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Effects of Bio-Ash Amendments on the
Metabolism of Ectomycorrhizal Fungi
– A Method Development and
Metabolomic Study
Ana Paola Vilches
Main supervisor: Professor Dan Bylund
Co-supervisor: Ph.D. Sara Norström
Faculty of Science, Technology and Media
Thesis for Doctoral degree in Chemistry
Mid Sweden University
Sundsvall, 2019-01-11
ii
Akademisk avhandling som med tillstånd av Mittuniversitetet i Sundsvall framläggs
till offentlig granskning för avläggande av filosofie doktorsexamen i kemi fredagen
den 11:e januari 2019, klockan 10:15, i sal O102 , Mittuniversitetet Sundsvall.
Seminariet kommer att hållas på engelska.
Effects of Bio-Ash Amendments on the Metabolism of Ectomycorrhizal Fungi – A Method Development and Metabolomic Study
© Ana Paola Vilches, 2019
Printed by Mid Sweden University, Sundsvall
ISSN: 1652-893X
ISBN: 978-91-88527-77-6
Faculty of Science, Technology and Media
Mid Sweden University, SE-851 70 Sundsvall, Sweden
Phone: +46 (0)10 142 80 00
Mid Sweden University Doctoral Thesis 290
iii
Oh que lindo eres librito
Junto a mi siempre te tengo
Me deleitan tus monitos
Y tus versos los aprendo
Anónimo
iv
Acknowledgement
Now that this project is complete, I can confirm that it has been a
very, very special journey. As in any journey of this nature, a union of
forces has made it possible for me to get here. I am very grateful to
many people:
Dan Bylund for giving me the opportunity to be a part of this project.
Thanks for the direction and the continuous discussions about the
many methods that I used in some way during all these years.
Sara Norström for your guidance, especially since you came back
from Umeå and for the contacts you made there, which made the
untargeted metabolomic study possible.
Petra Fransson, for sharing your knowledge in mycology, and for the
enthusiasm and will that you have shown from beginning to end.
Christine Gallampois, you are “simply the best”. You did an excellent
work and your way of explaining metabolomics made our discussions
so good. Your words were always so encouraging… cheers!
Madelen Olofsson for your knowledge and contribution, especially in
the siderophore project.
Charlotta Lindberg (SCA) for managing the ashes for my project and
for always being kind in answering my questions… by mail or by
telephone.
Håkan Norberg because you are so helpful; I guess you are tired of
reading the same sentence in every thesis… but it is true.
Matt Richardson, for the language revisions to all the manuscripts
and this thesis work. Thank you for your time and immediate
availability.
Håkan Edlund, for the factual review and useful comments to this
thesis work.
Christina Olsson, for being so thorough in the process of preparing
the thesis for publication.
v
Anna Haeggström thanks for your help with the many different
administrative and layout questions that you and only you can fix.
Dan, Sara and Madde: it has been an adventure (which I don´t regret)
to belong to the Environmental Analytical Chemistry group.
To all my colleagues: thanks especially for all the interesting lunch
conversations about everything but work, but also about work.
Special thanks to Johan, Niklas and Ran for answering all my
questions about Matlab, Excel, layout, etc. Fredrik C. for all the time we
spent with the bioactivity project. Although no article was produced, I
learned a lot. To Nassima for sharing the office and scary stories with
me, time at work has been funnier since then. To Krister A., Krister H.,
Benny T., Javier B. and Staffan R. from the electronic department, for the
most interesting conversations all these years. A special thanks to Britta
A., Karolina U. and Susanne B. for being at my side when I needed it
most.
To all my Spanish-Latin related speaking colleagues and friends
(you are plenty and cannot name you all ☺!). Because you make life in
Sweden much enjoyable! Mabel J. and Paola R. because you are so good
friends. Maria S, aunque ya no estés en Suecia, siempre te recordaré.
Por todos ustedes Salud!
Pamela, my sister, because no matter what, you are always on the
front line. Jorge, my brother, despite the distance, every day I think of
you. Naomi, my mother, somehow we will celebrate. Jorge, my father,
hoy puedo escribir el poema que aprendí de ti.
Family Berglund-Lodin, Sveriges bästa grannar. Because you have
been my rescuers time after time.
Lillemor and Bosse for all the logistic help during these years.
Hans-Erik, Alexander, Ian and Jack, because you are my family. For
the support I receive from you, I dedicate this work to you. Les amo
con todo mi corazón. Jag älskar er med hela mitt hjärta.
Whatever happens next, Salud!
vi
Table of contents
Abstract ................................................................................. viii
Sammanfattning ...................................................................... x
List of papers ......................................................................... xii
Author contributions ............................................................ xiii
1 Introduction ........................................................................... 1
2 Background ........................................................................... 2
2.1 Soil and forest ecosystem ........................................................................... 2
2.2 Role of mycorrhiza ....................................................................................... 3
2.2.1 Types of mycorrhiza ............................................................................. 4
2.3 Metabolites in the rhizosphere .................................................................... 6
2.3.1 Low molecular mass organic acids (LMMOAs) .................................... 6
2.3.2 Amino acids (AAs) ............................................................................... 7
2.3.3 Hydroxamate siderophores (HSs) ........................................................ 9
2.4 Effect of harvesting and acidification of forest ecosystem .................... 10
2.5 Management of acidification in the forest ............................................... 12
2.5.1 Wood ash composition ....................................................................... 12
2.5.2 Mechanism of ash action ................................................................... 14
2.5.3 Ash effect on soil biochemistry in the short and long term .......... 15
3 Existing analytical methods used ..................................... 18
3.1 Instrumental set up .................................................................................... 18
3.1.1 Liquid chromatography ...................................................................... 19
3.1.2 Interface ............................................................................................. 21
3.1.3 Mass spectrometry ............................................................................. 23
3.2 Analysis performed with existing methods ............................................. 26
3.2.1 Analysis of low molecular mass organic acids ................................... 26
vii
3.2.2 Analysis of hydroxamate siderophores .............................................. 26
3.2.3 Element characterization of stock ash solution .................................. 27
3.2.4 Metabolomics ..................................................................................... 29
3.3 Statistics and software .............................................................................. 32
4 Method development (Papers I and II) .............................. 33
4.1 Development of a method for analysis of amino acids (Paper I) ........... 33
4.1.1 Optimization of parameters and discussion ....................................... 34
4.2 Gallium exchange for siderophore screening (Paper II) ......................... 39
4.2.1 Work flow of the proposed screening method .................................... 41
4.2.2 Results and discussion ...................................................................... 43
5 Effect of ash on the metabolism of ectomycorrhizal fungi (Papers III and IV) .................................................................. 47
5.1 Fungal strains used in the studies ........................................................... 47
5.2 Ash solution used in the study ................................................................. 48
5.3 Experimental culture set up ...................................................................... 48
5.4 Targeted metabolomics (Paper III) ............................................................ 49
5.4.1 Parameters included in the study ....................................................... 49
5.4.2 Results and discussion ...................................................................... 50
5.5 Untargeted metabolomics (Paper IV) ........................................................ 54
5.5.1 General results ................................................................................... 55
5.6 Conclusions of the ash studies ................................................................ 59
6 General conclusions and future research ........................ 61
7 References .......................................................................... 63
viii
Abstract
Forest ecosystems have played a fundamental role in the development of
our society. Since the beginning of the civilization, forest have provided us
with wood as a product for construction, tools, furniture and domestic
heating. The well-being of the forest is therefore fundamental to our existence.
Today, our growing societies have increased energy needs; the resulting
depletion of fossil reserves and the effects of their use has again shown how
the forest is among the most important alternatives for sustainability of our
ecosystem. In order to responsibly make this resource a key part of our energy
and material supply, we need to understand how forestry practices influence
the different processes taking place in the forest ecosystems.
The use of raw material from forest as energy source produces huge
amounts of ash. The ash contains the base cations that have once been
translocated from soil to the upper parts of the trees. Ash recycling has
therefore been suggested as a measure to counteract soil acidification due to
extensive harvest. Since spreading of ash can have great effects on the forest,
it is important to understand which these effects are and how big they might
be.
