Santé INgéniérie Biologie St-Etienne) Campus

Santé Innovation, Université Jean Monnet, Saint-Priest-en-Jarez, France 2UMR5510 Mateis, CNRS/Lyon University/INSA-Lyon, Lyon, France

Introduction

Previous in vivo and in vitro studies showed that mother-of-pearl or nacre, a natural material of marine origin, influence bone health. Nevertheless,

limited studies investigated the effects of oral nacre supplementation in bone

loss model. In addition, the model of bilaterally ovariectomized (OVX) rats, which mimics the acceleration of bone loss observed in postmenopausal

women due to estrogen deficiency, is well established in investigations of

osteoporotic therapies.

Objective

This study was aimed to investigate the pharmacological effect of oral

administration with powdered nacre on ovariectomy-induced postmenopausal osteoporosis in female rats.

Materials & Methods: Eight-week-old 58 femal Wistar rats were purchased

in the study. At 16 weeks of age, the Baseline group (n = 10) was sacrificed; 48 rats were either sham- operated (Sham group, n=12) or ovariectomized

(n=36). These 36 ovariectomized rats were randomly divided into three

subgroups (n = 12 each): OVX group (the standard diet), OVX CaCO₃ group (250 mg CaCO₃ / kg body weight / day in the standard diet) and OVXB Nacre

group (250 mg Nacre / kg body weight / day in the standard diet). Their

corresponding treatments were administrated orally for 4 weeks. This study was reviewed and approved by the Institutional Animal Care and Use

Committee (IACUC) of St-Etienne Univ (Approval No.: #13401-

2018020615412128 v6). Changes in body weight as well as those in the uterus were measured. PIXImus measured BMD and % fat mass in vivo. The

bone microstructure of the proximal tibia was assessed by μCT imagery in

vivo and ex vivo. The level of mRNA expression of genes involved in bone formation and resorption was analyzed by qRT-PCR. The Kruskal-Wallis

and Mann-Whitney U-tests with the FDR adjustment were used to analyze

the different data.

Results

Changes in body and uterine weights confirmed the OVX model inducing estrogen deficiency (p <0.001). The increase in body weight of the OVX

Nacre group was significantly limited after 2 weeks and 4 weeks compared

with OVX group (p <0.01). The presence of extensive trabecular deterioration (μCT in vivo) of the three OVX groups confirmed the onset of

bone loss. The ex vivo study showed that mother-of-pearl treatment improves

the trabecular bone volume. Representative genes for bone formation and resorption increased significantly in OVX groups compared to the Sham

group (p <0.01). Some genes (e.g. OPN, RANK) are overexpressed in OVX

Nacre group compared with OVX and OVX CaCO₃ group (p <0.05).

Conclusion

The results obtained provide evidence of the protective effect of mother-of-

pearl on weight gain related to estrogen deficiency. We also found an improvement in the trabecular parameters which determined bone

microstructure. In conclusion, our study is the first step to advance towards

the development of a new osteoporosis therapy based on mother-of-pearl.

T 55

Trabecular bone growth and architecture in tibia defects

implanted with carbonated hydroxyapatite microspheres A. Malta Rossi*1, V. Martinez-Zelaya2, N. Lopes Archilha2, M. Calasans-Maia3, M. Farina4, A. Linhares Rossi1

1Brazilian Center for Research in Physics, Condensed Matter, Applied

Physics and Nanoscience, Rio de Janeiro, Brazil 2Brazilian Synchrotron Light Laboratory, Campinas, Brazil, Campinas,

Brazil 3Federal Fluminense University,Niteroi, Brazil 4Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

The trabecular bone architecture and its dependence on the mechanical load have been intensively reported in the literature. In recent work, Nathaly

Reznikov et al. (2016) introduced the concept of inter-trabecular angle to

describe the architecture of the trabecular network in adult human femora. The authors concluded that the trabecular topology present geometrical

motifs with tensegrity.

In the present work, we used Synchrotron Radiation-based X-ray microtomography (SR-μCT), SEM-FIB (Slice and View) and transmission

electron microscopy (TEM) to investigate the trabecular architecture induced

in rat tibia diaphysis defects in the early times of bone growth, in the absence (group 1) and presence of a nanostructured biomaterial (group 2).

The defects were induced in the rat tibia diaphysis for 07, 14 and 21 days. Microspheres (400-600 μm in diameter) composed of nanostructured

carbonated hydroxyapatite (CHA) and sodium alginate were implanted in

group 2 for the same experimental periods. SR-μCT analyses of the tibia defects were conducted in the IMX beamline

of the Brazilian Synchrotron Light Laboratory (LNLS) in Campinas/Brazil.

The reconstructed 3D images were segmented and skeletonized with Avizo (FEI, Oregon, USA), Fiji/ImageJ (http://fiji.sc/Skeletonize3D), PNExtract (

Imperial College and ITA App (Weizmann Institute of Science) software.

The 3D reconstruction of the trabecular network and its skeletonization

were used to determine and quantify morphological parameters such as the

number of edges and nodes, length and thickness distribution, planarity, intra-trabecular angles and the amount biomaterial surface covered with

new bone. After seven days surgery, a trabecular network consisting of

small trabeculae (≈ 5 μm) was already formed in group 1 and progressed to the center of the gap, which was covered with new trabecular bone in 21

days. The trabecular architecture was based on geometrical motives

consisting of 3, 4 and 5 trabecular edges interconnected to a node (3N, 4N and 5N, Nathaly et. al, 2016). The distribution of inter-planar angles (ITA)

had a maximum at 118 °, 106 ° and 96°, for 3N, 4N and 5N motifs,

respectively. After 21 of the CHA implantation, new trabecular bone was adhered to 70% of the spheres fragments surface and occupies the majority

of the gap and the tibia medullary cavity. The presence of the biomaterial

inhibited the trabecular growth (length and thickness) but did not modify

the inter-trabecular angle distribution. The trabecular architecture is lost

when new bone is very close (50-150 μm) and in contact with the biomaterial surface. SEM-FIB and TEM analyses showed that

bone/biomaterial interface was composed of disordered mineral tissue

grown close to the surface, and into the surface pores smaller than 1 μm. The results demonstrated that bone repair induced in rat tibia defects

initiated from interconnected edges and nodes structures, with triangular

planar, tetrahedral and pentahedral geometries. Since the tibia diaphysis was not submitted to extensive mechanical load during the 07 days of the

animal life, we may conclude that trabecular topology was an intrinsic

characteristic of the trabecular bone formation in rats. This inherent feature of the trabecular bone was not altered with the implantation of the

biomaterial, and the presence of numerous nanostructured fragments that

occupied the defect volume and interacted with the newly formed bone. Mineral tissue may be grown in sub-micron porosity of the biomaterial

surface.

T 56

In-situ hydrostatic compression and diffraction reveal

increased stiffness of ashed dentine apatite mineral J. B. Forien*1, C. Fleck2, C. Krywka3, A. C. Deymier4, P. Zaslansky5

1Lawrence Livermore National Laboratory, Livermore, United States 2Technical Universit Berlin, Berlin, Germany 3Helmholtz-Zentrum Geesthacht, Geesthacht, Germany 4University of Connecticut, Farmington, Germany 5Department for Operative and Preventive Dentistry, Charité - Universitätsmedizin Berlin, Berlin, Germany

Tooth dentine is a bone-like material containing carbonated hydroxyapatite

nanoparticles (cHAp) within and surrounding a network of collagen fibrils

with submicrometer diameters. The role of the mineral particles is to stiffen the nanocomposite and to enhance both stiffness and toughness of dentine so

that it can sustain decades of intense daily, cyclic mechanical stress. The

nanometer size of the mineral particles makes the measurement of their elastic properties technically challenging. Consequently, it is widely assumed

that the elastic properties of biogenic hydroxyapatites are identical to those

of geological apatite. However, in a previous study we found that pristine dentine apatite particles are about 20% softer than geological and synthetic

apatites and that the mineral has an average bulk modulus K=82.7 GPa.

In a new series of measurements, we investigate the effect of dentine heat

treatment on the response of the apatite phase of the composite to

compression. X-ray diffraction combined with in situ hydrostatic water-

mediated pressurization were used to track changes in the c and a crystal lattice parameters. Dentine samples were measured wet under pristine

condition, after annealing for 1 hour at 250 deg, and after ashing for 10 hours

at 550 deg. Results show a decrease of ~3% in Young"s modulus from pristine to

partially annealed dentine (250 deg, 1 hr), and an increase of ~7% from

pristine to fully annealed dentine (550 deg, 10 hrs). Similarly, bulk modulus decrease by ~3% and increase by about 7% from pristine to partially and fully

annealed dentine, respectively. The change of properties in cHAp upon heat

treatment is attributed to an initial partial degradation of the organic phase followed by ashing leading to loss of carbonate impurities.

20

T 57

Heat-induced changes in bone mineral structure and

chemistry: transformation from carbonate-apatite to

hydroxyapatite M. Greiner*1, A. B. Rodríguez-Navarro2, M. F. Heinig1, K. Mayer3, B.

Kocsis1, A. Göhring3, A. Toncala3, G. Grupe3, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department of Earth- and

Environmental Science, Munich, Germany 2Universidad de Granada, Departamento Mineralogía y Petrología,

Granada, Spain 3Ludwig-Maximilians-Universität München, Fakultät für Biologie, Anthropologie und Humangenomik, Planegg-Martinsried, Germany

Bone is a complex hierarchically structured composite material constituted of carbonated apatite (bioapatite) nanocrystals mineralizing collagen (type I)

microfibrils. Biological apatite chemical composition is crucially different

from stoichiometric hydroxyapatite being particularly susceptible to marked changes in ionic substitutions on the cationic, phosphate and channel anionic

sites during heating.

Experimental incineration experiments were conducted in order to better understand changes in the complex biomineral during heating. The

experiments were motivated by archeological and forensic research

questions, that can also shed important light on our understanding of the bone mineral structure and properties.

Bovine cortical bone pieces were heated at 100, 200, 300…1000 °C for 150

minutes. In addition, short-time incinerations (10, 20, 30… 60 minutes) at 650 °C and 700 °C were performed to understand the time-dependence of the

reaction in a critical temperature range. Original and heated bone samples were investigated using complementary analytical methods such as X-ray

powder diffraction (XRPD) with advanced profile deconvolution by Rietveld

refinement, Fourier-transform infrared spectroscopy (FTIR), and infrared-coupled thermogravimetric analysis (TGA-FTIR).

No hydroxyl ions are discernible in FTIR spectra of original bone mineral

while CO32- and H2O bands are prominent. Original bone mineral has a XRD

crystallite size with an average of 65 Å in the ab plane and 174 Å along the

c-axis. A pronounced increase of crystallite size at 700 °C after 30 minutes

of heat treatment can be observed, from 205 Å along [001] and an average of 134 Å in the (001) plane (700 °C/30 min) to 392 Å along [001] and an

average of 266 Å in the (001) plane (700 °C/40 min).

Rather than a simple recrystallization, this process can be better described as a reaction with a decrease of CO3

2- and H2O and an increase of hydroxyl

groups in the apatite lattice, as detected by FTIR. Thus, the bovine bone

mineral should definitely be referred to as "carbonate-hydro-apatite" rather than hydroxyapatite.

The carbonate content in bone decreases further with higher treatment

temperatures and only weak carbonate signals are observed in bone heated at 1000 °C. Above 800 °C, buchwaldite (CaNaPO4) is formed from the Na

component of the bone mineral. Signals of combusted organic compounds in

the TGA-FTIR spectra clearly diminish at temperatures higher than 650 °C. The time-dependence of the recrystallization-reaction at 650°-700°C is much

more complex than a simple Boltzmann/Arrhenius exponential relation.

From our data we can conclude that the recrystallization reaction from bioapatite to hydroxyapatite accelerates dramatically after the complete

combustion of all organic compounds of the bone, i.e., when the apatite

grains come into direct contact to each other (Greiner et al. 2019). Greiner, M., Rodríguez-Navarro, A., Heinig, M.F., Mayer, K., Kocsis, B.,

Göhring, A., Toncala, A., Grupe, G., Schmahl, W.W. (2019) Bone

incineration: An experimental study on mineral structure, colour and crystalline state. Journal of Archaeological Science: Reports, 25, 507- 518.

T 58

Mineral deposition and structure in the avian leg tendon E. Macias-Sanchez*1, Z. Zou2, T. Tang1, L. Bertinetti1, N. Tarakina3, W. J.

Landis4, P. Fratzl1 1Max Planck Institute of Colloids and Interfaces, Biomaterials, Potsdam,

Germany 2Wuhan University of Technology, State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan, China 3Max Planck Institute of Colloids and Interfaces, Colloid Chemistry,

Potsdam, Germany 4The University of California, San Francisco, United States

Introduction Mineral deposition in bone and mineralized tendon has been well described

in electron microscopy of longitudinal tissue sections, based on the banding

pattern of collagen and mineral platelets observed mainly parallel to the collagen fiber long axis1. Deposition pattern in transverse sections2-5 is

more uncertain and remains controversial. In particular, the question to which

extent mineral particles are predominantly intra- or extrafibrillar is still debated3-5. The cross-section deposition pattern has been described in terms

of a "lacy pattern" that consists of groups of concentrically arranged, curved

agglomerates of thin mineral particles surrounding unmineralized spaces 30 to 50 nm in diameter3-5. Both the filamentous (longitudinal) and lacy pattern

(transverse) are thought to be different projections of the same ultrastructure,

but the underlying 3D mineral arrangement is yet unclear.

Objectives

In order to understand the formation of these mineral deposition patterns, we

studied early stages of mineralization using a simplified but equivalent model system to bone: the avian leg tendon. Its collagen fibrils, arranged in a

parallel fashion, mineralize as the animal ages and permit the mineralization

process to be monitored precisely. Study aims were to increase understanding of collagen fibril mineralization and to compare resulting data with recently

described bone mineral patterns2-5.

Materials and methods The present work combines Transmission Electron Microscopy (TEM),

Scanning Transmission Electron Microscopy (STEM), and Selected Area

Electron Diffraction (SAED) to elucidate crystal distribution and orientation through the mineralization process. Elemental analysis by means of Energy

Dispersive X-ray Spectroscopy (EDS) was used to characterize the organo-

mineral interface of structures occurring during mineral formation and to asses Ca/P molar ratio.

Results Early mineral aggregates were found in extrafibrillar collagen spaces, following the contour of the fibrils. Crystallites then propagated within fibrils

(at sites where mineral was also outside the same sites) following curved

trajectories. As these foci of mineralization grew, they appeared as radially-oriented clusters of relatively large crystal aggregates. Mineral foci expanded

until they coalesced, encompassing several fibrils and forming a continuous mineral network. The resulting pattern strikingly resembles the lacy pattern

described for bone ultrastructure2-5.

Conclusion These analytical observations support the concept that early stages of

mineralization in avian leg tendon lead to formation of deposition patterns

that are similar to those documented in bone. This result would suggest a possibly common mineralization mechanism for type I collagen-based

materials.

1. Landis, Song, Leith, McEwen, McEwen. 1993. J. Struct. Biol. 110, 39-54

2. Rubin, Jasiuk, Taylor, Rubin, Ganey, Apkarian. 2003. Bone 33, 270-282

3. McNally, Schwarcz, Botton, Arsenault. 2012 PLosOne 7(1): e29258 4. Grandfield, Vuong, Schwarcz. 2018. Calcif. Tiss. Int. 103, 606-616

5. Reznikov, Bilton, Lari, Stevens, Kröger. 2018. Science 360, eaao2189

T 59

Infrared imaging analysis of collagen and hydroxyapatite in

sclerotic aortic valve tissue A. Mieting*1, C. Dittfeld1, A. Jannasch1, K. Plötze1, S. M. Tugtekin1, K.

Matschke1, G. Steiner2 1Dresden University of Technology, Faculty of Medicine Carl Gustav

Carus, Department of Cardiac Surgery, Dresden, Germany 2Dresden University of Technology, Faculty of Medicine Carl Gustav Carus, Clinical Sensoring and Monitoring, Dresden, Germany

Introduction

Aortic valve (AV) fibrosis is accompanied by collagen remodelling which

promotes accumulation of hydroxyapatite, resulting in a pathological

biocomposite. Biomineralization is the main characteristic of calcified aortic valve disease (CAVD) and in glutaraldehyde cross-linked AV bioprostheses

but pathophysiology is still not fully understood. Infrared (IR) spectroscopic

imaging yields spatial information e.g. of the collagen and mineralic composition due to unsupervised chemical information of the spectroscopic

data set by multivariate chemometrics. An IR-spectrum of biomineralization

shows vibrational band assignments for phosphate of the apatite structure and for the carbohydrate moieties of collagen at 950-1200 cm-1 and for the

secondary structure of collagen by amid bands at 1500-1700 cm-1.

Objective

Aim of the study is the investigation and association of different chemical

properties of hydroxyapatite and amid I and II alterations in biomineralized

collagen of calcification nodules in CAVD tissues by IR-spectroscopic imaging followed by multivariate chemometrics.

Materials & methods: Tissue samples of calcified human AV and

bioprostheses were cryosectioned and transferred on CaF2 slides. IR-microscopy: FT-IR spectrometer Vertex 70; infrared microscope Hyperion

3000; MCT focal plane array detector. Before multivariate analysis, the

spectra were evaluated for outliers, corrected for baseline and area normalized (Matlab). Representative parts of the sections were defined from

visible light image. K-means clustering analysis was used to group the

preprocessed infrared spectra. Principle component analysis (PCA) was calculated on correlation matrix of the imaging data set in the spectral ranges

of 950-1200 cm-1 and 1480-1800 cm-1.

Results

Spectral information of initial sample set were grouped by cluster analysis in

spatially distinct mineralic and organic regions with characteristic spectral

21

band features for each tissue group. PC1-PC4 of amid I and II bands of mineralized tissue areas reveal an increased content of β-sheet collagen

conformation demonstrated by signals at 1623, 1673 and 1690 cm-1. In

addition a signal at 1515 cm-1 is detected referring to an increased content of tyrosine residues in mineralized tissue areas of the human AV. The non-

mineralized organic AV tissue is characterized by α-helical collagen

conformation assigned to 1558 and 1662 cm-1. In comparison, glutaraldehyde cross-linked pericardial tissue of bioprosthesis exhibits additionally signals

at 1532 cm-1 for anti-parallel β-sheets and signals at 1684 cm-1 for β-sheets

of collagen. In contrast, changes in the range of 950-1200 cm-1 are similar in AV and bioprosthesis: non-mineralized tissue is characterized by

carbohydrate groups of collagen at 973, 1030 and 1075 cm-1 and mineralized

tissue is characterized by PO43- of apatitic structures at 996 cm-1 and an

assignment for poorly crystalline apatites at 1110 cm-1.

Conclusion

Collagen conformation is modified in calcified AV tissue and in bioprosthesis due to chemical pretreatment or due to the origin of the

pericardial tissue. An increased β-sheet collagen content and a higher

tyrosine content in collagen of mineralized human AV tissue is detected by IR-spectroscopy in an initial dataset. Also in pericardial bioprosthetic tissue

an increased β-sheet content was monitored that may promote degeneration

by calcification.

T 60

In situ nanoscale biomineralization of calcium oxalate kidney

stone in presence of molecular modifiers T. Shokuhfar*1, R. Shahbazian 1 1University of Illinois at Chicago, Bioengineering, Chicago, United States

Calcium oxalate (CaOx) is the primary constituent of kidney stones, calcium storage medium in plants, and may play a role in plant-mediated capture of

atmospheric carbon. Here, CaOx nucleation was studied in real-time with

nanoscale resolution via graphene liquid cell transmission electron microscopy. We observed the co-existence of both classical nucleation and

non-classical nucleation pathways by which the structure and morphology of

CaOx crystals show distinct characteristics. Interestingly, we observed that the addition of citrate to the CaOx solution significantly reduced the

thermodynamic stability of CaOx nuclei, interfering with CaOx nucleation,

and promoted the formation of CaOx dihydrate (COD). This study shows that manipulation of nanoscale formation pathways may influence macroscale

properties dependent on the mineral morphology, crystal structure, and hydration state, such as mechanical properties, solubility, and biological cell

attachment of CaOx.

In Situ GLC-TEM Imaging In situ TEM imaging was achieved via encapsulation of 0.1M CaCl2 and

0.1M NaOx in control samples and 0.1M CaCl2, 0.1M NaOx, and 0.1M

citrate in the citrate treated samples. The samples were added to microcentrifuge tubes and mixed. 0.5µl of the solution was added to a

graphene coated grid. A secondary graphene coated grid was placed

graphene side down on the liquid sample. The grid was then placed into a TEM sample holder. The TEM holder was placed into a vacuum pump to

remove any encapsulated liquid from the TEM holder and the sample. The

sample was then imaged in TEM at 80kV. The increase in the contrast of the particle throughout the video was measured as a function of the grey scale of

the image, where C is the measured greyscale:

∆C=C_particle/C_bacground -1. The electron dose rate was calculated by:

Ψ=(10^5 SI)/(πa^2 )

Where S is the stopping power ((MeVcm^2)/g), I the current, a the radius of the electron beam (m), and 105 converts units SI units to Grays/s.

The STEM-EELS data was acquired using a Hitachi HD2300 STEM

microscope operated at 200 kV. The energy range of 270-577.2 eV was examined with 0.30 eV energy dispersion and EELS aperture of 3mm.

Energy windows of 50 eV were used to compare the ratio of O:Ca using the

O K edge and Ca L edge. Electron exposure was set to 15 seconds. The EELS data collection electron dose rate was between of 0.08 to 38.20e-/Å2s while

the total dose was between 1.16 to 573 e-/Å2.

CONCLUSIONS Here, real-time nanoscale studies show the nanoscale nucleation of CaOx via

two pathways. Classical nucleation causes formation of rhombohedral COM,

the most thermodynamically favorable crystal structure of CaOx, whereas rectangular COM forms via non-classical nucleation. Further, formation of

both ex situ and in situ nanoscale COM via classical nucleation indicates the

lower ∆G and 〖∆G〗^‡ of COM as compared to COD. Citrate increases the

〖∆G〗_COM^‡ over the relatively high 〖∆G〗_COD^‡ to induce the

formation of COD via cyclic aggregation of CaOx rather than via classical

nucleation. The inhibition of CaOx formation by citrate occurs at the

nanoscale, which prevents formation of COM, but with high mixing this energy barrier is overcome to form COD in bulk solutions.

. Since COD does not bind to cell walls as well as COM, citrate may prevent

kidney stone formation by preferentially forming COD to promote CaOx

excretion. Here, the results suggest that this inhibition of COM does not only occur at the microscale but upon the very nanoscale nucleation of CaOx.

T 61

Precipitation mechanisms in renal calcium phosphate plaques

and their relevance for the growth of calcium oxalate kidney

stones I. Sethmann*1, G. Wendt-Nordahl2, T. Knoll2, H. J. Kleebe1 1Technische Universität Darmstadt, Institut für Angewandte

Geowissenschaften, Darmstadt, Germany 2Klinikum Sindelfingen-Böblingen, Urologische Klinik Sindelfingen, Sindelfingen, Germany

Introduction

Calcium oxalate monohydrate (COM) kidney stones often occur attached to

calcium phosphate (CaP) plaques that form in the interstitium of the renal

inner medulla and can come in contact with urine at lesions of the epithelium of the renal pelvis. These CaP precipitates, called Randall"s plaque (RP),

seem to be a precondition for the formation of these COM stones. RP, as a

form of pathological biomineralization, forms in the course of ion re-absorption in the nephrons at high levels of ion concentration in the

interstitial fluid and possibly significantly increased levels of CaP

supersaturation.[1] Comprehensive knowledge of the conditions and mechanisms of the formation of RP may be the key to inhibiting the

precipitation of CaP and preventing the growth of COM renal stones.

Objective

The aim of this study was a microstructural investigation of RP attached to

COM stones in order to conclude on conditions and processes that lead to RP precipitation and initial COM stone growth. Since direct observation of

plaque formation and analyses of the local conditions at the formation sites

is hardly possible, model experiments mimicking the precipitation process have to be designed to enable monitoring the mechanisms involved. The

synthesis of structural analysis and experimental simulation is expected to

lead to a better understanding of RP formation.

Materials and Methods

RP attached to surgically removed COM kidney stones was investigated

using scanning electron microscopy (SEM) and integrated energy-dispersive X-ray (EDX) spectroscopy. Model experiments were performed using

hydrogels with simulated body fluid in a double diffusion set-up for CaP

precipitation mimicking the formation of RP. The precipitation process and its products are currently being characterized using vibrational spectroscopy,

X-ray diffraction and SEM techniques.

Results

In RP, the mesh size of the interstitial tissue appeared to be positively

correlated to the local size of the CaP particles. Larger particles showed

spherical morphologies and laminated internal structures suggesting an initial precipitation as metastable amorphous calcium phosphate (ACP) which may

have crystallized at a later stage.[2] In COM stones, crystals in contact with

RP showed morphologies different from the bulk of the stones and in many places they were encrusted with a thin layer of CaP.[2] Model experiments

show that the density of the gel network is negatively positively correlated

with the waiting time for precipitation and negatively correlated with the spreading rate of the cloud of CaP particles. Further characterization of the

precipitates is in progress.

Conclusions

We conclude that locally differing spatial confinements in the tissue and the

related restrictions of ion diffusion have an impact on particle sizes and

arrangements in RP. Furthermore, we propose that CaP-(super)saturated interstitial fluid may diffuse through RP and into the urine, where it could

trigger COM and CaP precipitation at the RP–urine interface and, hence,

initiate stone growth.

References [1] A.P. Evan, F.L. Coe, J. Lingeman, S. Bledsoe, E.M. Worcester, Am. J.

Physiol. Renal Physiol. 315 (2018) F1236–F1242. [2] I. Sethmann, G.Wendt-Nordahl, T. Knoll, F. Enzmann, L. Simon, H.-J.

Kleebe, Urolithiasis 45 (2017) 235–248.

T 62

Molecular structure of osteopontin fragment at the interface

with calcium ion interaction and within calcium oxalate

monohydrate composite H. Lu*1, S. Alamdari2, S. Roeters3, J. Pfaendtner 2, T. Weidner3, M. Bonn1 1Max Planck Institute for Polymer Research, Molecular Spectroscopy,

Mainz, Germany 2University of Washington, Department of Chemical Engineering, Seattle,

Washington, United States 3Aarhus University, Department of Chemistry, Aarhus, Denmark

Introduction

Calcium oxalate monohydrate (COM) is the major inorganic mineral

component of kidney stone – the chronic human disease threatening peoples's

22

lives for years. Understanding molecular mechanisms of biological control over COM crystallization will promote development of effective stone

disease therapies and stimulate novel strategies for synthesizing biologically

inspired materials. The most potent kidney stone inhibitor is osteopontin (OPN). OPN protein and its peptide fragments are considered to be

intrinsically disordered and they have significant flexibility to bind calcium

ions. Despite previous extensive literatures all attest the great importance of direct interaction between OPN (or OPN peptides) and COM mineral surface

– due to the general difficulty in probing protein (peptide) surface structure

– the exact interaction mechanism and molecular picture of OPN at its mineralization interface are all the time missing.

Objectives

Here we focus on the functional OPN peptide domain (i.e. OPN62-85 H2NSNESHDHMDDMDDEDDDDHVDSQDCOOH). We combine

surface specific sum frequency generation (SFG) spectroscopy and

molecular dynamics simulations to probe peptide structure at the interface with calcium ion interaction and within its inherent COM composite.

Materials & methods

OPN peptides firstly absorbed at air-water interface, by injecting calcium cations and subsequent oxalate anions, we were capable to use SFG to in situ

probe the peptide at the interface with calcium ion interaction and within

mineral composite. SFG spectroscopy is a second order non linear optical spectroscopy by overlapping visible and infrared laser pulse. Its selection

rules dictate that SFG spectroscopy only probes the "interface" without

interference from bulk solutions. Molecular dynamics simulations were performed to complement SFG results. Calculated SFG spectra from

the simulated structure were compared with experimental spectra, thereby further verifying the simulated peptide structure.

Results

OPN peptides restructure strongly with Ca ions at their interface prior to calcium oxalate nucleation. In contrary to one disordered motif, we disclose

that – induced by the bidentate binding Ca ions – OPN peptides adopt highly

ordered beta turn motif. The ordered peptide structure is retained after COM mineralization.

Conclusion

Our results, on the one hand, provide direct molecular picture of OPN key fragment at their inherent relevant interfaces, and on the other hand,

demonstrate the strength of combining SFG spectroscopy and molecular

simulation in probing weakly folded peptides at their functional interfaces.

T 63

Improved biomineralization of lead in aqueous media by

microbial carbonate precipitation assisted with cationic

polypeptide J. He1, W. Li1, V. Achal*1 1Guangdong Technion Israel Institute of Technology, Environmental Engineering, Shantou, China

Introduction

Ever increasing urban expansion, industrial development and anthropogenic

activities are major sources of polluting environment with heavy metals.

Posing with serious environmental problems, heavy metals are the environmental priority pollutants and require ultimate remediation to protect

both health and environment. In recent years, biomineralization involving

ureolytic bacteria producing enzyme urease that leads to carbonate precipitation, chiefly calcite, was found effective in combating heavy metal

toxicity. This biocalcification process, widely known as "microbially

induced carbonate precipitation (MICP)" has the ability to immobilize heavy metals efficiently by precipitating them during urea hydrolysis.

Objectives

Lead (Pb) is widely recognized by its highly toxic and mobile nature, also brings severe negative effect on human health and the environment. Thus,

the process of MICP was adopted in presented research to immobilize Pb.

Further, considering the role of cationic polypeptide in CaCO3 crystallization, study aimed to improve the MICP efficiency to immobilize

Pb significantly.

Materials & methods

To get Pb tolerant bacterial strains, bacteria were isolated from metals

contaminated industrial sludge samples. Urease selection pressure media

were further used to screen urease producing bacteria. The efficient urease producing Pb tolerant bacterial strain was identified based on 16S rRNA gene

sequencing. The bacterial strain was grown in optimized MICP nutrient

media containing urea, CaCl2, and poly-Lys, supplemented with 50 mg/L and 100 mg/L of Pb, for a week. The experiments were also conducted in the

absence of poly-Lys. Urease activity, pH, Pb concentration were measured

at regular interval. The concentration of Pb was analyzed by ICP-MS. The FTIR spectra were recorded in order to know the functional groups involved

in Pb immobilization. The morphological and elemental analyses were carried out with SEM-EDX. The biominerals formed in the process of

remediation of Pb was identified by XRD.

Results

One of the most efficient urease producing and Pb tolerant bacterial strain,

identified as Lysinibacillus sp. was selected in this research. During seven

days of experiment, urease production was significantly more in media with poly-Lys with maximum activity on the fifth day and pH was also on the rise.

The concentration of Pb decreased significantly on the fifth day with removal

efficiency of 81% and 74% without poly-Lys, when the initial Pb concentration was 50 and 100 mg/L, respectively. The addition of poly-Lys

led to further decrease in Pb concentration, with removal efficiency of 87%

and 83%, respectively. The FTIR spectra revealed number of peaks indicative of CO3 bonding and formation of carbonate compounds in MICP

samples; however, such peaks were more intense in the presence of poly-Lys.

XRD identified peaks of calcite, in addition to confirmation on immobilization of Pb in the form of PbCO3.

Conclusion

The presented study improved biomineralization efficiency of carbonate precipitation in metal immobilization with poly-Lys addition.

T 64

Biosynthesis of copper nanoparticles from mine waste J. Ordóñez*1, L. S. Wong-Pinto1 1Universidad de Antofagasta, Department of chemical and mineral process engineering, Antofagasta, Chile

Introduction Chile is one of the most important worldwide producers of copper, which

results in the generation of a large amount of waste, such as ore tailings.

Latest reports indicate that eight billion tons of tailings have been generated from the copper mining industry. In tailings remain a broad spectrum of

species among them copper, that. On the other hand, nanosized materials

have unique properties compared to the macromaterial counterparts, due to their quantum confinement. For this, the application of nanoparticles (NPs)

in strategical, medical and renewable energy devices are in full development.

Cheaper and more eco-friendly techniques for nanoparticles synthesis is the current challenge to increase the application of these materials.

Objective This work aims to synthesise copper nanoparticles (CuNPs) from copper ore tailings, using bacterial biomass of Pseudomonas stutzeri.

Material and methods Samples of ore tailings were collected from porphyry copper deposits located in active mining operations in northern Chile. Mineralogical and chemical

characterisation was carried out by X-ray diffraction (XRD),

microfluorescence short-wave infrared reflectance spectrometry (SWIR), and by atomic absorption spectroscopy (AAS).

Biosynthesis of CuNPs was performed using 10 g/L biomass of P. stutzeri in

two different solutions: i) CuSO4 6 mM, and ii) leached tailings (copper

concentration of 1.4 mM), stirred at 150 rpm for 24 h. After biosynthesis,

biomass was discarded by centrifugation, and the supernatant containing

CuNPs was then analysed by microfluorescence, Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM), X-ray

photoelectron spectroscopy (XPS) and field-emission scanning electron microscopy coupled with energy dispersity X-ray spectroscopy

(FESEM/EDX).

Results Tailings have high levels of copper remaining about 1000 ppm, mainly as

chalcopyrite (CuFeS2). The absence of copper oxides and the predominance

of kaolinite in the clays was verified, which was useful to define the leaching conditions. CuNPs were obtained both synthetic CuSO4 solution and leached

tailing. Preliminary results suggest that the use of fresh biomass show best

biogenic activity than lyophilised one. Analysis of structural variations in the cell wall indicates biosorption and eventually, bioreduction, activity in both

treatments, related to hydroxyl, methylene and carboxyl groups. Various

characterisation techniques confirmed bioreduction; microfluorescence analysis revealed that after biosynthesis, on the surface of biomass, Cu, Fe

and S were detected. AFM images showed the formation of agglomerated

CuNPs. Finer visualisation by FESEM/EDX shows that CuNPs are spherical

with sizes between 20-250 nm. XPS spectra confirmed that CuNPs are not

only Cu0 but also as Cu1+ and Cu2+, being possible the coexistence of copper

oxides and sulphides with the metallic form.

Conclusions Biomass of P. stutzeri can biosynthesise CuNPs from tailings deposits, which

is a novel biotreatment of diluted industrial effluents that allow their valorisation in an eco-friendly way and extracting value from mining waste.

One of the challenges that must be faced is the separation of the nanoparticles

and the reduction of the agglomerated mass.

23

T 65

Bacterial effect on the crystallization of mineral phases in the

human urinary system (based on biomimetic syntheses) A. Izatulina*1, A. Nikolaev1,2, M. Kuz’mina1, O. Frank-Kamenetskaya1, V.

Malyshev3 1St. Petersburg State University, Crystallography, St. Petersburg, Russian Federation 2Grebenshchikov Institute of Silicate Chemistry of the Russian Academy of

Sciences, St. Petersburg, Russian Federation 3S.M. Kirov Military Medical Academy, Microbiology, St. Petersburg, Russian Federation

Urolithiasis is an example of pathogenic mineral formation in the human

body. It is well known that the presence of a variety of bacteria in the urine

is very likely and bacterial inflammation often accompanies stone formation. Under the conditions of model experiments, the effect of bacteria that are

present in human urine (Escherichia coli, Pseudomonas aeruginosa,

Klebsiella pneumoniae and Staphylococcus aureus) on the formation of the renal stone mineral phases, such as brushite, struvite, vitlocite, octacalcium

phosphate, apatite, whewellite, and weddellite, was studied in systems

simulating the composition of human urine and using two types of nutrient media (Muller–Hinton Broth and Meat–Peptone Broth). Multidirectional

changes in the pH values of the solutions were analyzed, which are the result

of all system components" interactions with the crystallization process. It was shown that the presence of bacteria has a different effect on the phosphate

and oxalate phases" formation. The presence of pathogens and nutrient media

significantly affect the precipitant phase composition and the position of the resulting phosphate phase"s crystallization boundaries, which can shift both

to more acidic (struvite, apatite) and more alkaline (brushite) areas. Under

conditions of oxalate mineralization, bacteria accelerate the nucleation of calcium oxalates by almost two times and also increase the amount of oxalate

precipitates along with phosphates and stabilize the calcium oxalate

dihydrate to weddellite. As it can be seen from the reported results and the available literature data, the bacterial effect on oxalate and phosphate phase

formation is different. Thus, in the case of oxalate mineralization, primarily

(most likely), the inflammatory process will contribute to the decrease of oxalate supersaturation in urine due to calcium oxalate crystallization. In the

case of phosphate mineralization, the change in urine pH and the products of

bacterial metabolism will be of major importance. Studies aimed at identifying the specific action of certain microorganisms on the

crystallization of certain mineral phases should serve to develop individual methods of treatment and prevention of urolithiasis. The obtained results

could be regarded as the scientific basis for understanding the mechanisms

of bacterial participation stone formation in the human urinary system and the creation of biotechnological methods for the prevention of this disease.

This work was supported by the Russian Science Foundation (no. 18-77-

00026). The XRD studies have been performed at the X-ray Diffraction Centre of St.Petersburg State University.

T 66

Bio-dynamics of copper sulfide minerals and K+-jarosite

probed by Raman and FTIR microspectroscopy C. Varotsis*1 1Cyprus University of Technology, Environmental Science and Technology, Lemesos, Cyprus

Introduction

Environmental friendly approaches for the proper management of copper production including processing of low grade ore and tails have been

developed. Heap bioleaching is the appropriate technology to handle low

grade copper sulfide ores bearing chalcopyrite, idaite, bornite, chalcocite and covellite. A variety of chemical and biological processes have been applied

towards establishing the most efficient environmental friendly bio-

hydrometallurgy treatment technique of low-grade copper mixed ores. High copper extractions are achieved in environmental acceptable conditions and

in low cost, under optimum redox potentials with isolated and mixed cultures

of A. ferroxidans and A. thiooxidans, The understanding of synergistic effects which have resulted from the presence of mixed ores in the bioleaching

procedure is important for understanding the bioleaching behavior between

single and mixed ores and the origin of the existing differences.

Objectives

Raman and FTIR microspectroscopies are structure sensitive techniques and

have been applied towards our understanding of the characterization of the structure as well the structure-function relationship in minerals. The

combined application of the techniques for the bioleaching behavior of

bornite, chalcocite and covellite and the comparison with other bioleached Cu/Fe/S systems such as chalcopyrite provides valuable information on the

whole bio-hydrometallurgy system and overall visualizations of the

dynamics of the bioleached mineral ore.

Materials and Methods

Unleached samples of bornite, chalcocite and covellite were collected from

the mines of HCM in Cyprus, and placed in different test tubes containing 30

ml of the corresponding growth medium and a total of 10% inoculation of cell suspensions from stock cultures collected from the mine at pH 1.8. FTIR

and Raman microspectroscopy.

Results

We report the μm-FTIR and Raman microspectroscopic detection of bornite

[Cu5FeS4] -, chalcocite [Cu2S] -, and covelitte [CuS] -bacterial interactions

by a consortium of microorganisms. The absorption signals of amide I, K+-jarosite and of the produced extracellular polymeric substances (EPS) from

the mixed culture as a function of position on the surfaces of the bioleached

bornite, chalcocite and covellite demonstrated their heterogeneity within the surface of the minerals. To our knowledge this is the first combined

application of μm-FTIR and Raman microspectroscopy for the bioleaching

behaviour of bornite, chalcocite and covellite and the comparison with other bioleached systems such as chalcopyrite [CuFeS2] provides valuable

information on the whole bio-hydrometallurgy Cu/Fe/S system.

Conclusions

We report for the first time the ν(Cu-S) of bornite and chalcocite which are

intermediates in the bioleaching of chalcopyrite and the bioleaching behavior

of bornite, chalcocite and covellite by a consortium of microorganisms present in HCM by Raman and FTIR microspectroscopies and compare it

with that previously reported for chalcopyrite. Based on our results we

propose a mechanism for the sequential steps for chalcopyrite bioleaching in which chalcopyrite is converted initially to Cu2S, and subsequently to CuS.

T 67

Influence of bacterial EPS on mineral organization in EPS-

hydrogel-calcite composite aggregates - the chelating effect of

Bacillus subtilis, Mycobacterium phley, Mycobacterium

smegmatis, and Pseudomonas putida EPS X. Yin*1, F. Weitzel1, E. Griesshaber1, C. Jiménez-López2, L. Fernández-Díaz3,4, A. Ziegler5, A. Rodríguez-Navarro6, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department für Geo- und

Umweltwissenschaften, München, Germany 2Universidad de Granada, Departamento de Microbiología, Granada,

Spain 3Universidad Complutense de Madrid, Departamento de Mineralogía y Petrología, Madrid, Spain 4Universidad Complutense de Madrid, Instituto de Geociencias (ICMM,

CSIC), Madrid, Spain 5Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm,

Germany 6Universidad de Granada, Departamento de Mineralogía y Petrología, Granada, Spain

Mineralized structures generated under biological control are hierarchical composites that consist of two distinct materials: biopolymer matrix that is

reinforced by mineral(s). The biopolymer matrix is developed within the

biological hard tissue as membranes or network of fibrils. The biopolymers affect mineral organization as well as material properties of the biological

composite material. Hydrogel systems can be regarded to some extent as

model systems for understanding the influence of biopolymer matrices in biologic structural materials on nucleation, crystal growth, mineral

orientation, and hard tissue organization. The fibrous fabric of agarose and

gelatin gels forms compartments with specific diffusion rates, local concentrations, and supersaturation of solutes. Hence, an environment is

formed in an artificial system that shows some common features to those that

are present at sites of mineralization in biological hard tissues. However, despite many similarities, major differences still remain.

Microbial cells surround themselves by a self-produced matrix of hydrated

extracellular polymeric substances (EPS), which protects the cells and enhances their physiological activities. Bacterial EPS is not a denaturalized

product if compared to agarose or gelatin hydrogels. To understand the directing influence of biopolymers on mineral organization and composite

material formation, we synthesized with Bacillus subtilis, Mycobacterium

phley, Mycobacterium smegmatis, and Pseudomonas putida EPS hydrogel-calcite composite aggregates and investigated the influence of EPS matrices

on aggregate formation, growth and calcite organization. EPS composition

was investigated with FTIR, aggregate morphologies and hierarchical mineral organization were characterized with scanning electron microscopy

(FE-SEM) and electron backscatter diffraction (EBSD). Bacterial EPS and

agarose hydrogel distribution were visualized with selective etching procedures, micro-Raman and kernel misorientation analysis derived from

EBSD data.

Relative to reference aggregates devoid of bacterial EPS, aggregates that contain bacterial EPS are reduced in size and, for the EPS of a specific

bacterium, have distinctive crystal morphologies. Polymer (bacterial

EPS/agarose hydrogel) distribution is highly inhomogeneous in aggregates that contain EPS. In the case of P. putida and M. phley, the occluded polymer

(EPS/agarose hydrogel) is mainly present as membranes, while for M.

24

smegmatis and B. subtilis the EPS/hydrogel mixture is occluded within the aggregate as membranes and as network of fibrils. Relative to reference

aggregates devoid of EPS, where subunit formation is either absent or

negligible, subunit formation in aggregates containing bacterial EPS is extensive. Subunits vary significantly in shape, size, and mode of

organization within the aggregate. For M. smegmatis and B. subtilis subunit

organization is radial to spherulitic, while for P. putida subunit organization within the aggregate is almost random, while for M. phley subunit

organization is highly co-oriented.

In conclusion, bacterial EPS changes the microstructure and texture of the mineral in a specific manner. This is a specific characteristic for a given

bacterium and is a feature that can be used as a tool for the recognition and

identification of bacterially mediated calcification in present environments as well as in the geological record.

T 68

The red algae mineralized tissue- a low weight high strength

material N. Bianco Stein*1, B. Pokroy1, P. Zaslansky2 1Technion, Materials Science and Engineering, Haifa, Israel 2Charité - Universitätsmedizin Berlin, Department for Operative and Preventive Dentistry, Berlin, Germany

Biomineralization, the formation of minerals by living organisms, has long attracted researchers' attention due to the enhanced properties of biominerals

compared to their synthetic and geological counterparts. For example, the

toughness of the lenses on the arm plates of the brittle star O. wendtii was found to be twice as high compared to geological calcite.

Calcite, the most thermodynamically stable polymorph of calcium carbonate,

is an abundant structural component in the skeleton of many marine organisms. In many cases Mg is incorporated into the calcite lattice with

levels reaching as high as 45 mol%.

In this study we investigate the structure of the coralline red algae at various length-scales. Their mineralized tissue is composed of high-Mg calcite. In

addition, an organic phase comprised of polysaccharides serves as a template

for crystallization. The structure of these algae has not been widely studied before and information on their nanostructure is highly missing.

Structural characterization was performed utilizing various high-end

techniques such as synchrotron radiation macro- tomography and nano- tomography. In addition, we used high resolution SEM and high resolution

TEM for the study of the morphology and the nanostructure. Coralline red algae exhibit a remarkable macrostructure. We have discovered

that the structure of these algae is in fact a highly porous structure, with

porosity reaching as high as 70vol%. In addition, we have shown that their structure is hierarchical with several orders from the nano to the macro scale,

formed by crystals with diameter in the nanometric size. Moreover, we have

discovered unique structural elements that have not been previously reported. We have shown that these unique structural elements provide the algae with

greatly enhanced mechanical properties that allow them to endure stresses

applied by the sea waves present in shallow waters. The combination of high porosity along with a unique macrostructure allows

these algae to obtain a low weight structure with enhanced mechanical

properties. This study can lead toward a better understanding of the structure function relation of biomineralized structures and toward the synthesis of

novel materials with improved mechanical properties.

T 69

Deep-water scleractinian corals and their skeletal organic

matrices: A source of phylogenetic data? J. Stolarski1, K. Janiszewska1, I. Coronado1, T. Takeuchi2, L. Ravet3, J.

Thomas3, F. Marin*3 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2Okinawa Institute of Science and Technology Graduate University, Marine

Genomics Unit, Onna, Japan 3University of Burgundy-Franche-Comté, Dijon, France

The reef-building scleractinian corals contribute massively to the global formation of biogenic calcium carbonate. Although the physiology of

calcification in corals has been well studied, the molecular mechanisms

underlying this process are still poorly understood. Similarly to other mineralizing metazoans, scleractinian corals build their exoskeleton by

secreting inorganic precursors of calcification together with an organic

matrix, which consists of proteins and polysaccharides. This matrix - the main regulator of the deposition of calcium carbonate - remains occluded in

the growing biocrystals. In recent years, high-throughput screening

techniques have allowed the identification of a large number of skeletal proteins. To date, however, these data were retrieved only from shallow-

water, photosymbiotic taxa such as Acropora, Stylophora or Porites.

The main objective of this study was to supplement the skeletal database with data from deep-water, asymbiotic scleractinians. The selected samples

represent 3 major clades i.e., Desmophyllum (the Robusta clade)

Anthemiphyllia (the basalmost position in Complexa clade), and Letepsammia and Gardineria, both representing basal scleractinians. Large

phylogenetic distances on molecular tree topology and unique

microstructural skeletal organization of each taxon suggest that the composition of skeletal matrices may differ accordingly to the phylogenetic

position.