This thesis focuses on the effects that such an ash recycling may have on
the metabolism of ectomycorrhizal fungi; that is, fungi that are able to
colonize root of trees, and contribute to the acquisition of nutrients and water
from soil. The work presented here utilized an in vitro metabolomic approach
on eight species of ectomycorrhizal fungi normally found in boreal forests. A
targeted metabolomic study addressed the effects of ash amendments on growth,
external pH and the exudation of low molecular mass organic acids, amino
acids and hydroxamate siderophores. This was complemented by an
untargeted metabolomic study to address the effects of ash amendment on the
general metabolism of the fungal species.
Analyses were performed with well-established chemical methods, and
some that had to be developed specifically for this thesis work. A method for
the analysis of amino acids without derivatization and yet compatible with
mass spectrometry had to be developed and validated. The result was a robust
ix
method that works well with external calibration, shows good long-term
stability, relatively low detections limits and high sample throughput. A
screening protocol for the determination of siderophores from mass
spectrometry data was also established.
The metabolomic studies showed that bio-ash amendment increased the
exudation of low molecular mass organic compounds from all the studied
species. This means that the species tended to exude more of the same
compounds compared to the controls without ash. In some cases, the bio-ash
also triggered the exudation of new compounds. There was some exceptions,
though; bio-ash amendment had negative effects on the exudation of certain
metabolites, but these negative effects were of lower magnitude compared to
the positive effects.
Both metabolomics studies showed a differentiation between the
ascomycetes and the basidiomycetes species. The targeted metabolomic study,
indicated a trade-off in the utilization of carbon for accumulation of biomass
or for the exudation of low molecular mass organic compounds, in which the
ascomycetes accumulated more carbon as biomass compared to the
basidiomycetes. According to the untargeted metabolomic study, the
ascomycetes species presented the greatest number of metabolites that were
influenced significantly by ash treatment, either as increase or decrease.
Adding extracted ash to the culturing medium at the beginning of the
experiment increased the pH, but this was counteracted by species
metabolism as exudation of organic acids correlated with a drop in external
pH. Ash treatment triggered the total exudation of low molecular mass
organic acids in five of the eight studied species and especially in Cortinarius
glaucopus. Ash treatment also triggered the exudation of amino acids from
Tomentellopsis submollis and ferricrocin from Hymenoscyphus ericae.
Of note was that no metabolite significantly influenced by ash was found
to be common to all species, indicating that the ash amendments mainly
affected the secondary metabolism under the culturing conditions used.
Additionally, the ascomycetes Hymenoscyphus ericae exuded the greatest
number of metabolites affected by ash that were exuded only by a single
species. Conversely, Piloderma olivaceum exuded the largest number of unique
metabolites not influenced by ash.
x
Sammanfattning
Skogsekosystem har genom alla tider spelat en central roll i utvecklingen
av vårt samhälle. Trä för konstruktioner, möbler och hushållsuppvärmning
har varit tjänster från skogar sedan civilisationens början. Skogens
välbefinnande kan därför anses grundläggande för vår existens. Idag har vårt
samhälle utvecklats och behovet av skogsråvara ökar kontinuerligt. Detta i
kombination med minskande fossila reserver och den inverkan som
användningen av dessa reserver har haft på vår miljö har återigen gjort
skogen till ett av de viktigaste alternativen för att åstadkomma en hållbar
utveckling. Detta innebär att vi måste öka vår kunskap om hur olika former
av skogsbruk påverkar miljön i skogsekosystemen.
Användningen av skogsråvara som energikälla ger upphov till stora
mängder aska. Askan innehåller höga halter av de baskatjoner som en gång
har transporteras från jorden till de övre delarna av träden. Återföring av aska
har därför föreslagits som en skogsbrukspraxis för att motverka den
försurning som en omfattande avverkning kan ge upphov till. Eftersom
återföring av aska kan ha stora effekter på skogen är det viktigt att förstå vilka
dessa effekter är och hur omfattande de kan vara.
Denna avhandling fokuserar på de effekter som askåterföring till
skogsmark kan ha på metabolismen hos ektomykorrhizasvampar, det vill
säga svampar som kan kolonisera rötter och bidra till trädens upptag av
näringsämnen och vatten från jorden. Arbetet uppnåddes med ett in vitro
metabolomiskt tillvägagångsätt på åtta arter av ektomykorrhizasvampar som
förekommer normalt i boreala skogar. En riktad metabolomisk studie
genomfördes för att studera effekterna av olika doser av aska på tillväxt, yttre
pH och utsöndring av lågmolekylära organiska syror samt aminosyror och
hydroxamatsideroforer. Denna studie kompletterades med en mer generell
metabolomikstudie där effekterna av aska på svamparnas totala metabolism
studerades.
I ett arbete som detta är det viktigt med tillförlitliga metoder. En stor del
av detta arbete har varit att utveckla nya kemiska analysmetoder. En metod
för analys av aminosyror utan derivatisering och samtidigt kompatibel med
xi
masspektrometrisk detektion behövde utvecklas och valideras. Detta
resulterade i en robust metod som fungerade väl med extern kalibrering och
uppvisade bra detektionsnivåer samt förhållandevis hög genomströmning.
Vidare så utvecklades även en metod för att kunna upptäcka förekomst av
låga halter av sideroforer i komplexa prover med stöd av masspektrometri.
De metabolomiska studierna visade att exponering for bioaska ledde till
ökad utsöndring av lågmolekylära organiska föreningar hos alla studerade
arter. Huvudsakligen tenderade svamparna till att utsöndra mer av samma
föreningar som registrerades för kontrollerna utan tillförsel av aska. I vissa
fall ledde exponeringen även till utsöndring av nya föreningar. Det fanns
också undantag där askan snarare hade negativ inverkan på utsöndringen av
vissa metaboliter. Generellt var dessa negativa effekter av lägre magnitud än
de positiva effekterna. Båda metabolomikstudierna visade en differentiering
mellan basidiomyceter och ascomyceter. Den riktade metabolomiska studien
antydde en kompromiss mellan användningen av kol för ackumulering av
biomassa respektive utsöndring av organiska föreningar med låg
molekylvikt, där ascomyceterna tycktes ackumulera mer kol som biomassa
jämfört med de basidiomyceter som ingick i studien. Den mer generella
metabolomikstudien, visade också att ascomyceterna var de arter som
utsöndrade flest antal metaboliter som påverkades av bioaska, antingen i
positiv eller i negativ riktning.
Tillsats av extraherad bioaska till odlingsmediet vid experimentets början
ledde till ökat pH, men detta motverkades med tiden av metabolismen genom
utsöndringen av organiska syror. Askbehandlingen resulterade i ökad
utsöndring av organiska syror med låg molekylvikt för fem av de åtta
studerade arterna och speciellt för Cortinarius glaucopus. Behandlingen
resulterade också i ökad utsöndring av aminosyror från Tomentellopsis
submollis och av ferricrocin från Hymenoscyphus ericae.
Ingen av de metaboliter som påverkades signifikant av bioaska visade sig
vara gemensam för alla studerade arter. Detta indikerar att under de
odlingsbetingelser som användes så var det huvudsakligen den sekundära
metabolismen som påverkades.
xii
List of papers
Paper I
Direct analysis of free amino acids by mixed-mode chromatography with
tandem mass spectrometry
Ana Paola Vilches, Sara H. Norström and Dan Bylund
Journal of Separation Science. 2017; 40: 1482-1492.
Paper II
A siderophore candidate screening protocol based on gallium exchange and
electrospray mass spectrometry
Ana Paola Vilches, Madelen A. Olofsson and Dan Bylund
Submitted
Paper III
Biofuel ash addition increases ectomycorrhizal fungal exudation in pure
culture
Ana Paola Vilches, Sara H. Norström, Madelen A. Olofsson, Petra Fransson
and Dan Bylund
Environmental Chemistry. https://doi.org/10.1071/EN18146. In Press
Paper IV
Effects of bio-ash on the exudation pattern of eight ectomycorrhizal fungi – an
untargeted metabolomic study
Ana Paola Vilches, Christine Gallampois, Madelen A. Olofsson, Sara H.
Norström, Petra Fransson and Dan Bylund
Manuscript
Papers I and III are reprinted with permission from Wiley and CSIRO
publishing.
xiii
Author contributions
Paper I
Planning, all laboratory work and data analysis. Validation of the method
together with Dan Bylund and Sara Norström. Principal author, writing in
consultation with all other authors.