Following thorough cleaning of the skeleton, the organic content was extracted, divided into soluble and insoluble fractions and submitted to

proteomic analysis. Since no genomic/transcriptomic data are available on

the 4 species, the in silico analysis was performed against a large data set comprising all "non vertebrate proteins" available and the transcriptome of

the coral Porites australiensis. Aliquots of the soluble fractions were

analyzed on SDS-PAGE and screened against a set of 21 lectins in order to obtain their saccharidic signature.

For each sample, several peptides were identified, matching with a large

number of protein hits (32 to 109). Many of them occured with P. australiensis proteins; hits with "other metazoans" proteins (including

Acropora millepora) were significantly reduced. Only 8 to 29% of the hits

corresponded to 2 or more peptide matches. In spite of potential bias, our data suggest that the protein matrices of Desmophyllum and Letepsammia are

more similar between them than from the matrices of Gardineria and

Anthemiphyllia. The saccharide analysis indicates that only 5 (Desmophyllum, Anthemiphyllia), 7 (Gardineria) and 8 (Letepsammia)

lectins reacted. The lectin profiles of all samples were rather similar and

almost superimposable for Gardineria and Anthemiphyllia. In all cases, the highest reactivity was obtained with LEL (an N-acetylglucosamine-binding

lectin); STL lectin gave high reactivity with Desmophyllum and Letepsammia and moderate with Gardineria and Anthemiphyllia.

The composition of organic matrices may provide some background

information about the evolution of biomineralization in scleractinian corals. The protein similarity between Desmophyllum and Letepsammia that seem

only distantly phylogenetically related or similar lectin profiles of

Gardineria and Anthemiphyllia provide a hint to seek deep homology/convergence of their biomineralization paths. Lack of close

similarity between two members of basal clade (Gardineria and

Letepsammia) may point to their early evolutionary divergence. Acknowledgments: This work was supported by the National Science Center

(Poland) grants 2011/03/N/ST10/06471 (KJ) and 2017/25/B/ST10/02221

(JS).

T 70

Molecular and skeletal fingerprints of scleractinian coral

biomineralization along the depth gradient T. Mass*1, A. Malik1, S. Einbinder1, P. Zaslansky2, B. Pokroy3, I. Polishchuk3, J. Stolarski4 1University of Haifa, Marine Biology, Haifa, Israel 2Charité - Universitätsmedizin, Department for Operative and Preventive Dentistry, Berlin, Germany 3Technion - Israel Institute of Technology, Department of Materials Science

& Engineering, Haifa, Israel 4Polish Academy of Sciences, Institute of Paleobiology,, Warszawa, Poland

Reef building corals, the major producers of biogenic calcium carbonate, form skeletons in plethora of morphological forms. The skeleton shapes and

sizes are distinct for individual species but show also phenotypic plasticity

along environmental gradients. A fundamental question remains how these phenotypic skeletal modifications are reflected in molecular

biomineralization program of the coral organisms? This study provides the

first comprehensive macro- and microstructural skeletal analysis of scleractinian coral Stylophora pistillata (clade 4) collected across the depth

gradient from the sea surface to mesophotic depths of 60m and reciprocally

transplanted from 5->60 and 60->5 m in the Gulf of Eilat (Red Sea, Israel). Morphological study was combined with expression pattern of distinct gene

ontology (GO) and of biomineralization "tool kit" genes. Macro- and

microstructure of S. pistillata colonies significantly change over a depth gradient from spherical (surface depths) to flat and branching morphotypes

(mesophotic depths) and from intense (surface depths) and weak (mesophotic

depths) development of fine-scale regions of enhanced skeleton growth ("calcification centers"). In parallel, the trascriptome composition shifts from

overrepresentation of genes associated with oxygen stress response and DNA

repair (shallow-water), to coral-biomeniralization, cilia, extracellular matrix, and immune response (mesophotic depths). Interestingly, at both the

phenotypic and the gene level, transplanted corals partly adapt the typical

depth-specific properties. Accordingly, we show that organic matrix fraction is enriched in the skeleton at deep-water, in parallel with overrepresentation

of biomineralization "tool-kit" structural extracellular genes in deep water

colonies. Our results provide insights into molecular mechanisms of coral calcification in changing environment which are also encoded in the

skeleton. As such they provide exciting perspective of new paleogenomic

interpretations of fossil corals that preserve fine-scale skeletal features.

25

T 71

Modern corals vs. ancient oceans N. Conci*1, S. Vargas1, E. Griesshaber1, W. W. Schmahl1, G. Wörheide1,2,3 1Ludwig Maximilian University, Munchen, Germany 2SNSB — Bayerische Staatssammlung für Paläontologie und Geologie,

München, Germany 3GeoBio-Center, Ludwig-Maximilians-Universität München, München, Germany

Biomineralization is a taxonomically ubiquitous process by which organisms form minerals that they use for support, protection, or nutrient storage, such

as shells, skeletons, and bones. Among corals (Class Anthozoa, Phylum

Cnidaria) biomineralization is widespread and both aragonite and calcite structures can be observed. Coral biomineralization has been extensively

studied, primarily due to the ecological role these organisms play in the

formation and accretion of coral reefs. Nevertheless, how corals acquired the ability to form skeletons characterized by different calcium carbonate

(CaCO3) polymorphs, i.e. calcite and aragonite, remains elusive. One

standing question revolves around whether corals can biologically control the deposition of either polymorph. This has important evolutionary implications

as changes in seawater chemistry through the Earth"s history, especially the

Mg/Ca ratio, appear to favor the deposition of either one polymorph or the other.

To address this question, we have set-up calcite-inducing, Cretaceous-like

marine aquaria experiments to grow coral species from both clades of

biomineralizing anthozoans, namely the subclass Octocorallia (Heliopora

coerulea) and the order Scleractinia (Montipora digitata). We employed a

diverse array of mineralogical analyses, including electron backscatter diffraction (EBSD) and energy dispersive spectroscopy (EDS), to determine

and examine the presence of environmentally-induced modifications within

the coral skeleton. We then used RNA sequencing to investigate the transcriptional response of the corals to different polymorph favoring

environments. In conjunction, these data provide insights on how the

diversity of skeletal structures in corals evolved and may inform our predictions about the response of these ecologically important organisms to

future changes in ocean chemistry .

T 72

Biomineralization pathways in calcifying dinoflagellates-

uptake, storage in MgCaP-rich bodies and formation of the

shell A. Jantschke*1, I. Pinkas2, A. Hirsch2, A. Schertel3, L. Addadi2, S. Weiner2 1Technical University Dresden, Bioanalytical Chemistry, Dresden,

Germany 2Weizmann Institute of Science, Rehovot, Israel 3Carl Zeiss Microscopy GmbH, Oberkochen, Germany

Introduction Dinoflagellates are one of the most important contributors to both marine and

freshwater productivity. They have two main life stages, the thecate/motile stage and the cyst/resting stage. One family of dinoflagellates, the

Thoracosphaeraceae, produce calcitic shells in their cyst stage.

Objectives Although dinoflagellates are the second most important calcifying

phytoplankton group after coccolithophores, their biomineralization mechanism is still not fully understood and considered as one of the most

serious gaps in knowledge. For this reason, the main objectives are to gain

insight in the mineral architecture and stabilization mechanism, subcellular structures that may play a role in biomineralization, as well as into cyst

formation mechanism.

Materials & methods We investigate calcitic cyst formation in two representative members

(L. granifera and C. operosum aff.) from different clades of calcareous

dinoflagellates using cryo-electron microscopy (cryo SEM and cryo FIB SEM) in combination with various spectroscopic techniques (FT-IR, Raman,

Fluorescence, EDS). These imaging techniques allow investigation of cells

as close as possible to the natural state.

Results Only calcein AM and not calcein enters these cells, indicating active uptake

of calcium and other divalent cations. In both species, we observed vacuoles containing crystalline inclusions using cryo-SEM. So far, crystalline deposits

in the family Thoracosphaeraceae were assigned to calcite and it was

assumed that they are involved in the calcitic shell formation. Surprisingly, using in situ Raman spectroscopic imaging we could identify these

crystalline inclusions as anhydrous guanine in the biogenic β-form using their

low-wavenumber Raman signature. Live-cell imaging of cells stained with Calcein-AM, the use of cryo-

sectioning and cryo-EDS shows the presence of small MgCaP-rich mineral

bodies within the same vacuolar enclosures. 3D cryo-FIB-SEM serial block face imaging of a calcifying cell of C. operosum aff. shows a remarkably

large number (353) of these bodies distributed in the cell volume. Out of

these bodies, 52 (about 15%) are located between the two inner organic layers. We suggest that these MgCaP-rich bodies are secreted into the outer

matrix and are part of a Ca-concentrating or transport mechanism.

Calcite formation occurs inside the outer matrix via multiple independent nucleation events. Based on 2D and 3D cryo-electron microscopic datasets

we show that individual calcite crystals grow with preferred orientation into

a dense reticular network that forms the calcitic cyst. In the final calcification stage the main structural motif are calcite plates with evenly arranged pores

that form the highly regular, porous calcite shell.

Conclusion In summary, our results show the presence of a multifunctional vacuole. In

calcifying cells of L. granifera and C. operosum aff. vacuolar MgCaP-rich

bodies seem to be part of a Ca-concentrating or transport mechanism, indicating there is a common biomineralization pathway in the family

Thoracosphaeraceae: (1) Uptake of Ca and other cations through the

membranes. (2) Deposition of Mg and Ca ions inside a disordered MgCaP-body. (3) Secretion of these bodies to the extracellular space between the

outer membranes. (4) Formation and growth of calcite bodies into a dense

reticulate network that forms the mature calcitic shell.

T 73

Marine temperature recorded in bivalve shell ultrastructures -

proxy potential and current limitations N. Höche*1, E. O. Walliser1, M. Peharda2, B. R. Schöne1 1Johannes Gutenberg University Mainz, Dept. of Applied and Analytical

Paleontology, Mainz, Germany 2Institute of Oceanography and Fisheries, Split, Croatia

Introduction Bivalve shells are increasingly exploited as high-resolution paleoclimate archives. These mollusks are globally distributed in aquatic ecosystems,

occur in large numbers and individuals of some species are very long-lived.

Periodic growth patterns can be used to precisely date each shell portion. Furthermore, the shells form in equilibrium with the oxygen isotope

signature of the ambient water, so that δ18Oshell provides information on

temperature during growth. However, oxygen isotope-based temperature estimates can be challenging if the isotope value of the water is unknown or

if the isotope signal is diagenetically altered. Evidently, there is a strong need

for independent temperature proxies. One potential candidate is the shell ultrastructure, specifically the size and shape of individual biomineral units

(BMUs). So far, BMU-based temperature reconstructions have only been tested in a few species from a limited number of localities. In addition, the

recognition and morphometric analysis of BMUs (in SEM images) needs to

automated through suitable image-processing algorithms.

Objective In this study, we assessed the effect of temperature on BMUs of

ultrastructures, which have hitherto not been studied (i.e. crossed-lamellar, homogenous and crossed-acicular). To advance objective and quantitative

temperature reconstructions, we developed new image-processing

algorithms for automated BMU recognition.

Material and methods We analyzed the marine bivalves Glycymeris bimaculata and G. nummaria

from the Adriatic Sea, and Arctica islandica from Iceland and the Baltic Sea. The two glycymerids were used to study potential species-specific

differences, and A. islandica was used to evaluate potential locality-specific

differences in recording environmental variables.

Results All studied species showed the same relationship between temperature and

ultrastructure as reported in previously studied bivalves, i.e., larger and more elongated BMUs were formed in warmer waters. We calculated transfer

functions for crossed-lamellar ultrastructures of Glycymeris spp. enabling

quantitative temperature estimates. The homogenous outer shell layer of A. islandica is challenging to interpret. Specimens from the Baltic Sea showed

significantly larger variation in BMU size than specimens from Iceland.

Conclusions Studied bivalve species recorded temperature changes in their shell

ultrastructure: BMUs were larger and more elongated when formed in

warmer waters. Transfer functions are species-specific. SEM-based quantification of BMU morphometry is challenging in case of the

homogenous ultrastructure of A. islandica.

26

T 74

Ocean acidification impacts European abalone (Haliotis

tuberculata) shell microstructure and mechanical properties S. Auzoux-Bordenave*1,2, S. Avignon1, P. Dubois3, N. Richard1, M.

Coheleach4, A. Badou1, S. Di Giglio3, L. Malet5, S. Martin2,6, S. Roussel4,

S. Huchette7 1Museum national d'histoire naturelle, Station de Biologie Marine,

Concarneau, France 2Sorbonne Université, Paris, France 3Université Libre de Bruxelles, Laboratoire de Biologie Marine, Brussels,

Belgium 4Université de Brest, CNRS, IRD, Ifremer, LEMAR, Plouzané, France 5Université Libre de Bruxelles, Service 4Mat, Brussels, Belgium 6Station Biologique de Roscoff, CNRS/CU, Roscoff, France 7Ecloserie France-Haliotis, Plouguerneau, France

Ocean acidification (OA) is of major concern for marine organisms,

especially for calcifying species such as mollusks, with their shell made of calcium carbonate. Indeed, OA has been shown to reduce survival and

growth, alter morphology and/or impair shell formation and mineralization

in marine molluscs. The European abalone Haliotis tuberculata is a commercially and ecologically important gastropod species with a calcified

shell mostly composed of CaCO3 under aragonite form. Since aragonite is

more susceptible to dissolution compared to calcite, the abalone shell provides a relevant model to study the impact of OA on

biomineralization. This study investigated the effects of pCO2 induced OA

on shell microstructure, calcification and resistance in adult abalone. According to the most pessimistic scenario (-0.3 pH unit by 2100), adult 3.5

year-old abalone were exposed to two pH conditions (pHT = 7.71 ± 0.06,

pCO2 = 988 ± 143 µatm, vs pHT = 8.01 ± 0.05, pCO2 = 449 ± 63 µatm) during four months by increasing pCO2 into five experimental tanks per

condition. Shell surfaces as well as cross sections were examined by

Scanning Electron Microscopy (SEM) to assess whether lowering the pH had an influence on shell microstructure. The thickness of the shell layers (i.e.

periostracum, spherulitic and nacre) was determined on cross-section images

using Image J software. Biomechanical tests (such as compression tests and nanoindentation measurements) were performed to compare the shell

properties (strength, hardness and elasticity) between the two pH treatments.

After four month of exposure to low pH condition, the periostracum of abalone shell exposed to low pH appeared lighter and exhibited a corroded

surface compared to those exposed to control condition. This corrosion was confirmed by the decreased thickness of the periostracum observed at pH 7.7,

while no difference in spherulitic and nacre layers thickness were observed.

SEM observation of nacre microstructure revealed irregularities with a heterogeneous aragonite platelets and a corroded texture surface in abalone

shells exposed to low pH. The shell resistance of abalone exposed to pH 7.7

was significantly reduced by 29 % compared to those exposed to pH 8.0. In addition, nanoindentation assays revealed significant differences in shell

hardness and elasticity for abalone exposed to pH 7.7. Shell corrosion and

microstructure changes would likely result from indirect effects of OA either on the carbonate chemistry of the calcification site or on physiological

processes driving CaCO3 deposition.

Our results highlighted that OA negatively impacted the shell microstructure and resistance in adult abalone, leading to more fragile shell. In their natural

environment, abalone may be at greater risk under future pH condition as

their shells may not offer sufficient protection from predators and other environmental stressors. As a consequence for the abalone industry, OA

might represent two challenges (i) by reducing abalone protection and

threatening wild population stocks, and (ii) by enhancing the cost of its aquaculture due to an increased time to reach a marketable size.

T 75

Oyster biomineralisation is maintained under ocean

acidification K. Chandra Rajan*1, T. Vengatesan1 1The University of Hong Kong, Swire Institute of Marine Science, School of

Biological Sciences, Hong Kong Island, Hong Kong

Introduction Ocean acidification (OA), the on-going reduction of oceanic pH and the carbonate saturation state (Ω), is known to affect biomineralisation in several

marine invertebrates. Ion transport is an important aspect of maintaining

biomineralisation under OA. There are two concepts, related to ion homeostasis, that explain the negative effect of OA on marine calcifiers:

1) Substrate limitation theory, where the reduction in the carbonate saturation

state is considered as the main negative stressor. 2) Proton flux limitation model, where more energy will be required for

active transport of H+ ions from the site of biomineralisation when the H+ in

the ocean increases. However, little is known about the epigenetic ability of the marine calcifiers

to adapt to these two challenges. Hence, in this study we have investigated

the epigenetic response of an ecologically and commercially important aquaculture oyster species - Crassostrea hongkongensis to OA.

Objectives Mantle tissue specialises in biomineralisation and is conserved throughout molluscs. Thus, in our study we focus over the epigenetic response of the

mantle tissue under OA. The objectives are:

1) Understanding the epigenetic response of mantle to OA (gene expression and DNA methylation) with a focus over ion transporters and shell matrix

proteins.

2) Characterising the changes in the shell quality under OA via shell microstructure, density, Mg/Ca ratio and hardness.

Materials & methods Oyster spats (one day post settlement) were grown under ambient pH (8.1) and OA conditions (pH 7.4 and 7.7) for five months, a significantly long

duration for an OA study. mRNA-seq and Methyl-RAD techniques were

used for gene expression and DNA methylation analysis. Shell properties were analysed using Scanning Electron Microscope (SEM), SEM-EDS,

Micro-CT and Vicker's Hardness test.

Results The shell growth under OA was surprisingly maintained, like the ambient

conditions, throughout the experimental duration. The Mg/Ca ratio and

hardness also remains unchanged. However, there was complete dissolution of the prismatic layer under pH 7.4. In-spite of severe dissolution, the overall

shell growth was maintained.

Preliminary analysis of the RNA-seq data shows that the expression of well-known biomineralisation related genes such as alkaline phosphatase,

carbonic anhydrase and tyrosinase were maintained. Among the differentially expressed genes, above 80% of the genes where

downregulated. The top downregulated genes under OA are related to

mitosis, ribosomes and various energy metabolism such as amino acid, carbohydrates and glycan. Among the few upregulated genes, the top

upregulated genes are related to plasma membrane and calcium binding. The

notable genes among the upregulated were voltage dependent calcium channels. Preliminary analysis of the DNA methylation data shows that top

down-regulated methylation sites are also related to voltage gated

calcium/sodium channels, indicating a strong epigenetic base for adaptation in oysters.

Conclusion Crassostrea hongkongensis maintains biomineralisation even under extreme ocean acidification scenarios. The adaptation strategy involves: 1)

upregulating the ion transporter system, for supplying required substrate and

proton homeostasis. 2) The energy spent on upregulating ion transporters is compensated by undergoing metabolic depression.

T 76

Effect of polycarboxylated eggshell membrane on in-vitro

mineralization J. L. ARIAS*1, K. SILVA1, A. NEIRA-CARRILLO1, L. ORTIZ1, N.

BUTTO1, M. S. FERNANDEZ1 1University of Chile, SANTIAGO, Chile

Biomineralization concerns not only inorganic ions formation, but also the

regulatory role of an organic matrix mainly related to the action of anionic groups such as sulfate in proteoglycans or aspartate and glutamate in

proteins. The eggshell membrane (ESM) is a network of a fibrillar

biopolymer organized as two layers of protein fibers. Main protein of the ESM is type X collagen. Mineralization of chicken eggshell started on

specialized negative charged sites of the ESM referred to as mammillae,

associated to the occurrence of mammillan, a keratan sulfate-rich proteoglycan. Growth of calcite crystals occurs from the mammillae upwards

in association with the sequential secretion of ovoglycan, a dermatan sulfate-

rich proteoglycan. Although 22% of the ESM aminoacids are glutamic plus aspartic, there is no mineralization on the surface of ESM fibers, but only on

the afore mentioned sulfate-rich mammillae. Therefore, as a possible

explanation we hypothesize that the occurrence of a competitive effect between sulfated and carboxylic groups on the surface of the ESM could be

involved in driving calcium carbonate crystal nucleation to privileged sites.

In order to answer this question, an experimental enrichment of ESM with polycarboxylic groups was tested on its ability to crystallize calcium

carbonate in vitro.

Polycarboxylated ESM was obtained by coupling polyaspartic or polyglutamic acids to ESM fibrils crosslinked with water-soluble

carbodiimide. Selective protection and cleavage of carboxylic groups during

synthesis were followed by FTIR spectroscopy. Crystallization assays were based on a gas-diffusion chamber method consisting of a chamber built with

a plastic Petri dish having a central hole in its bottom and glued to a plastic

cylindrical vessel. Polystyrene microbridges were settled on the bottom of the petri dish. Microbridges were filled with 200 mM dihydrate calcium

chloride solution in 200 mM Tris buffer, pH 9.0. The cylindrical vessel

contained 25 mM ammonium carbonate solution. Intact or polycarboxylated ESM strips were deposited on the top of each microbridge with the

mammillary side facing down. Five replicates of each experiment were

27

carried out inside the chamber at 20 °C for 24 h. After the experiments, eggshell strips were taken out of the microbridges, air-dried at room

temperature, mounted on aluminum stubs with scotch double-sided tape, and

coated with gold. Crystal morphology was observed and number and size estimated in a Jeol JSM-IT300 EDS-Oxford Instrument scanning electron

microscope.

When functionalized ESM were located upside down on the calcification solution on the top of the microbridges for 24 hours, higher amount of small

2-3 µm rhombohedral calcite crystals adhered to the fibers were observed

when compared with the intact ESM where 7-10 µm calcite crystals were located only on the mammillae.

It is safe to conclude that the occurrence of polycarboxylic groups

immobilized on ESM is a strong attractor for calcium carbonate crystallization on functionalized ESM used as a template scaffold. Funded

by FONDECYT 1180734

T 77

Fungi-induced mineralization in vitro- controlling the crystal

morphology and polymorph I. Polishchuk*1, A. Livne1, B. Pokroy1 1Technion - Israel Institute of Technology, Materials Science and Engineering, Haifa, Israel

Fungi live within diverse environments and survive well under extreme conditions, which are usually beyond the tolerance of many other organisms.

In various environments fungi are known to induce precipitation of a wide

range of minerals. Specifically, it has been shown that various species of fungi facilitate calcium carbonate mineralization. Inspired by examples of

needle-fiber calcite formed via fungal biomineralization typically observed

in soils and sediments, herein we utilized active and inactivated fungus to induce mineralization synthetically. For the first time we report the ability of

fungi mycelium to serve as a template for growth of chlorhydroxy-apatite

tubes and hopeite cylinders. We also demonstrate the feasibility of growing aragonite needles from a fungal source in vitro. The obtained needles are

curved, display hexagonal facets and demonstrate high aspect ratios close to

60. The size and the shape of the synthetic needles match that of the natural fungus" mycelium. The morphology, micro- and nanostructure of the grown

crystals were studied utilizing high-resolution characterization techniques.

The presented findings demonstrate that fungus present in the crystallization environment can induce the formation of high aspect ratio fibers and stabilize

metastable polymorphs.

T 78

Post-mortem recrystallization of biogenic amorphous calcium

carbonate and crystal tailoring by the inherited

macromolecular framework J. Stolarski*1, I. Coronado1, G. Luquet2, M. Potocka3, M. Mazur4, A.

Baronnet5, O. Grauby5, A. Meibom6,7 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2Muséum National d'Histoire Naturelle, UMR BOREA, Paris, France 3Institute of Biochemistry and Biophysics , Department of Antarctic Biology, Warsaw, Poland 4University of Warsaw, Department of Chemistry, Warsaw, Poland 5CNRS - Aix Marseille Université, CINaM - UMR 7325, Marseille, France 6Ecole Polytechnique Fédérale de Lausanne , Lausanne, Switzerland 7Université de Lausanne, Center for Advanced Surface Analysis, Lausanne, Switzerland

Assembly of mineral deposits from transient, disordered and nanometric-size

precursor particles is a widespread skeleton-formation strategy of many groups of organisms (e.g., corals, mollusks, echinoderms). It enables

efficient cellular transport of mineral components to the skeleton formation

site and their aggregation within biologically confined space. The disordered particles enclosed by the organic matrix are then transformed through local

dissolution–co-precipitation processes into stable and crystalline phases.

Consequently, biologically-controlled minerals, in contrast to abiotic (synthetic/geological) crystals, are organo-mineral nanocomposites with

uniform particle sizes, high levels of spatial organization, complex

morphologies, controlled aggregation and texture, preferential crystallographic orientation and form higher-order hierarchical structures.

Such criteria are often used to distinguish biogenic and abiogenic minerals in

the fossil record. In our experiment, we induced the formation of crystalline mineral phase

from lobster gastroliths, originally formed mainly by biogenic amorphous

calcium carbonate (ACC). In natural conditions (inside the lobster body) the amorphous gastrolith mineral does not crystallize and is assimilated by the

organism immediately after molting. Inducing crystallization of the gastrolith

amorphous phase, which acted as a crystallization "precursor", we found that the resulting mineral is calcite, which shows all aforementioned properties of

biominerals, including highly elaborated hierarchical organization. These

newly formed structures were characterized by FESEM, AFM, EBSD,

Raman spectroscopy techniques. Such "biogenic" organization of the post-mortem formed calcite is explained by the inheritance of the original

macromolecular framework, which acts as an organic template in precisely

orchestrated dissolution-co-precipitation process. The finding has several implications for traditional, structural criteria in interpreting the pristine

structure of biominerals in paleontology and astrobiology.

Acknowledgments: Acknowledgments: This work was supported by the National Science Center (Poland) grant 2017/25/B/ST10/02221.

T 79

Spinodal decomposition in the formation of biogenic single

crystals of magnesium calcite E. Seknazi1, S. Kozachkevich1, I. Polishchuk1, N. Bianco Stein1, P.

Zaslansky2, A. Katsman1, B. Pokroy*1 1Technion Israel Institute if Technology, Materials Science and Engineering, Hiafa, Israel 2Charite, Berlin, Germany

As organisms can form crystals only under ambient conditions, they

demonstrate fascinating strategies to overcome this limitation. Recently, we

reported a previously unknown biostrategy for toughening brittle calcite crystals by means of pre-compression of the material, using coherently

incorporated Mg-rich nanoprecipitates arranged in a layered manner in the

lenses of a brittlestar, Ophiocoma wendtii. Here we propose the mechanisms of formation of this functional hierarchical structure under conditions of

ambient temperature and limited solid diffusion. For the first time, we

propose that formation proceeds via a spinodal decomposition of a liquid or gel-like magnesium amorphous calcium carbonate (Mg-ACC) precursor into

Mg-rich nanoparticles and a Mg-depleted amorphous matrix. In a second

step, crystallization of the decomposed amorphous precursor leads to the formation of high-Mg particles-rich layers. The model was supported by our

experimental results in synthetic Mg-calcite, which reinforce the concept of

a spinodal decomposition in the amorphous precursor. We also show that organics suppress the spinodal decomposition and that most probably the

paucity of organics present in the brittlestar"s mineralized tissue allows it to

occur in this system. These new insights have significant implications for fundamental understanding of the role of Mg-ACC material transformation

during crystallization and its subsequent stability.

T 80

Bionic synthesis of a magnetic calcite skeletal through living

foraminifera G. Magnabosco1, H. Hauzer2, C. Albonetti3, V. Morandi4, J. Erez2, G.

Falini*1 1Alma Mater Studiorum - Università di Bologna, Chemistry "Giacomo

Ciamician", BOLOGNA, Italy 2The Hebrew University of Jerusalem, Institute of Earth Sciences, Jerusalem, Israel 3National Research Council, Institute for Nanostructured Materials,

Bologna, Germany 4National Research Council, Institute for Microelectronics and Microsystems, Bologna, Italy

The peculiar functional properties of calcium carbonate biominerals, such as

shells, echinoderm spines and brittle stars, have stimulated both fundamental research in biomineralization and in vitro bio-inspired synthetic processes in

material science. As a consequence of the latter, bio-materials having

different/additional properties with respect to the natural ones have been produced by cell-free laboratory activities. Despite this effort, the production

of materials having functional properties even similar to the natural ones

remains elusive. Here we demonstrate that new nano-composite materials can be prepared in vivo exploiting the special mineralization pathway of the

foraminifer Amphistrigina lessoni, a calcifying organism producing a calcitic

skeleton by vacuolization of seawater. Accordingly, a bionic skeleton possessing a chamber entrapping magnetic nanoparticles has been obtained

growing the organism in seawater containing such nano-particles. Such a

bionic synthetic approach differs and goes beyond the biologically inspired synthetic processes and the biosynthesis of nano-particles by bacteria. It

represents the first research in which a bionic calcified tissue has been

prepared in vivo. This represents a new powerful tool for the preparation of nano-materials exploiting the capability of organisms to control the

calcification pathway with an accuracy that is unparalleled in laboratory cell

free synthetic processes.

28

T 81

Biomimetic precipitation of carbonates in tubular materials

formed in flow conditions C. I. Sainz-Diaz*1, E. Esacmilla-Roa2, J. Cartwright1 1Instituto Andaluz de Ciencias de la Tierra, CSIC-UGR, Armilla-Granada,

Spain 2Lulea University of Technology, Lulea, Sweden

Biomineral tubes are often secreted around an organism, or part of it, that forms a template about which the tube assembles. Another method of

forming biomineral tubes can be templating mineral tube formation around a

fluid jet. Such self-assembled fluid-flow-templated tubes are found in the physical sciences, in the so-called chemical gardens[1], and also in such

geophysical cases as volcanoes, soda straws in caves, and brinicles under sea

ice. Chemical gardens of carbonates have been prepared at laboratory forming different nano-morphology and crystallographic transformations by

nanoscale precipitation of carbonates and other metal oxides grown in the

surfaces of tubular materials generated in flow conditions out of equilibrium. The formed materials were characterized observing nanocrystals with

different morphology and chemistry depending on the growth conditions.

The interaction of several organic polymers has been explored during the precipitation of carbonates.

Molecular modeling calculations have been performed related with the

interaction of organic molecules on mineral solid surfaces. The adsorption of organic compounds showed to be an exothermic process. This indicates that

these nanomaterials can be good absorbants for organic compounds. These

results open interesting applications of these materials to clean polluted soils and as nanocarriers of bioactive compounds for therapeutic and

environmental applications.

[1] Barge, L. M.; Cardoso, S. S. S.; Cartwright, J. H. E.; Cooper, G. J. T.;

Cronin, L.; De Wit, A.; Dolobo↵, I. J.; Escribano, B.; Goldstein, R. E.; Haudin, F. et al. From chemical gardens to chemobrionics. Chem. Rev. 2015,

115, 8652–8703.

T 82

Curving crystals in biominerals and biomimetic minerals H. Imai*1, Y. Yukimasa1, M. Takasaki1, M. Suzuki1, Y. Oaki1, T. Sasaki2 1Keio University, Applied Chemistry, Yokohama, Japan 2The University of Tokyo, The University Museum, Tokyo, Japan

Introduction

A wide variety of biominerals are comprised of micrometric and nanometric

building blocks. We frequently observed unusual curving crystalline units as a building block in the biogenic hierarchical architectures. However, details

of the curving morphologies including their crystallographic structures and

their formation mechanism have not been clarified sufficiently. The clarification of the detailed structures would be important for understanding

of biogenic mineralization and development of biomimetic material

processing.

Objective

We characterized several kinds of curving crystals in biominerals to clarify

their crystallographic structure. Here we focused on aragonite helix of a pteropod shell and hydroxyapatite around the enamel-dentin junction of a

bovine incisor as typical examples of curving morphologies. Moreover, we

tried to produce biomimetic curving morphologies with calcium carbonate and calcium phosphate in artificial systems to discuss the formation

mechanism of the specifically designed crystals.

Materials and method

A petropod shell of C. globulosa was used for structural analysis of aragonitic

helical structures. Bovine incisors of cattle were used for characterization of

hydroxyapatite in tooth enamel. We fractured the teeth with a hammer and prepared small pieces of the specimen that exposed their cross section for

electron microscopies. Several pieces were treated with solutions of sodium

hypochlorite or disodium ethylenediaminetetraacetate to reveal their microstructures. The morphology of the shell surfaces and grown crystals

was observed using scanning microscopes. The crystallographic structures

were revealed by transmission electron microscope with selected area electron diffraction using cross-sectional samples prepared using focused ion

beam milling.

Rod-like crystals of calcium carbonate and calcium phosphate were prepared on several kinds of organic and inorganic substrates in supersaturated

aqueous solutions containing specific organic molecules. Curving

morphologies consisting of rod-like crystals were successfully produced with change in the direction of an ion flow.

Results and discussion

We observed the parallel assembly of curving aragonite fibers ~200−300 nm in width on the mildly etched shells of C. globulosa. The rod-like building

blocks 1−2 mm in length were found in the curving fibers as parts of the

helical architecture. The curving fibers are deduced to be composed of several rod-like building blocks whose a and b axes of the orthorhombic

crystal are intermittently deviated. We also characterized the crystallographic

structures in the enamel-dentin junction region of bovine incisors. The

enamel prisms of hydroxyapatite are regarded as a bundle of nanometer-scale fibers that are elongated in the c direction. Curving parts were observed in

the radial arrangements of the nanofibers around the root of the prisms. A

gradual stepwise change of the c direction is found to be essential for the formation of the curving parts.

We produced highly ordered arrays of c-axis-elongated aragonite nanorods

~100 nm diameter similar to the microstructure in lamellae of gastropods in a supersaturated solution containing specific organic molecules. We also

fabricated enamel-like fluorapatite nanorod arrays on an organic substrate in

an aqueous solution system that was based on the simulated body fluid. A gradual change in the growth direction along the c axis was induced through

a stepwise growth behavior. The direction of an ion flow is deduced to affect

a gradual change in the growth orientation of nanometer-scale crystalline rods.

Conclusion

We characterized aragonite helix of a pteropod shell and hydroxyapatite around the enamel-dentin junction of a bovine incisor. We produced

biomimetic curving morphologies with calcium carbonate and calcium

phosphate in artificial systems. The crystallographic orientation in the curving crystals in biominerals and biomimetic minerals was changed in a

stepwise fashion along nanometer-scale rods and fibers.

T 83

Minerobiolization - biomineralization at the origin of life J. Cartwright*1 1CSIC, Granada, Spain

If, as seems likely, life developed in hydrothermal vents it first used mineral membranes within the vents. The first photo-cells must have learnt to

manipulate the mineral membranes that formed their compartments in order

to control their metabolism. There must have occurred a biological takeover of the self-assembled mineral structures in the first proto-cells, with the

incorporation of proto-biological molecules within the mineral membranes

to alter their properties for life"s purposes, so that, for example, passive osmosis in a mineral membrane gradually became active chemiosmosis in a

proto-cell membrane. This biological takeover of a mineral system is what

we term minerobiolization, in contradistinction to the usual biomineralization in which the biology controls and assembles the mineral.

T 84

Insights into the evolution of biomineral (calcium and silicon)

transporters in viridiplantae M. A. Nawaz*1, I. Zemchenko1, X. Lin2, A. Zakharenko1, R. Muhammad

Atif3, T. F. Chan2, K. S. Golokhvast1 1Far Eastern Federal University, Education and Scientific Center of Nanotechnology, Vladivostok, Russian Federation 2The Chinese University of Hong Kong, Shatin, Hong Kong 3University of Agriculture, US-Pakistan Centre for Advanced Studies in Agriculture and Food Security, Faisalabad, Pakistan

Introduction

Biomineralization is a ubiquitous adaptive strategy in living organisms for

sustenance and structural integrity. Members of viridiplantae and especially

the vascular plants have mastered the art of transporting and accumulating silicon and calcium biominerals. To this respect silicon transporters (SITs),

NOD26-like major intrinsic proteins (NIPs) are responsible for silicon

transport and Ca2+ ATPases (pumps) and Ca2+ exchangers (CAXs) are primarily responsible for calcium transport.

Question

To resolve the sequestration mechanisms behind the deposition of these minerals i.e. the formation of phytoliths and cystoliths, it is essential to

understand the transport mechanism and especially how the transporters

evolved in plants and diverged functionally.

Methods

We performed comparative genomic studies supported by phylogenetic

reconstruction, gene structure analysis, duplication analysis, divergence

analysis, codon bias analysis, molecular evolution analysis, co-expression

networking and phylostratigraphic analysis to provide evolutionary insights.

Results and Conclusions

Here, we identified SITs, NIPs, Ca2+ pumps and CAXs in viridiplantae. Our

evidence suggests that segmental duplication was a prevalent evolutionary force for the expansion of viridiplantae biomineral transporters. Non-

terrestrial plants lost many members of biomineral transporters suggesting

lower biomineralization need/capacity. We identified that angiosperms experienced speciation followed by species/lineage-specific gene

duplications and similar gene modules existed across monocots and dicots.

The duplicated gene pairs have resulted in substantial neo-functionalization. Silicon transport-related genes expanded after embryophyte split. Dicots had

higher biomineral transporter genes owing to lineage-specific and species

specific whole genome duplications.

29

T 85

Evolution of planktonic gastropod calcification over short and

long timescales P. Ramos-Silva*1, D. Wall-Palmer1, L. Mekkes1,2, F. Marin3, F. Marlètaz4,

K. Peijnenburg1,2 1Naturalis Biodiversity Center, Marine Biodiversity, Leiden, Netherlands 2University of Amsterdam, Institute for Biodiversity and Ecosystem

Dynamics (IBED), Amsterdam, Netherlands 3University of Burgundy-Franche-Comté, Biogéosciences UMR CNRS 6282, Dijon, France 4Okinawa Institute for Science and Technology, Molecular Genetics Unit, Okinawa, Japan

Calcification by marine organisms can be drastically affected by ocean

acidification (OA) due to a reduced availability of calcium carbonate in the seawater. Pteropods and heteropods are planktonic gastropods believed to be

among the most vulnerable organisms to the effects of OA because they live

at the ocean surface and build thin shells of aragonite. Shelled pteropods have received considerable attention and are reported to decrease calcification

rates and experience shell dissolution under high CO2 conditions. Shelled

heteropods have received much less attention, but are expected to be equally vulnerable. Both groups are proposed as bioindicators to monitor the impacts

of global change on open ocean ecosystems. However, their vulnerability is

based on short-term exposures to extreme OA conditions. Similar to other mollusks, planktonic gastropods build shells through a biomineralization

process, which is biologically controlled at the molecular level. Still, little is

known about their evolutionary potential in the long-term. Using shell proteomics and transcriptomics we are currently identifying the

"biomineralization toolkit" in pteropod and heteropod species. Extensins,

collagens and whey acidic domain proteins are part of the repertoire of shell matrix proteins (SMPs) that is being identified in planktonic gastropods for

the first time. Next, SMPs are used to understand how biomineralization

evolved in the two independent plankton groups over short and long timescales. Over long timescales includes the evolutionary analysis of SMPs

using a fossil-calibrated phylogenomics tree. Over short timescales involves

studies at the population-level by measuring gene expression under past, present and future concentrations of CO2. Combining macro- with micro-

evolutionary approaches will shed light on the processes that drive diversity

and evolution of calcification in planktonic gastropods and will allow more realistic predictions of the consequences of global change on marine

calcifiers.

T 86

Biomineral growth kinetics and thermodynamics as an

architectural constraint on the evolution of molluscan shells I. Zlotnikov*1, V. Schoeppler1, R. Lemanis1 1B CUBE - Center for Molecular Bioengineering, Dresden, Germany

Molluscan shells are a classical model system to study formation-structure-

function relationships in biological materials and the process of biomineralized tissue morphogenesis. Typically, each shell consists of a

number of highly mineralized ultrastructures, each characterized by a specific three-dimensional mineral-organic architecture. Surprisingly, in

some cases, despite the lack of a mutual biochemical toolkit for

biomineralization or evidence of homology, shells from different independently evolved species contain similar shell ultrastructures. In the

current study, using a recently developed physical framework, which is based

on an analogy to the well known process of directional solidification, we compare the process of ultrastructural morphogenesis of shells from three

major molluscan classes: a bivalve Unio pictorum, a cephalopod Nautilus

pompilius and a gastropod Haliotis asinina. First, we demonstrate that the fabrication of these highly biomineralized tissues is guided by the organisms

by regulating the chemical and physical boundary conditions that control the

growth kinetics of the mineral phase. Second, we expand our understanding of the theoretical macroscopic morphospace of possible molluscan shell

shapes (Raup's concept of morphospace) to the level of possible

ultrastructures that comprise them. Finally, we shed a new light on the evolutionary aspect of molluscan shell ultrastructural fabrication within the

framework of Seilacher's Constructional Morphology and Morphodynamics.

We suggest that the repeated "discovery" of some mineral morphologies reflects a series of architectural constraints provided specifically by

biomineral growth kinetics.

T 87

The shell microstructure of the species of Gigantoproductus

(carboniferous), the giants of the phylum brachiopoda L. Angiolini*1, G. Crippa1, K. Azmy2, G. Capitani3, G. Confalonieri4, G.

Della Porta1, E. Griesshaber5, D. Harper6, M. Leng7, L. Nolan8, M. Orlandi3,

R. Posenato9, W. W. Schmahl5, V. Banks7, M. Stephenson7 1Università degli Studi di Milano, Scienze della Terra A. Desio, Milano,

Italy 2Memorial University of Newfoundland, St. John's, Canada 3Università degli Studi Milano Bicocca, Milano, Italy 4Università degli Studi di Torino, Torino, Italy 5Ludwig-Maximilians Universität München, Munchen, Germany 6Durham University, Durham, United Kingdom 7British Geological Survey, Keyworth, United Kingdom 8University of Leicester, Leicester, United Kingdom 9Università di Ferrara, Ferrara, Italy

The species of Gigantoproductusreach over 30 cm in width and are considered giants within the Palaeozoic sedentary marine benthos. Their

shell thickness is remarkable, reaching over 1 cm and consisting of a thin

pseudopunctate laminar secondary layer and a very thick columnar tertiary layer, made of a few hundreds of micrometres long, substructured columnar

units. The microstructure of several specimens has been investigated by

multiple analyses [petrography, cathodoluminescence (CL), Scanning Electron Microscopy (SEM), Electron Backscatter Diffraction (EBSD),

Transmission Electron Microscopy (TEM)]. The analysed shells are

generally well preserved, but locally altered by authigenic silica replacement of brownish, fibrous chalcedony and anhedral to subhedral microquartz

forming concentrically laminated spherulites, and euhedral megaquartz

crystals with hexagonal basal section, which embed calcite crystals and growth lines of the brachiopod shells. Petrographic and isotope analyses

show that silica replacement of the gigantoproductid outer shell occurred

during early diagenetic phase. Despite this, EBSD analyses show that the shells retain the presence of large pristine columnar units with crystallites

being highly co-oriented. MUD values are 41 to 71, except for occasionally

overprinted shell margins with low MUD values. TEM analyses also show that the columnar layer is formed by nanoscopic, biocomposite mesocrystal

calcite, that is built up by grains approximately co-oriented and that it

contains nanometres inclusions, with a dark contrast, forming trails between calcite grain borders similar to those observed in Recent brachiopod shells.

These inclusions were analysed by Nuclear Magnetic Resonance (NMR), and Gas Chromatography Mass Spectrometry (GC-MS) analyses, which showed

that an occluded organic fraction is preserved in these ancient fossil shells,

and its preserved amino acid composition is comparable with that observed in Recent brachiopod taxa.

Finally, the analyses of the carbon- and nitrogen-isotopic compositions of the

occluded organic matrix within their calcite shells allowed to explain the gigantic size and thick carbonate skeleton of these Palaeozoic benthic

brachiopods, as the result of a mixotroph lifestyle, by which they could rely

on the energy and nutrients derived both from photosymbiotic microbes and from suspension feeding.

T 88

How did the carrier shell Xenophora crispa build its shell?

Evidence from the recent and fossil record G. Crippa*1, G. Pasinetti1, M. Dapiaggi1 1University of Milan, Milano, Italy

The animal kingdom offers several examples of organisms forming their

exoskeletons selecting and agglutinating objects from the surrounding

environment. The most famous and spectacular among these agglutinating organisms is probably represented by the carrier shell Xenophora Fischer von

Waldheim, 1807. The genus Xenophora comprises species of marine

gastropods, known from the Cretaceous to the Recent, which are able to agglutinate fragments of different origins to form their shells; they show

different agglutination potentials, from species lacking attachments to

species completely covered by agglutinated materials, as the Mediterranean Xenophora crispa. Here, we analyse Recent and fossil specimens of

Xenophora crispa from the Mediterranean area at the Scanning Electron

Microscope and X-Ray Powder Diffraction, to better understand its biomineralization pattern and the mechanisms leading to the agglutination of

shells and bio/lithoclasts. Also, we provide new data on the poorly described

gastropod shell microstructures which knowledge is generally limited to a relatively small number of specific taxonomic groups.

We conclude that: a) most of the Xenophora crispa shell is composed by an

aragonitic crossed lamellar fabric, but a fibrous to spherulitic prismatic fabric seemingly of calcite has been found in the columella and in the peripheral

edge, i.e. the thickest parts of the shell; b) the attachment of objects is

mediated by a prismatic microstructure, indicating that this is the most functional fabric in attachment areas in molluscs; c) the functional meaning

of the agglutination in Xenophora crispa is related to a snowshoe strategy to

30

successfully colonize muddy substrates. Indeed, this species secretes in the columella and in the peripheral edge a less dense, less hard and more organic

rich calcitic fabric, to lighten the thickest parts of the shells in order not to

sink in soft sediments.

T 89

Molecular evolution of the matrix proteins of shells and darts

in terrestrial snail Euhadra quaesita K. Shimizu*1,2, K. Kimura3,4, Y. Isowa5, K. Oshima2, M. Ishikawa2,6, H. Kagi2, K. Kito5, M. Hattori2,7, S. Chiba3, K. Endo2 1University of Exeter, College of Life and Environmental Sciences, Exeter,

United Kingdom 2The University of Tokyo, Tokyo, Japan 3Tohoku University, Sendai, Japan 4Kyungpook National University, Bukgu, South Korea 5Meiji University, Kawasaki, Japan 6Yamazaki University of Animal Health Technology, Hachioji, Japan 7Waseda University, Tokyo, Japan

Mollusks are among the most diverse calcifying animals and are well studied

for various aspects of their biomineralization. Although many shell matrix proteins (SMPs) have already been reported in mollusks, most reports have

focused on marine mollusks, and the SMPs of terrestrial snails remain

unclear. In addition, some terrestrial stylommatophoran snails have evolved a novel calcified apparatus, known as "love dart", used for mating behavior.

We identified 54 SMPs in the terrestrial snail Euhadra quaesita. The SMP of

the highest abundance index value in E. quaesitais similar to the most abundant SMP in the other hitherto studied pulmonate Cepaea nemoralis.

These proteins contain Pro- and Gly-rich regions, but have no known specific

domains and have no homologous proteins in the GenBank non-redundant protein database except for the proteins reported from pulmonates. These

novel proteins may have evolved in the last common ancestor of pulmonates

with a key role in their shell mineralization under terrestrial or freshwater environments. We then identified four dart matrix proteins (DMPs) and

found that two of them are the same proteins as those identified as SMPs in

this species. Our results suggest that some DMPs possibly have evolved by independent gene co-option from SMPs during dart evolution events. These

results provide a new perspective on the evolution of SMPs in the terrestrial

environment and the novel calcified feature of "love darts" in land snails.