Paper II
Part of the planning and all laboratory work. Data treatment together with
Dan Bylund. Principal author, writing in consultation with all other authors.
Paper III
Part of the planning and most of the laboratory work. Data treatment together
with Dan Bylund and Sara Norström. Principal author, writing in
consultation with all other authors.
Paper IV
Part of the planning and all sample preparation. Data treatment together with
Christine Gallampois, Dan Bylund and Sara Norström. Principal author,
writing in consultation with all other authors.
xiv
List of abbreviations
AAs Amino acids
ACN Acetonitrile
APCI Atmospheric pressure chemical ionization
APPI Atmospheric pressure photoionization
CE Collision energy (potential)
CXP Collision exit potential
DP Declustering potential
ECM Ectomycorrhiza
EP Entrance potential
ESI Electrospray ionization
FIA Flow injection analysis
FP Focusing potential
HILIC Hydrophilic interaction liquid chromatography
HPLC High performance liquid chromatography
HSs Hydroxamate siderophores
ICP-MS Inductively coupled plasma mass spectrometry
IE Ion exclusion
IEX Ion exchange
ISP Ion spray potential
LC Liquid chromatography
LMM Low molecular mass
LMMOAs Low molecular mass organic acids
LOD Limit of detection
LOQ Limit of quantification
MeOH Methanol
MRM Multiple reaction monitoring
MS Mass spectrometry
MS/MS Tandem mass spectrometry
NPLC Normal phase liquid chromatography
PCA Principal component analysis
pI Isoelectric point
Q Quadrupole
RP Reversed phase
RPLC Reversed phase liquid chromatography
RSD Relative standard deviation
xv
SIM Single ion monitoring
SPE Solid phase extraction
SRM Selected reaction monitoring
TOF Time of flight
UHPLC Ultra high performance liquid chromatography
WTH Whole tree harvest
1
1 Introduction
Ectomycorrhizal associations play a key role in forest ecosystems, as they
are involved in tree nutrition. At the same time, it is generally accepted that
the forest ecosystems are influenced by environmental perturbations resulting
from human activity.
Recycling of bio-ash through its spreading in forests has been proposed as
a measure to counteract soil acidification resulting from extensive harvesting.
Despite numerous studies addressing the effects of ash recycling on the
recovery of acidified soils and microbial communities, little is known about
the effects of ash recycling on the metabolism of the fungal species normally
involved in mycorrhizal associations.
The work presented in this thesis contributes to the knowledge on this
important matter. The exudation patterns of eight ectomycorrhizal fungi
species were investigated in vitro with both targeted and untargeted
metabolomic approaches. To achieve this, new methods had to be developed
which together with well established analytical methods were applied in two
metabolomic studies.
2
2 Background
2.1 Soil and forest ecosystem
Soil plays a key role in the forest ecosystem. It is essential for all plant
development, serving as a medium for the roots and as a nutrient source for
growth. Soil is also the habitat for an almost infinite number of small animals,
insects, nematodes, bacteria and fungi. Soil structure has massive influence
on hydrological properties and thus the life it supports – and which inevitably
contributes to its composition upon death. Soil is, in essence, the biggest
recycling system on earth – transforming complex material into available
nutrients [1].
Soils develop unique layers normally parallel to the soil crust known as
horizons, and these are given a single letter nomenclature. The O, A, E, B, C
and R horizons are the main horizons found in soils, in descending order. The
O horizon is the organic layer derived from dead plant and animal residues
and is common in forested areas (where it is known as the forest floor) but not
in grasslands regions. The dark-colored A horizon is the top mineral layer
containing highly decomposed humified organic matter. The E horizon, also
common to forests, is the eluviated/leached zone where clay, iron and
aluminum oxide migrate downward, leaving resistant materials such as sand
and silt. The eluviation of iron gives this horizon a grayish color. The B
horizon is the layer that receives all material from the above leached zones
and is therefore known as the illuviation zone. The C horizon has lower
biological activity and has not been weathered to any significant extent.
Finally, the R horizon supports the profile, has almost no biological activity
and exhibits very little weathering [1], see Figure 2.1.
3
Figure 2.1. Soil profile showing the different horizons. Image adapted from
Wikipedia Commons [2].
2.2 Role of mycorrhiza
A mycorrhiza is a mutually beneficial symbiotic association between
fungus and the roots of a vascular plant. In a mycorrhizal association, the
fungus colonizes the host plant at the root tissue, receiving access to simple
carbohydrates, such as glucose and sucrose that it cannot produce on its own.
The carbohydrates are translocated from their source (usually leaves) to the
root tissue and then into the plant´s fungal partners. In return, the extensive
fungal mycelium increases the plant’s water and nutrient absorption. This is
accomplished because the hyphae of the mycelium are much longer than root
hairs and so they produce greater surface area. As a consequence the hyphae
can mobilize soil minerals otherwise unavailable to the plant [3], see Fig. 2.2.
4
Mycorrhiza are found mostly in the organic layer, but as roots extend all
the way down from the O- to A- and B horizons, the presence and action of
mycorrhiza can be encountered in the entire soil profile [4]. However, the
majority of ECM fungi are found in the mineral part of the soil profile - logical
location as these organisms are specialized in the scavenging of nutrients [5].
2.2.1 Types of mycorrhiza
Colonization by mycorrhizal fungi can be extracellular (ectomycorrhiza)
or intracellular (endomycorrhiza), whereby the hyphae of ectomycorrhizal
fungi do not penetrate individual cells within the root, while endomycorrhizal
fungi penetrate the cell wall and invaginate the cell membrane (Fig. 2.3.). In
ectomycorrhiza (ECM), the fungus form a structure called mantle (or sheath)
which encloses the rootlet. The hyphae or rhizomorphs radiate from this
outwards into the substrate. Hyphae also penetrate inwards between the cells
of the root to form a complex intercellular system known as Hartig net.
Ectomycorrhiza typically form in approximately 10% of plant families
(generally woody ones) and fungi belonging to the phyla basidiomycetes,
ascomycetes and zygomycetes. Outside the root, ectomycorrhizal
extramatrical mycelium forms an extensive network within the soil and leaf
litter.
This thesis focuses mainly on fungi from phyla basidiomycetes and
ascomycetes that take part in ectomycorrhizal association with coniferous
trees. This kind of mycorrhiza are found mostly in the organic layer, but due
to their ability to mobilize minerals in rock they can also be found in the
mineral layer [4].
5
Figure 2.2. Representation of a mycorrhizal association. Artwork inspired
from diverse images in “The nature and properties of soils” [1] .
Figure 2.3. Differences between endo- and ecto-mycorrhiza formations.
Author’s illustration.
6
2.3 Metabolites in the rhizosphere
The rhizosphere is the narrow layer of soil that is directly influenced by
root metabolism. Its biomass and activity of microorganisms is enhanced
compared to the bulk soil, due to exudation compounds. Plant roots release
diverse substances such as organic acids, sugars, amino acids, vitamins, and
polymeric carbohydrates [6]. At the same time, microorganisms such as root-
colonizing bacteria and fungi can exudate sugars, polyols, low molecular
mass organic acids (LMMOAs), hydroxamate siderophores (HSs), amino
acids (AAs), peptides, proteins, antibiotics and pigments [4]. These are
produced and consumed by microorganisms in order to among other
functions, interact with plants via chemical signaling at the root vicinity [7],
aid in the acquisition of nutrients (e.g. LMMOAs and HSs) [8], enhance
tolerance to metal toxicity (e.g. LMMOAs and AAs) [9], or act as a defense
mechanism (e.g. antibiotics) [10, 11] etc.
A main focus of this thesis is the effect of bio-ash addition on the exudation
of LMMOAs, AAs and HSs by ectomycorrhizal fungi, and are described
below.
2.3.1 Low molecular mass organic acids (LMMOAs)
Low molecular mass organic acids (LMMOAs) are organic acids with a
molecular mass ≤ 300 Da containing a functional carboxylic group. They are
of either aliphatic type (mono-, di-, and tri-carboxylic acids, Fig. 2.4.),
containing unsaturated carbons, hydroxyl and ketonic functionalities, or
aromatic type with substituted forms of benzoic and cinnamic acids and most
commonly hydroxyl and methoxy substituent groups [12, 13].
The LMMOAs are produced in metabolic processes and are released into
soil from root exudates, microbial activities, and decomposition of organic
matter [14]. They are found in highest concentration in the upper organic layer
of forest soil, where they are also most rapidly decomposed [15-17]. Their
7
concentration is also significantly higher in close vicinity to roots and fungal
hyphae (in rhizosphere and mycorrhizosphere) when compared to the bulk
soil [18].
The LMMOAs play an essential role in nutrient acquisition, mineral
weathering and alleviation of anaerobic stress in roots. They can also serve as
carbon sources promoting growth of microorganisms [8] and affect sorption
and desorption [19, 20], precipitation and dissolution [21], and oxidation and
reduction [22] in soils. Additionally, LMMOAs alter soil pH [23] and directly
affect microbial activity and the growth of roots [24]. They can also form
complexes with ions in solution and on mineral surfaces. The dissociation
properties of the carboxylic groups and the number of such groups in the
molecule, determine the negative charge and stability of the ligand-metal
complexes [21, 25, 26]. The LMMOAs also play an important role in the
mobilization of trace elements and control the availability of them for plant
uptake in soil [8].
Figure 2.4. The structure of some aliphatic LMMOAs.
2.3.2 Amino acids (AAs)
Amino acids (AAs) are small molecules that are amphoteric - their charge
state can vary from positive to negative depending on the surrounding pH. In
a free AA, both the carboxyl group and the amino group of the general
8
structure are charged at neutral pH (the carboxylate portion negatively and
the amino portion positively, Fig. 2.5.). Amino acids without acidic or basic
groups in their side chain exist in neutral solution mainly as zwitterions,
having equal positive and negative charges. Every AA has a pI, which is the
solution pH at which the net charge of the amino acid is zero. Some AAs
(asparagine, glutamine, histidine, lysine, arginine, tyrosine and cysteine)
contain chargeable groups in the side chain that affect their pI [27].
As the building blocks of proteins, AAs are part of all living systems.
Twenty AAs are naturally incorporated into proteins; many can be
synthesized by the organism, but those that cannot are referred to as essentials
AAs. Others AAs exist and can be found in some proteins as well, although
these are modified from a common AA after the protein is synthesized by the
organism. Additional AAs are produced through metabolism of organisms
and function as intermediates or excretion products [28].
Figure 2.5. General structure of AAs and some examples.
9
Amino acids in soil can be found in “free” form, meaning not covalently
bound to other chemical entity, or in proteins or AA conjugates. They are key
intermediaries in the soil nitrogen cycle, and their abundance and diversity in
soil provides a status report of the processes resulting in their appearance,
namely organic matter degradation and release from cells [29-31]. On one
hand, root exudates, decomposition of organic matter by microorganisms and
exudation by these produce AAs in soil [9, 32]. On the other, plants and
microbes compete for the free AAs present in the rhizosphere [30-32], despite
their affinity for uptake of inorganic nitrogen.
2.3.3 Hydroxamate siderophores (HSs)
Siderophores are small (molecular mass ≤ 1500 Da), high-affinity iron-
chelating compounds secreted by microorganisms such as bacteria and fungi.
Siderophores are among the strongest soluble Fe3+ binding agents known,
with formation constants up to Kf 1052 [33, 34].
Hydroxamate siderophores (HSs), phenolate or catecholate siderophores
and mixed ligands are among the types produced by various bacteria and
fungi (depending on the actual functional group). Fungi produce mainly HSs,
and then mostly hexadentate ligands. Ferricromes, having a maximum of five
stereocentra, can give up to 32 stereoisomers (Fig. 2.6.) that can all act as metal
chelating agents [35]. They form six-coordinated octahedral complexes with
Fe3+, but they can also coordinate with other trivalent metal ions such as Al3+,
Ga3+ and In3+ [15, 34].
The prototype of HS is ferricrome (a tri-HS), a siderophore commonly
synthesized by several fungi species and used by bacteria if they cannot
synthesize it on their own. There are several compounds belonging to the
ferricrome family, including ferrichrome, ferrichrome A, ferrichrome C,
ferricrocin (Fig. 2.6.), ferrichrysin, ferrirubin, ferrirhodin and malonichrome
[33, 35].
10
Iron is an essential element for almost all organisms, due to its various
functions in biological processes [35]. Bacteria and fungi growing under
deficient conditions can synthesize and excrete siderophores, which can aid
in the acquisition of iron when its concentration is low. In this manner, they
fulfill their own needs but may also increase the iron availability for other
organisms that can utilize, but not produce, siderophores on their own [36].
Figure 2.6. Structure and base unit of a hydroxamate siderophore.
2.4 Effect of harvesting and acidification of forest
ecosystem
Plant metabolism generally leads to acidification. Plants need nutrients,
and a major source of these are base cations acquired by their roots from the
soil. Root cell metabolism delivers one or two protons to the soil for every base
cation that enters the root cell (Fig. 2.7.). When a system is in equilibrium, the
base cations that are translocated to the upper parts of a plant are returned to
the soil when the plant dies and decomposes. This balances acidity and make
base cations available to plants again – a recycling process. In commercially
11
managed forests, when whole tree harvesting is performed, little is left to
decompose in-situ. The concentration of base cations declines in the soil
solution and thus acidification results, with more intense harvesting resulting
in greater acidity [37, 38].
Figure 2.7. Representation of nutrient uptake by roots and respective charge
balance. The size of ions and charges has been enhanced for simplicity.
Author’s illustration inspired by diagrams in “The nature and properties of
soils”, [1].
Whole tree harvesting (WTH) in Swedish forestry has increased because of
the greater interest in renewable energy sources [39, 40] due to a progressive
depletion of conventional fossil fuels [41]. Previously, only stem wood was
harvested while branches, tips and foliage – the parts of the tree that contain
a majority of the mineral nutrients – were left to decompose at the harvest site,
returning mineral nutrients to the forest soils [39].
12
Due to intensive harvesting, the organic layer in the forest soil become
thinner, reducing available mineral nutrients and potentially future growth
[42]. In coniferous forests on acidic soils, the humus layer (the top part of the
O- horizon) is the soil layer most likely affected by WTH [38]. Several studies
and reviews comparing WTH with stem-only harvesting demonstrated that
lower soil base cation pools (Ca2+, Mg2+, K+, Na+) resulted after WTH compared
to stem-only harvesting. Additionally, H+ and Al3+ concentrations were
reportedly higher with more intense harvesting [43-46].
2.5 Management of acidification in the forest
Intensive use of forest bioenergy generates large amounts of ash. In the
past, wood ash was considered a waste product of the forest industry. Today,
sustainability goals have led to proposals to return this bio-ash to the forest.
Indeed, recycling the ash can potentially compensate for the loss of mineral
nutrients from the forest, thus neutralizing soil acidity and increasing nutrient
availability for the trees [47-53].
2.5.1 Wood ash composition
Ash is the inorganic incombustible part of the fuel left after complete
combustion, and contains the mineral fraction of the original biomass [54].
The ash composition of wood chips normally depends on the bark content of
the mixture, as the minerals are usually more concentrated there. Biomass
combustion produces two types of ashes: bottom ash and fly ash. The major
ash-forming elements in biomass of environmental significance include Ca, Si,
Al, Ti, Fe, Mg, Na, K, S and P [54, 55]. According to Nurmesniemi [56] fly ash
13
is a better plant nutrient and soil improvement agent than the bottom ash, due
to greater concentrations of Ca, Mg, K and P.
Table 2.1. Recommended minimum and maximum concentration of elements
in ash intended for spreading in the forest.
Macronutrients, g/kg DW Microelements, mg/kg DW
Element
Recommended
lowest Element
Recommended
concentrations
concentration lowest highest
Calcium 125 Boron 800
Magnesium 15 Copper 400
Potassium 30 Zinc 500 7000
Phosphorus 7 Arsenic 30
Lead 300
Cadmium 30
Chromium 100
Mercury 3
Nickel 70
Vanadium 70
Data according to the Swedish Forest Board [57].