P 01

Self-assembling block copolymers in the nucleation of

hydroxyapaptite Y. Jhons*1, N. Judge1, C. Fowler2, F. Nudelman1 1University of Edinburgh, Chemistry, Edinburgh, United Kingdom 2GlaxoSmithKline, GSK Consumer Healthcare, Weybridge, United Kingdom

Dental erosion is a worldwide pandemic with over 3.6 billion people

suffering from tooth decay, with 486 million children with caries on their permanent teeth.1 Enamel is the outer layer of the teeth, is highly mineralised

with around 95 % (by volume) of the structure being formed of carbonated hydroxyapatite (HAP), a form of mineralised calcium phosphate (CAP). The

exposure of enamel to acids from bacterial fermentation or from our diets

results in dissolution of the mineral and the formation of irreversible lesions that can result in tooth loss if left untreated. To address these issues, polymers

that are able to bind to enamel and prevent demineralization have been

investigated, however the effect of the polymer structure on the growth of HAP have not yet been studied.2

The aim of this project is to design and synthesise self-assembling

phosphorous-containing polymers that either prevent demineralisation or promote controlled nucleation and growth of HAP in enamel. We

hypothesize that controlling the self-assembling morphology can result in

controlled HAP growth, which could be key to remineralizing enamel. Here reversible addition fragmentation chain transfer (RAFT) polymerisation has

been used to synthesize phosphorous-based triblock copolymers. Using

dynamic light-scattering (DLS) and scanning electron microscopy (SEM), we observed that the triblock copolymer forms hollow spheres 300 – 700 nm

in size at concentrations above 6.2 x 10-3 mg cm-3. When incubated in tris

buffer pH 7.4 containing 2 mM of CaCl2 and 1 mM of K2HPO4 at 37 ºC, DLS and SEM demonstrate that the polymer at concentrations above 7.5 x 10-3 mg

cm-3 promotes the precipitation of calcium phosphate. Control experiments

without additives or using similar polymers that are either devoid of the phosphate group or that do not self-assemble did not result in calcium

phosphate precipitation. In conclusion, we synthesized a triblock copolymer

that promotes the precipitation of calcium phosphate. Both the self-assembly into micelles and the presence of phosphate groups in the polymer are needed

to promote calcium phosphate precipitation. Next steps will be to use

transmission electron microscopy to study their 3D morphology and characterize the form of calcium phosphate precipitated.

References

1 T. Vos, A. A. Abajobir et. al., Lancet, 2017, 390, 1211–1259.

2 Y. Lei, T. Wang, J. W. Mitchell, L. Zaidel, J. Qiu and L. Kilpatrick-

Liverman, RSC Adv., 2014, 4, 49053–49060.

P 02

The effects of protein incorporation and crosslinking on the

mechanical properties of mineralized chitin matrices J. Elias*1, L. Gower1 1University of Florida, Materials Science and Engineering, Gainesville, United States

Many natural protective systems, such as the exoskeletons of various

arthropods, use specialized organic templates and additives to guide the

mineralization of calcium phosphate, calcium carbonate or other mineral species. These natural mechanisms create mineral-organic composite

structures with attractive mechanical properties tailored to specific

environments, making them a subject of interest for many materials researchers. Even though there have been many attempts to mimic the

structure and properties of the crustacean cuticle in synthetic materials, there

are still interactions that are not fully understood because of the limitations of studying natural organisms. In vitro model systems, however, can provide

for the modification and isolation of system variables, making them valuable

for the understanding of various natural systems. The goals of this proposal are to: 1) Synthesize ordered chitin templates, then incorporate silk fibroin

or methacrylated silk fibroin into the templates to create chitin-fibroin

structures with varying degrees of crosslinking, 2) mineralize templates with calcium carbonate using a polymer-induced liquid precursor (PILP) process

and evaluate the effects of fibroin incorporation and crosslinking on the

mineralized structures, and 3) determine the effect of fibroin incorporation on the hardness, modulus, and fracture toughness of mineralized templates

using nanoindentation. The liquid crystalline ordering and protein

stabilization is expected to produce structures that mimic the helicoidal ordering of the chitin-protein phase in the arthropod exoskeleton, and the

biomimetic mineralization of these templates can create structures that mimic

the crustacean cuticle composite structure. This research can provide insight into the possible mechanisms used by biological organisms to create unique

hierarchical structures as well as investigating unique mechanisms that may

be used as inspiration for the synthesis of new composite structures.

P 03

Influence of bioaragonite microstructure in the kinetics of its

pseudomorphic replacement by apatite L. Fernández-Díaz*1,2, M. Greiner3, E. Griesshaber3, D. Reinares4, M. Zenkert3, X. Yin3, A. Ziegler5, S. Veintemillas-Verdaguer4, W. W.

Schmahl3 1Universidad Complutense de Madrid, Mineralogy and Petrology, Madrid, Spain 2Institute of Geosciences (IGEO) , Geomaterials, Madrid, Spain 3Ludwig-Maximilians-Universität, Geo-und Umweltwissenschaften, Munich, Germany 4Institute of Material Sience of Madrid (ICMM, CSIC), Madrid, Spain 5University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany

Numerous features of carbonate biological hard tissues are inspirational for

the development of new functional materials. Their characteristic internal

hierarchically arranged porosity and their microstructure are among these features. Both can partially or totally be preserved during the transformation

of carbonate biominerals into apatite after interaction with boiling phosphate-

bearing aqueous solutions. This carbonate-apatite transformation involves the development of interface coupled dissolution-crystallization reactions

(ICDR) and commonly takes place with the preservation of the biomineral

external shape as well. In this work we study the kinetics of the pseudomorphic replacement by apatite of the bioaragonite of the cuttlebone

of the cephalopod Sepia officinalis and the different portions of the shells of

the bivalves Arctica islandica and Hyriopsis cumingii. All these biominerals have distinct microstructures and distribution of occluded organic

biopolymers. The kinetics of the transformation is fastest for Sepia

officinalis cuttlebone, which after only 10 hours of interaction with a (NH4)2HPO4 boiling aqueous solution is converted in a 90 wt % apatite

scaffold [1, 2]. This conversion is accompanied by a significant increase in

the biomineral surface area, which is attributed to the generation of new porosity that facilitates the progress of the ICDR. All other biominerals

transform into apatite at a much slower rate [2]. Both, the inner and outer

layers of the shell of Arctica islandica transform into apatite at similar rates, but after 14 days of interaction with the phosphate-bearing solution only

reach transformation percentages as small as 10 wt% and 15 wt%,

respectively. The prismatic portion of the shell of Hyriopsis cumingii is more reactive than the shell of Arctica islandica, with transformation percentages

above 25 after 14 days interaction with the phosphate bearing solution. In

31

contrast, the aragonite of the nacreous portion of this shell is extremely unreactive and remains virtually pristine during the whole duration of the

interaction experiment (14 days). We interpret that the distinctly different

kinetics of the into apatite transformation reflect the different accessibility of the phosphate-bearing aqueous solution to the mineral component of the hard

tissues studied. Differences in fluid accessibility arise from the

characteristics of each hard tissue regarding their porosity, their content of biopolymers and their ultra- and microstructure as well as the specific

evolution of each of these features as the ICDR progresses.

References

[1] Reinares-Fisac, D.; Veintemillas-Verdaguer, S.; Fernández-Díaz, L.

Conversion of biogenic aragonite into hydroxyapatite scaffolds in boiling

solutions. CrystEngComm, 2017, 19, 110-116. [2] Greiner, M.; Fernández-Díaz, L.; Griesshaber, E.; Zenkert, M.; Yin, X.;

Ziegler, A.; Veintemillas-Verdaguer, S.; Schmahl, W. W. Biomineral

reactivity: The kinetics of the replacement reaction of biological aragonite to apatite. Minerals, 2018, 8(8), 315.

P 04

Mineral replacement of bioaragonite by apatite – differences

between symbiotic and asymbiotic corals P. Forjanes*1, M. Greiner2, E. Griesshaber2, L. Fernández-Díaz1,3, I.

Coronado4, J. Stolarski4, M. Zenkert2, D. Joester5, L. Stegbauer5, U.

Rameshbabu5, S. Veintemillas-Verdaguer6, W. W. Schmahl2 1Universidad Complutense de Madrid , Mineralogía y Petrología, Madrid,

Spain 2LMU Munich, Department of Environment and Earth Sciences, Munich, Germany 3Institute of Geosciences (CSIC, UCM), Madrid, Spain 4Institute of Palaeobiology,, Warsaw, Poland 5Northwestern University, Materials Science and Engineering, Evanston,

United States 6Institute of Materials Science (CSIC), Madrid, Spain

Scaffolds for bone tissue engineering can be obtained through the

pseudomorphic mineral replacement of carbonate skeletons of invertebrates by apatite upon interaction with boiling phosphate-bearing aqueous solutions

[1, 2]. These skeletons exhibit porosity at different length-scales, which is

preserved during the mineral transformation process. Their characteristic pore sizes and pore distribution are closely matching those ideals for bone

grafting. In this work, we assessed the kinetics of the aragonite-apatite transformation of skeletons of four coral taxa: Acropora, Porites, Lophelia,

and Madrepora. Coral skeletons are organic-inorganic composites that show

distinct function-related characteristics, regarding their microstructure, skeletal density, original porosity compositional and distributional

differences in organic biopolymers and geochemistry. These distinct

features, which are significantly different in some symbiotic (Acropora and Porites) and asymbiotic (Lophelia and Madrepora) corals, have a complex

influence on the skeleton reactivity. Skeletons of Acropora and Porites

contain larger volumes of macro- and mesoscale porosities and have rougher surfaces. In contrast, the skeletons of Lophelia and Madrepora are compact

and show smooth surfaces. Moreover, the bioaragonite of symbiotic corals is

Mg-richer and Sr-poorer than that of asymbiotic corals. Further differences regard the characteristics of their organic matrix. The highest content of

organics is found in the skeleton of Madrepora oculata, with 6.05%,

followed by Lophelia pertusa, with 4.93%. The two symbiotic corals, Acropora sp. and Porites sp., show lower contents of organics, with 2.64%

and 2.10%, respectively.

The skeletons of Madrepora and Lophelia transform at a slower rate in contrast to Porites and Acropora into apatite during the first 4 days of

interaction with the phosphate-bearing solution. This is followed by a

subsequent latent period, without apatite replacement, that lasts 7 to 9 days and afterwards, the transformation rate rapidly increases. The fastest

transformation kinetics corresponds to the skeletons of the two symbiotic

corals Porites and Acropora, whose apatite content after 14 days of interaction reaches 39 wt% and 60 wt%, respectively. After this period the

skeleton of Lophelia contains only 15.3 wt% of apatite. Interestingly, the

skeleton of Madrepora is the least reactive, reaching only a 2.2 wt% of apatite at the end of the experiment. We conclude that the transformation of

coral bioaragonite into apatite is facilitated by the higher porosity and

rougher surfaces of the skeletons of symbiotic corals. The higher content of organics of asymbiotic coral skeletons seems to prevent aragonite

transformation. Further differences in aragonite into apatite transformation

kinetics might be modulated by both, biopolymer decomposition rate and composition-related differences in aragonite solubility.

References [1] D. Reinares-Fisac, S. Veintemillas-Verdaguer, L. Fernández-Díaz. Conversion of biogenic aragonite into hydroxyapatite scaffolds in boiling

solutions. CrystEngComm 2017, 19, 110-116.

[2] M. Greiner, L. Fernandez-Diaz, E. Griesshaber, M.N. Zenkert, X. Yin, A. Ziegler, S. Veintemillas-Verdaguer, W.W. Schmahl, Biomineral Reactivity:

The Kinetics of the Replacement Reaction of Biological Aragonite to Apatite. Minerals 2018, 8, 315.

P 05

Synergetic organic-inorganic interactions regulate

mineralization Y. C. Huang*1, B. Wu2, M. Drechsler3, S. J. Huang4, A. Rao5, J. C. C.

Chan4, D. Gebauer1,6 1University of Konstanz, Chemistry, Konstanz, Germany 2Forschungszentrum Jülich, Jülich Centre for Neutron Science, Garching,

Germany 3University of Bayreuth, Bayreuth Institute for Macromolecular Chemistry, Bayreuth, Germany 4National Taiwan University, Chemistry, Taipei, Taiwan 5University of Twente, Faculty of Science and Technology, Enschede, Netherlands 6Leibniz University of Hannover, Institute of Inorganic Chemistry, Hannover, Germany

Biomineralization is regulated by biomolecular and ionic species in complex

physiological environments. However, the underlying mechanism of how organisms precisely exert control over mineralization still remains elusive.

In the present study, we address the fundamental aspects of nucleation and

crystallization of calcium carbonate (CaCO3), a vital biological and geological mineral. The bidirectional relations between specific protein

moieties (CTL domains) of the sea urchin spicule proteome and distinct

inorganic entities in the course of mineralization are elucidated. Given the pH-dependent speciation of (bi)carbonate ions, a potentiometric titration

methodology was implemented for quantitatively investigating CaCO3

mineralization at near-neutral pH levels (pH 7.5–9.0). Notably, our investigation brings forth the mechanistic contributions of HCO3

- ions in

mineral nucleation as active soluble species interacting with ionic

components and structural constituents of the emergent solid amorphous phases. Moreover, exploring the influences of recombinant proteins

associated with sea urchin skeletons (CTL proteins), biophysical properties

involving ion-complexation and self-association are found to impact mineralization. In particular, in the presence of Mg2+ ions at lower pH levels,

the investigated CTL proteins exert enhanced control over mineralization.

This implies that in physiological scenarios, with the synergy of "spectator" ion species (e.g. Mg2+ and HCO3

- ions), minute quantity of biomolecules can

profoundly govern mineral nucleation. Collectively, our findings suggest that biomineralization, emerging as a biologically programmed multistep

crystallization reaction, is a bidirectional process which encompasses (i)

ionic and biomolecular additives that regulate the nucleation and crystallization of inorganics and (ii) distinct mineral entities that tune the

self-association of biomolecules.

P 06

Green synthesis of silver nanoparticles using Crocus sativus

corms aqueous extract and evaluation of their antibacterial

activity and cytotoxic effect on human ovarian cancer cell line

(A2780cp) A. Taghva*1, M. Entezari2, S. Ghafoori3 1Islamic Azad University Farahan Branch, Microbiology, Tehran, Iran 2Islamic Azad University Tehran Medical Sciences branch, Biology,

Tehran, Iran 3Payame Noor University, Biotechnology, Tehran, Iran

Introduction Recently, the application of nanoparticles is grown in various fields such as

biotechnology, nanotechnology, physics, chemistry, materials science, as

well as other new commercial applications. Noble metal nanoparticles such as gold, silver, and platinum present unique physicochemical properties,

which are not observed in larger metal nanoparticles, therefore, the metal

nanoparticles are used in different fields such as optical devices, catalysis,

biological labelling, drug delivery system, and cancer therapy.

Objectives In the present investigation, biosynthesizing silver nanoparticles (AgNPs) via Crocus sativus corm aqueous was explored. Then, antibacterial and

mutagenicity potential of the silver nanoparticles was investigated by Agar

Well Diffusion and Ames methods, respectively. Finally, the cytotoxic effect of AgNPs against human ovarian cancer cells was examined by the MTT

method.

Materials and methods

Biosynthesis of silver nanoparticles has done by subjecting Crocus sativus

corm aqueous extract and 0.001 Mm silver nitrate solution. After color

changing, nanoparticle production has investigated via spectrophotometry, XRD, and TEM. Then, antibacterial properties of produced silver

nanoparticles have investigated by Agar Well Diffusion and MIC determining Methods. Also, mutagenicity potential of produced silver

nanoparticles has determined by Ames test. Finally, cytotoxic effects of the

32

created the nanoparticles on A2780cp cell line has checked by cell culture and MTT method.

Results

The results indicated that nanoparticles were produced in 45 minutes by reducing silver nitrate ions and color changing occurred during this time.

Spectrophotometry assay showed an absorbance peak at 425 nm wavelength.

Also, XRD test was confirmed nanoparticles production. TEM microscopy indicated that the size of produced AgNPs was ranging from 5 to 25 nm with

a spherical shape. Antibacterial properties of silver nanoparticles were

approved by both Agar Well Diffusion and MIC test methods. Moreover, No mutagenic activity was seen in produced AgNPs. MTT assay after treatment

of human ovarian cancer cell line (A2780cp) with AgNPs in 24 h showed

that there is a dose-dependent cytotoxic activity in produced AgNPs against cancer cell line.

Conclusion It is clear that silver nanoparticles have a high potential to be used in industry and medicine. However, they should be examined from various aspects such

as safety, toxicity, and so on.

P 07

Enhancement in the photobiological hydrogen production of

chlorella-material hybrids by dimethyl sulfoxide Y. Zhao*1, L. Shu1, C. Shao1, W. Xiong2, R. Tang1 1Zhejiang University, Hangzhou, China 2Nanjing University, Nanjing, China

Photobiological production of hydrogen is low energy consumption and environmental friendliness, ensuring the generation of clean and renewable

energies. It has been reported that Chlorella aggregates, which is induced by

silica-based materials, can continuously produce hydrogen photobiologically under normal aerobic conditions. Even so, the yield is relatively low, which

equals only 0.42 % of the light‐to‐H2 energy‐conversion efficiency.

However, we find that a simple addition of dimethyl sulfoxide (DMSO) into an aqueous environment (to 0.5 vol %) can significantly promote the H2 yield

of Chlorella aggregates, reaching 0.69 % of the light‐to‐H2 energy‐

conversion efficiency. The improvement is explained by a DMSO-induced increase in the cellular respiration rate. This will lead to a decrease of the

oxygen content within the aggregates, and subsequently resulting in the

activation of more hydrogenases. In general, this strategy exhibits a functional enhancement by a combination of small molecules and organism-

material hybrids.

P 08

Non-classical crystallization of calcium carbonate towards

single crystal formation Z. Liu*1, H. Pan1, Z. Zhang2, Z. Wang3, B. Jin1, R. Tang1, J. J. De Yoreo3 1Zhejiang University, Chemistry, Hangzhou, China 2Xiamen University, Xiamen, China 3Pacific Northwest National Laboratory, Richland, WA, United States

Crystallization by particle attachment or two step nucleation has been widely

observed in both natural and synthetic environments. However, this non-classical crystallization towards single crystal formation cannot be explained

by classical nucleation theory, and many mysteries are still remained to

understand this process. We herein use calcium carbonate (CaCO3), which is a typical geological and biological mineral, as a model to investigate its

crystallization. It is revealed that a random particle attachment followed by a

self-orientation could occur on CaCO3 particles, leading to single crystal formation. And this is attributed by its surface stress induced grain-boundary

migration. Besides, we find the crystallization pathway of amorphous CaCO3

can be controlled by magnesium ion (Mg), which is a widely observed impurity in biominerals. In most cases, the crystallization of amorphous

CaCO3 is dominated by dissolution/re-precipitation process, and the resulting

crystal exhibit expected morphologies. Only at high Mg concentration, the crystallization occurs in the absence of a morphological change to give

spheroidal single crystalline Mg-calcite. It is explained by the high water

content in Mg doped amorphous CaCO3, in which the reorganization of molecules in solid-phase becomes available. Our findings promote the

understanding of crystallization in nature, implying some potential

mechanisms in biomineralization. Most importantly, these results can shed light on the construction of functional crystals with controllable crystalline

orientation and morphology, by design.

P 09

Biosynthesis and evaluation of the characteristics of silver

nanoparticles using Cassia fistula fruit aqueous extract and its

antibacterial activity S. Ghafoori*1, A. Taghva2, Z. Tayebi3, M. Hashemi4 1Payame Noor University, Biotechnology, Tehran, Iran 2Islamic Azad University, Farahan Branch, Microbiology,, Farmahin, Iran 3Islamic Azad University, Tehran Medical Sciences, Faculty of Medicine, Microbiology, Tehran, Iran 4Islamic Azad University Tehran Medical Sciences branch, Genetics, Tehran, Iran

Introduction

There are several ways of nanoparticles production, but the biological method of nanoparticles production is under the attention of researchers due

to its eco-friendly and energy saving properties.

Objectives In the present study biosynthesis of silver nanoparticle by Cassia fistula fruit

extracts were examined and mutagenesis potential of nanoparticles was

investigated. Furthermore, the antibacterial effect of the produced nanoparticles was investigated on In vitro and In vivo.

Materials and methods

In order to nanoparticles production, the fruit extract was subjected to the silver nitrate aqueous solution at the final concentration of 1 mMolar. After

nanoparticles production, the color changed reaction mixture was used for

characterization with spectrophotometry, X-ray diffraction analysis (XRD), Transition electron microscope (TEM) microscopy and DLS. Then, the

antibacterial effect of the produced nanoparticles was investigated by agar well diffusion method against three bacterial pathogenic strains. Mutagenesis

effect of silver NP was investigated by the Ames test. At the final, wound

healing repair of silver Nano NP in vivo was examined.

Results After nanoparticles production, the color of the plant extract was converted

to dark green attributed to the surface plasmon resonance band (SPR) of the silver nanoparticles. Visible spectra of the color changed extract had

maximum absorption peaks around 418 nm. Furthermore, the presence of the

silver nanoparticles was confirmed by the XRD. TEM analysis revealed that the obtained silver nanoparticles were triangle, hexahedron and spherical in

their shapes. DLS test has shown that average sizes of nanoparticles were

around 3/6-4/5nm. Antibacterial assays revealed that the produced nanoparticles had suitable effects against all of the three bacterial strains.

Mutagenic effects of nanoparticles were not observed. The silver

nanoparticles have a suitable effect on preventing wound infection in mice were tested.

Conclusion

It seems that the biological production of nanoparticles with the usage of these plant extracts is able to enhance their medicinal effects. In this study, it

is indicated that the antibacterial property of the extracts containing

nanoparticles was promoted considerably. Promoting other effects of these nanoparticles can be prospective for future studies.

P 10

SEM/EDS analysis of microbial induced calcium carbonate

crystal formation in agarose hydrogels B. Christgen*1, H. Mitrani1, M. Zhang2 1Newcastle University, School of Engineering, Newcastle upon Tyne,

United Kingdom 2Northumbria University, Health and Life Sciences, Newcastle upon Tyne, United Kingdom

Introduction

Microorganism mediated processes are present in every environment on the

Earth. Understanding how bacteria interact with and use their environment to survive is essential to translate to and use this knowledge in sustainable

biotechnologies. Microbial induced calcite precipitation (MICP) as a

potential low cost and environmental sustainable process has gathered extensive interest in the geotechnical and construction community for soil

improvement and as a sustainable construction material in recent years.

Underground carbon storage, sustainable biobricks, and healing and corrosion protection of cracks in cement based building materials are

examples of the multitude of applications investigated. Hydrogels have long

been used to investigate chemical crystal growth and morphology for calcium carbonate. Additionally hydrogels can be used as soil analogues to study

bacterial growth and MICP in situ in a more confined and easily accessible

environment.

Objectives

In this study we investigated the influence of variations in the cementation

media on micro-scale calcite growth and distribution in agarose hydrogels over time using S. pasteurii as a model organism.

33

Materials and Methods

S. pasteurii was grown over night in liquid culture before suspension in

agarose hydrogels which contained a cementation media of nutrient broth,

urea, CaCl2 (at concentrations between 30 mM and 480 mM), NH4Cl and NaHCO3. The hydrogels were grown between two to nine days at 30 °C and

were then analyzed by SEM/EDS and the calcium carbonate content was

measured. SEM specimen were prepared by flash freezing in liquid nitrogen to avoid structural deformations during the freeze drying process and then

freeze dried for 24 hours at -80 °C under vacuum. Before SEM analysis, the

specimen were sputter coated with a 5 nm layer of platinum using a high resolution sputter coater. The specimen were visualized at low voltage (1.8

kV) to avoid damaging the hydrogel structure while the EDS X-ray spectrum

for elemental analysis was obtained using 20 kV afterwards.

Results

SEM analysis shows spherical crystal growth after three days days incubation

of the hydrogels at 30 °C. Crystal size varied between 10µm and 300µ in different specimen depending on incubation time and CaCl2 concentration.

Higher calcium chloride concentrations in the cementation media led to more

and larger crystal formation in the hydrogel. EDS X-ray spectra of the crystals confirmed the presence of carbon, oxygen and calcium, the elements

that make up CaCO3 which verifies that the formed crystals are calcite.

Control hydrogel samples incubated without bacteria and/or without calcium chloride which were treated the same as the main specimen showed no crystal

formation.

Conclusion

SEM and EDS anlysis have been used to investigate calcite formation in

hydrogels from MICP induced by S. pasteurii and cementation media over time. EDS confirmed that the spherical crystals observed in the hydrogels

were calcite and growth depended on incubation length as well as the

concentration of calcium chloride in the cementation media used.

P 11

Use of biomineralized microsilica and highly nanostructured

CaCO3 particles for biosensors and bioseparation S. Kim1, M. B. Gu*1 1Korea University, Department of Biotechnology, College of Life Sciences and Biotechnology, Seoul, South Korea

Introduction

The calcium carbonate (CaCO3), which is most abundant biomineral found

in nature, is commonly used as exoskeletons of many life forms such as algae, clam, and sea urchins. Recently, there has been a growing interest in using

CaCO3 for diverse applications due to their versatility and unique properties.

Especially, CaCO3 can be promising materials in biotechnological areas. Furthermore, the various hierarchical structures of biominerals make CaCO3

based materials more attractive.

Objectives

We report the application of CaCO3 based biomineral in the biosensing and

bioseparation using aptamer as a bioreceptor. For biosensing application,

CaCO3 structure derived from unicellular algae was utilized to enhance the analytical performance of the biosensor. For bioseparation application,

aptamer conjugated magnetically separable silica coated CaCO3

microparticles were developed for antibiotics removal.

Materials and Methods

For biosensing application, CaCO3 microparticle derived from algae was

applied for surface modification of electrode. Briefly, 1 μl of algae-derived CaCO3 structure (45 mg/ml) was applied to the substrate, and the gold

working electrode was formed by sputtering. Sandwich-type binding

aptamers were applied for sensing type-2-diabetes. For bioseparation application, aptamer conjugated magnetically separable silica coated calcium

carbonate microparticles were synthesized by simple procedures. Briefly, 20

mM of CaCl2 in Tris-HCl (1 M, pH 8.3) solution with Poly-acrylic acid (0.1 mg/ml) and 100 μL of magnetic nanoparticles was stirred in a CO2 chamber

and coated with silica by the modified sol-gel. Finally, oxytetracycline

binding aptamers were immobilized on to the particle for specific removal oxytetracycline.

Results

The morphological, electrochemical characteristics and the performances of the newly developed biosensor were investigated by SEM, EDAX, cyclic

voltammetry, and chronoamperometry. The electroactive surface area was

increased about 3.8-fold compared to a commercial screen-printed electrode (SPGE) which is likely due to the roughened surface provided by the

nanostructured CaCO3 microparticles. The analytical performance of the

newly developed biosensor was enhanced, when compared to the SPGE, by 1.16-fold and 3-fold in buffer and serum condition, respectively. The

improved analytical performance could be explained by the surface

properties of algae-derived CaCO3 microparticles which increased electroactive surface area affecting the diffusion of electrolytes and analytes.

Aptamer-conjugated magnetically separable silica coated calcium carbonate

microparticles for antibiotics removal were successfully synthesized and characterized by SEM, FT-IR, EDAX, BET, and CLSM. About 6 μm sized

aptamer modified CaCO3 microparticles showed low non-specific adsorption

to oxytetracycline (OTC) which was less than 15%. The selective capturing efficiency towards OTC in both buffer and tap water was about 85% and

73%, respectively. Moreover, these bio-hybrid mineral microparticles were

found to be stable, even after 5 repeated usages, maintaining the initial capturing efficiency of 72%.

Conclusion

Using the biomineralized and highly nanostructured CaCO3 microparticles for biosensing and bioseparation, we could successfully detect and capture

the target molecules with improved performances. With the advantages of

biominerals, these new studies could open up new possibilities for biotechnological applications of biominerals.

P 12

Calcium carbonate crystals formation under the influence of

otolithic and otoconial matrix collagen-like protein otolin-1 K. Bielak*1, A. Zoglowek1, J. Stolarski2, A. Ożyhar1, P. Dobryszycki1 1Politechnika Wrocławska, Department of Biochemistry, Wrocław, Poland 2Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland

The creation of mineralized structures in invertebrates and vertebrates

requires cellular regulations, where the action of distinctive proteins control the deposition of biominerals as they have an influence on the nucleation,

growth, localization and morphology of the growing structure. Many of them

belong to intrinsically disordered proteins (IDPs) considered as the major regulators of the process. However, significant role of matrix proteins in the

process of biomineralization cannot be neglected. Matrix proteins provide an

organic scaffold and a surface for mineral deposition as in an epitaxial nucleation. The possible interactions between matrix proteins and inorganic

elements suggest that also crystals supporting proteins may have an influence

on the polymorphic form as well as biomineral morphology. In the formation of fish otoliths and higher vertebrates otoconia, a protein

named otolin-1 is involved as the scaffolding and possible tethering element

of these calcium carbonate structures of the inner ear. Otolin-1 is one of a few non-IDPs proteins involved directly in the process of ear stones and ear

dust biomineralization.

In this study, we would like to test the influence of recombinant homologues of Danio rerio and Homo sapiens otolin-1 on the formation of calcium

carbonate crystals in slow diffusion method.

Recombinant zebrafish and human otolin-1 was expressed in the bacterial system composed of Escherichia coli Arctic Express cells transformed with

pQE80L plasmid containing cDNA of zOtol1 and hOtol1 separately. Protein purification and sample preparation involved affinity chromatography to

cobalt or nickel cations followed by size exclusion chromatography. Calcium

carbonate crystals were grown in the range of protein concentrations in 96-wells plate-based biomineralization chambers with the use of a slow

diffusion method.

The proposed method of production and purification of recombinant zOtol1 and hOtol1 was sufficient to obtain highly pure samples in the yields ranging

from 0.86 mg up to 1 mg of the protein from one liter of bacterial culture.

zOtol1 and hOtol1 affected the size and morphology of calcium carbonate crystals obtained by slow diffusion method. Micro-Raman microscopy

determined the polymorphic form of these biominerals.

Concluding, the presence of otolith and otoconia matrix recombinant collagen-like otolin-1 has an impact on the formation of calcium carbonate

crystals in the slow diffusion system for Danio rerio and Homo sapiens

homologues. Acknowledgments: This work was supported by the National Science Center

(Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity

subsidy from the Polish Ministry of Science and High Education for the Faculty of Chemistry of Wroclaw University of Science and Technology.

P 13

Cryo-TEM study of the growth and crystallization processes of

calcium phosphate G. Dalmonico1, M. Farina2, A. Rossi*1 1Brazilian Center for Research in Physics, Rio de Janeiro, Brazil 2Federal University of Rio de Janeiro, Biomedical Science, Rio de Janeiro, Brazil

Introduction

Calcium orthophosphates are a class of materials that attracts strong interest

in many research areas in the fields of chemistry, physics, biology and

medicine. They are one of the main inorganic constituents of the calcified tissues of vertebrates (bones and teeth) and can be used in bone implants due

to their remarkable biocompatibility and bioactivity. The synthesis of HA in

a controlled manner is an important requirement for different applications and can also be used for elucidating the mechanism of mineralization in

biological systems (biomineralization).

Objective

In this work, the morphology and structural characteristics of calcium

phosphate (CaP) nanoparticles were investigated at different steps of the

34

synthesis process by analytical cryo-transmission electron microscopy (Cryo-TEM) in combination with other analytical techniques that need large

amounts of material.

Materials and Methods

The synthesis of HA was performed by dropwise addition of phosphoric acid

to calcium hydroxide solution in order to obtain a final solution with a Ca/P

ratio of 1.67. Calcium and phosphate ions react to form CaP and water. The whole synthesis (dropwise addition and aging) occurred over a period of 24

h. The reaction was performed at room temperature (~25°C), and the pH was

not kept constant. Samples were collected after different periods of time (5 min, 10 min, 30 min, 1 h, 2 h, 7 h, 11 h, 15 h, 19 h and 24 h) for the study of

the process of HA growth and crystallization. The samples were ultrafast-

frozen to be analysed by transmission electron microscopy or freeze-dried to be analysed by XPS, XRD and FTIR.

Results and Discussion

We showed that the process of drying the sample, instead of analyzing the sample in vitreous ice, did not significantly alter the morphology of the

nanoparticles but did induce significant changes in their crystallinity. As

shown by Cryo-TEM, crystallization of the nanoparticles in the frozen hydrated state was detected only after a long period of synthesis. In the first

5 min of reaction, which corresponds to a low amount of phosphate ions in

solution, we observed nanoparticles characterized by a high Ca/P ratio that transformed after 30-60 min into needle-like amorphous nanoparticles with

a comparatively lower Ca/P ratio. The mechanism of HA formation using

these chemical routes will also be discussed in the presentation regarding new paradigms of (bio)mineralization on the basis of a nonclassic mechanism

of nucleation induced by nanocluster aggregation, amorphous calcium phosphate and multiple stages of crystallization.

P 14

Stabilization of amorphous calcium oxalate (ACO) particles

with poly(acrylic) acid in pre-nucleation essays F. Diaz-Soler*1, M. Weber1, A. Neira-Carrillo1 1Universidad de Chile, Santiago, Chile

Introduction

Calcium Oxalate (CaOx) is one of the most widely distributed biominerals in

nature and recently a non-classical crystallization route (CNC) via ionic

aggregates (clusters) as amorphous precursors particles has been reported. It is well known that acidic biomolecules present a key role in classical

crystallization (Biomineralization) and pathological mineralization of CaOx, present in plants and animals. Thus, poly(acrylic) acid (PAA) is an acidic

synthetic polymer, described as an inhibitor, widely used to understand how

biomolecules modulate nucleation and the stages of aggregation and crystalline growth of biogenic materials. However, the effect of PAA on the

kinetics of the CaOx CNC is currently unknown.

General Objective

To investigate the kinetics and PAA concentration effect on the CNC of

CaOx through in vitro pre-nucleation assays.

Materials and methods

The CaOx pre-nucleation assays were performed using a computationally

controlled automatic titration system (Titrando 907, Metrohm) and

commercial computational software (Tiamo ™, current version: 2.3). CaOx pre-nucleation assays were performed by using an automated titration of

5mM sodium oxalate solutions with 20mM calcium chloride solutions at a

rate of 0.06 ml per minute in the absence and presence of PAA at concentrations of 10, 50 and 100 mg/L. During the whole pre-nucleation test,

the monitoring of free calcium ion concentration was carried out with a

selective ion electrode kept constant at pH 6.7. The determination and characterization of amorphous precursors (cluster) was studied by using

dynamic light scattering system (Nanosizer), transmission electron

microscopy (TEM) with selected area electron diffraction and Raman and FTIR spectroscopic techniques.

Results

Preliminary trials showed that the concentration of free calcium detected in the absence or presence of PAA differs from the amount of calcium added to

the solution. When PAA was used as an additive at concentration of 100

mg/L, there is a decrease in the slope of the kinetic curve of the CNC prior to nucleation point respect to the kinetic curves obtained in the absence of

additives. PAA acted as an inhibitor of CaOx crystallization, stabilizing the

pre-nucleation cluster, delaying the nucleation stage at longer times, which was proportional to the additive concentration. In addition, PAA allowed to

tolerate a higher concentration of free calcium, increasing its super-

saturation. Finally, PAA induces the formation of more soluble solid phases after nucleation. Ultra-structural characterization by HR-TEM is under

progress.

Conclusion

The current experimental results regarding to the kinetics of CNC of CaOx

suggest that PAA has the ability to stabilize in vitro amorphous precursors

formed during the pre-nucleation assays. We believe that the electrostatic interaction of the acid groups of PAA with the ionic CaOx clusters would be

stabilized by steric repulsion, preventing their aggregation.

Acknowledgment

The authors are grateful for the funding provided by Fondecyt, Project N°

1171520.

P 15

Biomimetic supertough and strong hybrid macrofibers Y. Yu*1, R. Tang1 1Zhejiang University, Department of Chemistry, hangzhou, China

Inspired by the special organic-inorganic hierarchical nanostructure of bone, the block copolymer with crystalline and amorphous protein regions as well

as special structural orientation of natural spider silk and their excellent

mechanical strength and toughness, here we show a simple down-up self-assembly method to prepare organic-inorganic hybrid macrofibers with

ordered mineralized polymer chain alignment. Owing to the coexistence of

crystalline and amorphous domains and the special rivet structure in the hybrid nanostructured macrofibers, the resultant hybrid macrofibers exhibits

superhigh tensile strength to ~950 MPa which is close to natural spider silk

(1150 ± 200 MPa), special toughness to 221 J g-1 which is proved to be the world"s highest special toughness among the PVA based materials and

surpasses natural spider silk (195 J g-1) and large stretchability (80.6%) as

well as excellent knitting properties and dyeability. This supertough and strong biomimetic hybrid macrofibers hence has promising applications in

textile fields such as flexible ballistic fabric. This down-top biomimetic

preparation approach of nanostructured macrofibers with excellent

mechanical properties is simple, scalable, and cost-effective, representing a

promising direction for the development of fiber industry.

P 16

The synergic effect of Sr2+ and Mg2+ on the stabilization of

amorphous calcium phosphate W. Jin*1, R. Tang1 1Zhejiang Unniversity, Hangzhou, China

Amorphous calcium phosphate (ACP) is a meta-stable precursor phase for

bone formation and it can transform into the thermodynamic stable mineral phase, hydroxyapatite (HAP, the main inorganic phase in bone), under

physiological conditions. In biological system, it has been documented that

Mg2+ can effectively stabilize ACP to ensure a regulation of biomineralization kinetics. In this study, we investigate the effect of another

alkaline earth metal ion, Sr2+, on ACP stabilization. In the human body,

99.9% Sr2+ is located in bones, and administered Sr2+ is almost exclusively precipitated in bone. Sr2+ is vital to young bone tissues formation. The role

of Sr2+ on ACP formation and its transformation needs to be clarified. The

precipitation of ACP and the crystallization of HAP are accompanied by an

abrupt drop in pH. We find that Sr2+ itself have less stabilization effect on

ACP in comparison with Mg2+ pH curves. However, the presence of Sr2+ can

significantly enhance the stabilization of Mg2+ on ACP due to a synergic effect. The chemical analysis reveals more Mg2+ should be excluded from

ACP to initiate the crystallization of HAP when Sr2+ ions are co-existed in

the amorphous phase. This change results in additional energy barriers for the solid phase transformation to provide a better stabilization effect on ACP,

which benefit bone formation. The revealing of multiple ions effects on the

phase transformation of amorphous minerals might shed lights on the understanding of biomineralization process as well as the fabrication of

stable amorphous phase, which has wide applications in drug delivery,

vaccine reservations, environment treatments, and lithium ion battery.This finding highlights the process of dopant ion exclusion in ACP and its control,

which enriches our understanding on the bioinspired regulation of

crystallization by using the cooperation of multiple ions.

P 17

Understanding the phase transformation from amorphous to

crystalline calcium phosphate by in situ transmission electron

microscopy B. Jin*1, C. Shao1, Z. Liu1, Z. Mu1, R. Tang1 1Zhejiang University, Hangzhou, China

In nature, the growth front of delicate mineral structures is covered with an amorphous continuous layer which is regarded as a precursor phase of

crystallized phase. For example, amorphous calcium phosphate (ACP)

widely exists in the new bone where ACP can transform into hydroxyapatite (HAP). Although the local rearrangement mediated direct phase

transformation mechanism from ACP to HAP has been reported, the detailed

phase transformation process still remains to be a great challenge due to the lack of direct experimental evidences. Here, we use in situ transmission

electron microscopy (TEM) to investigate the phase transformation

mechanism by skillfully designing a system in where spherical ACP particle closely contacts with HAP. It is found that the phase transformation is

achieved via epitaxial growth. Furthermore, in situ high resolution TEM

results show that the epitaxial growth induced crystallization advances via

35

the formation of kinks and steps. It is proposed that these steps and kinks are formed by the local rearrangement of posner"s clusters within ACP. This

finding highlights the key role of physicochemical effects in phase

transformation, contradicting the prior assumed epitaxial match between the structural organic matrix and the new produced mineral, which provides an

important implication in biomimetic mineralization and materials science.

P 18

Influence of Equisetum arvense extract on calcium oxalate

crystallization S. POLAT*1, P. SAYAN1 1Marmara University, Chemical Engineering, Istanbul, Turkey

Introduction Calcium oxalate crystallization is of great interest in medicine because it is the main constituent in the majority kidney stones. Calcium oxalate exists in

nature as calcium oxalate monohydrate (COM, whewellite), calcium oxalate

dihydrate (COD, weddellite) and calcium oxalate trihydrate (COT, caoxite). Calcium oxalate monohydrate and dihydrate forms are the major constituents

in most kidney stones. In this study, the effects on Equisetum arvense extract

on calcium oxalate crystallization were investigated detailed.

Objectives The main aim of this study was to investigate the potential inhibition effect

on Equisetum arvense extract on calcium oxalate crystallization to prevent kidney stone formation.

Materials & methods The calcium oxalate crystallization as a result of the reaction between calcium chloride and sodium oxalate was carried out in a cylindrical glass

crystallizer. The experiments were performed in batch crystallization mode

and the conditions were set at 37°C and pH 7.4 at a constant stirring rate. The crystallizer was kept at a constant temperature using a thermostat. During the

experiment, a pH meter was used to monitor the solution pH.

To investigate the effects Equisetum arvense, the extract was fed into crystallizer via a syringe pump. In this study, the concentration of the

aqueous Equisetum arvense extract used was 10 wt%. The end-products were

characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning

electron microscopy (SEM) were used.

Results XRD results showed that the crystals prepared in pure media include only the

calcium oxalate monohydrate form. According to the SEM analysis, the crystals prepared in pure medium had a hexagonal prismatic morphology.

The calcium oxalate monohydrate crystals had very fine structures and their

surfaces were slightly rough. The surface morphology of the crystals prepared in the presence of the Equisetum arvense was changed. In addition,

the functional groups and thermal properties of the crystals were investigated

in Equisetum arvense extract medium.

Conclusion Equisetum arvense has been showed to affect the crystal structure and

morphology of calcium oxalate using in vitro experiments. The structural and morphological transformations were confirmed using XRD, SEM, and FTIR

spectroscopy. Our results are useful and will contribute to the research efforts

on kidney stone formation, a significant clinical disease and important area of research in the field of biomineralization.

P 19

Synthesis, characterization and photocatalytic properties of Ti-

bearing hydroxyapatites A. Korneev*1, O. Frank-Kamanetskaya1, M. Kuz'mina1, V. Ryabchuk2, E.

Sturm3 1Saint Petersburg State University, Department of Crystallography, Saint Petesburg, Russian Federation 2Saint Petersburg State University, Department of Photonics, Saint

Petersburg, Russian Federation 3University of Kontanz, Department of Chemistry, Konstanz, Germany

According to [Wakamura et al., 2003; Tsukada et al., 2011] Ti-containing hydroxyapatites have photocatalytic activity comparable to activity of

anatase. However, there are doubts if this activity related to incorporation of

Ti-ions into hydroxyapatite or to small concentrations of TiO2 in precipitate, which can be not detected by X-ray diffraction.

Two series of hydroxyapatites were synthesized from solutions containing Ti

ions. The 1st series was precipitated from CaNO3, (NH4)2HPO4 and TiCl3 solutions, the 2nd series was precipitated from Ca(OH)2, H3PO4 and

C12H28O4Ti. Synthesized precipitates were studied with a wide set of

methods (X-ray powder diffraction, Raman spectroscopy, SEM, EDX, TEM, diffuse reflection spectroscopy).

According to the study results, hydroxyapatite has been formed in all

synthesis. Also precipitates of 1st series contain anatase impurity. Formation of anatase starts at atomic ratio Ti/Ca=0.56 in solution by XRD data, and at

Ti/Ca=0.01 by Raman spectroscopy.

Precipitates of 2nd series do not contain anatase by XRD data. However, after annealing samples at 700 °C for 6 hours peaks of anatase appear at diffraction

patterns. It evidences of formation of amorphous TiO2 during precipitation,

also confirmed by EDX data. After annealing amorphous TiO2 transits into anatase. Samples obtained from solutions with Ti/Ca=0.01 – 0.03 do not

contain any titanium dioxide phases by all used methods.

The increase of Ti concentration is assotiated with cell parameters variations. For 1st series, in the interval of Ti/Ca=0 – 0.13 titanium enters at Ca-sites

and in the interval of Ti/Ca =0.13 – 0.15 titanium enters at P-sites. For 2nd

series, titanium enters at both Ca and P-sites, but substitution of P is prevalent. Maximum concentration of Ti in 2nd series (9.5 wt%) is

significantly higher than in 1st series (5.4 wt%).

Diffuse reflection spectra of precipitates become more similar to anatase spectra (KRONOClean 7050) with increase of Ti concentration. Band gap

energy estimated by Tauc method using Kubelka-Munk transformation of

experimental diffuse reflectance spectra R(λ) ranges from 4,34 to 3.25 eV and is less than band gap energy of pure hydroapatite (Eg = 6 eV).

The obtained data confirm that Ti-containing hydroxyapatites can show

photocatalytic activity which depends on the entrance of titanium into apatite. The next step of our work is estimating their photocatalytic properties

through decomposition of organics.

The research was supported by RFBR grant №19-55-45019 IND_a and following resource centers of SPBU: Geo-Environmental Research and

Modelling (GEOMODEL), X-ray Diffraction Studies, Nanophotonics,

Optical and Laser Matetials Research, Microscopy and Microanalysis References

Wakamura, M., Hashimoto, K., Watanabe, T. Photocatalysis by Calcium Hydroxyapatite Modified with Ti(IV): Albumin Decomposition and

Bactericidal Effect. Langmuir, 2003, 19, 3428-3431.

Tsukada, M., Wakamura M., Yoshida, N., and Watanabe, T. Band gap and photocatalytic properties of Ti-substituted hydroxyapatite: Comparison with

anatase-TiO2, J. Mol. Catal. A: Chem., 2011, 338, 18–23.

P 20

Immobilization of Amino Acid Dehydrogenases by GO-PEI-IL

Hybrid Material induced Biomineralization K. Liu1, S. Wang*1 1Xiamen University, Department of Chemical and Biochemical Engineering, Hamburg, Germany

Introduction

L-Homophenylalanine (L-HPA) is a chiral unnatural amino acid, which is a

key intermediate of several pharmaceuticals. Biosynthesis of L-HPA

catalyzed by phenylalanine dehydrogenase (PheDH) is an ideal route. There are problems with limited substrate solubility in aqueous system and enzyme

denaturation in organic solvents.

Objectives

Immobilization of halophilic phenylalanine dehydrogenase with hybrid

materials based on biomineralization in order to create biocompatible

microenvironment and improve its stability and catalytic efficiency.

Materials & methods

Halophilic phenylalanine dehydrogenase (PheDH) from Bacillus

nanhaiensis is expressed in E.coil and purified. PheDH was absorbed to GO-PEI-IL and formed GO-PEI-IL-PheDH-TiO2 with the addition of titanium

bis(ammonium lactato)-dihydroxide (Ti-BALDH). Enzyme activity were

studied with 2-oxo-4-phenylbutyrate as substrate and NADH as cofactor.

Results

The effect of ionic liquid on [EMIM]BF4, [BMIM]BF4, [BMIM]Cl enhanced

enzyme activity by 2.1, 1.5 and 4.6 folds, respectively, while enzyme entrapment efficiency was 92.3%, 82.9% and 99.0%. ILs not only increased

the substrate solubility and also activated the enzyme activity, which was

accord to the salt active property of PheDH from Bacillus nanhaiensis. Compared with free PheDH, GO-PEI-PheDH, GO-PEI-IL-

PheDH, thermostability were greatly enhanced by induced biomineralization

of Ti-BALDH. Titania provided a rigid cage pocket for the protection from structure unfolding. A mechanistic illustration of the formation of hybrid

nanoparticles were proposed based on the multi-level interactions of enzyme,

PEI and ILs.

Conclusion

Polyethylenimine (PEI) and ionic liquids (ILs) functioned graphene oxide

(GO) were applied for PheDH immobilization by induced biomineralization of TiO2 with improved stability and activity for biosynthesis of L-

homophenylalanine. Hybrid materials GO-PEI-IL-TiO2 with synergistic

effect is are highly potential for enzyme immobilization, especially for bioelectrocatalysis, due to its biocompatibility and conductivity.

36

P 21

Biomimetic synthesis of bovine serum albumin and cysteine

modified ubiquitin-templated fluorescent inorganic

nanocomposites O. Akyüz*1, H. Cölfen1 1Konstanz University, Chemical Biology, Konstanz, Germany

Introduction

Biomineralization which is one of the interesting strategy found in nature for

the fabrication of advanced inorganic materials, is a source of inspiration for

many scientists searching methods for the synthesis of functional and bio-friendly nanomaterials. Proteins which possess well defined three-

dimensional structures and self-assembly properties based on their specific

binding sequences have been used as templates for biomineralization of various noble metals to functional nanostructures. By combination of the

functionalities of nanostructures with the biomineralization strategies;

fabrication of convenient functional and bio-friendly protein-templated fluorescent inorganic nanocomposites (FINCs) has been possible to use them

in various biomedical applications[1].

To sum up, proteins are promising biomolecules serve as scaffolds for the fabrication of FINCs with marvelous functions via the biomineralization

process. Therefore, developing new strategies for the synthesis of FINCs are

crucial to extend their properties and potential usages in bio nanotechnology.

Objectives

Synthesis of FINCs has been getting attention by many scientists due to their

attractive functions and features. However, the traditional chemical synthesis routes of such FINCs require harsh reaction conditions which are the

drawbacks for their activities in biological applications. Therefore, developing biomimetic synthesis strategies for the fabrication of FINCs

which are templated with natural and recombinant proteins under mild

reaction conditions, is pivotal to improve their functionalities. The purpose of this study is biomimetic synthesis of FINCs (gold

nanoclusters (AuNCs), cadmium selenide and lead sulfide quantum dots

(CdSeQD and PbSQD)) by using genetically engineered and natural proteins as biotemplates and investigating their properties via spectroscopic and

microscopic methods.

Materials & Methods

Bovine serum albumin (BSA), Ubiquitin (Ub) and Cysteine Modified

Ubiquitin (CMUb) were used as natural and recombinant proteins for the

synthesis of AuNCs, CdSeQD, and PbSQD in aqueous solutions at physiological temperature under alkaline conditions. The proposed

stabilizing/reducing mechanism behind the protein-templated biomimetic

mineralization of FINCs is as follows: (1) proteins containing abundant metal binding sites, such as cysteine (Cys), histidine residues can covalently

interact with the metal ions, (2) when the pH of protein environment is

adjusted to alkaline conditions by adding NaOH, i) the conformation of the protein changes and forms such a cage-like structure which is suitable for the

accumulation of metal ions (nucleation), ii) the phenolic groups on tyrosine

residue can be activated to a phenoxide ion at alkaline pH and then reduces metal ions to metal nanoclusters (reduction).

Results

The biomineralization experiments with the natural Ub and recombinant CMUb have clarified the pivotal role of the presence of Cys in protein

sequence by resulting in unstable and stable FINCs, respectively. As a result,

BSA and CMUb stabilized, functional, and stable FINCs possess photoluminescence and crystal size between 570-700 nm and 3-4 nm,

respectively, have been synthesized and characterized.

Conclusion

Green biomimetic synthesis strategies have been used to direct the formation

of FINCs having sub nanometer size and stable photoluminescence.

[1] J.Huang, L. Lin, D. Sun, H. Chen, D. Yang, Q. Li, Chem Soc Rev 2015,44,6330-6374

P 22

The coupled influence of Mg content and humidity on the

transition from amorphous calcium carbonate to calcite R. Thuemmler*1, B. Purgstaller2, E. Griesshaber1, M. Zenkert1, M. Dietzl2,

W. W. Schmahl1 1Ludwig-Maximillian Universitaet, Muenchen, Germany 2University of Technology, Institute of Applied Geosciences , Graz, Austria

Amorphous calcium carbonate (ACC) shows a remarkable stability, which is initiated by incorporation of impurities, e.g. ions or/and organic ligands.

Some impurities, e.g. Mg, stabilizes amorphous calcium carbonate to a high

degree and prevents effectively its transformation to crystallized calcite or aragonite.

In this study we discuss the coupled influence of Mg and humidity on the

transformation of ACC to crystalline carbonate. We describe for ACC stabilized with 5 and 50 wt% Mg contents the newly-formed carbonate phase

and the mode and speed from the amorphous to crystalline transformation.

Experiments were carried out in three humidity environments: low (about

24%), high (about 90%) and a wet state, where destilled water was sprayed onto the ACC tablet. Transformation experiments lasted for one, two, six and

nine weeks. We characterize our reference and newly-formed carbonate

material with XRD, FE-SEM, and electron backscatter diffraction (EBSD). We find that in the low humidity environment, even with low Mg contents,

ACC is highly stabilized and, for time periods up to six weeks, does not

transform to calcite or aragonite. The most significant transformation takes place when ACC contains 5 wt% Mg but is exposed for a long time to a high

humidity environment. Spraying the surface of the ACC tablet with water

induces transformation and crystalline carbonate formation, however, to a lesser extent as it is the case when the ACC tablet is exposed high humidity

for a long time period.

P 23

Size matters- nucleation kinetics of amorphous calcium

carbonate in confinement D. Joester*1, N. Metoki1, J. Cavanaugh1, M. Whittaker1, J. S. Evans1, K.

Alvares1 1Northwestern University, Materials Science and Engineering, Evanston, Illinois, United States

Introduction

Biomineralizing organisms routinely assemble materials with sophisticated

design and advanced functional properties, often using amorphous precursors to access compositional and structural states far from equilibrium. Organic

macromolecules and inorganic additives are thought to play an integral role

in controlling phase transformations in these systems. However, it has proven extremely challenging to accurately describe pathways and determine

mechanisms, even for extensively studied system such as amorphous calcium

carbonate (ACC).[1] A particular problem is the determination of accurate nucleation rates for the transformation of metastable precursors such as ACC

into the final, crystalline minerals.[2]

Objectives

Inspired by work in protein crystallization,[3] and guided by theoretical

considerations,[4] we set out to expand our earlier work on nucleation and

growth in liposomes[5] by developing a platform for the determination of nucleation rates in confinement based on droplet microfluidics.[6]

Materials and Methods

We designed and fabricated microfluidic devices that enable in situ observation of the crystallization of amorphous precursors confined in

emulsion droplets. Using time lapse polarized light microscopy, XRD, and Raman spectro-microscopy, we estimated the timepoint of individual

nucleation events in hundreds of droplets. Finally, statistical analysis of

nucleation events allowed us to determine steady state nucleation rates for the conversion of ACC to vaterite and calcite, and the impact of

biomacromolecules and inorganic ions on these rates.

Results In the absence of additives, statistical analysis revealed kinetics consistent

with classical nucleation theory. A steady-state nucleation rate of 1.2 cm-3s-

1 for the crystallization from ACC was determined. This low rate has important implications for phase transformation in biological systems. We

find that protein extracts from the sea urchin tooth (S. purpuratus) greatly

accelerate the nucleation rate. Recombinant spicule matrix protein (rSM30B/C) shows complex, concentration-dependent impact on

crystallization rates. To our surprise, this includes a polymorph switch at

elevated concentrations. Finally, we find that the presence of barium cations in droplets has a dramatic impact on nucleation mechanisms and rates.

Conclusion

Droplet microfluidic devices are powerful means to dissect nucleation and growth of amorphous precursors, and the impact of additives relevant in

biology and bio-inspired materials chemistry. I will discuss implications of

the absolute rates we determined for biological systems as well as mechanistic insights into the acceleration by rSM30B/C and barium cations.

References [1] J. J. De Yoreo, P. U. P. A. Gilbert, N. A. J. M. Sommerdijk, R. L. Penn, S. Whitelam, D. Joester, H. Zhang, Jeffrey D. Rimer, A. Navrotsky, J. F.

Banfield, A. F. Wallace, F. M. Michel, F. C. Meldrum, H. Cölfen, P. M.

Dove, Science 2015, 349. [2] L. M. Hamm, A. J. Giuffre, N. Han, J. Tao, D. Wang, J. J. De Yoreo, P. M. Dove, PNAS 2014, 111, 1304-1309. [3] S. V.

Akella, A. Mowitz, M. Heymann, S. Fraden, Crystal Growth and Design

2014, 14, 4487-4509. [4] R. P. Sear, CrystEngComm 2014, 16, 6506-6522. [5] C. C. Tester, M. L. Whittaker, D. Joester, Chem Commun 2014, 50

5619 - 5622. [6] J. Cavanaugh, M. L. Whittaker, D. Joester, RSC Chemical

Science 2019, available online.

37

P 24

Polymorph control of anhydrous guanine crystals Y. Ma*1, F. Chen1 1Beijing Institute of Technology, School of Chemistry and Chemical Engineering, Beijing, China

Amorphous guanine was proposed to be the mineralization precursor of biological anhydrous guanine β phase in vesica of the Koi fish scales1.

Guanine is one of the most widespread organic crystal existing in organisms

to produce structural colors. Guanine monohydrate and two anhydrous

guanine phases (AG), aand b , are three well known crystalline phases of

guanine. The excellent optical performances of guanine crystals are mainly

attributed to the crystalline AG β micro-platelets exposed (100) plane and high refractive index (1.83) 2. Herein, a novel crystal phase of anhydrous

guanine consisting of N9-G purine ring was obtained by dehydration of

guanine monohydrate (GM) at 111 °C, termed as dehydrated-GM, which is the first reported tautomeric polymorph of AG (N7-G purine ring). The

Dehydrated-GM has very good water harvest ability (8 wt%) with a fast rate

( in 30 min) even at relatively low room humidity (below 20 %) and low temperature (40 °C). We realized the synthesis of three polymorphs of

anhydrous guanine, AG a, AG b and dehydrated-GM in this work. A pure

phase of the anhydrous guanine (AG) β form was obtained via transformation of a hydrated amorphous guanine phase (HAmG), demonstrating short-range

order in solvents such as formamide, DMSO and DMF. Solid-state NMR

(ssNMR) characterization indicates that the HAmG precursor has similar

short-range order as AG β, which might be the reason for the formation of

AG β instead of the thermodynamically more stable AG α. The AG β nano-

platelets obtained in DMSO expose (100) plane when polyvinylpyrrolidone (PVP) was applied as additive. The sizes of AG β nano-platelets were about

100 nm in length, 40 nm in width, and 10 - 20 nm in thickness. The delicate

control on the polymorph and morphology of guanine crystals via an amorphous phase strategy may inspire the formation of highly ordered

hierarchical structures of guanine crystals with unique optical properties.

References [1] D. Gur, Y. Politi, B. Sivan, P. Fratzl, S. Weiner, L. Addadi, Angew. Chem.

Int. Ed. 2013, 52, 388.

[2] A. Levy-Lior, B. Pokroy, B. Levavi-Sivan, L. Leiserowitz, S. Weiner, L. Addadi, Cryst. Growth Des., 2008, 8, 507.

[3] F. H. Chen, Y. R. Ma*, Y. X. Wang, and L. M. Qi*, Cryst. Growth Des.,

2018, 18, 6497. [4] F. H. Chen, B.B. Wu, N. Elad, A. Gal, Y.N. Liu, Y. R. Ma * and L. M.

Qi, CrystEngComm, 2019, DOI: 10.1039/C9CE00245F.

P 25

The incorporation of organic and inorganic impurities into the

lattice of metastable vaterite E. Seknazi*1, B. Pokroy1 1Technion - Israel Institute of Technology, Material Science and Engineering, Haifa, Israel

Most of calcitic marine biominerals were shown to incorporate intracrystalline organic and inorganic inclusions. Inspired by these previous

findings, biomimetic calcite, containing organic or inorganic intracrystalline

inclusions was successfully synthesized in various routes. In the present study, we consider this feature in another calcium carbonate polymorph,

namely vaterite. Vaterite is the least thermodynamically stable and the least

abundant anhydrous crystalline phase of calcium carbonate. It is also the least well understood, as its crystal structure is still a matter of debate.

In this study, we show that vaterite is able to sustain high lattice strains and

incorporate Mg, Ba, and aspartic acid in its lattice. We report, for the first time, the formation of substituted Mg- and Ba-vaterite. We characterized and

quantified the structural distortions caused by incorporation of aspartic acid,

Mg or Ba into the vaterite hexagonal pseudo-cell by means of high-resolution synchrotron X-ray diffraction and micro-Raman spectroscopy. We show that

Mg substituted for up to 12% of Ca, and Ba for up to 2.5% of Ca, to form

crystals of Mg-vaterite and Ba-vaterite with lattice distortions of up to 0.9% and 0.14%, respectively.

Interestingly, this study proves that the presence of Mg and organics in biogenic vaterite can be parameters which affect its reported structural

variability. Therefore, this study, if accompanied by analyses of the Mg and

organics present in biogenic vaterite, could help clarify the true structure of vaterite.

P 26

Novel nanoscopic pathways in crystallisation and selfassembly

of barium carbonate nanoparticles in microemulsions W. Sager*1 1Forschungszentrum Jülich, Peter Grünberg Institute - Microstructure reserch (PGI-5), Jülich, Germany

Different nanoscopic paths to witherite nanoparticle formation via the birth

and deterioration and/or transformation of meta-stable amorphous and, so far unreported, crystalline barium carbonate nanoparticles using nonionic water-

in-oil microemulsions as precipitation media have been investigated in detail

by transmission electron microscopy and selected area diffraction. Compared to precipitation from homogeneous media, the employment of

microemulsions allows for a more precise control of the ongoing nucleation

and growth processes, not only via adjusting the concentration of the reacting species involved and temperature, but also by tuning the size and the

exchange kinetics of the isolated and compartmentalised nanometre-sized

water domains. Here we present a comprehensive study on how tuning the properties of the parental microemulsion can be implemented in tailoring the

crystal structure, morphology and self-assembling properties of nucleating

and growing nanoparticles. At low water content a variety of different meta-stable nanoparticles forms that ranges from in the electron beam amorphous

filaments to monoclinic (m) cubes and hexagonally shaped, probably trigonal

(t), thin platelets, which assemble into micrometre-long stacks. Slowing down of the exchange kinetics permits us to study the early stages of particle

formation and to gain full insight into unrevealed crystal transformation and

re-crystallisation processes. The genesis of the, at ambient conditions thermodynamically stable, orthorhombic (o) polymorph (witherite) evolves

either via direct transformation (e.g. a→o transition from filaments into rods)

or via dissolution/re-crystallisation processes. In the latter case the crystalline pre-structure particles dissolve, thereby freeing material for the re-nucleation

of the witherite phase (m/t→o transition), emphasising the double role of the

microemulsion domains as nucleation site and host and thus transport medium for water-soluble species. Depending on the different crystallisation

paths taken and the growth conditions encountered, a rich variety in final

nanoparticle morphologies is obtained. The ubiquitous microemulsion droplets induce attractive depletion forces between the forming nanoparticles

leading to different types of particle selfassembly, such as columnar stack

formation of nanoplatelets that is additionally characterised by small angle x-ray scattering and electron tomography.

P 28

Organization of prismatic layers in mollusk shells - Pinctada vs.

Pinna Y. Dauphin*1, E. Zolotoyabko2, A. Berner2, E. Lakin2, C. Rollion-Bard3,

J. P. Cuif4, P. Fratzl5 1Museum national d'histoire naturelle, ISYEB UMR 7205, Paris, France 2Technion-Israel Institute of Technology, Department of Materials Science

and Engineering, HAIFA, Israel 3Université Sorbonne Paris Cité, IPGP UMR 7154, Paris, France 4Museum national d'histoire naturelle, CR2P UMR 7207, Paris, France 5Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Potsdam, Germany

Introduction

Cross-sections of calcitic prismatic layers in mollusk shells, cut

perpendicular to growth direction, reveal well-defined polygonal shapes

clearly resolved by light and electron microscopy. These observations are typical for the prisms of Pinna, Pinctada, Atrina, and Ostrea. Taking into

account the rhombohedral symmetry of calcite, often presented in hexagonal

axes, this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In other words, the c-axis of calcite is thought to be

perpendicular to the surface of the shell. This paradigm is mostly based on

the long-term studies (since 1840) of Pinna nobilis shells, revealing the largest prism size; however other shells are much less investigated.

In this research, we address the question whether or not the crystallography

of calcitic prisms in Pinctada shells is similar to that well established in Pinna. As working example, we focused on the structure of Pinctada

margaritifera shells.

Materials & methods

The shells of Pinctada margaritifera were collected in French Polynesia,

while those of Pinna nobilis came from the Mediterranean Sea. The shell

morphologies and finer structural features were imaged by optical microscopies, SEM and AFM; crystallographic dissimilarities were analyzed

by X-ray diffraction and EBSD; in situ composition was studied using

Raman and FTIR spectroscopies, EPMA, XANES and X-ray fluorescence. Results: There are two kinds of prisms in Pinctada margaritifera: small

simple prisms and large prisms comprising several sectors. Only simple

prisms are known to exist in Pinna and Atrina shells. All prisms are built of nano-granular calcite (still being nearly single-crystalline) and organic

matrix. In contrast to Pinna nobilis, we find that in both large and small

prisms of Pinctada margaritifera the c-axis is mostly perpendicular to their

38

long morphological axis, i.e., parallel to the surface of the shell. Furthermore, the degree of this preferred orientation is higher in large prisms.

We relate these growth dissimilarities to the striking differences in the

organic contents. In fact, the soluble organic matrix of Pinna nobilis is rich in acidic sulphated polysaccharides, whereas that of Pinctada margaritifera

is rich in proteins. Lipid contents of Pinna and Pinctada also differ, as well

as Mg and S concentrations. In addition, small prisms have higher organic and organics-related sulfur contents than the large ones. In particular, atomic

percentage of sulfur in Pinna nobilis is three and twenty times higher than in

small and large prisms, respectively, of Pinctada margaritifera.

Conclusion

We conclude that, despite their morphological similarities and common

mineralogy, the prisms of Pinctada and Pinna, both Pteriomorpha, are far from being identical. The comparison of the organic contents reveals

significant differences in their nature and quantities. Based on these findings,

we suggest that in mollusk shells, the morphology of calcitic prisms is not primarily determined by the crystallographic structure of calcite, but largely

by organic components, the presence or absence of which drastically change

the growth mode.

P 29

Biomimetic cross-lamellar structures fabricated by ordered

self-assembly of CaCO3 nanorods M. Takasaki*1, M. Tago1, T. S. Suzuki2, Y. Oaki1, H. Imai1 1Keio University, Yokohama, Japan 2National Institute for Materials Science, Tsukuba, Japan

Introduction Biominerals have excellent mechanical and optical properties as compared

with geologic balky crystals. Especially, cross-lamellar structures of seashells consisting of CaCO3-based nanorods have received attention as an

excellent model for lightweight and tough structural materials because of

their superior mechanical properties derived from oriented architectures. Ordered self-assembly of nanocrystals is a non-classical way to fabricate

analogues of biogenic oriented architectures. However, artificial cross-

lamellar structures have not been produced by the self-assembly technique.

Objectives The aim of our study is fabrication of biomimetic cross-lamellar structure by

an evaporation-driven assembly method. We utilized calcite nanorods as a building block and an intense magnetic field to enhance their oriented

attachment.

Materials & methods Calcite nanorods ~50 nm in diameter that were elongated in the c direction

were obtained by oriented attachment of calcite nanograins synthesized by carbonation of Ca(OH)2 aqueous dispersion. We demonstrated a millimeter-

scale ordered assembly of calcite nanorods dispersed in ethanol by

combining the arrangement with an evaporation-driven capillary force and alignment under an intense magnetic force. Layer-by-layer accumulation of

the ordered assembly of the calcite nanorods was performed with a rotation

of the substrate at a 90-degree angle.

Results Calcite films ~10 μm in thickness were formed by oriented assembly with

lateral stacking of the nanorods on a substrate. The orientation of the nanorods in the films was improved by application of an intense magnetic

field at 12 T. The single-crystalline feature of the films similar to biogenic

cross-lamellae was characterized by X-ray and electron diffraction. Artificial cross-lamellar structures with crystal direction switching at 90-degree were

obtained by layer-by-layer accumulation of the ordered calcite films.

Conclusions Biomimetic cross-lamellar structures were fabricated through oriented

attachment of calcite nanorods by evaporation-driven self-assembly. Our

results suggest that the self-assembly techniques are applicable for fabrication of excellent analogues of biogenic oriented architectures.

P 30

The phenotypic plasticity in shell microstructures of vent and

seep pectinodontid limpets K. Sato*1, C. Chen2, R. G. Jenkins3, H. K. Watanabe2 1Waseda University, Department of Earth Sciences, School of Education,

Tokyo, Japan 2Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan 3Kanazawa university, Kanazawa, Japan

Gastropods often show intraspecific variation in phenotypes among different

habitats. Recently, the authors carried out molecular phylogenetic analyses

of pectinodontid limpets in two genera, Bathyacmaea and Serradonta, commonly associated with vent and seep environments in the west Pacific

(Nakano & Sasaki, 2011). Instead of revealing distinct lineages

corresponding to morphological identification, the analyses showed that all individuals from both genera were mixed in a single nested monophyletic

clade, except for one undescribed species from the South Chamorro

Seamount. This result strongly implies that most previously recognized "species" in fact belong to one species that is highly morphologically plastic

in shell form. The shell microstructure is the micro-scale morphological trait

of molluscan shells (Carter, 1990) and can represent a strong tool to estimate phyletic relationships and physiology. Therefore, the purpose of this study is

to assemble how genetic constraints and environmental factors affect the

expression of shell microstructure of pectinodontid limpets. Shell microstructures of pectinodontids were observed by SEM and their mineral

composition was detected by Raman spectrophotometry. As a result, we were

able to estimate specimens to genus level classification based on their shell microstructural compositions. Furthermore, the proportion of shell

microstructures with two different mineral compositions (aragonite or

calcite) clearly corresponds to differences in chemosynthetic habitat. Our study reports that mollusks regulate the mineral polymorphism in response

to their chemosynthetic habitat. This phenotypic plasticity in shell

mineralogy is significant not only in the context of the adaptive radiation of pectinodontid linages to chemosynthetic environments, but also to shed light

on mineralogical aspects regarding the biomineralization mechanism of the

molluscan shells.

P 31

Immunological detection and LC-MS/MS analysis of the shell

matrix protein ICP-1 in brachiopods Y. Isowa*1, K. Kito2, H. Sawada1, K. Endo3 1Nagoya university, Sugashima Marine Biological Laboratory, Toba, Japan 2Meiji University, Department of Life Sciences, Kawasaki, Japan 3University of Tokyo, Department of Earth and Planetary Science, Tokyo, Japan

Introduction

Brachiopods are sessile marine invertebrates belonging to Lophotrochozoa,

and have two valves composed of calcium carbonate or calcium phosphate.

Recently, proteomic analyses of shell matrix proteins have been performed on several brachiopod species to understand the molecular mechanisms and

evolution of brachiopod shell formation. In a previous shell proteomic

study of the rhynchonelliform brachiopod Laqueus rubellus, we identified a complete sequence of ICP-1, which was originally characterised as a

pigment-carrying intracrystalline protein from the three rhynchonelliform brachiopods Neothyris lenticularis, Calloria inconspicua, and Terebratella

sanguinea using Edman degradation. We showed that it is the most

abundant protein in the shell of L. rubellus by LC-MS/MS analysis. However, further details of ICP-1 remained to be clarified.

Objectives Antibodies are useful for localization of proteins within tissues. As a first

step to localize shell matrix proteins in the shells, we prepared an antibody

against ICP-1 in this study, and verified the binding specificity of the antibody. In addition, we carried out LC-MS/MS analysis to characterize

post-translational modifications of ICP-1.

Materials & methods The shell extracts from L. rubellus were subjected to SDS-PAGE, and

Western blotting was performed using the antibody against a synthetic

peptide designed from the ICP-1 sequence of L. rubellus. LC-MS/MS analysis was also performed against three major bands in the SDS-PAGE.

Results Western blotting showed a single band of approximately 60 kDa in size. This is substantially higher than the molecular mass expected from the theoretical

amino acid sequence of ICP-1 (17.5 kDa). We inferred that LrICP-1

undergoes post-translational modifications. Indeed, searches using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc) indicated

that LrICP-1 has one potential glycosylation site. Meanwhile, LC-MS/MS

analysis showed that a peptide of ICP-1 was identified from a band under 6.5 kDa, which is smaller than the molecular mass expected from the theoretical

amino acid sequence of ICP-1.

Conclusion The synthetic peptide used for antibody generation is located in the N-

terminal region, while the peptide identified by LC-MS/MS is located in the

C-terminal region. Therefore, one possibility is that ICP-1 undergoes post-translational cleavage, and the N-terminal fragment undergoes other post-

translational modifications. To confirm this hypothesis, we plan to perform

additional LC-MS/MS analysis to identify peptides in the N-terminal region of ICP-1.

39

P 32

Exploring biomineral formation at the nanoscale by electron

energy loss spectroscopy (EELS) M. de Frutos*1, O. Stéphan1, W. Ajili2, T. Azaïs2, N. Nassif2, S. Auzoux-

Bordenave3, D. Bazin4, M. Daudon5, E. Letavernier5 1CNRS Université de Paris Sud, Laboratoire de Physique des Solides, Orsay, France 2CNRS Sorbonne Université, LCMCP, Paris, France 3CNRS Sorbonne Université, BOREA, Paris, France 4CNRS Université de Paris Sud, Laboratoire de Chimie Physique, Orsay,

France 5Hopital Tenon AP-HP, Service des Explorations Fonctionnelles, Paris, France

Understanding of the hierarchical organization of biominerals and the mechanisms involved in their formation implies to unveil their chemical

composition and structure at different scales. Of particular relevance is the

characterization of the organo-mineral interface to understand how the organic fraction regulates the initiation of the calcification (nucleation) and

direct the crystal growth. Conventional TEM (transmission electron

microscopy) is limited, since it cannot easily distinguish the nature of the different components. Compared to other spectroscopic approaches, EELS

in a STEM (scanning transmission electron microscope) offers the advantage

of an outstanding spatial resolution (at the nanometer scale) in both chemical analysis and imaging.

To illustrate the interest of EELS for biomineral studies, we will present

selected results related to two different systems: kidney calcifications and abalone nacre. EELS was used to analyze the specimens with a resolution

defined by the diameter of the electron probe (typically better than a

nanometer). Kidney samples were prepared by ultramicrotomy from small pieces of

human kidney chemically fixed and embedded in epoxy resin. Nacre thin

section samples were obtained by Focused Ion Beam (FIB) on a selected area of the nacre layer of one-year-old Haliotis tuberculata shell. EELS data were

recorded on a VG HB01 STEM operated at 100 keV. The measurements were

obtained using a nitrogen-cooled sample stage and with the minimum electron dose to limit the radiation damage induced by the electron beam.

During an acquisition, the electron beam was scanned over the specimen and

an EELS spectrum was acquired for each position, giving access to an elemental mapping.

EELS data were compared with different reference samples, including mineral and organic compounds, in order to identify the chemical species

present in calcified tissues. The relative distributions of mineral and organic

fractions were obtained from the signal of the calcium and phosphorus edges and by the gaussian fitting of the peaks associated to carbonate and organic

compounds on the carbon K-edge. Two main minerals were found: calcium

carbonate in nacre and calcium phosphate, sometimes associated to carbonate, in kidney calcifications. The biological fraction were clearly

distinguished from the embedding resin.

Our study shows that STEM-EELS allows a nanoscale analysis of the chemical composition of biominerals in different calcified tissues giving

access to the mineral/organic interface. These data provides new insights to

elaborate possible mechanisms involved in biominerals formation. Acknowledgements: This work was partially funded via the CNRS-CEA

"METSA" French network (FR CNRS 3507) and the EU grant ESTEEM.

P 33

Sub-structures in bivalve simple prisms - a record of species-

specific control over crystallization during shell growth J. P. Cuif*1, Y. Dauphin2 1Museum Histoire Naturelle, CR2P-Paleontology, Paris, France 2Museum National Histoire Naturelle, umr ISYEB, Paris, France

Introduction

Since their very first observation in the shell of the Pteriomorphid Bivalve

Pinna nobilis, (Bowerbank 1844) "simple prism" as a microstructural

category has been used for shell description in many biological taxa, with taxonomic and evolutionary purposes. Most of the comparative studies are

based on static description focusing on morphological patterns and three

dimensional arrangements of the crystal-like units observed in adult shells. Here attention is drawn to diversity of mineral phase organizations and

growth mode variations in two simple prisms: P. nobilis and Pinctada

margaritifera.

Materials and Methods

Series of Pinna shells were kindly provided by Pr. N. Vicente, head of the

Les Embiez laboratory at Toulon-Six-Fours (France). Shells of Pinctada margaritifera were collected in Polynesia during a five years research project

involving the research department of the Direction des Ressources Marines

at Tahiti. Structural patterns were made visible through polished surfaces prepared on variously located areas of the prismatic layers followed when

necessary by appropriate etchings. From these specifically prepared surfaces,

information was obtained by using optical observation (mostly epi-polarisation), SEM on both secondary and back-scattered modes,

synchrotron based XANES method (ESRF Grenoble France) and

fluorescence (SOLEIL Saint-Aubin France).

Results

In Pinna nobilis etching shows that prisms are built by closely associated 6

to 8 µm thick sub-units (about 8 to 15 for every prism depending on specimens) whose overall orientations is slightly oblique to longitudinal

growth axis. Close correspondence of these mineral sub-structures between

adjacent prisms shows that their formation at the growing edge of the mantle is related to the pulsed stepping extension of the shell.

Prisms of the Pinctada margaritifera exhibit important morphological and

microstructural changes during growth. After an initial monocrystalline status increase in prism diameter is correlated to passage to a polycrystalline

status. Additionally, orientation of the crystallographic c axes becomes

perpendicular to growth direction, a surprising contrast to common views. Shift from single crystal to polycrystalline structure of the prisms, then

regression of the diameter and biochemical change in the final stages suggest

a continuous evolution of the mineralizing phase related to shell overall elongation and aging of the mineralizing cell layer.

Conclusion

These two prisms exhibit very distinct and contrasting internal sub-structures, revealing the biological control exerted over crystallization by the

mineralizing secretions of the epithelial cells during shell growth. From a

methodological view point, note can be made that such biological control of the structural and crystallographic patterns rises question regarding models

based on simple physical process (e.g. crystalline competition as well as predictive capability of thermodynamic grain growth theory and self-similar

crystallization).

P 34

Correlative 3D Raman imaging- a powerful method for

comprehensive studies of biomineralized samples U. Schmidt1, J. Englert1, A. Richter1, M. Böhmler1, T. Olschewski1, K.

Hollricher1, H. Fischer*1 1WITec GmbH, 89081, Germany

Introduction Raman microscopy is a useful tool for analying biominerals, as it can reveal the chemical composition of composite materials. Even more information

can be obtained by correlating the acquired images with data from other methods, such as Atomic Force Microscopy (AFM) or Scanning Electron

Microscopy (SEM). Such approaches can yield information that would not

be available with only one technique. Thus, Raman Imaging in combination with AFM or SEM can provide new insights into the fundamental processes

by which organisms produce biocomposites.

Objectives In this contribution we will introduce the principles of state-of-the-art

confocal Raman Imaging as a tool for analyzing the chemical and molecular

characteristics of a sample. We will show how this technique can be used in combination with AFM and SEM to correlate chemical information with

structural properties. The aim of this contribution is to describe and highlight

the unique features of such combined scientific analysis instruments, based on examples from various nano-biomaterials.

Materials & Methods For this study we use a Raman Imaging system which combines a confocal microscope with a highly sensitive Raman spectrometer. It delivers an

excellent depth resolution that enables the acquisition of 3D Raman images.

Raman microscopy allows the analysis of the distribution of the chemical components in a sample without requiring specialized sample preparation or

risking damage to the sample. It can easily be combined with other

techniques, such as AFM or SEM for correlative measurements.

Results SEM provides information on morphology, elemental composition and

crystalline structure of a sample. Confocal Raman Imaging of the same sample area reveals the chemical composition as well as polymorphism,

stress states and anisotropies. The combination of SEM and confocal Raman

microscopy in a single instrument allows the nondestructive characterization of biominerals with the highest resolution. Measurements of pearls and nacre

illustrate the benefits of combining these methods for investigating

biominerals. For example, a biomineralization defect in pearls was studied, which leads to loss of its pearlescence ("milky pearl").

Conclusion This submission highlights the power of correlative Raman techniques, such as Raman-AFM and Raman Imaging and Scanning Electron (RISE)

Microscopy, for the analysis of composite materials.

40

P 35

Asymmetric distribution of the crystal particle implies the

potential mechanism of aragonite tablets formation in the

nacreous layer of Pinctada fucata T. Jiang*1, J. Huang1, L. Li1, L. Xie1, R. Zhang1,2 1Tsinghua University, MOE Key Laboratory of Protein Sciences, School of

Life Sciences, Beijing, China 2Yangtze Delta Region Institute of Tsinghua University, Department of Biotechnology and Biomedicine, Jiaxing, China

The structure of the pearl oyster shell is intriguing because it implies that there are two biomineralization strategies co-existed in one system. The

thermodynamic aspect of the prismatic layer formation has already been

discussed but that of the nacreous layer seems lost in the whole picture. The mechanism governing the formation of the nacreous layer is convoluted.

However, the discussion about the pivotal component in the process is still

enlightenment. Here, with the help of the scanning electron microscope (SEM) and the transmission electron microscope (TEM), the crystal particle

distribution on the shell surface and the arrangement of aragonite tablets in

Pinctada fucata have been carefully examined. Strikingly, contrary to intuition, particles do not uniformly accumulate outward from the center

which might imply the dominant role played by the Marangoni effect. The

thickness of the aragonite tablets is not identical. The organic frame has evenly shifted among different layers. Taken together, these results imply the

existence of the Marangoni effect in the process of the nacreous layer

formation and the asymmetric distribution of the particle may lead to the even shift of the organic frame.

P 36

Microstrured biomineral particles from Emiliania huxleyi as a

raw material M. Chairopoulou*1, M. Herrmann2, U. Teipel1 1TH Nürnberg Georg Simon Ohm, Mechanical Process Engineering,

Nürnberg, Germany 2Fraunhofer-Institut für Chemische Technologie, Pfinztal, Germany

Microstructured particles have a number of interesting assets that can be beneficial for certain applications. To that counts for example the specific

surface area, which tends to increase with decreasing particle size, the pore structure, promising for material loading, and also the wetting behavior of

fine powder materials. When such structures are pursuit in natural systems

the algae family of Coccolithophorida pose a good representative. The sophisticated structures, formed according to strict biomineralization

mechanisms, are called coccoliths and are composed mainly of calcium

carbonate (CaCO3). Since CaCO3 is broadly used in numerous applications the potential of coccoliths is anticipated to be promising. However, in order

to use those uniquely formed particles in any future applications basic

questions and challenges still need to be improved. In an effort to address challenges associated with their separation process this study focuses on

recovering coccoliths from freshly cultivated media. The biogenic broth for

the experimental part was taken from laboratory cultivations of an Emiliania huxleyi strain in a 5 L fed-batch cultivation system. To that system, strategies

to clean and isolate the particles were followed according to literature

references and were tested for the first time for the same system. Through characterization of the treated particles, the applied methods could be

compared and two separation concepts could be distinguished. For the two

concepts further optimization steps were followed concluding in still open challenges that need to be phased in the coccolith separation chapter.

P 37

Proteins involved in Cyprinus carpio otoliths biomineralization M. Kalka*1, A. Ożyhar1, J. Stalarski2, P. Dobryszycki1 1Wroclaw University of Science and Technology, Department of

Biochemistry, Wrocław, Poland 2Institute of Paleobiology, Polish Academy of Science, Department of

Environmental Paleobiology, Warsaw, Poland

Otoliths, the ear stones of teleost fish, are biomineralized structures whose

major role is sound transduction and sensation of linear acceleration. They are mainly composed of calcium carbonate, which is deposited onto an

organic matrix. The organic matrix, which consists of proteins,

polysaccharides and lipids, accounts for only a few mass percent of an otolith. Nevertheless, the organic fraction, especially proteins, have been

shown to be essential for proper biomineralization of otoliths. Our recent

proteomics studies have revealed that otoliths contain more proteins than previously thought.

The aim of our work was the isolation and characterization of

macromolecules found in common carp otoliths. We paid particular attention to structural properties and biomineralization activity of isolated proteins.

In order to isolate proteins, asteriscus otoliths were decalcified with EDTA.

Then, obtained macromolecules were separated by various chromatographic

technics and examined with electrophoresis. Proteins compositions were analyzed with LC-MS/MS. Their structural properties were investigated by

CD. Influence of isolated proteins on calcium carbonate crystallization was

studied by a slow diffusion method. Crystals morphology and polymorph were investigated by SEM and micro-Raman microscopy. Moreover, post-

translational modifications, such as phosphorylation and glycosylation, were

also studied. Developed methods allowed the initial separation of otolith's proteins.

Besides previously studied otolith proteins, proteomics identified several

new ones that could be connected to biomineralization. Isolated macromolecules strongly affect the morphology of calcium carbonate

crystals. Appropriate staining of separated fractions indicates a possible high

percentage of proteoglycans and glycosaminoglycans in the ear stones. In addition, one protein that has a particularly strong affinity to the

hydroxyapatite column was isolated. In vitro studies of this protein indicate,

that it occurs in phosphorylated form in otoliths. The protein changes calcium carbonate crystals polymorph from calcite to vaterite. Our results pointed out

that the protein is present in high-molecular-weight aggregates and peptides

identified with MS/MS demonstrated that it might be Starmaker-like protein. In conclusion, obtained results indicate that otolith biomineralization process

might be far more complex than previously claimed. Our research allowed

isolation, initial separation and characterization of individual components of the organic fraction. Most probably it was the first time when the Starmaker-

like protein was successfully isolated from otolith matrix. Our results

revealed that proteins from common carp could be especially prone to phosphorylation and glycosylation.

Acknowledgments: This work was supported by the National Science Center (Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity

subsidy from the Polish Ministry of Science and High Education for the

Faculty of Chemistry of Wroclaw University of Science and Technology.

P 38

Proteomic investigation of the blue mussel larval shell organic

matrix A. Carini*1, T. Koudelka2, A. Tholey2, E. Appel3, S. Gorb3, F. Melzner4,

K. Ramesh5 1The University of Hong Kong, School of Biological Sciences, Hong Kong,

Hong Kong 2University of Kiel, Institute for Experimental Medicine - Systematic

Proteomics and Bioanalytics, Kiel, Germany 3University of Kiel, Department of Functional Morphology and Biomechanics, Institute of Zoology, Kiel, Germany 4GEOMAR, Marine Ecology, Kiel, Germany 5University of Gothenburg, Sven Lovén Centre for Marine Infrastructure, Fiskebäckskil, Sweden

Introduction Shell matrix proteins (SMPs) are occluded within molluskan shells and are

fundamental to the biological control over mineralization. While many

studies have been performed on adult SMPs, those of larval stages remain largely undescribed.

Objectives Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell

proteomes.

Methods Following development of a method for cleaning minute larval shells of

tissue contaminants, the whole blue mussel larval shell proteome was

extracted and subsequently sequenced using shotgun proteomics.

Results Forty-nine SMPs were identified in total. Twenty-one proteins were common

to all samples including: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP

domain-containing sequence and a ferric chelate reductase 1-like sequence.

Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. This study demonstrates

that the mussel larval shell proteome is unique: while key predictable

mollusk shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized. Further, comparison to adult mussels reveals

that only four domains are conserved among species and developmental

stages and nine domains are specific to mussel larvae.

Conclusion The bivalve larval shell proteome is not a subset of the adult one: blue mussel

larvae assemble a unique and novel shell proteome that serves specific physiological needs for their development. Further, the observed differences

in shell proteomes between species and life stages support the idea that strong

species-specific and ontogenetic variation exists in molluskan shell proteomes.

41

P 39

Deciphering the intricate 3D Structure of growing calcite

crystals during coccolith formation E. M. Avrahami*1, L. Aram1, A. Gal1 1Weizmann Institute of Science, Plant and Environmental Sciences, Rehovot, Israel

Coccolithophores are a widespread and ecologically important group of

unicellular calcifying phytoplankton. They are distinguished by a complex exoskeleton composed of multiple interlocking calcite plates, known as

coccoliths. Coccoliths are produced intracellularly within a specialized

membrane-bound organelle, the coccolith vesicle, and are subsequently exocytosed to the cell surface. Although extensive research in the past

century has contributed greatly to our understanding of coccolithophore

physiology and ecological significance, not much is known about the mechanisms by which they precipitate and control coccolith assembly.

Nearly thirty years ago, the V-R model for crystal nucleation, orientation,

and growth was suggested based on light- and electron microscopy, and to this day it is the ruling paradigm. However, the path by which crystals grow

into their final morphology remains unclear. In this work, we chose

Cacidiscus leptoporus as a model organism for exploring the growth of intracellular coccoliths, given that this species forms relatively large

coccoliths in comparison to the commonly studied species Emiliania huxleyi

and Pleurochrysis carterae, simplifying their analysis. A simple, yet effective, protocol was established for extracting developing coccoliths at

initial formation stages while preserving the inorganic phases and their

ultrastructure. These coccoliths were then imaged ex vivo using several electron microscopy techniques, including high-resolution STEM. By

recording a tilt-series of the crystals, a tomogram was created, and a 3D

model of the interlocking crystals was constructed. This model has allowed us to visualize the native interlocked R and V units with clear

crystallographic planes. Applying this methodology for multiple samples

will enable us to reconstruct the entire sequence of crystal growth, thus linking shape control to the crystallographic driving forces, and yielding a

mechanistic understanding about the control organisms have on biomineral

formation.

P 40

Diagenetic alterations reshape the hierarchical organization of

Cretaceous brachiopod shells - from nanoscale to higher scales D. Gaspard*1, J. Nouet2, C. Rountree3 1Muséum National d'Histoire Naturelle - Sorbonne Université,

Département Origines & Evolution, Centre de Recherche en Paléontologie

de Paris, UMR 7207, CR2P, Paris, France 2Université Paris-Saclay, Earth Sciences, Orsay, France 3SPEC, CEA, Gif-sur-Yvette, France

Introduction/objectives

After the attempt to reveal and understand the hierarchical organization of

the inner layers of the calcite shell of modern rhynchonelliform brachiopods (Gaspard, 2006, Gaspard & Nouet, 2016), the purpose here is to highlight the

modifications introduced in Cretaceous shells since the death till the state of fossil. Conservation, alterations in different palaeo-environmental situations

for these witnesses of old times are to be uncovered from the nanoscale to

continuum scale.

Materials and Methods Two and three-layered shells [cf. Sellithyris cenomanensisGaspard,

Moutonithyris dutempleana (d"Orbigny) among others], from different Cretaceous Western Europe locations, were observed with comparable

modern ones (Liothyrella neozelanica, Aerothyris kerguelenensis, Tichosina

cubensis, Notosaria nigricans…) using complementary throughput techniques (scanning electron microscopy (SEM), mapping elements,

including in the foremost rank the Bruker atomic force microscopy (AFM).

Results The observations reveal that from the death of the organisms, the exoskeleton

(biomaterial) composed of low-Mg calcite and organic matrices is subject of

chemical and physical aggressions leading to light or heavy alterations. More often the organic matrices are partly or entirely destroyed. First, after the

disappearance of the organic sheathes, several fibrous elements merge. As

well, after partial disappearance of the intracrystalline organic matrix in intimate relation with the individual homogeneous nanoparticles (in the

ordinary course of shell construction) in extant shells, these later become

more angular and amalgamate to shape blocky calcite and heterogeneous structures in fossil shells. AFM Peak Force tapping modes provide

topographical and nanomechanical information. Nanomechanical

information includes adhesion, deformation…measurements highlighting the most viscoelastic components (organic compounds) with respect to the

nanoparticles.

Conclusion As a matter of fact, the hierarchical organization observed at sub-micrometric

levels in the modern shells can be more or less heavily rubbed out by a

recrystallization (secondary calcite or silicic nodules) that contributes to destroying or partly strengthening the fossil shells. This aspect can be due to

bacterial activity using the organic matrices as food supply, presence of

microboring organisms that induce dissolution and recrystallization, and weathering.

P 41

High resolution study of nacre formation and organo-mineral

interface in the shell of the European abalone Haliotis

tuberculata S. Auzoux-Bordenave*1,2, W. Ajili1, N. Menguy1, I. Estève1, M. De Frutos3, K. Benzerara1, N. Nassif1, T. Azaïs1 1Sorbonne University, Paris, France 2Muséum national d’Histoire naturelle, Concarneau, France 3University Paris-Sud, Physics, Orsay, France

The European abalone Haliotis tuberculata is a commercially and ecologically important gastropod species and represents a key-model to study

the basic mechanisms of shell formation. The abalone shell is composed of

two calcified layers underlying the periostracum, i.e. an outer spherulitic layer and an inner nacreous layer. The nacreous layer is exclusively

composed of aragonite platelets, 6–8 μm in diameter, horizontally aligned in

layers and vertically in columns, arranged with a small amount of organic material. This typical architecture provides to nacre exceptional mechanical

properties with potential uses in jewellery and biomimetics. The organic

phase, which represents less than 5 wt.% of nacre, is proposed to guide nucleation and control the growth and orientation of the mineral phase.

Although the nacre structure is studied since many decades, the formation mechanisms and the interaction between organic and mineral components

still need to be clarified.

In this study, we used juvenile shell of one-year-old abalone H. tuberculata to investigate nacre formation and organo-mineral interface through high-

resolution (HR) microscopies, at the nanometer scale. A growing nacre area

was selected in juvenile shell for further investigation of nacre surface using scanning electronic microscopy (SEM) and Field Emission Gun (SEM-

FEG). HR imaging of the juvenile abalone shell resulted in an in-depth

characterization of the growing nacre, from the spherulitic edge to the mature zone of the nacre surface, providing a detailed sequence of nacre formation.