Studies of wood ash with X-ray diffraction and infrared spectra show that
calcite (CaCO3) is the major compound in ash. Additional constituents include
lime (CaO), riebeckite ((NaCa)2(FeMn)3Fe2(SiAl)8), portlandite (Ca(OH)2),
calcium silicate (Ca2SiO4), hydrotalcite (Mg6Al12CO3(OH)16 ∙4H2O) and
serandite (Na(MnCa)2Si3O8(OH)). During the combustion of wood, organic
compounds are mineralized and the base cations are transformed to their
oxides and carbonates. Carbonates and bicarbonates predominate in
combustion temperatures under 500 °C, while oxides become prevalent above
1000 °C. After combustion, oxides are slowly hydrated and subsequently
carbonated under atmospheric conditions. Oxides can also be hydrated to
14
form hydroxides, which can subsequently react with CO2 in order to form
carbonates [58].
Ash also contains heavy metals that can have a negative environmental
impact by leaching into ground and surface waters [48]. Ash also affects
heavy metals already present in the soil, as their solubility decreases with
decreasing soil acidity [59]. Unfortunately, fly ash – which in general contains
greater amounts of metals of nutritional value - also contains greater amounts
of heavy metals (As, Cd, Cr, Cu, Hg, Pb, Ni and Zn) [56] compared to bottom
ash. On a positive note, heavy metals such as Pb, Ni and Cd, remain insoluble
due to the alkalinity of the ash [60].
The Swedish Forest Board has established the minimum and maximum
concentrations for both nutrients and heavy metals, to ensure the
sustainability of ash for spreading (Table 2.1.).
2.5.2 Mechanism of ash action
Hydroxyl and bicarbonate ions form because of the dissolution of oxides,
hydroxides and carbonates in the ash; the formed ions then neutralize the
protons in the soil solution. Simultaneously, the protons in cation exchange
sites are replaced by base-forming cations (Ca2+, Mg2+, K+ and Na+) [61].
Wood ash application may strongly affect soil texture, aeration, water
holding capacity and salinity, as the ash is essentially composed of fine
particles. It has been shown that wood ash particles swell in contact with
water and can obstruct soil pores. Consequently, this may reduce aeration and
increase water-holding capacity [58]. To minimize the negative effects of
powdered ash, pelletized or granulated forms of ash have been recommended
(Fig. 2.8.). Experiments have indicated that granulation of ash fertilizers
decreases their solubility compared with self-hardened and powdered ash
products, reducing their reactivity [62]. Additional studies have also noted
differences in the rates of dissolution between untreated, powdered ash
15
products and granulated fertilizers, and also differences between different
types of granular products [60].
Figure 2.8. Scheme showing the cycle of ash recycling to the forest. a)
Harvesting, b) ash in loose form, c) ash in pelletized form and d) ash return.
With permission of: a) Bo Norling, private image; b) Weda Skog; c) Hasse Dahlgren,
Lantbrukets Affärstidning; d) Rikard Flyckt, Skogsstyrelsen.
2.5.3 Ash effect on soil biochemistry in the short and long
term
The effects of ash recycling in the forest are both short term (1-6 years post-
distribution) and long term (18 years or more post-distribution). The
characteristics of these effects are largely dependent on the soil and tree
quality of the forest ecosystem and the physical and chemical properties of
the ash used [52, 63]. During biomass combustion, the organic material is lost,
16
and ash therefore lacks nitrogen - at least in significant amounts [64]. In
Nordic upland coniferous forests, as long as nitrogen remains the growth-
limiting nutrient, ash addition will not result in increased growth [52].
In a meta-analysis of studies on selected forest systems, it was found that
the effect of wood ash and lime on soil pH, base saturation, foliar Ca
concentration, tree growth, C:N, ectomycorrhizal fungi roots colonization,
and microbial diversity appeared highly variable [65]. A considerable number
of studies in that meta-analysis also showed no significant effect of ash
compared with controls. While the effect of wood ash application on soil
solution pH is rather weak, a consistent increase in the downward transport
of base cations has been observed. [61]. Reduced soil acidity due to ash
application affects the microbial activity and bacterial growth rates in the soil
[48, 59, 63, 66-68]. Fungal biomass, however , seems to be unaffected after
liming [69]. In general, the changes are related to the dose and form of ash
applied [63]. Although not all studies agree [70], changes in microbial activity
and community structure have been detectable 18 years after ash fertilization
[63] including decreases in the ratio of fungi to bacteria in boreal forest after
ash addition [71].
If the ash addition has the potential of altering such a wide array of soil
parameters, then it should be logically able to influence microorganism
metabolism at the exudation level. Considering the fundamental role of soil
microorganisms in C cycling in ecosystems, their responses to environmental
perturbation [72] are highly relevant. In soil ecosystems there is a constant
and complex interaction of exudates from roots and microorganisms, and the
influence is often reciprocal [73, 74]. One of the primary factors affecting the
ecosystem of the rhizosphere is the quantity and quality of exudates released
by mycorrhizal associations [72]. The exudates, especially LMM organic
compounds such as LMMOAs and AAs, support diverse microbial
communities, and enhance mineral weathering and nutrient uptake [21, 72,
75, 76]. Ash amendment has been reported to increase organic acid exudation
by ectomycorrhizal fungi, but in that study, the results were limited to the
analysis of species growth and formic, succinic and malic acids only [77].
Ectomycorrhizal fungi may exude LMM organic compounds under stress by
heavy metals [9, 78-80], a finding that may have particular relevance for ash
17
distribution, as it also contain heavy metals. Ectomycorrhizal fungi exude an
array of metabolites in response to different conditions, but little is known
about the of ash’s ability to influence their concentrations in exudates. This
thesis addresses the effect of ash amendment on the metabolism of different
ECM fungi normally found in boreal forests, using both targeted and
untargeted metabolomic experiments.
18
3 Existing analytical methods used
3.1 Instrumental set up
High performance liquid chromatography (HPLC) coupled to tandem
mass spectrometry (MS/MS) was the main technique employed in this thesis
work (See Fig. 3.1.). As HPLC separations are performed at high pressure and
MS/MS detection in high vacuum, a hyphenating interface between the two
systems is required – in this thesis in terms of an electrospray ionization (ESI)
source. The respective parts of the system are described below.
Figure 3.1. Representation of a hyphenated system for LC-ESI-Q-MS/MS
analysis with a triple quadrupole instrument.
19
3.1.1 Liquid chromatography
Chromatography encompasses a group of techniques that allow the
separation of closely related compounds in complex mixtures. In a
chromatographic separation, the sample is dissolved in a mobile phase, which
may be a gas, a liquid, or a supercritical fluid. The studies in this thesis,
employed liquid chromatography (LC), and as such the mobile phase was a
liquid pumped through an immiscible stationary phase, which is fixed in a
column, on a solid surface.
The components being separated are distributed between the phases at
different degrees. Those components strongly retained by the stationary
phase move slowly with the flow. In contrast, components that are weakly
held by the stationary phase travel rapidly. The different retention times
separate the sample components into discrete bands or zones that can be
analyzed qualitatively and quantitatively [81]. There are different types of LC
depending on the separation mechanisms involved and the polarity of the
respective phases. The main types are partition chromatography, ion
chromatography, multimodal chromatography, ion-pairing chromatography,
adsorption chromatography, size exclusion chromatography and affinity
chromatography. A description of the types of LC used in this thesis is
provided below.
3.1.1.1 Partition chromatography
Two types of partition chromatography are distinguishable based on the
relative polarities of the mobile and stationary phases. In normal phase liquid
chromatography (NPLC) separation occurs by adsorption of analytes to the
polar stationary phase, which can be unaltered silica, in combination with a
nonpolar mobile phase. More polar analytes are retained for a longer period.
The other type, reversed phase liquid chromatography (RPLC) employs a
bonded stationary phase, typically consisting of hydrocarbon chains of 8 or 18
carbon atoms length (C8 and C18) covalently bound to silica spheres. These
carbon chains form a hydrophobic layer which, when combined with a polar
aqueous organic solvent mobile phase, enables the separation of analytes via
20
partitioning chromatography. More nonpolar analytes are retained for a
longer period [81]. If the porous particles are less than 50 µm in diameter and
the pressure developed is 30-400 bars, then the technique is called reversed-
phase high performance liquid chromatography (RP-HPLC). If the particles
are sub-2 µm and the pressure developed is 600-1500 bars, then the technique
is called reversed-phase ultra-high performance liquid chromatography (RP-
UHPLC) [82-84].
3.1.1.2 Ion chromatography
Ion chromatography (IC) uses columns with relatively low ion-exchange
capacity to separate ions. Ion exchange chromatography (IXC) is based on
exchange equilibria between ions in solution and ions of the same charge
interacting with the surface of an essentially insoluble, high molecular-mass
solid. The most common active sites for cation-exchange resins are the
sulfonic acid group –SO3ˉ H+ - a strong acid - and the carboxylic acid group –
COOˉ H+ - a weak acid. Anionic exchangers contain strongly basic tertiary
amine groups –N(CH3)3+OH- or weakly basic primary amine groups –
NH3+OH- [81].
Ion–exclusion chromatography (IEC) is not directly a form of ion
chromatography as the ions being separated have the same charge as the
stationary phase. Nevertheless, IEC uses ion-exchange columns to achieve
separation. It is a useful technique for the separation of ionic and nonionic
substances, in which ionic substances are strongly rejected by the resin while
nonionic or partially ionized substances are rejected to a lesser extent. For
example, when analyzing organic acids via IEC, the column has a cationic
exchange stationary phase (with sulfonic groups). The sulfonic groups are
negatively charged, and repel the anions of the organic acids. Every acid has
a specific pKa and will be present in solution in both the ionic and nonionic
forms where both forms continuously inter-exchange. The ionic forms are
strongly rejected while the non-ionic forms are rejected less. Because different
organic acids have different pKa values, the overall degree of rejection will
differ and the acids will move through the system at different speeds [85].
21
3.1.1.3 Multimodal chromatography
Hydrophilic interaction liquid chromatography (HILIC) is considered as
multimodal technique and has similarities with both NPLC and RPLC. HILIC
is used in the separation of polar analytes via hydrophilic partitioning, polar
interactions and electrostatic interactions with polar or charged functional
groups covalently bound to a silica-based stationary phase [86]. Hydrophilic
partitioning is made possible by the layer of water that is retained on the
hydrophilic stationary phase when using aqueous-organic solvent mobile
phases with high organic solvent content [87].
Mixed-mode chromatography is a new type of multimodal
chromatography in which ion-pairing groups are embedded in an otherwise
nonpolar stationary phase. The mixed–mode HPLC can thereby retain both
hydrophobic compounds (by a RP mechanism) as well as hydrophilic
compounds (by an ion-exchange mechanism) at higher organic content in the
mobile phase. Depending on the pH of the mobile phase, the functional
groups in the mixed-mode column may be ionized or nonionized [88-92]. See
section 4.1. for the use of mixed-mode chromatography in method
development in Paper I.
3.1.2 Interface
There are different types of interfaces depending on the chromatographic
technique (liquid or gas chromatography). The most common interfaces for
LC are electrospray ionization (ESI), atmospheric pressure chemical
ionization (APCI), and atmospheric pressure photo-ionization (APPI). These
interfaces produce ions from a liquid in high-pressure environment (HPLC)
and transfer them in gas form to the mass spectrometer in high vacuum [93,
94]. The interface used in this thesis is ESI and an explanation about how it
works is provided in the next section.
22
3.1.2.1 Electrospray ionization
In ESI, the eluate is introduced via a capillary into a spray chamber, and a
potential difference is applied between the tip of this capillary and the
oppositely positioned counter electrode (the entrance to the MS). Depending
on the direction of the electrical field, the applied potential induces either
oxidation (positive mode) or reduction (negative mode) of the compounds
present in the solution within the capillary. The dominant reactions in
aqueous solution involves the following redox reactions:
6 H2O (l) → 4 H3O+ (aq) + O2 (g) + 4 e- (Positive mode, low pH)
2 H2O (l) + 2 e- → 2 OH- (aq) + H2 (g) (Negative mode, high pH)
Neutral bases or acids can react with these redox products to form
positively or negatively charged ions via protonation or deprotonation [95].
Depending on the pH, these ions may also already be present in the solution.
Furthermore, non-covalent associations between neutral and charged species
present in the solution may form the analyte ions in the ESI. This can produce,
for example, NH4+ or Na+ adducts in positive ESI mode [96].
Figure 3.2. Functioning of the electrospray ionization process [97].
23
The charged droplets produced in the so-called Taylor cone drift through
the air down field, towards the counter electrode. Solvent evaporation at
constant charge leads to the droplets´ shrinkage and an increase of the electric
field normal to the surface of the droplets. At a given radius, the increasing
repulsion overcomes the surface tension. Droplet fission occurs followed by
repeated solvent evaporation and repeated fission. The sequential fission
ultimately leads to gas-phase ions, which are accelerated to the entrance of the
MS system [98] (Fig. 3.2.). This process might also be assisted by a jet of
nebulizing gas along the tip of the capillary.
3.1.3 Mass spectrometry
A MS instrument contains a mass analyzer, which is the part of the system
where the separation by mass occurs. There are several types of MS
instruments depending on the mass analyzer, including time of flight MS,
double-focusing MS, ion-trap MS and quadrupole MS. Next is a brief
description of time of flight MS and then, a more detailed description of
quadrupole MS, the mass analyzer mainly used in this thesis work.
In the time of flight MS (TOF-MS), ions are accelerated by an electric field
and then passed into a field-free drift tube. In this tube, the ions are separated
according to their velocity. Ions entering the tube have (ideally) the same
kinetic energy, so their velocity in the tube varies inversely with their masses,
with the lightest masses arriving earlier to the detector than the heavier ones.
A quadrupole MS (which is the kind of analyzer used in the method
development described in Paper I) contains four parallel cylinders (rods) that
are connected in such a way that the pairs of opposite rods are connected to
two dc current sources, one negative and the other positive, as shown in Fig.
3.3. The ions being separated are accelerated in the space between the rods in
the direction to the detector due to a potential difference. As the ions travel
between the rods an ac current is superimposed to the pairs of opposite rods
changing the polarity of them. At the same time, the ac and dc voltages are
24
increased simultaneously maintaining the ratio. This make possible that, at
any certain moment only a specific m/z is able to stay in trajectory in direction
to the detector. All other ions will strike the rods and will be neutralized
[81].
Figure 3. 3. Representation of a quadrupole analyzer visualizing the ion
trajectory.
A single quadropole can be run in full scan mode or selected ion monitoring
(SIM). In full scan mode, all analytes in a specific m/z interval are detected. In
SIM only one specific m/z is detected. The hyphenation of three different
quadrupoles in what is called tandem mass spectrometry (MS/MS) is also
possible (Fig. 3.1.). The first, second and third quadrupoles are denominated
Q1, Q2 and Q3 respectively. From these, Q2 acts as a collision chamber filled
with a gas (nitrogen, helium, argon or xenon) where ions are activated by
collision and induced to fragment. There are four different ways of running
(MS/MS) with a triple-quadrupole: 1) In product ion scan, ions of a given m/z
are selected in Q1 and passed to Q2 where they are fragmented. Then, the
25
fragments are passed and scanned in Q3. A product ion spectrum called
daughter ion spectrum is obtained. 2) In precursor ion scan, Q1 is scanned over a
range of m/z and every ion passes on to Q2 and then fragmented. Q3 is set to
a specific m/z, which is the only ion allowed to reach the detector. Ions that
pass through Q1 are detected if they after fragmentation produce the pre-
selected m/z in Q3. 3) In neutral loss, Q1 and Q3 are scanned simultaneously
but with a constant m/z difference. All analytes scanned in Q1 pass through
Q2 and are then fragmented. Then, all fragments produced for every analyte
are scanned in Q3. This allow the selection of all ions, which, by fragmentation,
lead to the loss of a given neutral fragment. 4) In selected reaction monitoring
(SRM), Q1 is set to a specific m/z, which passes to the Q2 where it is
fragmented. The fragments are then passed to the Q3 where only a specific
m/z is selected to reach the detector [99]. Multiple reaction monitoring (MRM)
is an application of SRM to multiple product ions from several precursor ions
[100].