At a higher magnification, the tablet surface exhibits typical nanograins and

early stacks of aragonite platelets appeared separated by a porous layer of organic matrix. Additional focused ion beam (FIB) coupled with SEM

allowed the observation of the 3D organization of the aragonite platelets

stacks at early stages of their formation. Additional techniques such as high-resolution transmission electronic

microscopy (HR-TEM), scanning transmission X-ray microscopy (STXM)

and electron energy loss spectroscopy (EELS) were performed onto thin FIB section of aragonite platelets columns to gain local information on the nature

and atomic composition of the organo-mineral interface at the nanometer

scale. In growing nacre, large disordered organic inclusions were evidenced in the center of each aragonite platelet. These organic inclusions are oriented

along the c-axis direction, extending from one tablet to another through

organic bridges. It is suggested that these organic inclusions act as a vertical "backbone" controlling the longitudinal growth of the stacked tablets column

along the c-axis. In the case of lateral growth, a 20 nm thick disordered layer

was evidenced by HR-TEM at the surface of growing aragonite platelet. This disordered layer appeared mainly composed of an organic fraction where

carbonates ions appear to be absent. However, the presence of both calcium

and phosphate suggests the presence of calcium-binding acidic and phosphorylated proteins involved in the control of lateral growth of nacre.

Together, these results bring new insights to the fundamental mechanisms of

nacre formation and on the interaction between organic and mineral components involved in the control of aragonite platelets growth.

P 42

Growth dynamics and fine scale characterization of calcareous

granules of the annelid Lumbricus terrestris S. Mandera*1, I. Coronado1, M. Mazur2, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2University of Warsaw, Department of Chemistry, Warsaw, Poland

Calciferous glands of some species of earthworms (Annelida, Oligochaeta)

secrete amorphous calcium carbonate (ACC) particles that are transported toward a pair of oesophageal pouches where they coalesce and crystallize.

The final product is a solid calcareous granule that is excreted by the

organism to the soil. Spherical or sub-elliptical CaCO3 granules from 0.5 to 2 mm in diameter are composed mostly of calcite, although vaterite,

aragonite and ACC have also been found. The purpose of formation of such

mineralized structures still remains uncertain and possible hypotheses include: pH buffering of the internal body, adjustment of respiratory CO2,

and elimination of excess of Ca derived from diet and even spurious

mineralization. Little is known on the processes that control the final mineral

42

product secreted by earthworms, even though metals uptake by earthworms granules as a potential use for bioremediation have been studied in-depth and

the ACC formation by the calciferous glands have been characterized. The

main purpose of the current study is to elucidate the mechanisms of biocrystallization and the growth dynamics during granules formation.

Earthworm"s granules were obtained from laboratory culturing of fully

clitellate specimens of Lumbricus terrestris. Specimens were kept in artificial soil at a constant temperature, neutral pH, and controlled moisture. In order

to characterize the growth dynamics of the granules, an Mn-labelling

experiment in an amended artificial soil was carried out using two Mn-sources in separate lines of trials: 1) Mn-bearing CaCO3 (at 50 and 100 ppm,

respectively), and 2) a solution of MnSO4 (100 ppm of Mn). 15 specimens

were cultured during 28 days, 3 control specimens in an Mn-free soil and 12 specimens in amended soil (4 for each composition). Specimens were

exposed to amended soils and Mn-free soils every 2 to 7 days. An ulterior

structural, biogeochemical and crystallographic characterization of the granules was carried out, using petrographic microscopy,

cathodoluminescence (CL), SEM, AFM, EMPA, EBSD, and FTIR

spectroscopy. Growth dynamics experiment reveals that control specimens produce

between 6-9.8 mg of CaCO3 in 2-5 granules per week. Granules show a radial

texture formed mostly by calcite crystals with variable habits and some evidence of original concentric growth. The crystal habits are prismatic, lath,

and foliated–lamellar crystals, which occasionally are arranged forming

cross-lamellar-like microstructures. Moreover, some opaque, in transmitted light, and microgranular patches have been found. Organic matrix is

arranged, mostly, parallel to the growth axis of crystals and some calcite crystals surfaces exhibit nanogranular textures. CL data show a hidden

concentric sub-daily banding, with a thrombolite-like texture and kinked

zonation inside the calcite crystals. These features reveal a complex mechanism of formation that depends on physicochemical conditions

generated at microscale during the granule formation.

Acknowledgments: This work was supported by the National Science Centre (Poland) grant 2017/25/B/ST10/02221.

P 43

Towards identification of the protein machinery of coccolith

formation in the coccolithophore Pleurochrysis carterae E. Zschieschang*1, A. Skeffington1, M. Gorka1, A. Scheffel1 1Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany

Coccolithophores are marine unicellular algae that produce mineralized

scales called coccoliths, which are 3D arrays of complex-shaped calcite crystals. How the arrangement of the crystals is guided and how crystal

morphogenesis is controlled is of interest for bioinspired materials synthesis.

Coccolith formation involves an insoluble organic structure called the base plate that templates calcite nucleation, as well as soluble acidic

polysaccharides. Recent work on the coccolithophore Pleurochrysis carterae

(recently reassigned to Chrysotila carterae) suggests that the base plate provides an attractive model system for the identification of macromolecules

controlling the crystal nucleation pattern. These base plates are composed of

polysaccharides and proteins. The exact molecular composition and identities of these components remain unknown. Here we tested different

extraction approaches to isolate individual components of the base plates of

P. carterae coccoliths. One extract was subjected to proteomic analysis. As the proteomic analysis of the coccolith base plates is at its infancy, we tested

different proteases to generate peptides. For protein identification we used

the publicly available protein database of P. carterae, which has been generated from short Illumina reads and is likely to contain incorrect protein

models due to misassembly of the reads. To improve our confidence in the

identifications we also used a database which we generated from long-read cDNA PacBio data. A comparison of the different data sets will be presented.

The workflow we have established will greatly aid attempts to elucidate the

protein machinery involved in coccolith formation.

P 44

Biological patterning in calcareous biomineralization of

terrestrial gastropod eggs J. Stolarski*1, I. Coronado1, M. Mazur2, A. Sulikowska-Drozd3 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2University of Warsaw, Department of Chemistry, Warsaw, Poland 3University of Lodz, Department of Invertebrate Zoology and Hydrobiology, Lodz, Poland

Gastropod mollusks are known from the formation of elaborated calcium carbonate shell patterns. Calcified protoconchs (larval shells) and complex

hierarchical structures of adult shells (teloconch) have been described among

various gastropod clades that include marine, fresh-water and terrestrial taxa. Much less is known about the earliest developmental phase of calcification

associated with the formation of eggs, especially among the pulmonate land

snails. Such calcified egg shells may supply the snail embryo with calcium ions to enable the formation of the earliest shell before hatching, provide

mechanical support, and potentially (not confirmed) reduce the water

evaporation from the egg. It was shown, that in contrast to almost invariably aragonite mineralogy of adult gastropod shells, the egg membranes are

mineralized with calcite crystals, often showing tetrahedral morphology.

This suggests a lack of strong biological control on this type of mineralization. Nonetheless, some authors noted a surprising variation of

ultrastructural features among more heavily calcified eggs.

Here, we have examined with SEM, micro-Computed Tomography, Raman and FT-IR spectroscopy), various structural and compositional features of

egg membrane mineralization in representatives of a highly diversified

family of door snails (Clausiliidae). The eggs were taken from oviparous (Caspiophaedusa, Oospira), viviparous (Idyla), and taxa with egg-retention

reproductive strategy (Pontophaedusa, Zaptyx, and Formosana). The

mineralized egg membranes show different patterns of crystal distribution, which invariably are composed of calcite. The calcite crystals are typically

embedded in the organic multilayered membrane (mixture of proteins and

polysaccharides), which can sandwich the crystal stacks. No evidence of amorphous precursors was found (common in systems of biologically

controlled mineralization), however distinct patterns of distribution of

crystals point to biological coordination of this process. A unique, spiral arrangement of crystal clusters is typical to eggs of Caspiophaedusa, more

homogenous distribution of tetrahedral crystals is characteristic of eggs of

Idyla and Formosana, whereas in eggs of Zaptyx and Oospira, the calcite crystals form pyramidal aggregates. In Pontophaedusa homogenously

distributed calcite crystals form a dense cover. Biological patterning that governs the calcite crystal distribution in door

snails egg membranes points to the potential use of this character in a

phylogenetic context. In fact, well-differentiated molecular clades (such as Caspiophaedusa, Pontophaedusa) show distinct crystal distribution patterns,

irrespective of reproductive strategy. Further proteomic and organic content

analyses are required to elucidate the precise biological mechanism of crystal growth coordination.

Acknowledgments: This work was supported by the National Science Center

(Poland) grant 2017/25/B/ST10/02221 (to JS) and 2016/21/B/NZ8/03086 (to ASD).

P 45

Crystallographic texture and microstructure of modern

Glycymeris shells G. Crippa*1, E. Griesshaber2, A. Checa3, E. Harper4, W. W. Schmahl2 1University of Milan, Milano, Italy 2Ludwig-Maximilians Universität München, Munich, Germany 3Universidad de Granada, Granada, Spain 4University of Cambridge, Cambridge, United Kingdom

Mollusk shells are biocomposites made of calcium carbonate crystals

(calcite, aragonite or both) and intra- and intercrystalline organic matrix,

resulting in a lightweight product of highly elaborate architectures, endowed with unique structural properties (stiffness, fracture toughness, tensile

strength). Calcite and/or aragonite crystals show different crystallographic

arrangements, which are hierarchically organized at the nano-, micro- and meso-scale, forming different microstructures.

The most common fabric among mollusk shells is represented by the

aragonitic crossed lamellar one, which forms the shells of both marine and continental taxa, thus showing a great adaptive potential and evolutionary

success; this is one of the hardest microstructures which has the ability to

dissipate and stop cracks through the shell. Species belonging to the genus Glycymeris Da Costa, 1778 are excellent taxa

to investigate for this type of microstructure, as they have an aragonitic shell

with an outer and an inner crossed lamellar layers. Also, they are widespread and common in fossil faunas and their applicability in the fossil record spans

from paleoecology, paleoclimatology, sclerochronology to

archaeomalacology. Although Glycymeris shells and the crossed lamellar fabric have been widely

study and documented, up to now a detailed characterization of the

crystallography of both this taxon and this microstructure with Electron Backscatter Diffraction (EBSD) is lacking. Here, we analyzed modern

Glycymeris shells combining the use of the traditional and easily available

Scanning Electron Microscopy with the more advanced EBSD technique, to improve the knowledge of the crossed lamellar microstructure and of its

relationship with other shell layers (i.e., myostraca). With the present

contribution we will be able to provide: i) a modern unaltered reference sample for detecting diagenetic alteration in fossil shells, which, due to the

widely use of Glycymeris fossil shells in paleoclimatic and

paleoenvironmental reconstructions, is of crucial importance, and ii) new data to better understand mollusk biomineralization processes, as well as the

formation of the crossed lamellar microstructure and its correlation with

myostracal layers, here characterized in detail for the first time.

43

P 46

Formation of biomimetic agar gel carbonate composites –

Influence of Mg and Sr S. Rudin1, M. Greiner*1, E. Griesshaber1, L. Fernández-Díaz2,3, W. W.

Schmahl1 1Ludwig-Maximilians-Universität München, Department of Earth- and Environmental Science, Munich, Germany 2Universidad Complutense de Madrid, Departamento de Cristalografía y

Mineralogía , Madrid, Germany 3Ciudad Universitaria, Instituto de Geociencias (UCM, CSIS), Madrid, Spain

Hydrogels are excellent models for understanding extracellular matrix

microenvironments and are therefore highly suitable for mimicking

biomineralization processes. Recent studies (Greiner et al. 2018) showed the combined influence of reagent solution concentration and agar solid content

on the formation of calcite aggregates grown in double-diffusion systems.

Biologic calcite hard tissues e.g. sea urchin teeth can incorporate up to 45 mol% Mg (Ma & Qi 2010). Coral skeletons, both aragonitic or calcitic,

incorporate small amounts of Sr (Smith et al. 1979).

We present results on the effect of additives (Mg, Sr) in agar gel on the formation of carbonate agar composites in double-diffusion systems. We

vary the additive concentration, the agar solid content and the concentration

of reagent solutions and compare our results to previous findings where additives were not used.

The aggregates are characterized by X-ray powder diffraction (XRPD),

scanning electron microscopy (SEM), electron backscatter diffraction (EBSD) and energy dispersive X-ray spectroscopy (EDX).

The presence of magnesium results in the formation of dumbbell shaped and

spherical calcite aggregates with a vast variation of surface morphologies containing a spherulitic inner structure, whose subunits are crystals with a

slight mosaic structure. The incorporated magnesium is (almost) evenly

distributed within the composites; occasionally it is slightly enriched at the rim of the aggregates. Crystallization under high Mg to Ca ratios in the

aqueous solution (0.1M Mg: 0.5M Ca or higher) appears to promote the

stabilization of amorphous calcium carbonate (ACC) and the formation of aragonite.

The presence of strontium in the growth medium leads to the formation of

calcite aggregates with predominantly spherical morphologies. These aggregates are polycrystals. Individual subunits of the aggregates show a low

mosaic-spread (high co-orientation). Incorporated Sr shows a zoned pattern

within the calcite aggregates, regardless of the Sr concentration in the agar hydrogel. For Sr to Ca ratios of 0.01M Sr:0.1M Ca and 0.1M Sr:0.5M Ca we

observe calcite composites overgrown by strontianite. A high agar solid content (2 wt%) promotes this overgrowth compared to a low agar solid

content (0.5 wt%).

Mg or Sr in the growth medium promotes misorientation in calcite-agar gel composites. This misorientation most likely arises from split growth

phenomena as dislocations generate to release Mg or Sr incorporation-related

lattice strain. Greiner, M., Yin, X., Fernández-Díaz, L., Griesshaber, E., Weitzel, F.,

Ziegler, A., Veintemillas-Verdaguer, S., Schmahl, W.W. (2018) Crystal

Growth and Design, 18, 1401-1414. Ma, Y. & Qi, L. (2010) Biomineralization of sea urchin teeth. Frontiers of

Chemistry in China, 5, 299-308.

Smith, S.V., Buddemeier, R.W., Redalje, R.C., Houck, J.E. (1979) Strontium-calcium thermometry in coral skeletons. Science, 204, 404-407.

P 47

Observation and simulation of 3D pigmentation patterns of

molluscan shells H. Sato*1, T. Sasaki2 1The University of Tokyo, Earth and Planetary Science, Tokyo, Japan 2The University of Tokyo, The University Museum, Tokyo, Japan

Molluscan shells show highly diverse, variable and complex pigmentation

patterns. Morphogenesis of the patterns can be understood by a mathematical model which represents dynamics of pattern formation. Several models have

been proposed so far. The previous models assume that secretory organs of

pigment are distributed in line along mantle edge, and the pattern on shell are regarded as a track of a temporally changing pattern of pigment secretion

along mantle edge. However, an observation of mantle edge suggests that the

secretory organs are two-dimensionally (2D) distributed; hence, the patterns in shells should be three-dimensional (3D). Here, we confirmed if 2D-

distributed secretory organs generated 3D pigmentation patterns in shells, by

observation and simulation. First, we observed pigmentation patterns on cross sections of Conidae shells

to describe 3D patterns. We found that some species had 3D pigmentation

patterns and different surface patterns had different patterns on cross sections, which cannot be explained by simple dynamics such as simple

diffusion in the direction of inner shell surface. This suggests that pigment

secretion may interacts mutually not only in the direction of mantle edge but also in the perpendicular direction.

Second, we proposed a new model assuming 2D-distributed secretory organs

and simulated the model numerically. Then, the results were compared with actual shell patterns. The new model was based on a reaction-diffusion

system. A simulation of the model generated triangles and stripes on surface,

which appear different patterns on cross sections respectively. The generated patterns corresponded with observed patterns of C. textile shells.

The correspondence between the observation and the simulation suggests

that Conidae species have mutually interacting secretory organs of pigment which are 2D-distributed, and form 3D pigmentation patterns. The new

model for 3D patterns provides a new interpretation of morphogenesis of

pigmentation patterns.

P 48

When predators become prey- shell repair patterns in Nucella

lapillus (Linnaeus, 1758) D. Mayk*1,2, L. Peck2, E. Griesshaber3, W. W. Schmahl3, E. Harper1 1University of Cambridge, Earth Sciences, Cambridge, United Kingdom 2British Antarctic Survey, Cambridge, United Kingdom 3Ludwig-Maximillian Universitaet, Muenchen, Germany

Introduction

The common intertidal gastropod Nucella lapillus (also referred to as dog whelk) is a predatory species feeding on e.g., blue mussels and barnacles. It

builds a massive calcium carbonate shell that shelters it from predators and

other environmental hazards. Young dog whelks which have not been able to grow thick shells yet may be subject to attacks from crabs, birds and even

conspecifics. If an attack turns out to be non-lethal it is of great importance

for the individual to repair the damaged shell as fast as possible to be fit for potential future attacks. It is very likely that the need for rapid repair goes

jointly with an increase in the energy requirement per unit calcium carbonate

formed due to [1] an elevation of the saturation state of the calcifying fluid or [2] an increase in the amount of organics used. Previous studies have

shown that increased calcification rates at damaged sites often result in a

disturbed microstructure and crystal alignment which possibly affects the shell integrity.

Objectives

We investigate the ability of N. lapillus specimens from a broad shell size range (i.e., juvenile to adult specimens), to repair artificially damaged shells

and compare the microstructure of the repaired regions with those of naturally occurred damage in conspecifics. We investigate the rate and mode

of shell repair in an attempt to link the capability of shell repair and mortality

rate of the specimens to energy availability among shell size/age groups.

Materials & methods

120 specimens of N. lapillus were collected from the rocky shore in Whitby,

UK in spring 2019 and reared in a seawater aquarium setup at the British Antarctic Survey [BAS] in Cambridge. Specimens were starved throughout

the experiment. After an acclimation phase of two weeks, long notches were

cut perpendicular to the aperture rim into the shells of ~60 specimens using a milling bit. This was done with great care to prevent damages of the

specimen's tissue. Notch repair increments were monitored weekly.

Subsequent analysis of the repaired notch regions of sections under the scanning-electron microscope [SEM] and microstructure orientation

mapping by means of electron-backscatter diffraction [EBSD] was carried

out.

Preliminary results

Our results suggest that smaller individuals (shell length < 1.5 cm) are readily

capable of repairing shell damage and start the repair earlier than larger individuals. Absolute shell repair measured as length, however, is greater in

larger/older individuals. The mortality rate during the experiment was > 10

times higher in < 1.5 cm long specimens than in > 1.5 cm long specimens suggesting that energy reserves in the younger dog whelks are smaller and

faster depleted by the repair process than in larger animals. The shells of N.

lapillus consist of three layers, namely cross-lamellar aragonite, homogeneous calcite, and the periostracum. The microstructure analysis

showed that N. lapillus produces a more regulated shell structure in the

repaired regions than is normally secreted in the undisturbed shell. We distinguish four steps observed in the repair process: [1] the damaged shell

surface is lined with a thin layer of cross-lamellar aragonite, [2] a mound-

shaped calcite structure is built on to the aragonite lining exhibiting a specific upward grading of crystal sizes from small to large and with a highly co-

orientated crystal orientation, [3] homogeneous and disorientated calcite is

built on top in a rather elongated way with a strongly frayed surface to the outside. This step happens rather quickly and covers up most of the damaged

area. [4] A lining of cross lamellar aragonite is laid over the inside of the

entire notch covering the inner surface.

Outlook

Thermogravimetry measurements of the notch material will be carried out to

investigate the organic content of the repaired shell regions. Results will be compared to the organic content of the shell layers in undisturbed shells.

44

Further SEM and high-resolution EBSD measurements will be carried out to further characterize N. lapillus' microstructure of the repair margins.

P 49

Shell-muscle attachment in the bivalves Ostrea stentina

(Payraudeau, 1826) and Anomia ephippium (Linnaeus, 1758) J. D. Castro-Claros1, C. Salas*1, C. Lucena2, A. Checa3 1Universidad de Malaga, Biologia Animal, Malaga, Spain 2Universidad de Malaga, SCAI, Malaga, Spain 3Universidad de Granada, Estratigrafia y Paleontologia, Granada, Spain

Introduction The muscles of the mantle in bivalves attach to the shell valves and retract

the mantle edges. Among them, the adductors are particularly important for

the survival of the animal. Most studies have addressed the type and morphology of the muscular fibers and filaments (Paniagua et al 1996), and

a few have focused on the attachment of these muscles to the shell (Nakahara

& Bevelander 1970). However, the mechanism of transport through the muscle to the myostracum was never addressed. The goal of this research is

to describe ultrastructure and the transport of material across the adductor

muscle-myostracum attachment in Ostrea stentina and Anomia ephippium.

Material and methods We examined 10 specimens of O. stentina and 10 specimens of A. ephippium,

collected in the littoral of Málaga (south Spain). Specimens were fixed in 2.5% glutaraldehyde (4 °C), decalcified in 2% EDTA, post-fixed in OsO4

(2%) and embedded in epoxy resin Epon 812 (EMS). Samples for calcium

detection were post-fixed in a mixture of OsO4 (2%) and potassium hexahydroxoantimonate (2%) in PBS. Ultra-thin sections (50 nm) were

stained with uranyl acetate (2%). They were observed in a TEM JEOL-

JEM1400 and a TEM-STEM FEI Talos 200X, with EDX analyzer. Collagenase was used to test the presence of collagen. One specimen of each

species was embedded in methacrylate (Technovit 7200 VLC). These

sections (50 μm) and the semi-thin sections (~0.5 μm) were stained with toluidine blue (1%)

Results The adductor muscles in both species are composed of "smooth" and "striated" muscles. The outer epithelium of the mantle continues across the

adductor muscle area and their cells contain many vesicles. Bundles of

filaments connect the muscular cells with the extrapallial space through hemidesmosomes. The extrapallial space (ca.100-150 nm thick) is filled with

organic secretions from the vesicles of the mantle cells. Additional bundles of microfilaments of collagen fibers cross the extrapallial sheet, from the

hemidesmosomes to inside the myostracum, where they form a network.

EDX analysis shows the presence of calcium inside vesicles from the mantle cells, the extrapallial sheet and the myostracum network.

Conclusion The presence of a layer of cells between the muscular cells and the shell was indicated by Nakahara & Bevelander (1970), who called them "adhesive

cells". Subsequently, Bubel (1984) called them "tendon cells". Our

observations confirm the existence of this cell layer, which is the continuation of the outer mantle epithelium across the adductor muscle. The

extrapallial space is replenished with secretions of the mantle cells and

collagen fibers, seemingly originated in the hemidesmosomes. The presence of numerous vesicles extruding from the mantle cells were probably mistaken

as a microvillous border by Nakahara & Bevelander (1970). However, no

microvilli have been observed in the basal mantle cell membranes. The presence of calcium inside some vesicles suggests that part of the calcium is

transported thereby. It would be interesting to study whether calcium is

transported as amorphous calcium carbonate or as a crystalline phase. Bubel A. 1984. Epidermal cells. In: Bereiter-Hahn et al. (eds.) Biology of the

Integument 1, Springer: 400-447.

Nakahara H., Bevelander G. 1970. Texas Rep. Biol. Med. 28(3): 279-286. Paniagua R., Royuela M., García-Anchuelo R.M., Fraile B. 1996. Histol.

Histopathol. 11(1): 181-201.

P 50

Structure - material property relationships in marine bivalve

and gastropod calcite and aragonite Z. Tang*1, E. Griesshaber1, E. Harper2, S. Zaefferer3, M. Zenkert1, N.

Lagos4, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department of Geoscience and

Environmental Sciences, Munich, Germany 2University of Cambridge , Department of Earth Sciences, Cambridge, United Kingdom 3Max-Planck-Institut für Eisenforschung, Department of Microstructure

Physics and Alloy Design, Düsseldorf, Germany 4Universidad Santo Tomás, Centro de Investigación e Innovación para el Cambio Climático, Santiago, Chile

Carbonate biological hard tissues are composite materials consisting of

biopolymer and biomineral components. Both material classes are present in

the composite hard tissue with complex hierarchical structures and architectures.

In this study we investgate the interlinkage between mineral organization

(microstructure, texture), biopolymer matrix distribution and biomaterial properties for some marine gastropods (Haliotis glabra, Patella vulgata,

Tegula sp.) and some marine bivalves (Mytilus edulis, Chama arcana). The

hard tissues of all these organisms consist of calcite and aragonite, however in characteristic organizational patterns. Haliotis laevigata and Mytilus

edulis contain two shell layers: an outer calcitic and an inner aragonitic shell

region. The aragonite in both organisms is nacreous aragonite, in Haliotis laevigata the calcite is prismatic, while in Mytilus edulis it is fibrous. In

Patella vulgata and Chama arcana the aragonitic shell portion is sandwiched

between two calcitic shell layers. These two calcitic shell portions have distinct microstructures; are neither prismatic (as it is the case in Haliotis

laevigata) nor fibrous (as it is the case in Mytilus edulis). Tegula sp. also has

an outer calcitic and an inner aragonitic shell portion. However, the aragonite in Tegula sp. is not developed in a nacreous microstructure; it is an assembly

of aragonitic prisms that increase in size towards the soft tissue of the animal.

Microstructure and texture characterization was done with SEM imaging and electron backscatter diffraction (EBSD), biopolymer content was analyzed

with Thermo-Gravimetric- Analysis (TGA), biopolymer distribution within

the biological hard tissue was visualized with various etching protocols and SEM imaging, biomaterial properties were measured for two hierarchical

levels with instrumented nanoindentation and microindentation.

We find that biopolymer contents is fairly similar for the hard tissues of the investigated species, however, mineral organization differs significantly

between the species, for both, the calcite and the aragonite. Accordingly, for the selected hard tissues, mineral organization exerts the strongest effect on

the observed variation of nano- and micro-hardness as well as elastic

modulus. With the wide range of selected microstructures we aim to highlight in this contribution the relation between specific microstructural construction

principles, biomaterial properties and biomaterial functions.

P 51

Pattern formation of interlocking calcite fibre hybrid

composite in brachiopod shells W. W. Schmahl*1, E. Griesshaber1, A. Ziegler2, M. Simonet Roda1, D.

Henkel3 1LMU Munich, Earth and Environmental Sciences, Munich, Germany 2University of Ulm, Central Facility of Electron Microscopy, Ulm,

Germany 3GEOMAR Helmholtz Centre for Ocean Research , Marine Biogeochemistry/Marine Systems, Kiel, Germany

Fibrous biological hybrid composites are an important class of materials.

Aragonite or calcite fibers are embedded in a pliant biopolymer matrix, the

latter being always cross-linked within the hard tissue. Most biological carbonate hard tissues are subject to compressive, bending and shearing

forces. As fibers within a matrix cannot be reorganized once they endure

these forces, they must be properly packed and oriented within the hard tissue from the onset of their formation. This is accomplished by the formation of

stacks of parallel fibers with interlocking concave/convex cross-sectional

shapes. On the next hierarchical level, the stacks are twisted in a plywood-like arrangement. This ensures that all components of the composite are

interleaved in three dimension and on all length scales.

The fibrous shell layer of modern brachiopod shells is such a hybrid composite material. The interlocked concave-convex packing morphology of

the calcite fibres is more sophisticated than the simple "brick and mortar"

pattern of mollusc nacre or the random cellular polygonal convex cross-sectional of columnar calcite of many invertebrates. In the fibrous calcite

composite, the convex surface of each fibre is lined by an organic membrane,

which is intricately connected to the fibre calcite. The four concave surfaces are adjacent to the linings of the convex sides of neighboring fibers.

When brachiopod fibre and nacreous tablet formation are compared,

significant differences emerge in biopolymer/mineral deposition and, hence, microstructure generation. In the case of modern brachiopod fibers, during

secretion, mantle epithelium cells are always in direct contact with the

mineral, whereas in molluscs the nacreous tablets are never in direct contact with epithelial cells. There is always an interlamellar (in bivalves) or surface

(in gastropods) membrane between secreting mantle cells and the growing

aragonite platelets. When brachiopod fibers form, secretion of the biopolymer membrane covering the convex surface of the fibre is the last and

terminal step in fibre growth. In contrast, when nacre forms, aragonite tablet

formation starts with the consecutive self-assembly by liquid crystallization of interlamellar membranes. This leads to the formation of compartments that

become successively infiltrated by aragonite. Accordingly, we find for

modern brachiopod shell and molluscan nacre development two divergent microstructure generation processes. One is biologically controlled through

direct cellular contact and activity with the mineral as it is the case for

brachiopods, the other is physically controlled through the self-organization of extracellular matrix membranes as it is the case for molluscan nacre.

45

P 52

Formation and mosaicity of coccolith segment calcite of the

marine algae X. Yin*1, A. Ziegler2, K. Kelm3, R. Hoffmann1, P. Watermeyer3, P. Alexa1,

C. Read2,4, L. Schlüter5, T. B. H. Reusch5, E. Griesshaber1, P. Walther2, W.

W. Schmahl1 1Ludwig-Maximilians-Universität München, Department für Geo- und

Umweltwissenschaften, München, Germany 2Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm, Germany 3Deutsches Zentrum für Luft- und Raumfahrt, Institut für Werkstoff-

Forschung, Köln, Germany 4Universitätsklinikums Ulm, Institut für Virologie, Ulm, Germany 5GEOMAR Helmholtz-Zentrum für Ozeanforschung Kiel, Marine Evolutionary Ecology, Kiel, Germany

Coccolithophores belong to the most abundant CaCO3 mineralizing

organisms.Coccolithophore calcification is a complex and highly regulated process, resulting in a biogenic product that differs in both, morphology and

chemical composition from the abiogenically produced equivalent. Unlike

extracellularly formed biological carbonate hard tissues, coccolithophore calcite is neither a hybrid composite, nor is it distinguished by a hierarchical

microstructure. This is remarkable, as the key to optimize crystalline

biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the specific microstructure.

To get insight into the pathway of biomineralization of E. huxleyi coccoliths

we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of

the calcite in the coccolith vesicle within the cell. With TEM diffraction and

annular dark field (ADF) imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic-block-boundaries.

As at these only minor misorientations occur, we attribute them to dislocation

networks creating small-angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E. huxleyi calcite mosaicity is not

caused by occluded biopolymers, as it is the case in extracellularly formed

hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical atom-by-atom growth

mechanisms. Using cryo-preparation techniques for SEM and TEM we found

that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well-controlled constant gap

of about 4 nm between them. We describe this conspicuous connection as a not yet described inter-organelle junction, the "nuclear envelope junction".

The narrow gap of this junction likely facilitates transport of Ca2+ ions from

the nuclear envelope to the coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear

envelope – endoplasmic reticulum Ca2+-store of the cell for the transport of

Ca2+ ions from the external medium to the coccolith vesicle and that E. huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle

accretion mechanism.

P 53

Skeletal repair of modern brachiopod shells E. Griesshaber*1, J. H. Robinson2, E. M. Harper3, M. Lamare4, M.

Williams4, M. Zenkert5, W. W. Schmahl5 1Ludwig-Maximilians University Munich, Department of Earth and

Environmental Sciences , Munich, Germany 2University of Otago, Department of Geology, Dunedin, New Zealand 3University of Cambridge, Department of Earth Sciences, Cambridge,

United Kingdom 4University of Otago, The Department of Marine Sciences, Dunedin, New Zealand 5LMU Munich, Department of Earth and Environmental Sciences, Munich, Germany

Invertebrates, brachiopods, molluscs, echinoderms, are known to repair

damage to their shells. This is either inflicted by predators or by physical

environmental impact, e.g. ice blocks or, in high surf regions, e.g. rocks.

Shells often show various and distinct repair traces. This indicates that a specific organism is often attacked by a range of animals as well as different

environmental impacts.

In this contribution we describe new shell formation at damaged shell portions for the modern terebratulid brachiopod. We investigate how

Calloria inconspicua copes with an injured shell, how quickly it repairs its

shell and how much control the animal exerts on the organization of the newly-formed mineral, the mineral that is secreted for repair. Optical and

electron microscopical techniques were used for imaging the shells, electron

backscatter diffraction analysis (EBSD) was applied for the determination of calcite organization and measurement of calcite crystal co-orientation

strength. Live Calloria inconspicua specimens were collected at Otago

harbor, New Zealand and were placed into aquaria. Animals were damaged mechanically either by drilling 1 to 2 mm sized holes into their shells or by

cutting slices off the shell. Subsequent to damage formation, brachiopods

were placed back into the tanks for repairing their shell. Most specimens survived and, thus, coped well with injured shells. They repaired damaged

shell parts quickly, within three to five months.

Calloria inconspicua secrets two-layered shells, a primary, outer, and a fibrous, inner, shell portion with, in the latter, well-aligned calcite fibres (Ye

et al. 2018, Journal of Structural Biology). As brachiopod fibres occlude very

minor amounts of biopolymers, calcite crystallites within the fibres are highly co-aligned (e.g. Casella et al. 2018, Biogeosciences). At repair of the

hole drilled into the shell, the mantle tissue secrets first a thin layer of calcite

that is added onto inner shell surfaces at still undisturbed shell portions. Relative to shell calcite secreted under normal living conditions, the calcite

that forms first at repair has an almost random microstructure and texture. It

is a thin layer of calcite that appears to serve as stabilization for the subsequently secreted calcite, the calcite that covers the hole drilled into the

shell. The microstructure and texture of the first-formed shell calcite is

significantly different to that secreted under normal conditions. In the former (the first-formed calcite) typical morphologies of mineral units forming the

primary and the fibrous shell layers are entirely absent as well as the high co-

orientation strength of calcite crystallites within a particular mineral unit. MUD values (indication for the strength of crystallite co-orientation, Casella

et al. 2018, Biogeosciences) of undisturbed shell portions of modern

Callorica inconspicua range between 60 to 70, while for the first-formed calcite and the calcite that covers the hole drilled into the shell MUD values

are between 18 and 20.

Despite low MUD values microstructure recovery starts soon when the shell is repaired. For the shell portion that covers the hole drilled into the shell, we

can clearly distinguish between primary and fibrous layer mineral units. With ongoing repair these gain more and more their normal morphologies. Recent

studies of cell ultrastructure secreting the calcite of the modern terebratulid

brachiopod Magellania venosa (Simonet Roda 2019a, Nature Scientific Reports; Simonet Roda 2019b: Journal of Structural Biology) showed that at

active secretion outer mantle epithelium cells are in direct contact to the

forming fibre ant the primary layer calcite. Taking this into account it can be assumed that the mantle portion beneath the drill-hole only secretes the

calcite that is needed for repair of that shell portion.

P 54

The role of innate immunity of aortic valve cells in human

aortic valve calcification X. Meng*1, L. Ao1, D. Fullerton1 1University of Colorado Denver, Surgery, Aurora, United States

Chronic inflammation and progressive calcification of the aortic valve

leaflets lead to calcific valvular heart disease (CVHD) that is common in the elderly. Currently, pharmacological prevention of CVHD progression is

unavailable although there is a wide window for intervention. In addition, the

interaction between the pro-inflammatory and pro-osteogenic mechanisms in aortic valve calcification is not well understood. Aortic valve interstitial cells

(AVICs) are actively involved in valvular calcification. Initial studies by our

group demonstrate that human AVICs express pro-osteogenic proteins (including BMP-2 and TGF-β1) in response to stimulation of Toll-like

receptor (TLR) 2 or 4. Further, the TLR-mediated innate immune response

in human AVICs leads to pro-osteogenic reprogramming characterized by the expression of Runx2 and alkaline phosphatase, and formation of calcium

deposits. These studies uncovered a novel mechanistic role of the AVIC

innate immunity in aortic valve calcification. Our recent work identified several endogenous factors that can elicit the osteogenic response in human

AVICs through TLR2/4, including oxidized low-density lipoprotein,

biglycan and matrilin 2. These endogenous factors (damage-associated molecular patterns, DAMPs) induce the osteogenic response in human

AVICs mainly through the NF-κB and ERK1/2 pathways. Together, our

findings demonstrate that DAMPs are capable of inducing osteogenic response in human AVICs and that the innate immune receptors have novel

functions in modulating the osteogenic response in human aortic valve cells.

These findings suggest that AVIC TLRs may play an important role in the pathogenesis of CVHD and that modulation of the signaling pathways

utilized by TLRs may have therapeutic potential for suppression of CVHD

progression.

46

P 55

Structural characterization of acellular components in

Ariolimax californicus (Gastropoda; Stylommatophora)

D. Montroni*1,2, X. Zhang1, J. Leonard3, M. Kaya4, C. Amemiya5, G.

Falini2, M. Rolandi1 1University of California Santa Cruz, Department of Electrical Engineering, Santa Cruz, California, United States 2Alma Mater Studiorum Università di Bologna, Department of Chemistry

“Giacomo Ciamician”, Bologna, Italy 3University of California Santa Cruz, Joseph M. Long Marine Laboratory,

Institute of Marine Science, Santa Cruz, California, United States 4Aksaray University, Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray, Turkey 5University of California, School of Natural Science, Merced, CA, United States

Biological materials such as the chiton"s tooth, the squid"s beak, and the

byssal threads of bivalves have inspired the development of new materials. In this regard, we have characterized the acellular components of the

terrestrial slug Ariolimax californicus (banana slug), which are three matrices

in the buccal mass and the internal shell. The buccal mass is an apparatus, very similar in function to the human mouth,

whose role is processing food. The components observed in the buccal mass

were the radula, the jaw, and the odontophore. In many marine mollusks the radula is a well studied organ, but no study has ever been done on terrestrial

soft food eating mollusks. Moreover, the jaw and the odontophore have never

been described before under a structural point of view. The other matrix studied was the internal shell. Along with the evolution,

slugs have lost their external shell to adapt to calcium poor environments, but

some of them maintained a vestigial internal shell. Differently from mollusks shells, which role, composition and mechanisms of formation have been

widely investigated, the internal shell of slugs has been poorly studied and

many aspects are still unknown. It has only been observed that mineralizes and demineralizes depending on the life cycle of the animal.

The research involved the sectioning of the matrices and the study of their

morphological structures using electron scanning and optical microscopy. Spectroscopy, X-ray diffraction, and chemical analysis were also used to

investigate the composition and the structure of the material in its different

regions. In the radula, calcium-rich denticles were tightly interlocked one to the other

on top of a nanofibrous chitin membrane. The jaw was observed having a nanostructured morphology made of chitin to achieve compression resistance

and is directly linked to the foregut cuticle, which has a protective

nanofibrous structure. Finally, in the odontophore, we observed a structurally elastic microstructure that interfaces soft tissues with a highly stressed radula

membrane. Based on those observations, we discussed the interaction

between these components and highlighted how the materials in these components have evolved together to perform their tasks.

The study of the internal shell showed two different calcite crystal

morphologies on the two faces of the shell. Those crystal phases grew at the interface of three chitin-protein based layers: a thick internal layer and two

external membranes on each side of it. In the ventral face spherulite crystals

were observed, while lamellar crystals were observed in the dorsal face. This polymorphic asymmetry was observed also in the organic matrix hosting the

crystals. Those observations led to the hypothesis that this matrix act as

calcium storage with a face working as short-term calcium reserve, while the other as a long-term one.

In conclusion, considering the structural characterization of the A.

californicus" matrices in relation to their functions, this study could give a new perspective on fabricating bioinspired materials.

P 56

Structure-function relationships in carbonate endo- and

exoskeletons E. Griesshaber*1, A. Checa2, A. Ziegler3, M. Zenkert1, W. W. Schmahl1 1Ludwig-Maximilians University Munich, Department of Earth and

Environmental Sciences , Munich, Germany 2Departamento de Estratigrafía y Paleontología Facultad de Ciencias

Universidad de Granada Avenida Fuentenueva s/n Granada, Granada,

Spain 3University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany

In this contribution we highlight and discuss differences in mineral

organisation of biocarbonate hard tissues used by organisms for diverse

purposes: as a buoyancy device, for protection of the soft tissue and as enforcement of the crustacean cuticle. We observe a huge diversity of

microstructure and texture patterns that range from almost unaligned over

graded to co-aligned crystal assemblies. Hence, a high order as well as a high disorder in mineral organization is advantageous and is fabricated by the

organism. Carbonate organization patterns were measured with conventional

and high-resolution EBSD (electron backscattered diffraction) and transmission Kikuchi diffraction (TKD); differences in structural

organization of the mineral were imaged with FE-SEM and STEM. We

characterize carbonate assembly in the buoyancy device of the cephalopod Spirula spirula, the shell of the molluscs Mytilus edulis and Haliotis glabra,

and the calcite occlusions within the cuticle of the isopods Tylus europaeus

and Porcellio scaber. The cephalopod Spirula spirula possesses a light-weight internal shell that is

divided into discrete chambers. These are separated from each other by

partitions, the septa. A thin organic siphuncle runs through all chambers and facilitates that liquid and gas diffuse into and out of the chambers, hereby

allowing for buoyancy adjustments. Spirula sp. covers daily almost 1 km

difference in altitude as the animal spends the night in deep waters, while for the day it traverses the water column up to about 200 to 300 km depth below

the water surface. The shell is coiled in a single plane, resembling a

logarithmic spiral, the coils do not touch one another. Both the shell wall and the septum are mineralized with the walls being almost devoid and the septa

consisting of a large proportion of organic matter. The septa allow the shell

to function as a buoyancy device. They are curved, with the concave side pointing towards the first chamber and consist of organic laminae enforced

by intercalated nanometric granular aragonite. In contrast, aragonite crystals

that form the shell walls are micrometer sized and are irregular in size and morphology. Their arrangement is structured, such that large crystals line the

outer sides, while an assembly of small aragonite crystallites form the inner

regions of the shell wall. Crystal axes co-orientation strengths is high for both the aragonite of the walls as well as for the granular aragonite within the septa

(MUD values scatter between 150 and 200). Aragonite c*-axes are perpendicular to the shell vault and rotate with its curvature.

The shell of the molluscs Mytilus edulis and Haliotis glabra consist of three

distinct layers separated from each other by either organic membranes or, for a given genera, by characteristic transition zones. The mineralized shell

portions consist of dense assemblages of carbonate mineral units. For both

organisms calcite forms the seaward pointing layer, while nacreous aragonite constitutes the shell part that is next to the soft tissue of the animal. In Mytilus

edulis the calcitic shell portion comprises calcite fibres, each of them

sheathed in organic substance. In Haliotis glabra the calcitic shell section is formed by irregularly sized and shaped calcite units that decrease

consistently in size towards the periostracum, the outermost layer of the shell.

For a given age, shell thicknesses are comparable for the two animals. Mytilus edulis lives attached to the substrate, in very shallow waters and in

high surf regions, while the benthic Haliotis glabra lives in water depths

between 50 to 80 m. Mytilus edulis developed a thin, flexible and tough shell, while Haliotis glabra protects its soft tissue with a hard and stiff shell. The

calcitic shell portion is rigid in Haliotis glabra, while it is ductile in Mytilus

edulis. For adult specimens and within the central portion of a cross-section through the shell in Haliotis glabra we find an about 60 to 70 mm thick layer

of prismatic calcite and of an about 280 to 300 mm thick layer of nacreous

aragonite. In a comparable section of the shell the calcitic shell region of Mytilus edulis comprises about 150 to 160 mm of fibrous calcite and an about

80 mm thick layer of nacreous aragonite. Thus, in Haliotis glabra nacreous

aragonite prevails, while in Mytilus edulis fibrous calcite forms the major part of the hard tissue. The outer shell portion of Haliotis glabra with its

irregularly shaped and sized mineral units (arranged with a low degree of co-

orientation; MUD below 20) provides the necessary stiffness, while in Mytilus edulis the (highly co-oriented; MUD above 300) calcite fibre

arrangement, with each fibre being sheathed by biopolymers, provides the

tensile strength and ductility. The common rough woodlouse Porcellio scaber and the sand burrowing

species Tylos europaeus reinforce the exo- and endocuticle layers with

mineral, amorphous calcium carbonate, calcite and phosphate. The amount of incorporated calcite as well as the pattern of calcite organization differs

significantly between the species. While the calcite layer within the

exocuticle of Tylos europaeus is thick and consists of a multitude of highly misoriented calcite domains, the calcite in the exocuticle of Porcellio scaber

is significantly thinner and is composed of a few large domains containing

highly co-oriented calcite crystallites. Differences in mineral incorporation and patterns of crystal assembly are relatable to functional and habitat

adaptations: The tergite of Porcellio scaber is thin and flexible. Upon

predation the animal either runs away or clings still and firmly to the substrate, hence it needs a lightweight and highly flexible cuticle. In contrast,

the beach dwelling isopod Tylos europaeus rolls into a sphere upon threat

and the animal relies on its thick cuticle for protection of its soft body tissue.

47

P 57

Nanoscale structuring of modern brachiopod calcite fibres E. Griesshaber*1, R. Schmidt2, T. Schmidt2, R. Steffen2, H. Gnägi3, M.

Simonet Roda1, A. Ziegler1, W. W. Schmahl1 1Ludwig-Maximilians University Munich, Department of Earth and

Environmental Sciences , Munich, Germany 2Hitachi High-Technologies Europe GmbH, Krefeld, Germany 3Diatome Ltd, Biel-Bienne, Switzerland

Organization of nanosized entities across many length scales poses a major challenge in the development and production of man-made materials with

advanced functions. In contrast, in biologically formed hard tissues, this

design feature and its formation principle is intrinsic. It began already with the emergence of first skeletal hard parts in late Precambrian times and, since

then, it was diversified by evolutionary adaptation.

Modern terebratulid and rhynchonellid brachiopod shells consist of up to three mineralized shell layers: the outermost primary, the inner fibrous, and,

where developed, an innermost columnar layer. In two-layered shells the

fibrous layer forms an extensive part of the shell. The fibers are hundreds of micrometers long and are almost single-crystalline mineral units. The shape

of brachiopod fibers is unique and was already developed in Lower Cambrian

times. In recent brachiopods, the morphology and dimension of fibers is characteristic for a given brachiopod species and is evolutionary adapted to

the animal"s habitat.

The aim of our study was to detect and visualize a possible nanostructuring

of modern brachiopod fibres. We investigated the fibrous layer of the modern

brachiopod Magellania venosa from micrometer to nanometer scale levels

with FE-SEM and EBSD, STEM imaging and TKD. Conventional EBSD measurements prove a strong co-orientation strength of calcite crystallites

within the fibres and document on micrometer scales their clossness to being

single crystals. However, with AFM and STEM imaging and transmission Kikuchi diffraction (TKD) measurements we are able to demonstrate the

internal nanoscale structuring of the fibres. Co-oriented nanosized calcite

crystals are stringently assembled to individual rows, a multitude of the latter comprise individual fibres.

Hence, the almost single crystalline calcite brachiopod fibres are

substructured internally. They comprise highly co-oriented calcite crystallites that are placed in a thin network of biopolymer fibrils, a finding

that has not yet been shown for modern brachiopod calcite fibres. Individual

fibres in modern brachiopod shells are not single crystals.

P 58

Diversity and function of biominerals in Ciliates M. L. Lemloh*1 1University of Stuttgart, Materials Testing Institute, Stuttgart, Germany

Eukaryotic single-celled organisms like Ciliates (Protista) represent an

excellent model system to discover biomineralization principles with respect

to intracellular mechanisms involved in ion enrichment, vesicular mineral transport and biomineral formation. There are over 8,000 species of the

Phylum Ciliophora and since Ciliates naturally occur in freshwater, brackish,

and marine habitats as well as in extreme environments, they can be used to study the impact of different environmental conditions, e.g. available mineral

sources. Although it is known that Ciliates form diverse intracellular mineral

structures, little is understood about their biomineralization processes. Therefore, we combine dynamic in vivo studies of mineral forming Ciliates

together with high-resolution methods like analytical electron microscopy, to

identify common principles and to distinguish diverse mechanisms with specific adaptations involved in mineral formation. The presentation will

outline selected examples of biomineralizing Ciliates with respect to the

characteristics and various functions of the formed minerals.

P 59

Effect of eggshell on polymorphic transformation of calcium

carbonate S. Polat*1, P. Sayan1 1Marmara University, Chemical Engineering, Istanbul, Turkey

Introduction Calcium carbonate precipitation has drawn a lot of research attention over

the last few decades due to its widespread industrial applications and

scientific value in the field of biomineralization. Calcium carbonate crystals can exist as three different forms, namely, calcite, vaterite, and aragonite.

Different polymorphs tend to have different chemical and physical

properties, which can strongly influence the properties of the products; thus, it is important to have control of the crystal form. This study investigated

precipitation of calcium carbonate in the presence of eggshell.

Objectives The purposes of this study were to determine the influence of eggshell on the

morphology of calcium carbonate and to describe the effects of eggshell on

the polymorphic transformation process and the thermal decomposition behavior of calcium carbonate.

Materials & methods Calcium chloride dihydrate and sodium carbonate were used as the reactants in the calcium carbonate precipitation process. The experiment was

conducted in a 1-L double-jacketed crystallizer. The temperature was

controlled using a thermostat and the experiments performed at 30 °C. During the experiments, the pH of the solution was monitored consistently

and fixed at pH 8.5. The solution was mixed at 500 rpm using a three-blade

propeller and mechanical stirrer. The calcium carbonate suspensions obtained as a result of the experiments were removed from the crystallizer,

filtered and then dried. The as-prepared products were characterized by X-

ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy

(SEM) were used.

Results To investigate the structure of the calcium carbonate formed in the absence

and the presence of additive, calcium carbonate crystal forms were

investigated using XRD and FTIR spectroscopy. All the diffraction peaks for the crystals obtained in pure media were attributed to the existence of calcite

crystals with a rhombohedral structure. The calcite form was fully converted

into the vaterite form in eggshell media, which was in agreement with the FTIR results.

SEM and particle size analysis were performed to investigate the effect of

the eggshell on the morphology and the size of the calcium carbonate. The crystals formed in pure media mainly consisted of uniform crystals with a

cubic-like shape and smooth surface. When using eggshell in the crystallization media, nearly all of the crystals occurred in vaterite form and

their appearance was spherical and the specific surface area of calcium

carbonate increased significantly. The filtration characteristics of the calcium carbonate crystals were

determined and the mean specific cake resistance and the mean cake porosity

of the crystals were calculated based on Darcy"s Law. In addition, the thermal decomposition of the calcium carbonate samples was analyzed using

thermogravimetric analysis.

Conclusion The present study investigated the effect of eggshell on the precipitation of

calcium carbonate. In the eggshell medium, the diffraction peaks of the

calcite crystals disappeared and only vaterite crystals were observed. SEM images illustrated that eggshell significantly improved the rounded and

spherical crystals. This study demonstrated the modification of the structure

and morphology of calcium carbonate as well as the formation of its vaterite form were possible using eggshell.

P 60

Crustacean cuticular matrix in transition- spatial and temporal

diversity N. Žnidaršič*1, P. Mrak1, J. Štrus1, K. Žagar Soderžnik2, M. Čeh2 1University of Ljubljana, Department of Biology, Biotechnical faculty,

Ljubljana, Slovenia 2Jožef Stefan Institute, Department for Nanostructured Materials, Ljubljana, Slovenia

Biological mineralized matrices display inhomogenity in structure and

composition. Coexistance of different mineral forms in well defined

functional layers was characterized in detail in several calcified and other biomineralized structures. Complex assemblages of hierarchically organized

organic macromolecules in biomineralized matrices also show structural

alterations in distinct regions. Additional complexity of biomineralized matrices refers to the time scale alterations in structure and composition due

to tissue differentiation during animal development and during replacement.

To address the questions in biomineralized matrix elaboration it is necessary to determine the ultrastructure and composition of the sample at the specific

site(s) and at the specific time. In addition, for microscopical characterization

it is beneficial to preserve both, mineral and organic components. We have applied a correlative microscopic approach that combines structure

determination with elemental and molecular localization data to characterize

the crustacean cuticle differentiation during development and to characterize the interface between calcified and non-calcified layers in adult animals.

Localizations with specific ligands, transmission electron microscopy and

block face imaging in BSE mode by scanning electron microscopy were supplemented by EDXS analyses. Different specimen preparation

procedures were implemented, including methanol fixed and resin embedded

specimens. This enables to obtain information on elemental composition in selected regions of the sample that was not exposed to water solutions and to

correlate this chemical information with ultrastructure as revealed by SEM

imaging of the block surface and by TEM imaging of the corresponding ultrathin section. We have focused to characterize the interface between the

two horizontal regions of the cuticle in the adult specimens, i.e. between the

calcified endocuticle and non-mineralized membranous layer that faces epidermal cells. This is a complex transition zone from the architectural and

compositional point of view. Next, the cuticle of different developmental

48

stages was analysed to determine the modifications of exoskeletal cuticle during differentiation. The earliest stage with an epidermal matrix displaying

typical structure of adult exoskeletal cuticle is the prehatching embryo. In

early postembryonic stages a new cuticle is formed and calcium sequestration in the matrix is evident. Major alterations of epidermal matrix correspond to

the transit from embryonic to postembryonic period that is accompanied by

embryo hatching from the vitelline membrane.

P 61

Crystallographic and biomineral organization of the cuticle of

Devonian trilobites- tailored armours of the past I. Coronado*1, J. Esteve2, J. A. Cruz3, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2Los Andes University, Geosciences Department, Bogotá, Colombia 3Complutense University, GEODESPAL, Madrid, Poland

Systematic characterization of well-preserved fossils can help to trace back

evolutionary changes in biomineralization processes that occurred along the Earth history, but also to provide inspiration to create biomaterials of unique

properties. These tasks can be reached insofar as the original crystallo-

chemical properties of biominerals have not been obliterated by diagenesis. Biominerals formed in biologically controlled process by marine and

terrestrial organisms are hierarchical organo-mineral composite

nanomaterials, with distinctive microstructures and well constrained crystallographic arrangements.

Trilobites are marine fossil arthropods that inhabited seas from Cambrian to

Permian, which have a characteristic articulated carapace. Trilobite cuticle is heavily mineralized assembled by layered structures that form a dense

framework, providing them tailored armours for protection. Cuticles moult

during ontogeny (creating exuviae) and they represent majority of trilobite finds, whereas only exceptionally the full skeletons are preserved. Although

microstructure of trilobite cuticles have been vastly described in the

literature, still, little is known about the mechanisms that ruled their formation and their characteristic biomineral properties. The purpose of this

work was assessed for the first time, the structure of Devonian trilobite

cuticles at micro- and nano-scale levels, as well as the crystallographic arrangement of selected parts of carapace.

Two complete trilobite carapaces belonging to the genera Phacops sp. and

Scabriscutellum sp. from Middle Devonian of Hamar Laghdad Formation (Erfoud, Morocco) were studied. The material was observed under

cathodoluminescence (CL) with a special emphasis to the preservation state. Micro- and nanostructure was characterized by SEM and AFM and

subsequently crystallographic arrangement of four skeletal parts was studied

(EBSD): cranidia, pygidia, articulated thoracic segments and hypostome. The calcitic cuticle is a hierarchical self-assembled structure composed by a

taxa-specific microstructure, dense packaging, forming curved and

interwoven textures. Although, some evidence of diagenesis have been observed in Scabriscutellum sp. cuticle (CL and EBSD data), most of

microcrystals are well preserved in both taxa, and they are composed of

nanogranules coated by dark envelopes, at nanoscale. Cuticles exhibit the c-axis of crystals oriented perpendicular to skeletal surface and although the

analysis shows a common crystallographic arrangement between both taxa,

small differences have been observed in the supra-specialized structures. External structures of carapace exhibit a dense and well-constrained

crystallographic organization in comparison with internal ones. Such

structural organization of trilobite cuticle may explain its mechanical resistance and contribute to a better understanding of the success of this

group during almost 350 million years.

Acknowledgments: This study was supported by the Spanish "Ministerio de Economía y Competitividad" (research projects CGL2016‐78738‐P).

P 62

Solid-state NMR studies of 13C, 15N,29Si-enriched biosilica of

the diatom Cyclotella cryptica F. Kolbe*1, H. Ehren2, M. Baldus2, E. Brunner1 1TU Dresden, Faculty of Chemistry and Food Chemistry, Dresden,

Germany 2Utrecht University, Bijvoet Center for Biomolecular Research, Utrecht, Netherlands

Introduction

Diatoms are unicellular algae producing micro- and nano-structured cell

walls consisting of amorphous silica. The shape and pattern of these cell walls is species-specific. Although some compounds which are involved in

the process of biomineralization like polycationic peptides called silaffins or

long-chain polyamines (LCPAs) are known, the process of biomineralization is not fully understood until today.

Objectives

Solid-state NMR spectroscopy is used to gain a deeper insight into the interactions at the organic/inorganic interface of the diatom cell wall. Special

solid-state NMR methods, i.e. the REDOR experiments, can be used for

distance determination between hetero nuclei. (REDOR: Rotational Echo DOuble Resonance)

Materilas & methods

Within the present contribution, the biosilica of Cyclotella cryptica, a centric maritime diatom, is studied. The silica phase of the purified cell walls can be

characterized using 29Si solid-state NMR spectroscopy whereas the organic

compounds are investigated by 13C, 15N, and 31P solid-state NMR spectroscopy. The sensitivity of the experiments can be greatly enhanced

through isotope-labeling of the diatoms during cultivation in isotope-

enriched culture medium. Moreover, DNP-supported solid-state NMR experiments (DNP: Dynamic Nuclear Polarization) provide further signal

enhancement, which is especially interesting for 2-dimensional NMR

experiments like PDSD (Proton-Driven Spin Diffusion) or DQSQ (Double Quantum Single Quantum).

Results

One important feature of C. cryptica is a massive insoluble organic matrix. Solid-state NMR spectroscopy is used to investigate the insoluble organic

matrix in C. cryptica as well as the silica/organic interface. The different

organic compounds like sugars as well as proteins and LCPAs are studied by the observation of hetero nuclei like 13C, 15N and 31P. In addition, the distance

between different organic compounds and the silica phase is determined

using a 1H-13C-29Si REDOR experiment. It can be shown, that the shortest distances between silica and organic compounds appear for different signals

in the 13C-chemical shift range of 40 – 60 ppm, which is typical for LCPAs.

This indicates a very close contact between the LCPAs and the biosilica. Moreover, DNP-supported NMR measurements confirm the presence of

different sugars. Especially, the presence of chitin is verified using a PDSD experiment. The calculated enhancement factors for different signals and

thus, for different organic compounds can help to get an idea for the

supramolecular architecture of the biosilica. Furthermore, a comparison to the biosilica of Thalassiosira pseudonana reveals both, similarities as well

as differences between the organic matrices of these diatom species.

Conclusion

Our studies show that solid-state NMR is one powerful technique to learn

more about biomolecules and their arrangement in diatom biosilica. NMR

experiments help to identify different organic compounds. Moreover, we get a deeper insight into the supramolecular arrangement of the different

biomolecules in the biosilica.

P 63

Biosilicification- phytolith formation in plants M. A. Nawaz1, I. Zemchenko*1, A. Zakharenko1, K. S. Golokhvast1 1Far Eastern Federal University, School of Engineering, Vladivostok, Russian Federation

Silica is deposited extra and intracellularly in plants in solid form as

phytoliths. Phytoliths have emerged as accepted proxies for reconstructing

ancient flora, agricultural economies, environment, climate, and taxonomic

tools. The discovery of silicon transporter genes has aided in understanding the mechanism of silicon transport within the plant body and reconstruct

plant phylogeny based on the ability of plants to accumulate silica. However, a clear understanding of silica deposition and the formation of phytoliths is

still an enigma and the information on the proteins involved in plant

biosilicification is still scarce. With the observation of various shapes and morphologies of phytoliths, it is essential to understand which factors control

this mechanism. During the last two decades, much research has been done

in this regard and silicon research has expanded as an earth-life science superdiscipline. We integrated the recent knowledge and new concepts on

the evolution of biosilicification in plants; uptake, transport and deposition,

shape, size, and chemistry. We also consider the questions such as how phytoliths of fixed shape are biogenically produced and discuss their

implications in taxonomy, palaeoenvironment, and palaeoflora. Finally, the

applications of phytoliths in the fields of agricultural and biomedical nanotechnology are discussed.

P 64

Regioselective immobilization of an enzyme cascade on diatom

biosilica E. kumari*1, N. Kröger1 1Technische Universitaet Dresden (BCUBE), Dresden, Germany

Introduction

Diatom biosilica is a favorable support material for enzyme immobilization,

as its hierarchical mesoporous structure provides a large surface area for the attachment and allows the efficient reactant diffusion. The immobilization of

multi-enzyme systems is an emerging technology that enables the rapid

multi-step conversion of a substrate into a desired product. Previously it was shown that the activity of immobilized glucose oxidase is strongly influenced

by the species-specific diatom biosilica structures. However, it has remained

unknown, whether the activity of an enzyme cascade is influenced by its positioning within structurally different regions of the biosilica from the

same diatom species. Thalassiosira pseudonana biosilica, which served as a

49

model diatom for this project, is composed of two morphologically distinct building blocks: girdle bands and valves. Girdle bands biosilica rings which

a smooth surface and perforated by uniformly sized mesopores (20 nm

diameter). In contrast, valves have very rough surfaces with a hierarchical pore patterns.

Objective

The aim of the present work is to test the hypothesis that silica structure is a key parameter for the activity of an immobilized enzyme cascade.

Furthermore, we investigated whether the activity of the cascade is

influenced by the relative positioning of the two enzymes on the biosilica surface.

Materials and methods

We have used an in vivo method (LiDSI) to immobilize on biosilica the enzymes glucose oxidase and horseradish peroxidase. Together they

constitute a minimal enzyme cascade. LiDSi is based on introducing into the

diatom T. pseudonana synthetic genes that encode the desired enzymes fused to a fluorescent proteins and a regioselective silica targeting peptide. The

enzymes were localized on the biosilica surface of transformant diatoms

using confocal fluorescence microscopy. After biosilica isolation the catalytic activity of the immobilized cascade was determined through a

colorimetric assay.

Result

We generated four T. pseudonana strains each carrying the same enzyme

cascade, but with different locations of the two enzymes inside the biosilica.

The four enzyme cascade configurations exhibited striking differences in specific catalytic activities (i.e. catalytic activity per immobilized enzyme

molecule). Transformants with both enzymes immobilized in the valve exhibited the lowest specific activity, whereas the specific activity of

transformants containing both enzymes in the girdle band region was 3- to 6-

fold higher. Further investigations into the cause for the observed differences in the cascade activity ruled out influences of quantity, surface exposure and

stability of the enzymes.

Conclusion

Our results confirm the remarkable, yet little understood, influence of

biosilica architecture on enzyme activity.

P 65

In vivo incorporation of iron oxide into diatom biosilica S. Machill*1, J. Kaden1, S. I. Brückner1, L. Köhler1, R. Reichenbächer1, M.

Schumacher1, E. Brunner1 1TU Dresden, Chair of Bioanalytical Chemistry, Faculty of Chemistry and Food Chemistry, Dresden, Germany

Introduction

Diatom biosilica is a species-specific, micro- and nanostructured material

which consists of silicon dioxide and tightly attached organic compounds.

Diatoms can accumulate "foreign" elements like germanium, aluminum or

gold and incorporate or attach them to the silica. These in vivo synthesized

nanocomposite materials can be used as catalysts, adsorbents or biosensors. The role of iron in the biosilification of diatoms is not fully

understood. Iron is an essential nutrient in the metabolism of diatoms and plays a central role for photosynthesis. Genomic analyses indicate a close

connection between the metabolism of silicon and iron in diatoms. Therefore,

the role of iron in biosilification should be further elucidated.

Objectives

The aim of this work is to examine weather or not iron can be accumulated

by diatoms and to determine the chemical state it is associated with the siliceous diatom cell walls. In order to quantify the maximum amount of

incorporated iron, we offered excessive iron concentrations up to the

tolerance limit in the culture medium during diatom growth. The morphology and the structure of the obtained materials were characterized by microscopic

and spectroscopic techniques to answer the question if iron is dispersed in

the biosilica or preferentially clustered. As a defined reference material, silica precipitates with different amounts of iron were also studied.

Materials & methods

The diatom species Stephanopyxis turris and Thalassiosira pseudonana were

cultured in an artificial seawater medium with increased iron concentrations.

To remove the physically bound organic matter and ions, the cell walls were

extracted by a lysis buffer. The obtained biosilica as well as samples of the reference material were analyzed by ICP-OES in order to determine total

Fe:Si ratios. For morphological studies, scanning electron microscopy

(SEM) was used. The chemical state of iron was characterized by Fourier transform infrared (FTIR), Raman, 29Si magic angle spinning (MAS) nuclear

magnetic resonance (NMR), as well as electron paramagnetic resonance

(EPR) spectroscopy.

Results

The amount of iron incorporated in the biosilica of Stephanopyxis turris does

not linearly increase with the amount of iron offered in the growth medium. It is concluded that iron deposition in biosilica is obviously regulated and

limited. The spectroscopic investigations revealed that iron within the silica

framework of the synthetic silicagels is preferentially dispersed leading to Si-O-Fe bond formation. In contrast, biosilica-associated iron in Stephanopyxis

turris exists almost exclusively as Fe2O3 clusters/nanoparticles which are tightly associated to the cell wall. T1 measurements in 29Si MAS NMR

experiments revealed that only a minor amount (below 5 %) is dispersed over

the biosilica. That means that iron does not replace silicon atoms at tetrahedral sites as it is known for aluminum incorporation.

Conclusion Iron is not mainly coprecipitated in dispersed form within the biosilica during cell wall synthesis of Stephanopyxis turris. It rather exists as Fe2O3 clusters.

The obtained material is thus not suitable for technical applications due to

the low amount of iron incorporation. A suitable material may therefore be an iron-silica-nanocomposite.

P 66

New data on the structure of the giant basal spicule of

monorhaphis sponge A. Pisera*1, M. Łukowiak1, K. Tabachnick2 1Institute of Paleobiology, PAS., Warszawa, Poland 2P.P. Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russian Federation

The skeletal elements of sponges are called spicules and are made of silica or calcium carbonate. They are usually small (micrometers to millimetres)

and situated inside the sponge body. But the basal (siliceous) spicule of deep

water hexactinellid sponge Monorhaphis can reach almost 1 cm in diameter and over 2 meters of length being probably the largest single biosilica-based

structure on Earth. The spicule protrude from the sponge body and anchors

it in the sediment. This giant spicule became a model of hexactinellid spicules structure and

formation. According to previous studies (Wang et al. 2011 with references)

the majority of the spicule is composed of regular smooth siliceous laminae, but two most external layers differ in morphology and structure. The most

external layer called "banded ribbon layer" was first described as made of a

solid fibrous collagen but later its siliceous composition has been proven. The sculpture of the second layer, occurring directly below, was described as

depressions with elevated rims that housed sclerocytes.

The aim of the study was to better understand the structure of this spicule, and to interpret the observed features.

We have examined several spicules of Monorhaphis along the whole length

under a light microscope. Then the fragments which were different in appearance were studied under SEM. Mineral composition of the studied

samples was determined with EDAX. Our studies have shown that the two most external layers are not occurring

along the whole spicule length. All three structurally different units

superimposed occur only in the lower part of the spicules, the uppermost part display only central core of smooth solid silica. We have confirmed that the

banded ribbon layer is siliceous, not organic, but, composed of granular silica

and porous, in opposition to a solid glassy structure of all more internal lamellae. The structure of the penultimate layer is exactly the same as the

core of the spicule. i.e. glassy and without visible porosity. We also

demonstrated that this layer is covered with regularly developed and distributed elevations, not with depressions as previously thought. For this

reason they cannot house sclerocytes; additionally in late ontogenetic stages

in hexactinellids occurs only sclerosyncytium. The elevations in penultimate layer are developed by progressively stronger folding of the surface of

superimposed thinner layers. They fit tightly into depressions of the banded

ribbon layer, developed its lower surface. It is not clear why and how the banded ribbon and tuberculated layers

of Monorhaphis basal spicule have developed, but we speculate that the

banded ribbon surface may be an adaptation to help the sponge to stay fixed on the spicule, not to glide down on very smooth silica surface. The

elevations of the penultimate layer that fit into depressions of the lower

surface of banded ribbon layer help to stabilize (fix together) the two most external layers.

References 1. X. H. Wang, M. Wiens, H. C. Schröder, K. P. Jochum, U. Schloßmacher, H. Götz, H. Duschner, W. E. G. Müller, J. Experimental Biol. 214 (2011)

2047-2056.

Financial support by the Polish National Science Center Grant 2016/21/B/ST10/02332 to A. Pisera.

50

P 67

The accumulation of aluminum in diatom frustules and

modifications of the resulting alumosilicate L. Köhler*1, S. Machill1, A. Werner2, C. Selzer2, S. Kaskel2, E. Brunner1 1Chair of Bioanalytical Chemistry, Faculty of Chemistry and Food

Chemistry, Technische Universität Dresden, Dresden, Germany 2Chair of Inorganic Chemistry I, Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, Dresden, Germany

Introduction

Diatoms stand out from other unicellular algae through their uniquely

patterned, siliceous cell walls. These so-called frustules contain mainly amorphous SiO2, but also foreign elements such as metalloid and metal

atoms. Aluminum is a particularly interesting constituent because it directly

replaces silicon in the silica framework, forming an alumosilicate.1,2 This aluminum insertion leads to a negative charge, which is probably

counterbalanced by cations like calcium.1

Objectives

In the present study, several ways of aluminum addition to the living diatom

cultures were evaluated. The structure model from literature1 was reviewed

with respect to the coordination of aluminum and the type of counter ions. Varying growth parameters were assessed to tailor materials properties of the

frustules, i.e., the molar ratio of aluminum to silicon. This characteristic

property affects possible future applications in sorption and catalysis. Besides the changes in vivo, frustules were further modified in vitro to

enhance porosity and acidic strength.

Materials & methods

The fast growing and resistant diatom species Thalassiosira pseudonana was

cultivated in aluminum-enriched media. After harvest, frustules were cleaned

with lysis buffer and calcined. This biosilica was characterized by various methods including infrared, nuclear magnetic resonance and optical emission

spectroscopy as well as nitrogen and ammonia sorption. To exchange the

counter ions created by aluminum incorporation, the biosilica material was stirred in an ammonia solution and subsequently heated. Furthermore,

frustules were etched with a mild base to increase the specific surface area.

Catalytic activity was investigated by an acid-catalyzed alkylation of aromatic compounds, which was monitored by gas chromatography coupled

with mass spectrometry.

Results

Diatom incorporated aluminum into their frustules, resulting in n(Al):n(Si)

ratios up to the scale of 1:10. With further in vitro modification steps, acidity was more than doubled to 320 µmol NH3 g

-1 and the specific surface slightly

increased to 70 m2g-1. Ion-exchanged biosilica was shown to be catalytically

active in contrast to non-modified biosilica.3

Conclusion

The current study shows that the siliceous cell walls of Thalassiosira

pseudonana can be aluminum enriched in vivo while preserving their structure. Through in vitro modification, the acidity and specific surface area

of the biosilica can be enhanced. As successful catalytic tests show, diatoms

are a possible source for "green" catalysts. (1) Gehlen, M.; Beck, L.; Calas, G.; Flank, A.-M.; Bennekom, A. J. Van;

Beusekom, J. E. E. Van. Geochim. Cosmochim. Acta 2002, 66 (9), 1601–

1609. (2) Machill, S.; Köhler, L.; Ueberlein, S.; Hedrich, R.; Kunaschk, M.;

Paasch, S.; Schulze, R.; Brunner, E. BioMetals 2013, 26 (1).

(3) Köhler, L.; Machill, S.; Werner, A.; Selzer, C.; Kaskel, S.; Brunner, E. Molecules 2017, 22 (12).

P 68

The role of aluminum in diatom biosilicification M. Soleimani*1, S. Maddala 1, A. Akiva1, L. Rotten1, I. Zlotnikov2, R.

A.T.M. van Benthem1, H. Friedrich1, N. A. J. M. Sommerdijk1 1Eindhoven University of Technology , chemical engineering and chemistry , Eindhoven, Netherlands 2 CUBE Center for Molecular Bioengineering, Dresden, Germany

Introduction Diatoms are unicellular photosynthetic algae which live in nearly every aquatic territory. Their unique morphological characteristic is the silica cell

wall (frustule), which is formed from two halves, like a petri dish, joined

together by girdle bands. Besides silicon, diatoms incorporate aluminum into their silica cell wall, impacting the solubility and dissolution rate of the

frustule. Due to the complexity of the structural arrangement of diatoms, the

chemical connectivity between aluminum and silicon inside the frustule has not yet been discovered. Here, we use various analytical techniques to get

insights into how aluminum plays a role in frustule formation.

Materials and methods Pinnularia sp (P.sp) and Thalassiosira pseudonana (T.p) were grown in

filtered, autoclaved seawater amended with nutrients according to Guillard"s

f/2-recipe. To synchronize cells at cytokinesis arrest, silicon starvation was applied with an incubation time of 48 h in Si free medium. Starvation was

terminated by adding Guillard"s (F/2) marine water enrichment solution and

varying amounts of aluminum to have Si and Al precursors simultaneously in the culture. After 12 days of cultivation, cells were harvested and washed

by centrifugation and kept at -20 for future characterizations. In order

to monitor the frustules formation inside the silica deposition vesicle (SDV) with fluorescence microscopy, synchronized growth was

initiated by repleting the culture with a final concentration of 100 µM silicic

acid and 1 µM PDMPO as a staining agent.

Results Fluorescence microscopy images show valve formation starts from the

central nodule and proceeding toward the outer valve edges. Following one hour the full two-dimensional size of the new valve has been reached. After

6 hours the fluorescence intensity of the SDV reaches the maximum,

implying at this point the two cells are relatively separated and cell division is taking place. EDX measurements of P.sp and T.p unveil the presence of

aluminum in the frustules. SEM images of P.sp show that the pore diameters

seem to decrease by increasing the concentration of aluminum in the cultures. The Solid-state NMR spectra show that the sample contained both

four and six coordinated Al (III) as indicated by peaks at 7.99 and 55.03 ppm,

respectively. Six coordinated Al is the most predominant Al (III) species in our sample which could be from our precursor bonded Al to organic

compounds inside and outside of the cells or adsorbed Al (III) on the surface

of frustules. However, the peak at 55.03 ppm shows the presence of four-fold coordinated Al suggesting it is incorporated inside the silica networks of the

frustules.

Conclusion EDX measurements show the presence of aluminum in both species. SEM

images of P.sp show that the pore size decreased by increasing the AlCl3 in the growth medium. Cell division in P.sp as shown in fluorescence

microscopy images takes six hours. Solid State 27Al NMR spectra show four

and six coordinated aluminum in the structure of T.p. In the future, we aim to perform 29Si and 27Al solid-state NMR of the frustule to not only

evaluate the coordination environment of aluminum but also to pinpoint the

effect of aluminum on cross-linking of silica. In addition, some mechanical properties of intact cells and frustule will be measured to investigate the

effect of aluminum on the mechanical characteristic of diatoms.

P 69

Highly efficient encapsulation of biocatalyst in diatom-inspired

silica nanoparticleforenzymatic CO2 capture and utilization B. H. Jo*1

1Gyeongsang National University, Division of Life Science, Jinju, South Korea

This study reports on the development and characterization of a carbonic anhydrase (CA)-based biocatalyst encapsulated in a biosilica matrix for use

in CO2 capture an utilization. Encapsulation occurred simultaneously with

autonomous silica synthesis by diatom-derived silica-condensing R5 peptide, which was genetically fused to recombinant CA. The encapsulation

efficiency was greater than 95%, and the encapsulated CA was not leached

from the silica matrix, demonstrating the highly efficient R5-mediated auto-encapsulation process. The catalytic efficiency for CO2 hydration was pH

dependent, suggesting that proton transfer from silica to water is a rate

limiting step. In addition to good reusability, the encapsulated CA exhibited outstanding thermostability, retaining 80% activity after 5 days at 50˚C. The

thermoactivity was also remarkable, showing ~10-fold higher activity at

60˚C compared to that at 25˚C. The physical structure was observed to be highly compact with a low surface area, stressing the importance of the

outermost surface for catalytic performance. We also demonstrated the

applicability of the silica nanoparticle to the sequestration of CO2 in carbonate minerals. The rate of CaCO3 precipitation was remarkably

accelerated by the encapsulated biocatalyst. Thus, this silica-CA

nanocomposite, efficiently synthesized via a biomimetic green route, can be successfully used as a robust biocatalyst for biomimetic sequestration of the

greenhouse gas CO2.

P 70

Design of biosilica-enveloped R5 ferritin cage for dual drug

delivery system development M. R. Ki1, M. A. A. Mohamed1, K. B. Yeo1, S. H. Kim1, K. H. Min1, S. P.

Pack*1 1Korea University, Biotechnology and Bioinformatics, Sejong, South Korea

Silica has unique properties such as good biocompatibility, excellent pH and thermal stability, as well as high porosity, low toxicity, and superior

mechanical stability. Consequently, silica-based nanomaterials have gained

considerable interest for their potential biotechnological applications such as in drug delivery, enzyme immobilization, and biocatalysis. However, the

classic approaches to silica synthesis require harsh reaction conditions such

as extreme pH, high temperature, and toxic chemicals. One approach to alleviating these limitations is to mimic the silica mineralization seen in

biological systems. In one of well-known examples, silaffin polypeptides

derived from diatom biosilica are a class of massively post-translationally

51

modified (PTM) proteins that are responsible for initiating silica formation at ambient temperature and pressure. Interstingly, an unmodified silaffin R5

peptide is capable of mediating silica precipitation in vitro under specific

conditions. Using recombinant DNA technology, R5 allows for in situ formation of biosilica matrices containing the R5-fused recomabinant

proteins. Here, we designed R5 peptide-fused ferritin (R5FT) for the

developement of an advanced dual drug delivery system (dDDS). Since the fused R5 can mediate biosilica deposition on the ferritin surface

(SiO2/R5FT), we could load two types of molecules into the core inside

(ferritin cage) and the shell outside (biosilica matrix), respectively. One model (D#1) was loaded into the cage by the reassembly of R5FT to obtain

R5FT(D#1), and then the other model drug (D#2) was captured in situ by

biosilica matrix formation to prepare SiO2(D#2)/R5FT(D#1). The captured D#2 in the shell exhibited a short-term release, while D#1 in the

core showed a long-term sustained release. This dDDS system with the

additional release of D#2 decreased the IC50 value of D#1 by two-fold compared to the use of only D#1 in dDDS. We further optimized the size and

performance of biosilica-enveloped ferritin cage in terms of drug delivery

system. The developed dDDS using biosilica-enveloped ferritin cage can provide more efficient combinational drug therapies.

P 71

Biological silica formation in diatoms - mineralization outside

the box? B. Mayzel*1, S. Wolf2, A. Gal1 1Weizmann Institute of Science, Department of Plant & Environmental

Sciences, Rehovot, Israel 2Weizmann Institute of Science, Department of Chemical Research Support, Rehovot, Israel

Introduction

Diatoms are unicellular algae, abundant in all aquatic environments. The

hallmark of diatoms is their mineralized cell wall, made of amorphous silica. The morphology of the cell wall is species-specific, forming distinct 3D

micro- and nano-metric architectural features. The prevailing paradigm to

explain the biological control over the formation of the inorganic phase is that each silica element is formed intracellularly inside a silica deposition

vesicle (SDV), and once completed it is exocytosed. The complex cellular

processes within the SDVs are thought to regulate the mineralization process. Chaetoceros tenuissimus is a diatom species with a cell size of 5µm in

diameter, which is characterized by four long extensions, called the setae, radiating from the main cell body. The setae are 15-30µm long and 250-

300nm in diameter and are covered by a silica shell. Seta growth commences

after cell division and continues at the seta tip until its full length is reached. Therefore, the growing tip of the C. tenuissimus seta is a unique system to

study the cellular process of silica formation.

Objective

Identifying the cellular organelles and structural elements responsible for

silica mineralization of C. tenuissimus setae.

Methods

C. tenuissimus cells were grown in culture and their cell cycle synchronized

to increase the abundance of growth-stage seta in cells. CryoTEM

tomography was used to study the 3D structure of C. tenuissimus setae at various growth stages. Samples were plunge-frozen in liquid ethane to

preserve the native structure of the setae. Tomograms of seta tips were

acquired using the 200kV Tecnai F20 and the 300kV Titan Krios G3i microscopes. IMOD software was used to reconstruct the tomograms from

the data.

Results

24 tomograms of setae were generated, 11 are assigned to growth-stage setae.

These tomograms clearly show the complex helical architecture of the thin

silica fibers enveloping the seta. A thin extra-cellular polysaccharide layer covering the mineralized silica cell wall was observed. The cytoplasmic

extension within the growing setae contained a single microtubule filament

inside the cell membrane. In some cases, small vesicles were noted in the cytoplasm. Importantly, none of the tomograms showed an SDV or other

associated cellular structures at the tip of the setae.

Conclusions

These results suggest a silicification mechanism in diatoms that is not SDV

(silica deposition vesicle) dependent, but rather a continuous process of

extracellular silicification. This mechanism may explain other silica elements in diatoms and solve fundamental unknowns about the biological process of

silicification.

P 72

Interactions between gold nanoparticles and the diatom

Stephanopyxis turris N. Pytlik*1, S. Machill1, B. Klemmed2, A. Eychmüller2, E. Brunner1 1Technische Universität Dresden, Bioanalytical Chemistry, Dresden,

Germany 2Technische Universität Dresden, Physical Chemistry, Dresden, Germany

Introduction

Diatoms are unicellular algae, which contribute very significantly to the

global carbon fixation by photosynthesis. They are especially famous for

their cell wall made from amorphous biosilica which presents a spectacular example for the beauty of biomineralization. The cell walls are highly

structured and exhibit regular pore patterns at micro- and nanoscale. Each

diatom species is characterized by a unique cell shape and pore pattern. However, the understanding of the complex process of three-dimensional cell

wall formation is still limited.1

Objectives

Diatoms, furthermore, find increasing research interest in the field of

catalysis, solar cells or as templates. For several potential applications, the

cells are combined with gold nanoparticles either in vivo or in vitro. However, only few information is available about the interactions and effects

of gold nanoparticles on the diatom cells.

Materials & methods

We have recently exploited the potential of the gold nanoparticles to generate

a SERS (surface enhanced Raman spectroscopy) effect for an in vivo

monitoring. Using a 3D setup, it was shown that gold nanoparticles, which were biosynthesized by the diatom Stephanopyxis turris, can even occur

inside the diatom cells.2 Consequently, these intracellular gold nanoparticles

must either a) be taken up by the cell after extracellular gold ion reduction or b) be produced inside the cell after the uptake of gold ions.2 To answer the

question whether or not gold nanoparticles can generally be taken up by

diatom cells, chemically synthesized gold nanoparticles were added to living diatom cells and their interactions were observed by 3D SERS.

Results

Gold nanoparticles with diameters ≥ 50 nm could clearly be localized inside the cells, whereas smaller gold nanoparticles were never detected in the cell

interior. This indicates a size-dependent uptake mechanism that comes along

with different toxicities.3

Conclusion

The consequences of gold nanoparticle contact with diatom cells have not yet been fully understood and long-term effects for the organism itself as well

as for the environment are hardly predictable. Having in mind the raising

gold nanoparticle implementation in industry, our findings point out the responsibility to intensify research concerning nano-bio interactions –

especially focusing on diatoms, as an essential biomineralizing organism for

our ecosystem. 1 N. Pytlik and E. Brunner. MRS Commun. 8, 2018, 322-331. 2 N. Pytlik et al. Algal Res. 28, 2017, 9-15. 3 N. Pytlik et al. Algal Res. 39, 2019, 101447.

P 73

Macrobiomineralogy- principles of biomineralization in giant

whale bones and perspectives for bioinspired materials science M. Wysokowski*1 1Poznan University of Technology, Faculty of Chemical Technology, Poznan, Poland

Introduction Nowadays, biomineralization is defined as ancient fundamental biological

process with high dynamics by which living organisms produce minerals with multifunctional properties including to harden existing subcellular

organic matrices and tissues, to produce protective armors and shells against

external damages of diverse origins (predators, UV irradiation, toxic metals, etc.), as well as to carry out magnetic navigation. Diverse biominerals and

biomineral-based skeletal structures are occurring in nature on nano-(virus

and bacteria), micro- (diatoms, coccolothophores) and macro- (up to 7-meter-long whale bones) levels. The mechanisms of biomineralization

remain hotly debated. The best way to understand the basic principles of

biomineralization on both molecular and macro-scale levels is by a coherent synergetic collaboration using explicit reasoning and well-tested explanatory

principles of multidisciplinary experience, knowledge and new technologies.

Giant bones of whales (Cetacea) represent the largest biomineral-based constructs known. The existence of such macrobiominerals with still

unknown mechanisms of their origin and development is an example of a

ground-breaking phenomenon unexplored in nature. In this study, the concept of macrobiomineralization is proposed for the first time.

Objectives The main objective is to discover the fundamental mechanisms of biomineralization in selected giant whale bones. This includes the analysis

of the structure-function relationship between organic (collagen, lipids) and

52

inorganic mineral phases in selected lipid-rich whale bones (mandibles, ribs and vertebrae).

Materials & methods Selected whale bones from registered museum collections have been gentle demineralized using EDTA-based solution at pH 7.2 at 37°C to extract

corresponding organic phases and to carry out their identification using

modern bioanalytical tools including GC, HPLC, ESI-MS, FTIR, Raman, CARS, 13C solid state NMR, XPS, XRD, HR-TEM and NEXAFS. Modern

3D stereo microscopy together with fluorescence microscopy and BET

measurements are used for additional characterization of porosity, inner structural organization and special surface area of the biomaterials studied.

Results Biomineralizers like whales, which are able to produce hydroxyapatite (HAP)-based skeletal structures in lipid-rich (hydrophobic) environment, are

of crucial scientific interest. For the first time we show that millimeter-sized

fibers of collagen remain to play significant role in formation of highly carboxylated HAP, especially in vertebrae. Strong difference with respect to

organic phases has been shown between the whale vertebrae and ribs using

NEXAFS spectroscopy. Phospholipids in ribs fraction have been found tightly bound to collagen fibres. It is suggested that in contrast to human

bones,calcium oleate can be the precursor of HAP in the lipid-rich bones of

whales.

Conclusion The fact that giant whale bones represent examples of large-scale

biocomposites which has been synthesized in situ at 36.6 °C is very motivating to resolve the outstanding questions. Consequently, based on

obtained results the objective with regard to "large-scale biomimetics" includes the goal to develop the key way for industrial production and design

at ambient conditions of "3D Ca-phosphate-lipid-collagen composites" using

naturally occurring sources of each component.

P 74

The unstructured proteins in biological structures- the case of

human teeth from a protein chemist's perspective V. Sharma*1, A. Srinivasan2, A. Roychoudhary3, F. Nikolajeff4, S. Kumar1 1All India Institute of Medical Sciences, Biophysics, New Delhi, India 2Jamia Hamdard University, Biochemistry, New Delhi, India 3All India Institute of Medical Sciences, Oral and Maxillofacial Surgery, New Delhi, India 4Uppsala University, Engineering Sciences, Uppsala, Sweden

Introduction

Proteins are the biomacromolecules that work downstream of genes in every

living system. It is these protein molecules that are responsible for a plethora of functions in our body, ranging from building skeleton tissues to

transmitting signal from one place to others. There was a period when

biologists have the view that for a protein to function, the structure is of supreme importance. Nevertheless, this structure-function paradigm is fairly

obeyed by most of the proteins, if not all of them. Last two decades have

witnessed the increased reports of proteins that do not have a stable three-dimensional structure but are very much indispensable for the cellular and

biological activities. These unstructured proteins are now known as

"Intrinsically disordered proteins" (IDPs). Interestingly, these IDPs function not only in isolation (e.g. transcription factors) but helps in the formation of

hard tissues like teeth, bone, and mollusk shell, by governing a process called

biomineralization. It is the process by which living organisms produce minerals, often to harden or stiffen existing tissues. Such tissues are called

mineralized tissues. The process of biomineralization is still considered as a

scientific puzzle.

Aim

To investigate the structural differences of protein extracts from four types

of human teeth (i.e. Molar, Premolar, Canine, and Incisor) and their implication in regulating the calcium phosphate mineralization in vitro.

Methods

1) Isolation and purification of proteins from different types of human teeth. 2) Characterization of tooth proteins by (a) Fourier transform Infrared

Spectroscopy(FTIR); (b) Circular Dichroism (CD)

3) In vitro calcium phosphate biomineralization assay followed by size measurement experiments by Nanoparticle tracking analysis and analysis by

scanning/transmission electron microscopy.

Results and Discussion

Protein extract was successfully purified from different types of human teeth

(i.e. Molar, Premolar, Incisor, and Canine) which were quantified and run on an SDS PAGE and subsequently stained by silver staining. FTIR was done

to understand the differences present among the different teeth extract and

there secondary structure characteristics. CD was done to validate the protein secondary structure details obtained by FTIR. We have also investigated the

effects of these protein extracts on mineralization of calcium phosphate

through in vitro biomineralization assay followed by size-based measurement analysis via Nanoparticle tracking analysis (NTA).

Conclusions

1) Secondary structural characteristics of different protein extracts were

studied using various biophysical techniques. The presence of IDPs in the

majority was confirmed within these protein extracts. 2) We have also investigated the effects of these protein extracts on

mineralization of calcium phosphate through in vitro biomineralization assay

followed by size-based measurement analysis via Nanoparticle tracking analysis (NTA).

This is the first time the effect of the whole protein extract was taken into

account which in our view more closely imitates the in vivo process of tooth biomineralization.

P 75

Brachiopod shells can be folded when wet F. Nudelman*1, J. Ihli2, A. Schenk3, M. Holler2, K. Wakonig2, M. Duer4, M.

Cusack5 1University of Edinburgh, School of Chemistry, Edinburgh, United

Kingdom 2Paul Scherrer institut, Villigen, Switzerland 3University of Bayreuth, Faculty of Biology, Chemistry and Earth Sciences,

Bayreuth, Germany 4University of Cambridge, Chemistry, Cambridge, United Kingdom 5University of Stirling, Biological and Environmental Sciences, Stirling, United Kingdom

Question

For hundreds of millions of years, nature has evolved a large assortment of

organic-inorganic hybrid materials that are optimally adapted for a wide range of functions, including navigation, protection, mechanical support and

protection. These materials not only exhibit exceptional material properties

but also display multifunctionality, including features such as adapting, sensing and self-healing. Among the most remarkable biominerals found in

nature are the shells of the phosphatic brachiopod Discinisca tenuis. We

empirically observed, for the first time, that the mechanical properties of these shells vary according to their water content. While they are hard and

stiff when dry, they become flexible when hydrated, to the point that the shell

can be folded in 2 without fracturing. Such capability of an organic-inorganic composite to switch reversibly between stiff and flexible and in real time,

adapting to changes in the environment that demand a different set of

mechanical properties, is truly unique among both biological and synthetic materials. The aim of this research was to characterise, from the molecular

to the micron-scale, how the structure the brachiopod shells changes as a

function of hydration, leading to changes in mechanical properties.

Methods

We used cryo-ptychographic X-ray computed tomography (PXCT) on dry,

partially hydrated (exposed to 70 % relative humidity) and fully hydrated

shells (exposed to 100 % relative humidity) to characterise how the structure

of the shell at the sub-micron and micron levels changed as a function of

hydration. Cryo-scanning electron microscopy (cryoSEM) measurements were performed on dry and wet shells to analyse changes at the shell nano-

structure as a function of hydration. Solid-state NMR (SSNMR) was used to determine the effect of hydration on the molecular conformation of the

organic components.

Results

PXCT measurements demonstrated the organic-rich layers in the shell

expanded in thickness, increasing from ~160 nm in the dry sample to ~180

nm in the partially hydrated and ~340 nm in the fully hydrated sample. CryoSEM suggests that the organic components of the shell swell upon

hydration. Using SSNMR, we identified that the macromolecular chains of

proteins, and in particular the methyl groups in proximity to the mineral, become more mobile with hydration.

Conclusions Our results show that the changes in the mechanical properties of the shell as it absorbs water arise from modifications in the mobility of the organic

components and structural changes, at the sub-micron and micrometer

levels, caused by the swelling of the organic layers.

P 76

Ion substitutions in apatites of human urinary stones A. Korneev*1, A. Izatulina1, O. Frank-Kamanetskaya1

1Saint Petersburg State University, Department of Crystallography, Saint

Petesburg, Russian Federation

Hydroxyapatite is the main mineral component of phosphate stones in human urinary system according to the powder XRD data: it also occurs as an

impurity in oxalate and urate stones. According to our collection of renal stones of St. Petersburg and the Leningrad region residents, apatite is present

in 64,7% stones. Using SEM and EDX it was revealed that individual apatite

grains are present almost in all oxalate urinary stones, which confirms the hypothesis of initializing calcium oxalates crystallization by hydroxyapatite.

Due to the low degree of crystallinity of bioapatites the conclusion of their

composition can be made according to the detailed EDX analysis and values

53

of lattice parameters (Frank-Kamenetskaya et al. 2011). According to our data values of the a unit cell parameter of urinary stones apatites vary from

9.395(2) to 9.457(4) Å, i.e. can be higher or lower than that of stoichiometric

hydroxyapatite (a = 9.418, JCPDS № 9-432). Values of the c unit cell parameter (6.849– 6.885(4) Å) can be significantly lower than in

stoichiometric hydroxyapatite (c = 6.884 Å, JCPDS № 9-432). The increase

of a parameter of urinary stone apatites, are associated with substitutions of OH- ions by H2O molecules in channels of crystal structure, the decrease - by

F- ions. EDX data show that fluorine is present in trace amounts in apatite of

phosphate kidney stones, and in apatite of oxalate stones its concentration significantly higher (1.6 – 4.2 wt.%). Reduction of c parameter relative to

stoichiometric hydroxyapatite is due to the large portion of vacancies in Ca

sites. Their appearance can be associated with substitutions of OH- ions by H2O molecules and PO4

3- ions by CO32- and HPO4

2- ions.

The range of urinary stones apatite lattice parameters variations is

significantly higher than in other pathogenic apatites that are formed in human organism, which indicates the essential variation of their forming

conditions.

This work was supported by the Russian Science Foundation (no. 18-77-00026). The XRD studies have been performed at the X-ray Diffraction

Centre of St. Petersburg State University. Scanning electron microscopy and

EDX analysis have been performed at «Center for Geo-Environmental Research and Modeling (GEOMODEL)» of St. Petersburg State University.

References

Frank-Kamenetskaya O., Kol'tsov A., Kuz'mina M., Zorina M., Poritskaya L. Ion substitutions and non-stoichiometry of carbonated apatite-(CaOH)

synthesised by precipitation and hydrothermal methods // Journal of Molecular Structure. 2011. Vol. 992. P. 9-18.