Independently of the mode used, the voltages used to produce and direct
the ions through the system must be optimized individually for every
analyte/fragment. For the instrument used in Papers I and III, these voltages
were: ion spray potential (ISP), which determines in which mode (positive or
negative) the system is run; entrance potential (EP), which serves to guide and
focus the ions into the mass spectrometer; declustering potential (DP), which
is applied to the entrance orifice to avoid clustering of ions; focusing potential
(FP), which focuses the beam of ions before they pass through the skimmer;
collision energy (CE), which refers to the rate of acceleration of the ions
entering into Q2; collision exit potential (CXP), which focuses and accelerates
the ions from Q2 into Q3. After separation in the mass analyzer, the ions are
directed into the detector that transforms the ions in an electrical signal.
26
3.2 Analysis performed with existing methods
Paper III addresses the effect of ash on ectomycorrhizal fungi exudation
with a targeted metabolomic study. The study included AAs (method
development described in Paper I), low molecular mass organic acids
(LMMOAs) and hydroxamate siderophores (HS). In Paper IV, the effect of ash
was studied in a more broadly aspect in an untargeted metabolomic assay.
Development of new methods is described in section 4. Here, additional
existing methods that were employed in this thesis are described.
3.2.1 Analysis of low molecular mass organic acids
The analysis of LMMOAs was performed using the method developed by
Bylund et al [101]. In this method, the sample is separated by ion exclusion
chromatography (see section 3.1.1.2) using a cross-linked sulfonated
polystyrene/divinylbenzene column (Supelco C610-H, 300 x 7.8 mm, 9 µm).
The injection volume was 100 µL and the pump was operated at 0.4 mL/min
with a mobile phase composed of MeOH-0.01 % formic acid (10:90). The
method employed an effluent split with a Valco tee union after which
approximately 50 µL/min were directed into the ESI interface. The system
was run in negative mode, yielding precursor ions in the form [M-H]– and
using MRM to select specific fragments. The dwell time used was 200 ms.
3.2.2 Analysis of hydroxamate siderophores
The analysis of HS was performed using the method developed by
Olofsson et al [102]. In this method, the sample was passed through a pre-
column (Hypurity Aquastar column, 50 x 2.1 mm, 5 µm) for concentration on-
line with a 10-port switch valve. After the concentration step, the sample was
27
directed into a RP chromatographic column (Kinetex C18, 100 x 2.1 mm, 2.6
µm) for separation. The switch valve was operated in two positions. A volume
of 100 µL of sample was injected into the pre-column using a loading solution
containing 99 % of ammonium formate buffer at pH 4.0 and 1 % MeOH. The
flow rate was set to 400 µL/min and the valve was set to “position 1st”. During
the first 2.5 min of loading the effluent was directed to waste. After this time,
the valve was switched to “position 2nd” and the concentrated sample was
directed into the chromatographic column, where the flow direction was the
opposite. For the chromatographic separation, the flow rate was set to 200
µL/min and the mobile phase consisted of a gradient composed of mobile
phase A (90 % ammonium formate at pH 4.0 and 10 % ACN) and mobile phase
B (100% ACN). After the column switch, the mobile phase B increased linearly
from 0 % to 10 % over a period of 6 min. B was subsequently increased linearly
from 10 % to 35% over 5 min. Then, over a 2 min period, the mobile phase B
was raised to 85 % to clean the column after which it was returned to the
original conditions (100 % mobile phase A). After the separation, the effluent
was directed into the ESI interface in positive mode to yield precursor ions in
the [M+H]+ form, M corresponding to “siderophore + Fe(III) -3H”. The system
was run in SIM with a dwell time of 100 ms, and MRM was performed for
confirmation of samples that yielded a signal in SIM mode. Analytes were
quantified via external calibration with standards of the respective analytes.
An excess of FeCl3 was added to standards and samples to ensure the complex
form between iron and the HS.
3.2.3 Element characterization of stock ash solution
The ash solution described in Paper III contained all the non-combustible
metals left after biomass combustion. To characterize the composition of the
ash solution regarding metals, inductively coupled plasma-mass
spectrometry (ICP-MS) was employed. For the remaining content of nitrogen
and phosphorous, flow injection analysis (FIA) was employed. Next is a
description of both methods.
28
In ICP, the sample, usually in liquid form, is pumped into an introduction
system composed of a nebulizer and a spray chamber. The sample is pumped
in to the nebulizer at 1 mL/min via a peristaltic pump. Argon gas is pumped
at 1 L/min perpendicular to the nebulizer flow changing the liquid into an
aerosol of tiny droplets. This aerosol is then directed through a sample injector
into the base of the plasma. The plasma has different heating zones and as the
droplets travel through the plasma, they are dried, vaporized, atomized and
ionized. By this point the sample has been transformed from a liquid aerosol,
to solid particles and finally into a gas. When the sample arrives at the
analytical zone of the plasma, at approximately 6000-7000 K, the ground state
atoms are converted to ions via collisions with energetic electrons (and some
argon ions). This process strips one of the orbiting electrons in the outer shell
of the atoms, producing positive ions. The positive ions are then directed into
an interface region, which transfer the ions from the plasma at atmospheric
pressure (760 Torr) to a mass spectrometer at vacuum (10-6 Torr). The interface
in this case consists of two metallic cones with very small orifices (at 1-2 Torr
achieved with a mechanical roughing pump). The first cone, known as the
sampling cone, has an orifice of 0.8-1.2 mm i.d. From here, the ions travel a short
distance to the second skimmer cone that has a smaller orifice (0.4-0.8 mm i.d).
The ions are then directed through the ion optics, and finally into the mass
analyzer [103], which in this thesis was a quadrupole MS. Finally, the ions
were directed into an electron multiplier for detection.
Prior to ICP analysis, the sample is normally digested to eliminate the
organic material. This was not necessary, as in this study the ash solution was
prepared from the residual material from biomass combustion. The elements
analysed with ICP were K, Na, Ca, Mg, Al, Cu, Zn, Cr, Fe, Mo, Ba, V, Ag, Se,
Ni, Mn, Sb, As, Cd, Co, Pb and U. Prior to quantification with external
calibration, both standards and samples were acidified with HNO3 to a
concentration of 2 % v/v.
In FIA, the sample is aspirated with a peristaltic pump into a carrier
solution. The sample is then mixed by a radial and convection diffusion with
one or several reagents. The mixing duration will depend on the flow rate,
and length and diameter of the coil (the tube bearing the reagent). After
29
mixing with reagents, the flow is directed into the detector, a
spectrophotometer set at a specific wavelength [104].
For analysis of N and P, the sample is digested in an autoclave for 30 min
at 120 °C with a solution containing K2SO4 and H2SO4/K2SO4. After this
oxidation procedure the sample is aspirated in the FIA instrument and mixed
with the carrier (ultrapure water), and then with specific reagents [105, 106].
In the case of N analysis, the sample is mixed with ammonium chloride buffer
at pH 8.5, and then directed in to a cadmium column for reduction of nitrate
into nitrite. The flow is then mixed with sulfanilamide where the nitrite is
transformed into a diazo compound. The flow is then mixed with N-(1
naphthyl) etylenediamine- dihydrochloride after which it is transformed into
a pink colored azo-compound which absorbs at 540 nm [107, 108]. In the case
of P analysis, the flow is instead mixed with ammonium molybdate to form
heteropoly molybdophosphoric acid and then with stannous chloride to
produce phosphomolybdenum blue which absorbs at 720 nm [109].
The quantitation of the total amount of N and P, is achieved through
external calibration of standards made of N-NO3 and KH2PO4 respectively,
which go through the same procedure as the samples.
3.2.4 Metabolomics
Metabolomics is an analytical approach for the identification and
quantification of small metabolites (<1500 Da). Mixtures of metabolites are
known as metabolomes and these can provide insight on the biological and
biochemical processes in a complex system [110].
A metabolomic analysis can be targeted or untargeted. The targeted
method focuses on a group or classes of metabolites that are related to a
specific pathway. The untargeted method is a general analysis to detect
changes in patterns in the metabolome due to diseases, environmental or
genetic perturbations. Normally, an untargeted approach is run first for
30
hypothesis generation, and then followed by a targeted approach for more
specific quantification of relevant metabolites [110-112].
Due to the complexity of the metabolome of any biological sample, a
metabolomic analysis requires multiple analytical platforms. Mass
spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and
Fourier transform infrared (FT-IR) spectroscopy are the most common
techniques used [111].