P 77

TEM study of compositional and structural changes of bone

crystals by heating M. Yosikawa*1, M. Kakei2 1Meikai University School of Health Sciences, Department of Oral Health

Sciences, Urayasu, Japan 2Medical English Institute[NPO], Saitama, Japan

Introduction

Octacalcium phosphate (OCP) is considered to be a candidate for the central dark line (CDL) which is observed in tooth enamel, dentin and bone crystals.

On the other hands, it has been reported that the formation of huntite minerals occurred prior to the nucleation of apatite crystal (Casciani et. al., 1979).

Regarding the physical property of OCP, it has been reported that OCP was

decomposed completely up to 150 ℃ (Bigi et. al., 1990), while the thermal decomposition of huntite minerals has been reported to begin from around

550 ℃ (Földvárl, M. 2011). From the ultrastructural viewpoint, transmission

electron microscopic (TEM) study has shown that after heating at about 600 ℃, a small number of crystals still preserved CDLs in their structures as

reported previously (Kakei et. al., 2005). This finding suggested that CDL

known as the nucleation site of biological apatite crystals might consist from huntite minerals and a subsequent induced initial lattice line of apatite.

Furthermore, the lattice line of CDL did not create two lattice lines of apatite,

meaning that CDL is not identical to OCP. Based on the previous findings, the present study was aimed to further clarify the involvement of huntite

minerals to the CDL formation. Also, we conducted to examine the structural

change of crystals by heating at over 700 ℃.

Objective

The present study is conducted to distinguish between CDL in apatite crystal

and OCP, and examined the structural changes of apatite crystals by heating.

Material and Methods

To clarify the composition of CDL, the results of both thermal analysis of

huntite minerals and TEM observation of bone crystals treated by heating at different temperatures were compared. After removing the surrounding soft

tissues, rat calvaria were treated with a plasma reactor to remove organic

substances. Thermogravimetry/Differential thermal analysis (TG/DTA) analysis of huntite minerals was conducted in air atmosphere with heating at

20 °C/min. For TEM study, samples were heated at 600, 700 and 1,000 ℃,

respectively, for one hour in the muffle furnace.

Results

Fig.1 showed the TG and DTG derivative curves of huntite minerals. The

decomposition of huntite minerals began from around 500 ℃ (Fig. 1). TEM study of the control of calvaria showed numerous tiny crystals showing the

presence of CDL (inset) (Fig. 2). After heating at 600 ℃, each crystal seemed

to be obscure and some crystals amalgamated with each other, though some crystals still preserved CDL (Fig. 3). After heating at 700 and 1,000 ℃,

respectively, TEM observations revealed that large crystals were formed

(Figs 4a and b). CDLs disappeared completely.

Conclusion

The preservation of CDLs by heating at around 600 ℃ clearly reveals that

CDL is not identical to octacalcium phosphate, meaning that two different mechanisms for apatite formation exist. Creating larger crystals by heating

at above 700 ℃ may contribute to the increase of crystallinity.

P 78

Correlation of bone morphology and quality with melatonin

intake in pregnant and lactating rats N. Ishikawa*1, H. Mishima2, A. Hattori3, Y. Maruyama3, N. Suzuki4, Y.

Matsumoto5

1Kagawa University, graduate school of Agriculture, Division of applied

biological and rare sugar sciences, MIki, Kita gun, Kagawa prefecture,

Japan 2Tsurumi University School of Dental Medicine, Dental Engineering,

Yokohama, Japan 3Tokyo Medical and Dental University, Biology, Ichikawa, Japan 4Kanazawa University, Nature and Environmental technology, Housu-gun,

Japan 5Kagawa University faculty of agriculture, Life Science Course, MIki, Japan

Introduction

Melatonin (MEL) is a hormone that plays a role in the circadian rhythm.

MEL increases bone density and inhibits the differentiation of osteoclasts

(Koyama, 2002; Hattori, 2017). In 6-day-old rats, MEL participates in the formation of incremental lines and calcification of dentin (Mishima et al.,

2018). However, the effects of MEL intake during osteogenesis on bone

formation in rats have not been clarified. Therefore, we analyzed the bone morphology and quality based on MEL intake in lactating rats.

Objectives

The present study evaluated the effects of MEL intake in lactating rats on the bone morphology and quality during lactation.

Materials and methods

Six SD pregnant rats were divided into 3 groups: (1) control group (Con; 0.5% alcohol-containing water), (2) low-concentration group (Low; 0.5%

alcohol + 20 µg/mL MEL-containing water), and (3) high-concentration

group (High; 0.5% alcohol + 100 µg/mL MEL-containing water). MEL was administered for 9 days, the last 3 days of pregnancy and the 6 days after

birth. Slaughter was carried out at midday and midnight on 6-day-old. The

concentrations of plasma melatonin metabolites (serotonin: 5HT, N-acetyl serotonin: NAS, melatonin: MEL, and 6-hydroxymelatonin: HaMT) were

measured by LC-MS/MS. The trabecular area ratio was observed and analyzed using a fluorescence microscopy. The calcification, crystallinity

and collagen maturity of bone were analyzed by FTIR. The bone composition

was analyzed by SEM-EDS.

Results

5HT was significantly higher in the MEL-treated group at midday and

midnight (p < 0.05). MEL was significantly higher in the High group than that of the Con group at midnight (p < 0.05). HaMT value increased in the

proportion with the MEL dose. The trabecular area ratio was significantly

higher in the Low group than that of the Con group at midday (p < 0.05). Calcification was significantly higher in the High group than that of the Con

group at midday (p < 0.05). The crystallinity and collagen maturity were

significantly lower in the MEL-treated group at midnight (p < 0.05). The mass ratios of Ca and P were significantly higher in the MEL-treated group

at midday (p < 0.05). At midnight, the mass ratio of P was significantly lower

in the High group than that of the Con group (p < 0.05).

Conclusion

In blood analysis, 5HT is secreted in the breast milk during lactation, thus we

think that 5HT was transferred from breast milk to rat pups and NAS was confirmed in rat pups plasma. Further, we think that MEL metabolism was

activated by MEL intake and 5HT in breast milk increased. The value of

MEL and HaMT in plasma suggested the transfer of MEL to rat pups through breast milk. In the MEL-treated group, the trabecular area increased, and

calcification was promoted, however bone was immature. Thus, bone

maturation probably did not catch up with bone formation in the MEL-treated group. The SEM-EDS analysis suggests that MEL may have promoted bone

formation and affected bone composition. The present results suggest that

MEL intake in the pregnant rats was transferred to rat pups through breast milk to promote bone formation and calcification. From Mishima's (2018)

finding that MEL changes the structure of dentin apatite crystals and collagen

fibers, the results of this study suggest that the mechanism of MEL effects on the bone calcification may differ from the dentin calcification.

54

P 79

Crystallographic and chemical vital effects in Dendrophyllia

skeleton - a by-product of the biocrystallization I. Coronado*1, A. Pérez-Huerta2, J. A. Cruz3, J. O. Cáceres4, S. Moncayo5,

V. Motto-Ros5, F. Trichard5, G. Panczer5, F. Pelascini6, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2The University of Alabama, Department of Geological Sciences,

Tuscaloosa, United States 3Complutense University, GEODESPAL, Madrid, Spain 4Complutense University, Departamento de Química Analítica, Madrid,

Spain 5Institut Lumière Matière, UMR5306 – UCBL- CNRS, Lyon University, Villeurbanne, Germany 6CRITT Matériaux Alsace, Schiltigheim, France

Coral skeletons are natural archives of the climate variability and a potential

proxy for paleoenvironmental reconstructions. Paleoclimate reconstructions

are based on changes in isotopic ratios and trace element uptake by bio-aragonite skeletons, as a response to environmental changes such as

temperature and ocean circulation . Unfortunately, corals are not longer

considered as passive recorders of environmental changes because the geochemical composition of their skeletons does not precipitate in

thermodynamic equilibrium with seawater. This desequilibrium has been

attributed to vital effects. Two classes of vital effects have been recognized: those derived from

physiological processes and those related to biocrystallization, from crystal

nucleation to stabilization of the mineral phases. Those vital effects linked to biocrystallization have not been studied in-depth in corals.Their

interpretation requires the incorporation of groundbreaking knowledge from

recent discoveries in coral biomineralization i.e., identification of amorphous precursor phases that fulfills a new paradigm of crystallization by particle

attachment (CPA) and biocomposite nature of coral skeleton. Therefore, in

order to retrieve a "purely" environmental signalfrom coral skeletons, chemical variations linked to precursor phases and the organic templates

involved in bio-aragonite crystallization have to be understood.

The purpose of this study is to identify and characterize the vital effect derived from biocrystallization processes. We performed a detailed study of

a cold-water scleractinian coral species Dendrophyllia ramea skeletons,

analyzing the micro- and nanostructures (using SEM and AFM), the crystallographic arrangement and lattice parameters (using EBSD and XRD),

and the biogeochemical composition (using FTIR of isolated organic matrices, Raman mapping, TG-analysis and a multi-elemental imaging using

LIBS).

The Dendrophyllia skeleton has a hierarchical mesocrystalline organization formed by aragonite fibers in the Thickened Deposits (TD) and

microgranular aggregates of amorphous nature in the Rapid Accretion

Deposits (RAD). We recognize several structural and biogeochemical heterogeneities in the different skeletal parts derived from biocrystallization

processes: a variable amount and composition of organic matrix and water in

septa and coenosteum, which may cause marked distortions in the orthorhombic constant ratios. Also, the edge layers of coenosteum (in

contrast to those formed earlier) are poorly crystallized, and show

enrichments of Mg, Sr and H. Acknowledgments: This work was supported by the National Science Centre

(Poland) grant 2017/25/B/ST10/02221.

P 80

The role of lacuna and canalicular network morphology in

osteocyte mechanosensation A. H. van Heteren*1, Y. Nakajima2, T. Näreoja3 1Ludwig-Maximilians-Universität München, Department Biologie II, München, Germany 2Tokyo City University, Department of Natural Science, Tokyo, Japan 3Karolinska Institutet, Department of Laboratory Science, Huddinge, Sweden

Introduction

There are differences in bone structure between anatomical sites and species

and these are reflected in canalicular networks and the lacunar shape of

osteocytes. For example, bone microanatomy is different in terrestrial and aquatic vertebrates. In bone, osteocytes are intertwined between type-1

collagen lamella and hydroxyapatite crystals that reform countless times as

the bone adapts to loading applied on it. Differently shaped or oriented osteocyte lacunae likely have a different volumetric deformation under a

specific load, which will change the load-induced fluid flow that osteocytes

feel (e.g., round osteocytes are more mechanosensitive than flat ones). The precise influence of the canalicular network has not yet been assessed either.

Objectives

Our primary goal is to establish how lacunar and osteocyte cell shape impact mechanosensation, using long bones of extant vertebrates, as well as

palaeontological specimens for a deep-time perspective. We aim to test the

mechanosensory response in 3D in vitro osteocyte cultures. Variations in osteocyte shape between bone regions with different loading modes raise the

question whether osteocyte shape represents an adaptive response.

Materials & methods

Osteocyte morphology will be modified by 3D bioprinting of polymers into

patterns that control lacunar shape. After we have differentiated the osteocyte cultures in these networks, we apply mechanical compression load

(nanoindentation) and fluid-flow shear stress to measure their ability to

produce a mechanosensory response. Lacunar shape will be digitized using high-resolution X-ray tomography in a Synchrotron. The shape of the

lacunae will be quantified in 3D using geometric morphometrics. The

canalicular network will be analysed in terms of connectivity, canalicular thickness, canalicular spacing, etc. Multivariate statistics will be used to

assess the correlation between osteocytic mechanosensation and morphology

in extant vertebrates. Extreme changes in mechanical loading at the evolutionary transition from land to water will be used to test whether

osteocyte shape is an adaptive response.

Results

We hypothesise that osteocyte morphology is predictive of mechanosensory

ability of the bone. We expect to find that osteocyte markers (e.g., sclerostin,

RANKL, osteoprotegerin, and extracellular vesicles) will vary with lacunar morphology and canalicular network topology in extant vertebrates. We also

expect to find a correlation between the morphological parameters and the

marker variations, as well as differences between secondary aquatic vertebrates and their land inhabiting counterparts.

Conclusion

If our hypotheses prove to be correct, we will have established that osteocyte

shape is an adaptive response to mechanical loading. We also expect to

establish that the osteocyte and the canalicular network change across extreme changes in loading modes, such as during the land to water transition

of secondary aquatic vertebrates.This information could be used to interpret

the loading regimes of fossils.

P 81

First finding of mineralized primary layer in Lingula anatina

(Lamarck, 1801) Brachiopoda, Lingulida A. Madison*1, T. Kuzmina2 1Paleontological Institute, Moscow, Russian Federation 2Lomonosov Moscow State University, Moscow, Russian Federation

The shell secretion of recent brachiopods remains poorly known, especially

if compared with the studied in detail biomineralization processes in recent

mollusks. The lingulids are best known of all brachiopods in microstructural sense as they possess the unique shell composed of alternating

biomineralized and organic layers that remained structurally stable

throughout the Phanerozoic. Three parts are distinguished in the lingulide shell: a periostracum and primary and secondary layers. The mineralized part

of the linguloid shell consists of up to 10 nm in diameter nanogranules of

apatite, specifically the fluorapatite francolite, aggregated into up to several microns in size spheroids or variously long rods. Though the superfamily

Linguloidea appeared in the Early Cambrian, only two genera, Lingula

Bruguière, 1797 and Glottidia Dall, 1870, survived up to recently. Studies on the linguloid primary layer provided quite controversial data on its presence

and structure. Watabe and Pan (1984) described 40–50 µm thick primary

layer composed of aggregates of spherulites for Glottidia pyramidata (Stimpson, 1860). However, Iwata (1981) did not find any mineralized

primary layer in Lingula unguis (L.) (this species was considered as a

synonym of Lingula anatina Lamarck, 1801 by Emig (1982). Williams et al. (1994) reported a 40 µm thick organic primary layer in L. anatina composed

mainly of glycoaminoglycans (GAGs).

We studied ten specimens of L. anatina from Vietnam and the Philippines. The Vietnam specimens were preserved in a 4% formaldehyde solution in

filtered sea water and the Philippine specimens were dried without any

chemical treatment. All specimens were studied with SEM first with periostracum and then after bleaching with collagenase/proteinase mixture.

The shell substance was studied externally and along the fractures. In many

places on the shell outer surface after bleaching preserved a well-ordered sheets of about 0.5 µm high and 0.2 mm thick cylindroids directed

perpendicularly to the valve surface. Such dolioform crystals were previously

unknown for linguliform brachiopods. The sheets look as a thin light film in SEM images in contrast with the underlying compact lamina and cover the

whole shell surface including the protegulum and brephic shell. On the

brephic and adult shells, the cylindroids are neatly packed and their outer and inner ends are obtuse while on the protegulum they are more random and

fusiform and somewhat resemble the acicular primary layer of

rhynchonelliform brachiopods with carbonate shell. The external surface of the sheets of cylindroids bear reflections of the radial striation of

periostracum and thus this is the first shell layer underlying the periostracum,

i.e. the primary layer. As it preserved after the bleaching, it is mineralized. Thus three types of the primary layer are known for recent linguloids: 40–50

µm thick primary layer composed of aggregates of spherulites in G.

55

pyramidata (Watabe and Pan 1984), about 40 µm thick primary layer composed of GAGs in L. anatina from Japan (Williams et al. 1994) and about

1 µm thick layer of cylindroids in L. anatina from Vietnam and the

Philippines. One of the possible explanations for strongly differing primary layers in one species is that in fact they are different taxa but additional data

on the molecular phylogenetics of these brachiopods are needed in order to

resolves this problem.

P 82

Guided mineral growth on amelogenin scaffolds promoted by

amelotin B. Ganss1, A. Danesi1, A. Mansouri1, A. Phen1, J. Holcroft*1, J. Bonde1, K. Carneiro1 1University of Toronto, Faculty of Dentistry, Toronto, Canada

Introduction

Enamel, the outermost layer of the tooth, is composed of intertwined

hydroxyapatite crystals; it is the hardest, most highly mineralized tissue in the body and incapable of regeneration. Amelogenesis is the matrix-guided

process of enamel formation, but its detailed mechanism is not well

elucidated. The most abundant protein present in the developing enamel matrix is amelogenin (AMEL). AMEL acts as a scaffolding protein that

templates hydroxyapatite (HA) crystal organization. In vitro, recombinant

AMEL self-assembles into nanoribbons in the presence of calcium and phosphate ions. Amelotin (AMTN), a recently discovered enamel matrix

protein essential for proper enamel mineralization and has been shown to

promote mineral formation in vitro and in vivo. The functional relationship between AMTN and AMEL as the major enamel matrix protein has yet to be

studied. We hypothesize that AMTN can promote mineralization on AMEL

structures.

Objectives

This study aims to 1) determine the self-assembly behavior of AMTN and 2)

determine the effect of self-assembled AMTN and AMEL, alone and in combination, on calcium-phosphate mineral formation and growth.

Materials & Methods

Human recombinant AMEL and AMTN proteins were expressed in Escherichia coli. Proteins were self-assembled in a calcium phosphate

solution for up to 28 days before being co-incubated for 20 minutes. Self-

assembled AMEL and AMTN, as well as AMEL-AMTN were characterized by Atomic Force Microscopy (AFM) and Transmission Electron Microscopy

(TEM).

Results

AMTN self-assembled into ribbon-like nanostructures over a period of up to

3 weeks. . Mineral deposits formed on top of these ribbons 14 days after assembly. Co-incubation of self-assembled AMTN with AMEL resulted in

the formation of needle-like crystals along AMEL nanoribbons. No

mineralization was observed on AMEL alone.

Conclusion

The co-incubation of self-assembled AMTN to self-assembled AMEL

nanoribbons promotes the formation of guided mineral growth along the AMEL template. This combined activity provides new insights on

mechanistic details of enamel biomineralization. Understanding this

combined effect will open opportunities for developing biological and/or synthetic regeneration strategies for dental enamel.

P 83

Calcium phosphate granules in hypodermal cells participate in

the mineralization of crustacean mandible incisors A. Ziegler*1, B. Nutz1, U. Rupp1 1University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany

The incisive regions of the mandibles, the pars incisivae (PI), of the terrestrial

crustacean Porcellio scaber can be subdivided into three regions, according to differences in mineral content. The distal tip of the PI is not mineralized,

the middle region is mineralized with amorphous calcium phosphate (ACP)

and the base, that connects the incisive region with the corpus of the mandible, with calcium carbonate (Huber et al., 2014). The presence of both,

ACP in the middle region and CaCO3 in the adjacent base and corpus is an

ideal situation to study mineral phase specific differences in epithelial calcium transport pathways.

Which intracellular compartments are involved in calcium transport is

currently the most important question to understand the contribution of cells in biological mineralization processes. Knowing the pathway of how

intracellular calcium is directed through the cell is indispensable for

understanding, which molecular transport mechanisms are possibly involved in biomineralization. How mineralization with calcium phosphate instead of

calcium carbonate affects the structural differentiation of the mandible

epithelial cells and transport of mineral through it, is completely unknown. Therefore, we investigated the hypodermis cells of the PI with the aim to

describe calcium compartmentation and cell structure differentiations for

epithelial calcium phosphate transport in comparison to those in CaCO3 transport.

We used TEM and STEM to investigate the ultrastructure of epithelial cells,

and X-ray spectral mapping to detect mineral containing organelles within cells. For elemental mapping, we used high pressure frozen material, freeze-

substituted in acetone containing uranyl acetate and glutaraldehyde. Thin

sections of resin embedded material were floated on propane-1,2 diol instead of water to minimize loss of diffusive elements within the sample.

The results show that the hypodermis of the PI consists of two different cell

types. The P-cells and the C-cells that secrete and mineralize the calcium phosphate and calcium carbonate containing part of the PI cuticle,

respectively. The P-cells have up to 400 µm long extensions that originate

from the cell somata that are situated in the base region and corpus and extending to the forming cuticle of the middle region. The extensions, of P-

cells contain numerous clathrin-coated vesicles and non-coated vesicle along

microtubules. Near the cuticle, the extensions contain calcium phosphate granules up to 1µm in diameter, likely of endosomal origin, that are filled

with calcium phosphate. The element composition of these granules

resembles that of the ACP in the PI cuticle. The C-cells have no large extensions and contain no mineral granules. They resemble the hypodermis

of the sternal integument for which the cellular mechanism for epithelial

calcium transport are well studied. The results suggest that within the PI the cellular mechanism for

mineralization of the cuticle follows two different pathways. In P-cells

mineral formation takes place within organelles of the endosomal pathway that contribute to cuticle mineralization, while in C-cells mineral is

transported across the epithelial cells by ion channels and carriers, and intracellular compartmentation of calcium ions within the endoplasmic

reticulum.

Huber J, Fabritius HO, Griesshaber E, Ziegler A (2014) Journal of Structural Biology 188, 1-15

Supported by DFG ZI 368/11-1.

P 84

Physical and anatomical variation of mammalian bone

bioapatite structure and composition B. Foley*1, M. Greiner1, G. McGlynn2, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department of Earth- and Environmental Science, München, Germany 2Staatssammlung für Anthropologie und Paläoanatomie München, München, Germany

Bone is a hierarchical composite material primarily consisting of an

inorganic calcium phosphate phase—a widely substituted variant of apatite commonly referred to as bioapatite—dispersed in an organic collagen matrix.

Structural variation of the bioapatite lattice occurs with substitution of PO43-

and OH- by carbonate ions, which results in changes of lattice parameters (Handschin et al. 1995). Changes in structure and chemical composition of

bioapatite have been linked to aging and nutrition (Boskey et al. 2010).

Lattice parameters, crystallinity and ionic substitution of bioapatite also vary with anatomical location, pathological conditions, and physical stress in vivo

and with the environment during fossilization and cremation post mortem

(Greiner et al. 2018). As such, detailed investigation of bone bioapatite structure and chemical composition is essential to uncovering the stories of

forensic and archaeological bone samples and understanding bone resorption

processes in contexts such as disease pathology and tissue engineering. Human bone samples were analyzed by X-ray powder diffraction and

Fourier-transform infrared spectroscopy (FTIR) to investigate bone

bioapatite structure and composition. Specifically, Rietveld refinement of X-ray diffraction data was used to determine lattice parameters and crystallite

size. Samples of the femur, clavicle, sternum, talus, calcaneus, and parietal

bone were analyzed from the skeletal remains of three middle-aged human subjects: an average-build male, a robust-build male, and a petite-build

female. Lattice parameters and crystallite size were shown to vary with

respect to anatomical position, gender, and build. Bone samples from the robust-build male showed consistently larger a (=b) lattice parameters than

did samples from the average-build male and petite-build female for all

anatomical positions. FTIR spectra of analyzed samples showed consistent ratios of organic to inorganic content.

Preliminary results show consistent proportion of organic content but

structural variation with respect to physical build.

Boskey, A., & Coleman, R. (2010). Aging and Bone. Journal of Dental

Research,89(12), 1333-1348. doi:10.1177/0022034510377791 Greiner, M., Kocsis, B., Heinig, M. F., Mayer, K., Toncala, A., Grupe, G., &

Schmahl, W. W. (2018). Experimental Cremation of Bone: Crystallite Size

and Lattice Parameter Evolution. Biomineralization,21-29. doi:10.1007/978-981-13-1002-7_3

Handschin, R., & Stern, W. (1995). X-ray diffraction studies on the lattice

perfection of human bone apatite (Crista Iliaca). Bone,16(4). doi:10.1016/s8756-3282(95)80385-8

56

P 85

Infrared nanoscopy of biomaterials A. Cernescu*1, S. Amarie1, A. Huber1, F. Keilmann2 1neaspec GmbH, Haar, Munich, Germany 2Ludwig-Maximilians-University, Munich, Germany

Introduction

Scattering-type scanning near-field optical microscopy (s-SNOM) has

become a key technology to study the chemical composition of inorganic and

organic materials at the nanoscale.

Objectives

The investigated samples are nanocomposite biomaterials, namely human

bone sections [1], human tooth specimens [2] and mollusk shell which contain mineral nanocrystals in organic matrices [3]. The mineral parts are

highlighted by their resonantly enhanced contrast due to phonons.

Materials & methods

Our method is surface-sensitive, probing to a depth of about 20 nm.

Spectroscopic near-field imaging is enabled by combining 20nm-resolving

tip-scattering near-field microscopy (s-SNOM) with an infrared continuum source. Specific contrasting of biomineral components is enabled by simply

choosing the appropriate "fingerprint" infrared region that as in traditional

FTIR (Fourier-transform infrared spectroscopy) identifies virtually any chemical compound. Hence nano-FTIR stands for the successful realization

of combining s-SNOM and FTIR [3].

Results

Mineral distribution across dentine/enamel interface is resolved with 20nm

spatial resolution showing a gradient in the hydroxyapatite concentration.

Relatively large Ca2PO4 crystals (size >100 nm) are observed in both dentin and enamel regions, as well on the human bone samples (Fig. 1).

Fig. 1: Minerals in human bone. High resolution IR nanoscopy image

obtained simultaneously with the AFM topography shows the nanoscale distribution of Calcium phosphate and collagen within the cross section of a

human bone lamella extracted from a hip joint. Imaging at different

wavelengths maps different minerals within the tissue, which reveals important information about the bone structural integrity and could provide

details about the process of demineralization due to diseases, such as

osteoporosis. Interestingly, phosphate nano-crystals are also found in mollusk shell

specimens, close to the interface between calcite and aragonite biominerals.

Conclusion

The benefits of using the s-SNOM technology are the nanoscale resolution

combined with the chemical spectroscopic identification of molecules.

Straightforwardly applicable in many fields of biomaterials, general mineralogy and solid state research, this technology is becoming one of the

most powerful tools in nanoscale analytics.

References

[1] T. Geith et. al, Visualisation of methacrylate-embedded human bone

sections by infrared nanoscopy, J. of Biophotonics (2012);

[2] T. Sui et. al, Structure-Function Correlative Microscopy of Peritubular and Intertubular Dentine, Materials (2018);

[3] S. Amarie et al, Nano-FTIR chemical mapping of minerals in biological materials, Beilstein J. Nanotechnol. (2012).

P 86

Discovery of long-chain polyamines and their biosynthetic

enzyme in the biosilicifying bacterium Bacillus cereus T. Ikeda*1, K. Yamamoto1, R. Hirota1, A. Kuroda1

1Hiroshima University, Hiroshima, Japan

Introduction

Although silica biomineralization (biosilicification) has been intensively

investigated in several eukaryotes, such as diatoms, sponges, and higher

plants, prokaryotic biosilicification was not studied until recently. In 2010, we reported that biosilicification occurs in the gram-positive, spore-forming

bacteria, Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acids (Hirota et

al. 2010 J. Bacteriol. 192, 111). In a recent study, we demonstrated that the

spore coat protein, CotB1, which carries a characteristic C-terminal zwitterionic sequence, plays an essential role in biosilicification; however,

the underlying mechanism was not elucidated (Motomura et al. 2016 J.

Bacteriol. 198, 276).

Objectives

This study aimed to further investigate bacterial biosilicification

mechanisms. Since most eukaryotic silica-precipitating peptides and proteins were discovered by dissolving biosilica in hydrofluoric acid (HF) or

ammonium fluoride (NH4F), we hypothesized that the organic compounds

embedded in the B. cereus biosilica also play an important role in silica formation.

Materials & methods

A suspension of B. cereus spores was mixed with nitric and sulfuric acid and then boiled to degrade organic matter. Insoluble biosilica were harvested by

ultracentrifugation and repeatedly washed with distilled water until the pH of

the supernatant became neutral. The biosilica pellet was suspended in 6 M NH4F solution to dissolve the silica. After incubating at room temperature

for 30 min, the solution was repeatedly dialyzed against 200 mM ammonium

acetate to remove NH4F. The solution was then freeze-dried to remove the solvents and the remaining substances were dissolved in a small amount of

distilled water. Finally, the extract was analyzed by SDS-PAGE and mass

spectrometry.

Results

The extract from the biosilica showed a low-molecular-weight band on SDS-

PAGE gels. Mass spectrometric analysis revealed that this band contained long-chain polyamines (LCPAs) with long repeats of the -CH2CH2CH2NH-

unit. Notably, LCPAs were also identified in silica-accumulating eukaryotes

such as diatoms and siliceous sponges, although their chemical structures are slightly different from those of B. cereus LCPAs. These findings strongly

suggested that LCPAs play a common and important role in silica formation

in these organisms. Moreover, the chemical structure of B. cereus LCPAs strongly suggested that they were synthesized via repetitive aminopropyl-

transfer reactions. We also identified a plausible candidate for LCPA

synthase via homology searches using bacterial aminoproplytransferases as query sequences. Furthermore, gene disruption experiments showed that the

candidate gene is indeed essential for LCPA synthesis. Unexpectedly, the

disruptant lacking LCPAs formed biosilica around the spore coat, similar to the wild type, when cultivated in the presence of silicic acid, thereby

suggesting that LCPAs are not essential for biosilicification in B. cereus.

Conclusion

In this study, we demonstrated the presence of LCPAs in the B. cereus

biosilica and found a putative gene encoding LCPA synthase. Despite the common presence of LCPAs in different evolutionary lineages of silica-

accumulating organisms, gene disruption analysis unexpectedly showed that

LCPAs are not essential for biosilicification in this bacterium.

P 87

Crystallization of calcium oxalates inherent in mineralized

biofilms A. Rusakov*1, M. Kuz’mina1, O. Frank-Kamenetskaya1 1Saint Petersburg State University, Crystallography, Saint Petersburg, Russian Federation

Biofilms containing colonies of microscopic fungi (micromicetes) can often be found on the surface of carbonate rocks (marble, limestone etc.) in urban

environment. Micromycetes excrete substantial amounts of organic acids (oxalic, citric, malic, succinic, fumaric, lactic etc.) in the course of their

metabolism. These acids solubilize carbonate rocks and induce calcium

oxalate (calcium oxalate monohydrate - whewellite, calcium oxalate dihydrate - weddellite) crystallization. These minerals as well as micromicete

metabolites and several other chemical additives which come from the

environment form the so called oxalate patina. The aim of this work was to study the influence of different organic and inorganic biofilm components on

phase composition and the morphology of the formed calcium oxalates in

vitro. Calcium oxalate crystallization was performed by decantation of a mixture

of sodium oxalate and calcium chloride aqueous solutions with pH variation

(4.0-7.0) at room temperature (22-25С). Na+, K+, Mg2+, Fe3+, PO43-,SO42-, CO32- ions were added to the solution as the impurity components

as well as the organic acids, excreted by micromicetes. The compound

concentrations were close to the ones observed in the biofilms. It was found that whewellite crystallizes in the presence of all inorganic and

organic components. Weddellite formed in the presence of citric ions of

varying concentrations with no dependance on the presence of inorganic components, as well as in the presence of three acids (malic, succinic and

fumaric) with an equal ratio of their concentrations in the absence of

inorganic components. Weddellite also solely formed when Ca2+ ions to the oxalate ions concentration was set close to stoichiometry (1.5:1) at pH≥6,

and at an increased ratio of more than 7.5:1 at pH≥5. The addition of iron

cations to the system significantly increases the amount of weddellite in the sediment, while the admixture of carbonate ions or phosphate ions, on the

contrary, decreases the amount of weddellite in the sediment. The presence

of phosphate and carbonate ions in the crystallization medium can lead, in appropriate conditions, to the formation of very small amounts of additional

mineral phases, such as brushite and calcite, for example.

Whewellite crystallized in the following forms: small elongated lamellar crystals with side faces not very pronounced (in syntheses without any

additional impurities); large aggregates consisting of oval petals (in systems

with additional inorganic impurities); small spherical spherulites consist of small curved plates (in systems citrate ions); small spherical spherulites (in

systems with citrate ions and inorganic impurities); elongated cylindrical or

"kidney-shaped" aggregates which consist of thin plates (with the addition of additional organic acids).

Weddellite crystals had tetragonal dipyramid facetes (in systems with citrate

ions and inorganic impurities), and with the increase of Ca2+ concentration (while C2O42- concentration was set to 3 mmol/l) a transition from

dipyramidal to skeletal crystals was observed. In systems with Fe3+, Mg2+,

57

PO43- ions weddellite precipitated as real crystals with small tetrahonal dipyramid in the center of a large tetrahonal plate with an uneven edge. The

formation of prism facets of weddellite crystals was observed only in the

presence of citrate ions and MgSO4 in the solution. The results contribute to the research of the oxalate patina formation and give

evidence on the specific patina components which influence the mineral

composition and the crystal morphology. The obtained results allow to recreate the original pattern of biofilm

mineralization based on the morphology of calcium oxalate crystals found in

biofilms. Acknowledgements: This work was supported by the Russian Science

Foundation (no. 19-17-00141). The laboratory researches were carried out in

the Research Resource Centers of Saint Petersburg State University: SEM investigations - in the "Resource Center Microscopy and Microanalysis

(RСMM)" and "Interdisciplinary Resource Center for Nanotechnology"

"XRD measurements – in the «X-ray Diffraction Centre".

P 88

Modifying the marble surfaceinfluenced bybacteria Bacillus

spp. and bacterial-fungal associations K. Sazanova*1, O. Frank-Kamenetskaya2, D. Vlasov1,2, A. Vlasov2, M. Zelenskaya2, A. Rusakov2 1Komarov Botanical research institute RAS, laboratory of analytical

phytochemistry, Saint-Patersburg, Russian Federation 2Saint-Petersburg State University, Saint-Petersburg, Russian Federation

Metabolism products secreted by the microbial lithobiont community are a powerful factor in modern mineral formation. Most of the time rock surfaces

in natural systems are inhabited by the multi-species communities (mainly

fungi and bacteria). The biochemical activity of microorganisms in communities differs significantly from monocultures. The patterns of

microbial crystallizationunder influence of several species of

microorganisms are practically unexplored. The aim of this study was to receive a mophogenetic patterns of calcium

carbonates and oxalates crystallization on marble surface induced by

metabolism of bacteria and bacterial-fungal associationsin experimental conditions

Two series of experiments were performed with microorganisms: 1-

monoculture of bacteria Bacillus spp., 2 - co-culture of Bacillus subtilis and fungus Aspergillus niger.

Cultivation of microorganisms was carried out in a liquid medium and in wet chamber.Czapek-Dox medium with different glucose concentration (1, 5, 10,

30 g/l) was used as a nutrient medium. In the experiments in liquid medium

marble blocks were put on the bottom of Petri dishes and 15 ml liquid Czapek-Dox medium were added. The cultivation time: 14, 21, 30, 60

days. In experiments in wet chamber microorganism cell suspension was

applied on the marble block surface.The cultivation period for different marble blocks was 21 and 90 days.

Scanning electron microscopy (SEM) and energy-dispersive X-ray

spectroscopy (EDXS) were used for the identification of phase composition of crystallization products and examination of crystal morphology. The

determination of the metabolite composition was carried out in a liquid

culture of microorganisms using gas chromatography-mass spectrometry (GC-MS) by use of an Agilent mass spectrometer (MSD5975 mass selective

detector), column HP-5MS, 30 m 9 0.25 mm. The analysis of the EPS content

in the liquid monoculture of Bacillus subtilis and in co-culture of Bacillus subtilis and Aspergillus niger was performed by precipitation in cold ethanol,

centrifugation and weighing.

In a liquid medium with slightly alkaline pH values, the release of EPS by bacteria leads to crystallization of calcite, the intensity of which increases

with increasing sugar content in the crystallization medium. In oligotrophic

conditions of a moist chamber the acidifying activity of Bacillus subtilis prevales which leads to crystallization of calcium oxalate dehydrate

(weddellite). The metabolic activity of B. subtilis and A. niger association is

vastly different from the activity of the separate monocultures. The metabolic activity of micromycetes can suppress the formation of bacterial EPS and

prevent the formation of calcite crust. In a liquid medium, as the sugar

content increases, the acidifying activity of the fungus increases which leads to a shift of pH of the medium to the acidic region, carbonate crystallization

attenuation, and oxalate crystallization activation - the formation of calcium

oxalate monohydrate and dihydrate (whewellite and weddellite). The phase composition of microbial crystallization products is vastly

different in association of microorganisms from the activity of monocultures.

In the case of Bacillus subtilis and co-culture of Bacillus subtilis-Aspergillus niger it determined by the ratio of the concentrations of EPS and oxalic acid

in the crystallization medium.

This work was supported by the Russian Science Foundation (project No. 19-17-00141)

P 89

Cyanobacteria-related carbonate sedimentation in modern

rivers, Leningrad region, Russia O. Rodina*1, O. Vereshchagin2, D. Vlasov1, M. Nikitin3, M. Zelenskaya1 1Saint Petersburg State University, Biological, Saint Petersburg, Russian

Federation 2Saint Petersburg State University, Institute of Earth Sciences, Saint

Petersburg, Russian Federation 3The Bonch-Bruevich Saint-Petersburg State University of Telecommunications, Institute of Military Education, Saint Petersburg, Russian Federation

Introduction

Today, cyanobacteria (CB) continue to play a central role in the carbon and

nitrogen cycle. They are considered to be the pioneers of the colonization of mineral substrata. CB are not only biodegradation agents, but also they take

important place in biomineralization of carbonate in fresh water. CB are

common habitants of modern rivers riched in carbonates of the south of the Leningrad region, Russia. However, cyanobacteria-carbonates relation is still

in focus of scientific debates.

Objectives

Minerals composition of carbonate sediments from modern rivers of the

south of the Leningrad region was studied. The CB diversity was described.

An experiment of the community growing in cumulative culture for studying cyanobacteria-carbonates relation was performed.

Materials & methods

Thirty one samples of layers (biofilms) from the stone surfaces were selected in Leningrad region. Samples were dried for 3 month. X-ray diffraction

patterns (XRD) were recorded on a Rigaku Miniflex II diffractometer. The

chemical composition and micromorphology was studied by means of an Hitachi S-3400 N scanning electron microscope equipped with an Oxford

Instruments AzTec Energy X-Max 20. For the identification of CB, direct

microscopy was carried out after their settling in distilled water for a month. The cumulative culture of the microorganism community was grown in

distilled water for a year. Identification of species was carried out using light

microscopy (Leica DM 1000).

Results

The mineral composition of carbonate deposits is represented by calcite and

aragonite. The appearance of aragonite in the sediment is associated with an increased content of magnesium in calcite (MgO> 1 wt.%). CB recovered

and started to grow in solution only from samples, which contain aragonite. Twenty-nine taxa were identified in these samples. CB taxa are belonging to

5 orders, 10 families, 17 genera. The most widely distributed species were

from the genus Phormidium. Only Calothrix (C.) sp. from one sample has mucous cover with mineral crystals on it. This sample was used for further

community growing in cumulative culture. The sample of the community

with C. sp. started to be green after one week settling in distilled water and after that, it grew at room temperature in a 100 ml glass beaker under a glass

cover (ventilated) for a year. After a year of cultivation, the C. sp. began to

dominate in the community, and it was almost completely covered with mineral particles. Result of XRD showed that Mg-calcite is main phase of

this cover. However, no aragonite was found.

Conclusion

The communities of CB associated with the deposition of carbonates in the

modern rivers of Leningrad region was described. The fact of the association

of mineral particles with mucous covers of C. sp. is confirmed by means of optical and SEM microscopy, as well as by XRD. The natural samples of

carbonate sediments with CB contain calcite and aragonite. However, only

the mineral case of cyanobacteria grown in distilled water is composed of calcite. It is necessary to conduct further experiments for establishing the

relationship between cyanobacteria and carbonates. The authors thank the

Resource Center for X-ray diffraction studies, Geomodel Resource Centre, Resource Center for Microscopy and Microanalysis (RСMM) of Saint

Petersburg State University for providing instrumental and computational

resources. This work was supported by the Russian Science Foundation (project No. 19-17-00141).

P 90

Biodegradation of 2 - methoxyethanol by a new bacterium

isolate Pseudomonas sp. strain VB under aerobic conditions F. O. ekhaise*1 1University of Benin, Microbiology, Benin, Nigeria

Microbial biodegradation of 2-methoxyethanol also known as Methyl glycol

(MG) under anaerobic conditions has received much attention during the past

decade. However, not much is known about the aerobic degradation of 2-methoxyethanol. Samples from various environmental niches were enriched

to isolate and determine bacterial isolates capable of utilizing 2-

methoxyethanol as a sole source of carbon and energy under aerobic conditions. A 2-methoxyethanol degrading bacterium was isolated from

anaerobic sludge of a municipal sewage from a treatment plant in Bayreuth,

58

Germany by selective enrichment techniques. The isolate was designated strain VB after it was shown by the 16S rRNA phylogenetic sequence

analysis as belonging to the genus Pseudomonas. Under aerobic conditions

Pseudomonas sp. strain VB was capable of mineralizing 2-methoxyethanol and its intermediary metabolites. Stoichiometrically, the strain utilized one

mole of oxygen per one mole of 2-methoxyethanol instead of four mole

oxygen per one mole of 2-methoxythanol for the total oxidative metabolism

P 91

Formation of humboldtine-group oxalates Me2+(C2O4) 2H2O

(Me = Fe, Mn, Mg) under the influence of fungus Aspergillus

niger M. Zelenskaya*1, O. Frank-Kamenetskaya2, A. Izatulina2, A. Rusakov2, D.

Vlasov1 1St. Petersburg State University, Department of Botany, Saint Petersburg,

Russian Federation 2St. Petersburg State University, Crystallography , Saint Petersburg, Russian Federation

The interaction between the products of lichen metabolism and the rocks underlying the lichens leads to processes of modern mineral formation. The

present work describes the results of the laboratory experiments on the

formation of humboldtine-group oxalates under the influence of the fungus Aspergillus niger on the surface of todorokite (Ca,Sr)0.3-

0.5(Mn4+,Mn3+,Mg)6O12•3-4.5H2O, kutnohorite Ca (Mn,Mg,Fe)(CO3)2,

siderite (Fe,Mg,Mn)CO3, as well as ankerite Ca(Fe,Mg)CO3. The experiments were held at room temperature in liquid Czapek-Dox

medium. The synthetic products were studied under scanning electron microscope, as well as by EDX analysis and X-Ray powder diffraction

methods.

Magnesium oxalate dihydrate (glushinskite) formed under the influence of Aspergillus niger in the initial stages of the experiment with todorokite and

kutnohorite. The sequence of transformations of todorokite and kutnohorite

by fungus Aspergillus niger, leading to the formation of insoluble Mn2+ oxalates of different water content (mycogenic analogues of the minerals

falottaite and lindbergite), is different and depends on the valence of

manganese ions in the underlying mineral substrate. Due to the dissolution of todorokite by organic acids excreted by fungi, manganese ions (mostly

Mn3+ and Mn4+) get into the crystallization medium, where are then reduced

to Mn2+ along with a gradual pH increase, which all leads to oxalate crystallization. Kutnohorite on the other hand initially contains only Mn2+

manganese ions. The formation of manganese oxalate here is preceded by the

oxidation of Mn2+ ions to Mn3+,4+ during the initial stage of the experiment and the following crystallization of micogenic Mn-oxide (a todorokite

analog). Under the action of fungi micogenic todorokite immediately begins

to dissolve, and only after the reduction of manganese back to Mn2+ the oxalate crystallization begins. During todorokite transformation, manganese

oxalate trihydrate (falottaite) appears in the medium first followed by

manganese oxalate dihydrate (lindbergite), which we can explain by falottaite dehydration which feeds lindbergite crystallization. In the case of

the mycogenic transformation of kutnohorite, falottaite was not formed and

the crystallization of lindbergite occurs without the participation of falottaite. We can assume that the formation of manganese oxalates of different water

content is regulated by chemical compounds of their crystallization media,

compositions of which are substantially different for todorokite and kutnohorite.

Iron oxalate dihydrate (humboldtine) was first obtained in the experiments

with fungus Aspergillus niger on the surface of iron carbonates: siderite and ankerite. It formed plate-like crystals, close in habit to glushinskite: on

siderite surface it had an admixture of magnesium and manganese, on

ankerite surface - an admixture of magnesium. Acknowledgements: This work was supported by the Russian Science

Foundation (no. 19-17-00141). The laboratory researches were carried out in

the Research Resource Centers of Saint Petersburg State University: SEM investigations - in the "Resource Center Microscopy and Microanalysis

(RСMM)" and XRD measurements – in the «X-ray Diffraction Centre».

P 92

Improved CaCO3 biomineralization under extreme alkalinity

conditions of two native microbial consortia from extreme

Chilean ecosystem S. Marín*1, V. Zetola2, S. Olivares1, C. Demergasso*1 1Universidad Católica del Norte, Biotechnology Center, Antofagasta, Chile 2Universidad Católica del Norte, Facultad de Ciencias de Ingeniería y Construcción, Antofagasta, Chile

Introduction Biologically induced, controlled and influenced mineralization are three

avowed microbe-mineral interactions with noted implication in ecology, astrobiology, biogeochemistry and paleontology sciences. In the last decade,

the benefits of microbe-mineral interactions for the biocementation and

building materials industry are under extensive investigation and gradually being disclosed. One of the known challenges for reaching an effective

application of this kind of biotechnology in the concrete industry has been

the dramatical loss of cell growth and ureolytic activity under the extreme alkaline environment compatible with concrete sustainability. Obtaining

microorganisms naturally adapted to conditions of extreme alkalinity could

facilitate the adaptation process and improve the in situ mineralization performance on the concrete matrix. The aim of this study was to obtain and

adapt novel microbial consortia, native from extreme ambient of Chile, with

improved capacities to produce CaCO3 by biologically induced and controlled mineralization under extreme alkalinity conditions (pH > 12) and

useful for in situ concrete repairing and improving.

Materials and methods Ten microbial consortia with proven ability of ureolytic activity at laboratory

scale were obtained from different extreme alkaline lagoons (pH > 8.5), salt

flats and soils from the Altiplano, Chile, and immediately characterized by massive sequencing of 16S-rRNA gene. Consortia were enriched in both

Tryptic Soy Broth (TSB) and specific ureolytic culture media. Enriched

consortia were characterized again by DGGE. A gradual adaptation to extreme alkalinity conditions from pH 8.0 to 12.5 was performed for seven

months. Urease activity, pH and cell growth were constantly monitored. One

of the best representative species of CaCO3 biomineralizing microbes, Sporosarcina pasteurii (DSM 33), was used as a positive control. Mixes of

the native consortia were performed in an attempt to increase the adaptation

ability. Sand conglomeration and concrete repair ability for the best alkaline adapted and most efficient mineralizer consortium were evaluated. Obtained

CaCO3 mineral was characterized by X-ray Diffraction and Scanning Electron Microscopy.

Results and Conclusions

Natural consortia obtained from extreme alkalinity zones presented an interesting microbial diversity and confirm the microbial adaptation to these

extreme environments. Microbial consortia enriched with TSB medium was

more diverse than those enriched with ureolytic medium. Species that grow in ureolytic specific medium are not necessarily present in TSB cultures or

natural samples meaning that some ureolytic identified species are present in

natural environments but with low abundance. New native strains of Sporosarcina, Bacillus and Lysinibacillus species were obtained from

ureolytic cultures. All ureolytic consortia were active until pH 11.5 but only

two of them were able to actively growing and do hydrolyze urea at pH > 12. Moreover, the mix of these two native microbial consortia achieved a higher

ureolytic and mineralizing performance under all the evaluated conditions

showing an even better adaptive ability respect to each separate consortia and respect to control, S. pasteurii. According to the authors, this is the first work

performing successfully biologically induced CaCO3 mineralization at the

extreme alkaline conditions analyzed, with a great perspective in the concrete repair application.

P 93

Bacterial choreography- designing interactions through

biological induced mineralisation T. H. Arnardottir*1 1Newcastle University, Architecture, Newcastle, United Kingdom

This research explores the potential design role of bacterial-induced

biomineralisation. It sits within the speculations regarding our changing

relationship with nature through engineered biological systems and new material processes. In the convergence of design and biological fabrication,

the role of the designer is shifting from assuming the role of the sculptor to

adopting the perspective of the cultivator in the study and production of new living material assemblages. In this intersection, we are moving away from

the mass production of fast, cheap and repetitive elements by enabling the

production of biologically made materials. Much like properties of natural materials such as bones, which are not designed but instead and are shaped

by a set of natural constraints, biomineralisation has opened up the possibility

of utilising nature"s intelligence for the built environment. Such process has precise applications in the fabrication of materials as there has been extensive

research on utilising bacteria to induce biological cementation through

engineering solutions, such as crack repair in concrete, soils" improvement or forming energy-efficient bricks. By harnessing the biomineralisation

process, bacterial-induced fabrication can be used in the creation of new

materials with functionally graded and variable properties. This research aims to explore material- and fabrication methods that

incorporate living cells as an inherent part of the process and to outline

parameters that facilitate the synthesis of this biomaterial. It focuses on the assemblages of biologically fabricated matter through the interaction of

bacterial agents in a complex system, by challenging the designer"s thinking

from the application of an ideal form on an inert matter, to the shaping of a bottom-up emerging form. The goal is to set out material and fabrication

processes to enable designers to engage with these living systems as

resources. This research concentrates on structuring a biofabrication process whose

purpose is to partly control the physical geometry of a microbial induced

59

mineralised structure. The experimental approach entailed an exploration of established procedures and the testing of novel physical apparatuses that

were part bioreactors and part moulding vessels. These enabled the

biomaterial synthesis through the alteration and control of chemical, spatial and structural compositions of the environment. In this setting, the bacteria

Sporosarcina pasteurii was mixed in with sand in nutrient solution and

grown overnight. This allowed the culture to situate itself within the grain before being flushed at intervals with the cementation media of nutrient

broth, urea and CaCI2 over a few days.

The change in compositions led to a framework to reach biomineralisation at given points and, by optimising the biofabrication process, obtaining fully

cemented pieces through the hybrid moulding/bioreactor technique.

Based on these findings, this design framework can enable designers to visualise and generate interruptions and interactions with the mineralised

material. Designers can think of this process as an interactive, alive network

that can be used to choreograph parameters to produce form alongside microbes. By utilising this induced material, the framework can demonstrate

different ways the design paradigm can potentially shape living matter and

inherently evolve our relationship with biological design in new ways.

P 94

The directing effect of bacterial EPS on calcite organization

and EPS-carbonate composite aggregate formation X. Yin*1, F. Weitzel1, E. Griesshaber1, C. Jiménez-López2, L. Fernández-Díaz3, A. Rodríguez-Navarro4, A. Ziegler5, W. W. Schmahl1 1LMU Munich, Department of Earth and Environmental Sciences, Munich,

Germany 2Universidad de Granada, Departamento de Microbiología, Granada,

Spain 3Universidad Complutense de Madrid, Departamento de Mineralogía y Petrología, Madrid, Spain 4Universidad de Granada, Departamento de Mineralogía y Petrología,

Granada, Spain 5Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm, Germany

Mineralized structures generated under biological control are hierarchical

composites that consist of two distinct materials: a compliant biopolymer

matrix that is reinforced by stiff and hard minerals. The biopolymer matrix in the biological structural material is developed as a membrane or as a

network of fibrils and has structural as well as functional roles for the architecture and material properties of the composite hard tissue.

Microbial cells surround themselves with a fibrous biopolymer matrix (EPS)

for protection, orientation of cells and enhancement of physiological activities. For understanding the influence of biopolymer matrices on mineral

organization and composite material formation we conducted growth

experiments with the extracellular polymeric substance (EPS) of the gram negative bacteria Pseudomonas putida. We synthesized EPS-agarose

hydrogel-calcite composite aggregates and characterized aggregate

morphologies, EPS/hydrogel distribution, mineral organization and co-orientation strength.

We find that P. putida EPS exerts a tremendous influence on aggregate

morphology, pattern of polymer distribution and mode of mineral organization. Contrasting to reference aggregates that are devoid of bacterial

EPS, in aggregates that occlude EPS the pattern of polymer distribution is

highly inhomogenous and is developed mainly as membranes. Accordingly, subunit formation in these is extensive. Subunits are irregular in shape, size

and distribution and are highly misoriented to each other. Aggregates that

contain P. putida EPS are radial mosaic polycrystals, while the reference aggregates, devoid of EPS, are branched dendrites with the branches being

highly stepped and the calcite well co-oriented.

Incorporation of P. putida EPS into calcite changes the microstructure and texture of the mineral in a specific manner. This is a characteristic that can

be developed and used as a tool for the recognition and identification of

bacterially mediated calcification.

P 95

Development of CaCO3 materials through bacteria embedded

hydrogels R. Boons*1, G. Freitas1, G. Nyström1, A. Studart2, T. Zimmermann1 1EMPA, Dübendorf, Switzerland 2ETH Zürich, Zürich, Switzerland

Numerous types of mineral-based structures are used nowadays in a variety

of applications, ranging from construction to medicine. One possible way to

create a mineral formed structure is through a biomineralization process, which is utilised in this research to create mineral-based products.

Biomineralisation is a widespread phenomenon in several phylogenetic

groups including bacteria that are able to control, influence and induce mineralisation. Examples of the latter are certain Bacillus strains which are

known to induce calcium carbonate formation. This process is called

microbiologically induced calcium carbonate precipitation (MICP). Here the carbonate formed in presence of the bacteria will react with calcium in the

surrounding, resulting in precipitation of CaCO3. In order to obtain a specific

CaCO3–based structure using these strains, the bacteria have to be placed in a shape of interest. In this study, Sporosarcina pasteurii bacteria, formerly

known as Bacillus pasteurii, are embedded in bio-based polymer hydrogel

matrices (e.g. sodium alginate) together with the appropriate nutrients. The hydrogel should possess certain properties, such as biocompatibility,

porosity and high water content, optimised for the survival of the bacteria

and production of mineral crystals. The bacteria have to be provided with molecules specific to the bacterial strain, as well as supplementary products,

such as calcium sources, that are necessary for the mineralisation process.

The mineral formation is distinctively affected by different components added to the matrix and by the residing bacteria. Examining liquid media

cultures, this is observed as variations in S. pasteurii induced CaCO3

polymorphisms upon addition of different calcium sources. Additionally the ratio and transformation from one polymorph to the other are investigated

over time using several analysis methods, e.g. XRD and SEM. Finally, the

bacteria are embedded in the hydrogel and are shown to also induce CaCO3 formation in this condition.

P 96

Mineral deposition by lichens F. Bachmair*1, G. Lehrberger2, A. Beck3, M. Zenkert1, E. Griesshaber1,

W. W. Schmahl1 1Ludwig-Maximilians University Munich, Department of Earth and

Environmental Sciences , Munich, Germany 2Ingineurgeologie TU Munich, Munich, Germany 3Botanical State Collection Munich, Munich, Germany

Litchens deposit a variety of minerals, in particular, calcium oxalates in two hydration states: the monohydrate whewellite and the dehydrate weddelite.

Litchens release oxalic acid that reacts with the substrate mineral. Hence,

litchens etch and weather the surfaces of the rocks on which they grow. Etching rock minerals results in the extracellular formation of new mineral

deposits that most probably have no organic matrix associated with the

crystals. The purpose of our study is to identify and characterize those minerals that

are formed with the activity of litchens. In particular we aim to distinguish

between grains that constitute the substrate and the crystals that are deposited through litchen activity. We investigated Caloplaca falvescens litchens that

colonize limestone of the Jurassic Kehlheimer Kalkstein formation present

in the surroundings of Regensburg, Germany. Mineral characterization was performed with FE-SEM imaging, EBSD, EDX, XRD and FTIR. With the

applied combination of methods we detected both, chemical and

morphological differences between minerals and grains forming the substrate

and the minerals that were secreted by the litchens. While the limestone

substrate is composed of large, irregularly oriented carbonate grains,

substrate portions where the litchens settled are infused with a multiude of minute holes containing organic substance. In the latter regions we find

circular structures consisting of layered calcite, with the layers being assembled of minute calcite crystallites.

In conclusion, the distinctness of grain morphologies and crystallite sizes

coupled with some chemical characteristics enabled the clear distinction between original sedimentary carbonate deposits and carbonate minerals

formed as a mineralization product of litchens when colonizing and etching

the substrate mineral components.

P 97

Examination of the periodicity of incremental lines observed in

the otolith fossils of fishes from Nobori formation, Japan H. Mishima*1, Y. Kondo2, F. Ohe3, Y. Miake4 1Tsurumi University School of Dental Medicine, Department of Dental Engineering, Yokohama, Japan 2Kochi University, Science and Technology Unit, Natural Sciences Cluster, Kochi, Japan 3Nara National Research Institute of Cultural Properties, Nara, Japan 4Tsurumi University School of Dental Medicine, Oral anatomy, Yokohama, Japan

Introduction

The main component of the otolith in fishes is composed of a crystal of

calcium carbonate (CaCO3). As compared with the teeth and bone composed

of apatite crystal, the otolith is a different hard tissue. However, the incremental lines in the tissue of the otolith are formed in the same manner

as the teeth and bones. It has reported that the periodicity of the incremental

lines of otolith has a daily (circadian rhythm), a tidal, a lunar, a seasonal, and an annual periodicity (circannual) (Pannella, 1980). The ages of the fish were

estimated through the annual incremental lines. Little information is

available regarding the ultrastructure, chemical composition and the periodicity of incremental lines of otolith fossils of fish.

60

Objectives

The purpose of the present study is to examine the structure and composition

of incremental lines in the otolith fossils of fishes through the histological

and analytics studies.

Materials and methods

In this research, the otolith fossils of fishes (Nobori formation, Pliocene,

Muroto, Kochi Prefecture, about 3 million years ago) were used. The ten otolith fossils samples were the different fish genera habitats and used the

following samples (different fish genera).

(1) 1) Macrouridae spp. indet., 2) Cetonurus noboriensis (Aoki and Baba), 3) Caelorinchus anatirostris Jordan et Gilbert, 4) Ventrifossa sp. A, 5)

Coryhaenoides cinereus Gilbert. The habitat was continental shelf and slope

at a depth of 200m or deeper. (2) 1) Myctophidae spp. Indet., 2) Lobianchia gemellarii (Cocco), 3) Diaphus

gigas Gilbertt. The main habitats were the diurnal vertical movement.

(3) Sebates scythropus (Jordan and Starks). The habitat was continental shelf to upper slope (water depth 300m).

(4) Congriscus megastomus (Günther). The habitat was continental shelf -

upper slope habitat. Polyester resin-embedded samples and non-embedded samples were used in

this study. One-sided polish specimens and ground specimen were observed

and analyzed using light microscopy, digital microscopy, polarizing

microscopy, scanning electron microscopy（SEM), SEM-EDS analysis,

electron-probe microanalyzer (EPMA), laser Raman microprobe spectrometry, and X-ray diffraction method.

Results

The otolith crystals were aragonite by the X-ray diffraction method and the laser Raman microprobe spectrometry. The inside of the otolith was found to

consist of needle-like crystals crossed by incremental lines. The circadian

incremental lines (2-5 μm), and several long periods of incremental lines (tidal rhythm: about 14 days interval, monthly rhythm: about 28 days

interval, circannual rhythm) were observed in the otolith. By SEM images,

the incremental lines were observed as the dark bands. The incremental lines had content of C, O, and Ca, and additionally contained Si as a trace element.

Both Lobianchia gemellarii and Diaphus gigas were unclear in the circadian

incremental lines. Their habits were diurnal vertical movement. By comparison, Ventrifossa sp., Cetonurus nobonesis, Sebates scythropus, and

Congriscus megastomus were evident in the circadian incremental lines.

Their habitats were the slope from the continental shelf.

Conclusion

It is possible that the periodicity of the incremental lines of otolith changed

with the ecosystems.

P 98

Misorientation driven morphological evolution of the prismatic

ultrastructure in mollusc shells D. Stier*1, D. Zöllner1, I. Zlotnikov1 1TU Dresden, B CUBE - Center for Molecular Bioengineering, Dresden, Germany

Molluscs are a well-established model system to study biomineralization and

the process of biomineral morphogenesis. In addition to our growing

understanding of the different biochemical mechanisms that are responsible for mineral formation, a number of recent studies suggest that the deposition

of the various shell ultrastructures is a thermodynamically driven self-

assembly process. Specifically, it was shown that the formation of the prismatic ultrastructure, consisting of elongated mineral columns glued

together by an interprismatic organic membrane, can be quantified by

classical models of grain growth and coarsening. In this model, morphological evolution of the entire prismatic ultrastructure, which

proceeds parallel to the long axis of the prisms, is driven by the reduction of the amount of interfaces that separate the individual prisms. However, so far,

the energy of those interfaces was considered to be uniform. Therefore, the

influence of the crystallographic misorientation between adjacent prisms on the interface energy and thus, the morphogenesis of the corresponding

prismatic units, was never previously considered. In the current study, we

examine the prismatic ultrastructure in three mollusc species from the genus Pinctada. In these bivalves, the prismatic assemblies not only demonstrate a

coarsening behavior, which is predicted by the recently developed self-

assembly models, but also exhibit gradual changes in the crystallographic orientation of the individual prisms. Using electron backscatter diffraction

analysis (EBSD) and synchrotron-based X-Ray tomography to follow the

crystallographic properties and the shape of the prisms at different stages of growth, respectively, we show a correlation between the textural and

morphological evolution of the prismatic ultrastructures. In fact, we

demonstrate the key role of misorientation between neighboring mineral building blocks in the growth kinetics of biocomposite architectures.

P 99

The evolution of Theicideid brachiopods shell microstructure M. Simonet Roda*1, S. Milner2, E. Griesshaber1, H. Jurikova3, C. Rollion-

Bard2, L. Angiolini4, F. Ye4, A. Ziegler5, A. Bitner6, D. Henkel3, A. Einsenhauer3, W. W. Schmahl1 1Ludwig Maximilians Universität München, Department of Earth and

Environmental Sciences, Munich, Germany 2Institut de Physique de Globe de Paris, Department of Geochemistry and

Cosmochemistry, Paris, France 3GEOMAR Helmholtz Centre for Ocean Research, Marine Biogeochemistry/Marine Systems, Kiel, Germany 4University of Milan, Department of Earth Sciences "A. Desio", Milan, Italy 5University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany 6Polish Academy of Sciences, Institute of Palaeobiology, Warsaw, Poland

The interpretation of geochemical proxies allows the reconstruction of past

and present seawater chemical conditions and evolution with time. As proxy

data are obtained from geological archives, e.g. carbonate shells of marine invertebrates, a substantial understanding of shell microstructures is of

immense importance. With that knowledge a better understanding and

interpretation of geochemical proxy data is possible. Their abundance in the geological record together with the chemical and

structural stability of their low-magnesium calcite shells render brachiopods

to be an important group within the invertebrates. Further, within the phylum

Brachiopoda, the investigation of thecideidine shell morphology and

structure, histological characteristics of the mantle and metabolism in general

has been the subject of research for several decades and revealed the distinctiveness of this group of brachiopods relative to species of other

extinct and modern brachiopod genera (e.g. Pajaud, 1970, Grant, 1972,

Williams, 1972). Previous work suggests a progressive loss of the fibrous shell layer and the development of a "granular" primary layer, a process that

started in the Jurassic and carries on until now. Comparing FE-SEM, EBSD

and AFM results, on shell microstructure and texture, between modern rhynchonellide, terebratulide and thecideide brachiopod shells show that

mineral unit organization in modern thecideide brachiopod shells is entirely

different than the one present in the other groups. The study was completed with stable isotope and element analyses.

The objective of this study is to discuss in detail patterns of shell

microstructure of fossil and modern representatives of theicideid brachiopods and trace the evolution of shell organization and microstructure development

from the Triassic to the present time. Instead of the clear distinction between

an outer primary and an inward fibrous shell layer and the presence of stacks of fibers, as it is the case in modern rhynchonellide and terebratulide

brachiopod shells, we do not find any obvious mineral unit organization in

the shell of modern thecideides. The presence of fibers is clearly visible for

the Triassic and Jurassic specimens. It disappears subsequently, and from the

Cretaceous, to modern times granular microstructures prevail. The latter

consist of highly irregularly shaped mineral units with calcite being assembled with a low degree of co-orientation. Larger calcite granules,

acicular crystallites, and pseudo-polygonal mineral units are embedded in a matrix of small crystallites.

In modern species as Pajaudina atlantica and Kakanuiella chathamensis we

observe within the shell an extracellular organic matrix; however, this biopolymer matrix does not have a regular structure such as that of organic

sheaths encasing the fibers. We consider the granular microstructure of

thecideide shells as a recently evolved feature, followed its development with time, and discuss its interlinkage with the fibrous fabric from a

microstructural point of view.

Grant R. E. 1972. The lophophore and feeding mechanism of the Productidina (Brachiopoda). Journal of Paleontology, 46, 213- 249.

Pajaud D. 1970. Monographies des Thecidees (Brachiopodes). Memoire

Societe Geologique Francaise, 49, 112, 1-349. Williams A. 1972. The secretion and structural evolution of the shell of

Thecideidine brachiopods. Phil. Trans. Roy. Soc. B, 439-478.

P 100

Pineal gland calcification under hypoxic conditions M. Kopani*1 Bronislava Vraníková1, Daniel Kosnáč1, Michal Zeman2,

Vladimír Šišovský3

*1Comenius University, Institute of Pathological Anatomy, Faculty of

Medicine, Bratislava, Slovakia 1 Institute of Medical Physics, Biophysics, Informatics and Telemedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia 2 Department of Animal Physiology and Ethology, Faculty of Natural

Science, Comenius University, Bratislava, Slovakia 3 Institute of Pathological Anatomy, Faculty of Medicine, Comenius

University, Bratislava, Slovakia

Introduction The pineal gland (glandula pinealis) is neuroendocrine gland secreting melatonin. This hormone is involved in many physiological processes

61

including regulations/controls the circadian rhythm, body temperature, immunity and antioxidant activity.

Objectives The aim of this study is investigated the effect of hypoxia on the occurrence of pineal gland calcification. Distribution of calcerous material by light (LM)

and transmission electron microscopy (TEM) was investigated. Chemical

composition of foreign material by scanning electron microscopy with energy-dispersive microanalysis (SEM-EDX) was done. Melatonin

concentrations in blood plasma by direct radioimmunoassay were measured.

Materials & methods The experiments were performed on 24 adult male Wistar rats. First group

was exposed to prenatal hypoxia for 12 h at 20th day of development (n = 9),

second group to prenatal hypoxia for 2x8 h at 19th and 20th days of development (n = 5) and third group was control group (n = 10). All

procedures were conducted in accordance with the Declaration of Helsinki.

Results Scattered vacuoles and large focal calcium-rich particles in the pineal gland

and at the surface of pineal gland by LM were observed. The size of particles

is around 3 µm, sporadically up to 30 µm. SEM-EDX reveals that they consist of Ca, P, Na, S and Fe. TEM reveals vacuoles in the cytoplasm of

pinealocytes type I filled with both flocculent and fibrous material in 12h

hypoxia group. No significant differences in melatonin concentration among groups were found.

Conclusion Pineal gland consists of pinealocytes type 1 and type 2. Their function is secretion of melatonin. It was observed that the cytoplasm of pinealocytes

contains vacuoles filled with flocculent and fibrous material. Welsh (1984) showed that vacuoles play important role in calcification process. Our TEM

results confirmed the presence of flocculent and fibrous material in the

vacuoles. Our previous study revealed that calcerous material was mainly amorphous with the presence of calcite, aragonite and vaterite (nano)crystals

in the samples (Tofail et al. 2019). It is suggested that mineralization process

of pineal gland is similar like bone mineralization and hypoxia favors calcification under hypoxic conditions (Tan et al. 2018). Physical properties

(pyro-, piezo- and ferroelectricity) of crystals may play the important role in

the function of pineal gland.

References TAN DX, CHEN LD, POEGGELER BEA. Melatonin: a potent, endogenous

hydroxyl radical scavenger. Endocr J 1: 57– 60, 1993. TOFAIL SAM, MOURAS R, McNAMARA K, PATYK-KAZMIERCZAK

E, GEANEY H, ZAWOROTKO M, RYAN KM, SOULIMANE T, SILIEN

C, KOPÁNI M. Multimodal surface analyses of chemistry and structure of biominerals in rodent pineal gland concretions. Appl Surf Sci. 469: 378-386,

2019

WELSH MG. Cytochemical analysis of calcium distribution in the superficial pineal gland of the Mongolian gerbil. J Pineal Res. 1: 305-316,

1984.

P 101

Influence of organic additives on the crystallisation of gypsum

and portlandite C. Pritzel*1,2 1Building and Materials Chemistry University of Siegen, Chemistry, Siegen, Germany 2University of Siegen, Chemistry, Siegen, Germany

Gypsum (CaSO4*2H2O) and portlandite (Ca(OH)2) are used in inorganic

binding materials, in those materials organic additives are used to influence

technical properties. Most of the used additives are influencing the morphology of the formed crystals. This influence of organic additives on

the morphology of gypsum or portlandite was tested in crystal growth

experiments. Gypsum was prepared from Na2SO4 and CaCl2 or from calcium sulphate hemihydrate with and without different additives like betain

(used as foaming agent), fruit acids (used as retarder), poly carboxylate ethers

(used as superplasticizers) for example. The different additives influence the crystals morphology, because they are occupying different crystals surfaces

preferred. In case of citric acid the crystals morphology is changed from long

needle like crystals with a large aspect ratio and having more ramifications with the lack of acid to shorter, narrower crystals and less branches. The

smaller diameter of the crystals can be explained because the c-axis is firstly

covered by citric acid and as the consequence the fastest growing axis grows much slower. Owing to the collection the Ca2+ ions from the crystallization

solution leads to the lower ratio Ca/SO4 and the fewer branches can be

observed. These phenomena caused the first forming SO4-backbone which results in the fewer branches in the crystals afterward. The citric acid

adsorbed on the c-axis was shown by Raman-microscopy and AFM. The

calcium sorption was proved by Ca-ion selective electrode. The changed morphology of the created gypsum crystals is decreasing the strength of the

formed gypsum stone and increasing the forming accuracy. It was found that

the Polycarboxylate ethers are influencing the crystal morphology in the same way. The crystallisation of gypsum with and without additives from

hemihydrate was investigated with in-situ optical microscopy, ESEM, in-situ

XRD, particle size distribution measurement, ion chromatography, calcium sensitive electrode, ultra-sonic wave measurement and heat flow calorimetry.

The different techniques are needed to understand the process in detail.

The influence of betain on the crystallisation of portlandite was checked in a crystallisation experiment. Portlandite was produced from CaCl2 and NaOH

during a diffusion experiment with and without betain. The formed crystals

without betain are needle-like with a hexagonal shaped base. With betain thinner layers of those crystals grew together in different steps turned to each

other. The crystals morphology was investigated by ESEM and the sorption

of betain by RAMAN spectroscopy. The used techniques can also be applied to understand the crystal growth of different crystals in the presence of

organic additives. The influence of different organic substances which can

be found in bio-minerals should be investigated in future.

P 102

Reading between the lines in biomineralizations reaching out

for clues of environmental impact - case reports from studies

based on analytic spectroscopy, isotope geochemistry and

proteomics methods S. Berland*1, J. Arivalagan1, E. Feunteun1, A. Bartolini1, C. Thaler1,

A. Marie1 1MNHN, Adaptation du Vivant, Paris, France

Living organisms and mineral collectively speak in a rich and ancient story. Along evolution, complex biomineralized structures have appeared in a range

of species. These structures develop during ontogenesis under the control of

metabolism. The morphology and chemical composition of these structures are known to be shaped by environmental (external) factors under the limits

of functional outcomes. Here we present cases that illustrate calcium

carbonate biomineralisation responses to environmental factors, with special emphasis on technological investigations range, limits and stumbling blocks.

Calcareous tests of foraminifera were investigated. Among biomineralizing

organisms, foraminifera (unicellular eukaryotes) have been producing carbonates since geological time. Their tests (or shells) of calcium carbonate

trace environmental signals, such as ocean temperature, oxygen level,

contamination, salinity, etc. Our approach was to investigate the molecular and cellular machinery of biocalcification based on a benthic calcitic

formainifera, protozoan unicellular model. Coupling geochemical and

biological perspectives on foraminifera will enhance interpretation of the proxies used for environmental and climatic reconstructions.

The second case report arise from the Sub-Antarctic islands, in which severe

environmental conditions drive discrete ecosystems at meso-scale level. In the native land snail Notodiscus hookeri specific populations have evolved

into two ecophenotypes suited with shell either mineral-rich or organic-rich

flexible shells. The soil environment and especially the availability of

exchangeable calcium was the driving factor for ecotypes. Several

methodologies were applied: scanning electron microscopy of the shell, X-

Ray diffraction analysis of the soil, Solid-state Nuclear Magnetic Resonance and shell proteomics. This model enables to follow up the time over which

such adaptive response may have occurred towards phenotypic radiation.

In the context of global change, Baltic Sea was considered as a workshop area to study how native bivalve molluscs (Mytilus sp) can form shells under

lower calcium carbonate saturation states. A proteomic approach was

performed on the shells to learn about the composition of the nested matrix proteins, the local conductor of biomineralization. Result have shown

patterns of modulation in shell proteins which could be correlated to different

shell phenotypes in relationship with shell capacity to offer protection to the individuals.

Finally stable isotopes geochemistry (oxygen and carbon isotopes) in otolith

of glass eels (post larvae of European eels) was used with nanoscale mass spectrometry techniques (NanoSIMs) to track changes of the environment

along the early life history of this diadromous teleost fishes. The otolith

contains visible morphological checks that indicate life cycle key event, e.g hatching, first feeding, and metamorphosis. Minute differences in oxygen

isotopic ratios of oxygen (d18O) in otolith allow addressing the changes in temperature encountered by the larvae along their early life history.

Therefore, these analyses provide environmental information to trace

spawning places and dissemination routes of the eel larvae from the spawning places to the estuaries of Europe.

At the scale of the individual, community or species, biominerals stand as a

repository of environmental information alongside with evolution arrangements from lineage or acquisition. Enclosed time-frame range for the

process to apply needs to be looked for now.

References

Sabbatini A., Bédouet L., Marie A., Bartolini A., Landemarre L. , Weber M.

X., Gusti Ngurah Mahardika I., Berland S., Zito F., Vénec-Peyré M.T. (2014)

Biomineralization Of Schlumbergerella Floresiana, A Significant Carbonate-Producing Benthic Foraminifer. Geobiology: 12, 289-307

Charrier M, Marie A, Guillaume D, Bédouet L, Le Lannic J, Roiland C.,

Berland S., Pierre J.S. , Le Floch M. , Frenot Y., Lebouvier M. (2013) Soil Calcium Availability Influences Shell Ecophenotype Formation in the Sub-

Antarctic Land Snail, Notodiscus hookeri. PLoS ONE 8(12): e84527.

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Arivalagan, J., Yarra, T., Marie, B., Sleight, V. A., Duvernois-Berthet, E., Clark, M., Berland S. (2016). Insights from the shell proteome:

biomineralization to adaptation. Molecular Biology and Evolution, vol.34,

pp. 66-77

P 103

Role of biogenic carbonates in the sediment balance of

accumulative beaches of the Anapa Bay Bar (the Black Sea) A. Kosyan*1 1A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of

Sciences, Laboratory of morphology and ecology of marine invertebrates, Moscow, Russian Federation

Question

Sandy beaches of the Anapa Bay Bar are unique nature landscapes, possessing great esthetic and recreational value, as well as essential

significance for the economy of the Krasnodar region (Russia). The stability

of the beaches is determined by balance and dynamics of sediments. Biogenic sediments (carbonates) are provided by populations of coastal shelly

mollusks, mainly bivalves. In some sites, the share of biogenic carbonates

reaches 90%. Within the framework of the long-scale monitoring of the Anapa Bay Bar,

having been conducted by the researchers from IO RAS and IPEE RAS and

aimed to complex studying of lytho- and hydrodynamic processes, we assessed the quantitative role of mollusks in the sediment balance of the Bar.

Methods

We studied samples of benthic mollusks at five sections with stations at 2, 6 and 10 m depths (deeper mollusks cannot be transferred to the shore even by

stormy waves), taken in 2016-2018. Selected mollusks were counted and

measured. The age was determined by the lines of shell external growth. Determination of carbonate and mineral components of the sand samples was

carried out using 30% hydrochloric acid solution.

Results

The productivity of the two most widespread and abundant in the area species

of bivalves Chamelea gallina and Donax trunculus is not the same in

different years at different sections and depths. The contribution of C. gallina shells to the total carbonate sediments at depths of its mass

development 6-10 m was 327.2 g/m2 in 2016, 127.1 g/m2 in 2017, and 179.3

g/m2 in 2018; the contribution of D. trunculus shells at depths 2-10 m was 123.7, 35.9 and 31.3 g/m2, respectively. Gastropods Rapana venosa had an

order less number and biomass than bivalves, the biomass of other mollusk species was negligibly low. Carbonates content in the bottom sediments

varied from 3 to 30 % (average 11%). An increase in carbonates in bottom

sediments was not directly related to an increase in biomass of living mollusks on the same depths (r = -0.22). Thus, the distribution of carbonates

is rather explained by the movement of sediments, than the distribution of

live mollusks at the bottom. The data on the size-age structure of the mollusk population and earlier observations (Kosyan, 2016) show that the vast

majority of C. gallina within the Anapa Bay-bar (60%) die as a result of

rapana predation at an age of 2-3 years; most D. trunculus die at an age of one year (94% of the population). The width of the zone of mass development

of C. gallina at depths of 6–10 m is approximately 400 m, the width of such

zone of D. trunculus (2–10 m) is 900 m. Taking all above mentioned into account, we may calculate the annual income of the carbonates of biogenic

origin on the studied 45 km long site of the Anapa Bay Bar: 7834.5, 2227.5,

and 4257 tons in 2016, 2017 and 2018 respectively, or on average 4773 tons per year. There are about 1.0 to 4.0 million tons of sediments in constant

motion over the underwater coastal slope, thus restoring of the beaches by

means of shelly material amounts from 0.1 to 0.5%.

Conclusions

The annual contribution of shelly material to the total sediment balance of

the Anapa Bay Bar is on average 4773 tons per year which comprises from 0.1 to 0.5% of total amount of moving sediments. This indicates the

importance of taking measures to protect and restore the population of

mollusks living on the underwater coastal slope. The work was supported by RFBR grant No. 19-45-230001.

P 104

Biominerals in aquatic geosystems under extreme conditions L. Reykhard*1, N. Shulga1, N. Kozina1, Y. Novichkova1, O. Dara1, A.

Boev1, P. Sapozhnikov1, A. Izhitskiy1, N. Belyaev1, V. Gordeev1, O. Kalinina1, A. Reikhard2 1Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow,

Russian Federation 2State Secondary General School № 2086, Moscow, Russian Federation

Authigenic biominerals and their associations with minerals of other origin are used as indicators of the geological, hydrological, and ecological

characteristics of ancient and modern aquatic geosystems. Extreme

environments of biomineralization lead to the appearance of specific structural and chemical features of minerals that must be considered when

using the biomineral indicators. This work aimed to identify the properties

and the origin of biominerals under extreme conditions (high pressure and salinity, low temperatures, high content of hydrogen sulfide and methane,

etc.) in the aquatic geosystems.

Biominerals were studied during scientific surveys in the Arctic and the Antarctic, as well as the Caspian, the Black and the Aral seas. Biominerals

in ferromanganese nodules of the Clarion-Clipperton Fracture Zone (Pacific

Ocean) were also studied [1]. The sampling of biominerals was carried out from the ice cover, water column, bottom and coastal sediments; temperature

and salinity of the water column were measured in situ. Laboratory research

included light and scanning electron microscopy, electron probe microanalysis, X-ray diffraction, geochemical analyses,

micropaleontological and microbiological studies.

It was found out that specific associations of authigenic biominerals are formed as a result of biogenic and abiogenic interactions in each studied

geosystem. However, some biominerals, such as opal, framboidal pyrite (FP)

and some Fe-Mn oxyhydroxides behave like «cosmopolitans». Opal is formed under different extreme conditions and in different mineral

associations, including cryogenic and chemogenic minerals. Thus, opal

frustules of diatoms are formed in the ice cover and under-ice water of the Central Arctic Ocean [2]. In the hypersaline residual basins of the Aral Sea

(the Lake Tshchebas and the Chernyshev Bay) with a high content of

hydrogen sulfide and methane [3], opal is actively generated in the frustules of euryhaline diatoms in the water mass and involved in the structure of

coastal microcrystalline cortical deposits composed of chemogenic crystals

of konyaite, thenardite, blödite, eugsterite, halite, and gypsum. FP was found in bottom sediments of the areas under the variety of extreme

factors, such as low temperatures, high concentrations of hydrogen sulfide and methane [2, 4-6]. Influence of the hydrological and biogeochemical

conditions on the depth of formation, size, morphology, and structure of FP,

as well as the type of mineral/organic substrate, was determined for each studied area.

«Endemical» biominerals, such as discovered vivianite and polycrystalline

Mg-calcite are formed in very specific conditions during the diagenetic transformation of organic and mineral matter. Thus, in association with

aragonite and FP, Mg-calcite forms microcrystalline cement of the carbonate

concretions at a cold methane seep site in the Laptev Sea [6]. The results of the study can be used in the interpretation of the oceanographic

data, paleogeographic reconstructions, environmental monitoring, mining of

the oceanic ore deposits and development of new biocomposite materials. Acknowledgments. The work has been realized in the framework of the state

assignment (theme № 0149-2019-0004).

References [1] Reykhard, L.Ye., Shulga, N.А. (2019). Ore Geology Reviews. Vol. 110.

102933

[2] Reykhard, L.Ye. et al. (2018) Acta Cryst. A74, e250 [3] Izhitskaya, E.S. et al. (2019). Environmental Research Letters. ERL-

105929.R2

[4] Novichkova, Ye.A. et al. (2017). Doklady Akademii Nauk, Vol. 474, No. 3, pp. 365–369

[5] Kozina, N.V. et al. (2018). Russ. J. Earth. Sci., 18, ES6003

[6] Kravchishina, M.D. et al. (2017). Oceanology. V. 57. No. 1. P. 174–191

P 105

Biomineralization plasticity can maintain mechanical stability

of scallop shells exposed to ocean acidification and warming N. Lagos*1, A. Rodriguez-Navarro2, J. Vivanco3, C. Garcia4, C. Duarte5, M. Lardies6 1Universidad Santo Tomas, Centro de Investigación e Innovación para el

Cambio Climático, Santiago, Chile 2Universidad de Granada, Petrology and Mineralogy, Granada, Spain 3Universidad Adolfo Ibañez, Bioingenieria, Viña del Mar, Chile 4Universidad de Santiago de Chile, Ingenieria Mecanica, Santiago, Chile 5Universidad Andres Bello, Dept. Ecología y Biodiversidad, Santiago,

Chile 6Universidad Adolfo Ibañez, Fac. Ingeniería y Ciencias, Santiago, Chile

Ocean acidification (OA) is projected to impact the physiology and shell

carbonate precipitation in mollusks. However, warming may confer resistance to these impacts, and mollusks may trade-off growth and

calcification for maintain shell functionality under the influence of climate

stressors. In this study, we test this hypothesis by assessing the organic composition, crystallography and mechanical properties in shells of

Argopecten purpuratus juvenile scallops exposed to increased temperature

and pCO2-driven ocean acidification. Shell organic matter (%, TGA) increased at cold (14°C) and acidified (7.7) seawater condition. Discriminant

function analysis indicates that organic composition of the shells shown

systematic differences among treatments, with high levels of success (93%) in reclassifiyng the individuals to the combination of

temperature/acidification treatment at which were exposed based on their

organic composition data (ATR-FTIR). Amide and carbonate groups showed significant increments under warming (18ºC) and acidification conditions,

while sulphates and polysaccharides decreased under the same conditions.

63

Crystallographic orientation (XRD) of mineral phase was also variable, showing a significant reduction in the angular spread at the

warming/acidification treatment. In spite these changes in the biopolymer

and mineral phases of the shells of A. purpuratus, the structural resistance (elastic module) do no was affected, but a significant reduction in

microhardness was recorded in individuals shell exposed to

cold/acidification conditions. SEM and micro-CT observations indicate that exposure to these conditions promotes the erosion of the shell periostracum

and dissolution of microstructures associated to shell ribs of the outer surface.

Our results suggest that plasticity in both organic composition as mineral phases of the A. purpuratus shells could be a general compensatory

mechanism to confront climate stressors in order to maintain the shell macro-

structure functionality, but micro-scale dissolution and lesser hardness may compromise further ecological functions and the overall adaptive response.

P 106

Impedance-spectroscopy in biology and health-science I. Tobehn-Steinhäuser*1, A. Winzer1, T. Frank1, S. Herbst1, S. Görland1, T.

Ortlepp1 1CiS Forschungsinstitut für Mikrosensorik, Design, Erfurt, Germany

Introduction

We are partners for industrial research and development of silicon-based

sensors. The main areas of focus are MEMS and MOEMS with highest

stability and reliability and solutions for special photonic detectors and

detector arrays, as well as radiation and particle detectors. You will receive

customized solutions for your sensor systems as well as production in small

series and the qualification of reliability and service life. The contributions show the possibilities of impedance spectroscopy (IS) and

fluorescence in the field of microsystems technology with regard to

biological and medical applications.

Objectives

With the help of IS three projects were worked on. The first involved the

online determination of the diclofenac concentration in wastewater without major laboratory work (BioSam). Another project was the online detection

of mastitis pathogens in cow's milk directly at the milking process (Mastitis).

In addition, a project was conducted to enable the testing of new products for biocompatibility using IS and mouse fibroblasts (3DCellSens).

Materials & methods

To detect toxins, simple biological systems such as yeast cells are suitable (BioSam). These can be genetically modified so that they emit fluorescent

light when certain substances appear. To properly quantify this detectable

light, the number of yeast cells must be known. For this purpose one uses in this case the IS. In order to be able to combine both sensor principles,

interdigital structures made of ITO were realized on glass substrate, so that

the light can be detected simultaneously throw the optically transparent

structure.

In the project Mastitis the pathogens should be detected directly at the

milking process. without too much laboratory infrastructure. The structure consists of different variants of a switchable electrode array. It is possible to

interconnect random patterns to adapt the electric field to the measuring task (cell geometry). The switchable electrodes enable a fast changeover of the

electrode geometry. This makes it possible on the one hand to hide the

properties of the cells from the measurement signal (small electrode distances) and, on the other hand, to measure the polarization of the cells

directly (interconnection to electrode blocks).

From the behavior of certain biological cell cultures on different environmental influences and substances, conclusions can be drawn and on

the interactions in other applications. An important task here is the

determination of the biocompatibility of materials. The given new method allows the evaluation of biocompatibility already during the experiment. The

measuring device consists of a sensor with evaluation electronics and records

the data by means of IS. The basis for this is formed by adhesion coupling between the cells and the sensor. Vital cells behave like an insulator in the

nutrient medium, which binds to the cell carrier through adhesion. Upon the

onset of cell death, the cell membrane breaks and, as a result, the adhesive

contacts disapear.

The cell no longer acts as an insulator. As a result the impedance changes. 4.

Results Fully functional samples or prototypes could be produced in all three

projects. The results will be shown during the conference.

Conclusion

IS, also in combination with other measuring methods, is one powerful

method to caracterise biological and medical systems. With our contribution,

we may be able to show a way to solve an existing measurement task.

P 107

Fluorescence lifetime- sensor components I. Tobehn-Steinhäuser*1, A. Winzer1, C. Möller1, C. Heinze1, H. G.

Ortlepp1, T. Ortlepp1 1CiS Forschungsinstitut für Mikrosensorik, Design, Erfurt, Germany

Introduction

Determination of fluorescence lifetime is a key tool in the investigation of

cell metabolism and cell growth. The time decay of the fluorescence of

functionalized dyes facilitates the understanding for instance of intracellular temperature fluctuations originating from chemical processes within the

cells.

Objectives

For the development of our sensor components we establish the following

key requirements:

The excitation of the fluorescent dyes must be done with light

pulses as short as 1 nanosecond. And the excitation wavelength

must be about 370 nm, i.e. suitable functionality confirmation with a dye.

Our design of the optics module makes it possible to measure an

array of probe chambers in titer plates in parallel. Therefore the

width must not exceed 8 mm.

As detectors silicon photomultipliers (SiPM) are used which also

enable for miniaturization and thus for parallel measurements.

An application-specific integrated circuit (ASIC) allows for a fast

and accurate collection of the fluorescence decay statistics.

Materials & methods

The optical behavior of the lighting module was simulated by a ray tracing software tool (ZEMAX). For this purpose, a model of the UV 370nm LED

was established and adjusted to the measured LED radiance. For beam

shaping, UV antireflection-coated quartz lenses were used. The mounting of the optical elements (lenses, filters and aperture) was done with 3D printed

brass housing. As excitation filter a commercial bandpass filter is used.

A UV enhanced photodiode was integrated on the pulse excitation component for monitoring the radiated power of the LED.

For the fast determination of the fluorescence lifetime, we designed an ASIC

for the SiPM detectors. The functionality of the excitation module as well as the detection module is

demonstrated with the fluorescence dye ATTO 390.

Results

The possibility of generating nanosecond pulses with a LED is known in the

literature. However, the existing concepts need 200 to 400 V as supply

voltage for the avalanche transistors used in the circuitry. Our solution only needs 9 V of supply voltage, which is of benefit for assembling a compact

array of such sensors. The design allows a pitch of the PCB of 8 mm.

The generated light pulse of the LED was experimentally determined via a time-correlated single photon counting (TCSPC) measuring. The FWHM of

the LED pulse determined from the measurement is around 1 ns.

The functional test of the excitation component yielded a time constant of τ = 4.6 ± 0.3 ns for the used fluorescent dye ATTO 390. This value agrees

very well with τ = 5.0 ns given by the manufacturer.

The ASIC performance was determined and improved by simulations. Which suggest a high suitability for fluorescent lifetime measurements. Key

parameter is a time measurement accuracy of 80 ps and a measurement time

of up to 20 ns.

Conclusion

An illumination unit for fluorescence lifetime determination operates at a

wavelength of 370 nm and has a pulse width of around 1 ns. The width (8 mm) of the entire module is suitable for parallel measurements on titer

plates. As a confirmation example, the fluorescence lifetime of the dye

ATTO 390 was determined. Further, an ASIC for the TCSPC measurement with a SiPM was developed. The ASIC specifications as well as the first real

test results are discussed.

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P 108

Extracellular vesicles involved in the guinea fowl eggshell

quantitative proteomics yield new findings related to its unique

structure N. Le Roy1, L. Combes-Soia2, V. Labas2, A. Rodriguez-Navarro3, M.

Hincke3,4, Y. Nys1, J. Gautron*1

1French National Institute of Agricultural Research (INRA), Bird Biology

and Poultry, Nouzilly, France 2French National Institute of Agricultural Research, Physiology of

Reproduction and behaviour, Nouzilly, France 3Universitad of Granada, Departemento de mineralogia y petrologica, Granada, Spain 4University of Ottawa, Department of cellular and molecular medicine,

Ottawa, Canada Question The Guinea fowl (Numida meleagris) eggshell is a bioceramic material with

the remarkable mechanical property of being twice as strong as the chicken eggshell. In both species, the eggshell is composed of 95% mineral in the

form of calcite polymorph and 3.5% organic matrix including shell

membranes. However, the eggshell ultrastructure and microstructure are significantly different between these species. In the chicken, the eggshell is

made of columnar calcite crystal units arranged vertically. In the Guinea

fowl, the same crystal architecture is observed in its inner half, followed by a dramatic change in crystal size and orientation. The unique ultrastructural

characteristics of Guinea fowl eggshell confer a superior resistance to

breakage compared to eggshells from other bird species. In order to understand the underlying mechanisms controlling the formation and

structural organization of this highly resistant material at five key stages of

the mineralization process (4 h, 10 h, 11 h, 12 h and 18 h post-ovulation).

Methods FormingGuinea fowl Eggs were sampled at five stages: 4 h (n=5), 10 h (n=6),

11 h (n=6), 12 h (n=6) and 18 h (n=6) post-ovulation (p.o., i.e. time after the previous egg) when the nucleation sites appear and early mineralization starts

(4 h p.o.), or just before (10 h p.o.), during (11-12 h p.o.) and after the shift

in crystal orientation (18 h p.o.). The organic matrix was extracted from each eggshell and analyzed by a bottom-up quantitative proteomics approach. We

the used statistical and bioinformatics tools to determine the protein related

to the main structural shift and their functiuons.

Results The present work is the first Guinea fowl eggshell proteomic study, which

allowed the identification of 149 proteins. Comparison of the Guinea fowl eggshell proteome with that of other bird species leads to the identification

of 9 proteins that are only present in Guinea fowl. Among them Protein S100-

A6 and GDF6 are notable as they exhibit potential functions related to shell mineralization and especially at the point of the modification in crystal size

and orientation that confers remarkable strength on the Guinea fowl eggshell.

In addition to these two proteins, we also report that 61 proteins are more abundant during the secondary nucleation events associated with the change

in crystal orientation and the formation of the new layer. Additionally, we

identify the most abundant proteins involved in the different phases of Guinea fowl shell formation, from the first events of biomineralization until

the deposition of the new layer. Our study showed that proteins associated

with early mineralization are similar in chicken and Guinea fowl, but also revealed candidate proteins, which may be involved in a dramatic shift in

eggshell microstructure that is unique to Guinea fowl. The proteins more

abundant during the shift were ANXA2, S100A6, CALB1, TSKU, FAM20C, GPC-4, DCA-1-like and GDF6. Amongst them are calcium binding proteins,

protein cores of proteoglycans, proteins involved in the regulation of proteins

driving the mineralization.

Conclusion This is the first proteome survey in Guinea fowl eggshell which exhibits

exceptional mechanical proeprties. These data enriched the huge number of matrix proteins identified in various bird"s eggshell proteomics studies and

will allow genomic improvements and will give insights for material sciences. Genes coding matrix proteins will be used as biological markers for

genomic selection to reinforce eggshell breaking strength. The corresponding

transcripts will be associated with published and private SNPs and mapped in QTLs related to shell quality. They will constitute candidate genes to gain

precision for genomic selection to reinforce shell mechanical properties.

Industrial ceramics are made in high temperature and pressure. Material science explores the biomineralization to investigate how living organisms

build their shell in physiological conditions. Amongst various biominerals,

the bird"s eggshell are the most widely documented. Information on shell matrix proteins and how they contribute to the mechanical properties, gives

a chance to establish a list of natural organic compounds of benefit usable in

the fabrication of calcium carbonate materials/ceramics.

65