In contrast to targeted metabolomics, untargeted metabolomics datasets
are extremely complex (in the order of gigabytes per sample). In LC-MS
analysis, changes in retention time due to temperature variations, column
degradation and mobile phase fluctuation among other factors make
interpretation of data more difficult. Manual interpretation of the information
is therefore not feasible, and sophisticated software has become a routine part
of metabolomic studies [111].
This thesis included both targeted and untargeted approaches. The
targeted metabolomic study (Paper III) was performed with liquid
chromatography (LC) hyphenated with a triple-quadrupole MS/MS system
via an ESI interface. The untargeted metabolomic study (Paper IV) was
performed with LC hyphenated with time of flight (TOF) MS also via an ESI
source. The methods used in Paper III are described in section 3.2 (existing
methods) and section 4.1 (method development Paper I). The method used in
Paper IV is explained in the next paragraph.
3.2.4.1 Untargeted metabolomic assay
An untargeted metabolomic approach was used in the investigation
presented in Paper IV and included four fundamental steps: acquisition, data
processing, statistics, data prioritization and results (Fig 3.4.).
For acquisition, an instrumental set-up composed of a LC-ESI-Ion
Mobility-TOF-MS was used. The samples were run in both positive and
negative ESI modes. Sample volumes of 20 µL were first separated on a
Kinetex C18 (150 x 2.1 mm, 2.6 µm, 100 Å) column at 40 °C. Mobile phase
composition depended on the ESI mode used, but in both cases, a gradient
31
Fig 3.4. Overview of the different steps included in the untargeted
metabolomic assay described in Paper IV.
32
elution was applied. For analysis in positive mode, mobile phase A consisted
of 0.1 % formic acid in H2O and mobile phase B of 0.1 % formic acid in MeOH;
in negative mode, pure H2O as mobile phase A and pure MeOH as mobile
phase B were used. In both cases the gradient was the same, starting with 10
% mobile phase B directly followed by a linear gradient over 30 min to reach
95 % of mobile phase B. These conditions were maintained over 10 min. A
post run equilibrium time of 5 min was allowed to bring the system to the
original conditions. The flow was kept constant at 0.5 mL/min. After
chromatography, the eluent was directed into the ESI source for ionization
and then directed into the TOF part of the instrument. Mass spectra (MS) were
acquired in scan mode within a m/z range of 50-1700, and a scan rate was 4
spectra/s. The source parameters were: gas temperature at 225 °C, gas flow at
5 L/min, Nebulizer at 30 psig, sheath gas temperature at 350°C and at a flow
of 12 L/min, VCap at 4500V, nozzle voltage at 500V, fragmentor at 275 V and
the octopole at 550 V.
3.3 Statistics and software
The program Analyst 1.6 (AB Sciex, Ontario, Canada) was used for the
integration process in Papers I and III. The program LabSolutions (Shimadzu,
Tokyo, Japan) was used to obtain data in Paper II. In Paper IV, several Agilent
programs (Agilent Technologies, Santa Clara, California) were used. Features
extraction was performed with Mass Hunter Qualitative Analysis (B.07.00)
and alignment by species (creation of “cef” files) with Mass Hunter Profinder
(B.08.00). General alignment (all species) was performed with Mass Profiler
Professional (14.09). Microsoft Excel (Microsoft, Palo Alto , California) was
used for statistical evaluation in papers I, III and IV. Minitab 17.3.1 (Minitab
Inc, State College, Pennsylvania) was used for principal component analysis
in Papers III and IV. Matlab (Mathworks, Natick, Massachusetts) was used for
data treatment in Paper II.
33
4 Method development (Papers I and II)
4.1 Development of a method for analysis of amino
acids (Paper I)
Prior to the first studies in this thesis, almost all published methods for the
analysis of AAs included a derivatization step, and/or the use of ion-pairing
agents. Some methods like the one developed by Piraud et al. in 2003 [113]
used FIA-MS/MS in ESI positive and negative mode to study the
fragmentation patterns of molecules. Later the same authors published the
application of this method hyphenated with ion-pairing LC [114]. In 2008,
Thiele et al. [115] published a method with similarities to the one presented in
this thesis but the time of analysis of that method was considered too long.
Derivatization is a time-consuming process and a potential source of error
due to the possibility that the derivatization reagent may not react completely
with the AA. Further uncertainty can arise from the instability of the
derivatized compounds and interference with the sample matrices. Ion-
pairing agents are not fully compatible with the MS system as they may cause
signal suppression, and hamper the long-term stability of the MS interface.
The need for a quantitative analytical method for AA analysis that was
highly compatible with ESI-MS detection and that did not involve a
derivatization step was addressed in the method development presented in
Paper I [116]. The twenty AAs naturally incorporated in proteins, and
hydroxyproline, ornithine and cystine were included in the method, which
employed mixed-mode HPLC-ESI-MS/MS and external calibration.
34
4.1.1 Optimization of parameters and discussion
The column used (Primesep 100 from Sielc technologies, Prospect Heights,
Illinois) combined RP and IEX chromatography via a stationary phase
containing a hydrophobic bed formed by C12 carbon chains with substituted
carboxylic acids attached to them (See Fig. 4.1.). The dynamic of the separation
is more complex than in RP chromatography due to the charge state of the
substituted carboxylic acids and the charge state of the AAs, which are both
dependent upon the pH of the mobile phase. In Paper I, the best
chromatography was obtained with a pH gradient from 2.97-6.00 composed
of mobile phase A (20 mM formic acid/ammonium formate at pH 2.97) and
mobile phase B (20 mM acetic acid/ammonium acetate at pH 6.00) at the flow
of 200 µL/min.
Figure 4.1. Representation of the Primesep 100 column with a hydrophobic
bed conformed of C12 carbon chains, which in turn contain embedded
carboxylic acids.
After chromatography, the eluent was transformed from liquid-phase to
gas-phase in an ESI interface, where the AAs were ionized before they entered
the mass analyzer. The parameters for mass spectrometry were optimized
with single standards and direct infusion of the respective AAs. For
information on all the optimized parameters and more details, see Paper I.
35
The stationary phase was negatively charged during the entire gradient
due to the low pKa of the carboxylic acids embedded (according to personal
communication with Sielc Company).
Aspartic acid was the first AA eluted. At the start of the gradient, the
conditions were such that the pH of the mobile phase was close to the pI of
aspartic acid (see Table 1 in Paper I for pI values) which thus was mostly
uncharged while the majority of the other AAs were positively charged. At
this stage, aspartic acid interacted mainly with the hydrophobic part of the
stationary phase. As the gradient proceeded, the system become less acidic
and aspartic acid more negatively charged. Thus, aspartic acid started to be
repelled by the stationary phase and move rapidly through the column. As
the pH increased, the other AAs approached their respective pI, thus became
less charged. At this point, the RP interaction mechanism had a crucial role.
Since most AAs have similar pI values, their polarity played an important role
for their separation. An example of this was valine and threonine that
separated on the column even though their pI values are very similar. Most of
the AAs eluted at pH values close to the respective pI and during the first 15
min of run time. Arginine (highest pI) was the last AA being eluted (38 min),
immediately before the gradient reached pH 6 (Fig. 4.3.).
With the exception of ornithine, all the investigated AAs produced charged
fragments due to a neutral loss of H2O + CO upon collision-induced
fragmentation. In general, the fragment produced after this neutral loss was
also the most abundant one. In some cases, the neutral loss of H2O + CO + NH3
resulted in a fragment with stronger signal, and was thus selected for
quantification. Some of the possible structures produced after fragmentation
are shown in Fig. 4.2.
36
Figure 4.2. Possible structures for some of the AA product ions produced in
the collision chamber Q2.
37
Figure 4.3. Gradient program and mass chromatogram for the AAs included
in the LC-MS/MS method developed.
38
Since the chromatography was not able to separate all the AAs, the mass
analyzer was crucial, and by using MRM, almost all AAs could be quantified
without ambiguity. One exception was glutamic acid, which suffers from
isotopic interference of glutamine. This interference happens because the
difference in the respective nominal molecular masses is 1 (glutamine 146
g/mol and glutamic acid 147 g/mol). Due to the natural abundance of 13C,
which is 1.1%, it is expected that about 5 % of the molecules of glutamine have
a nominal molecular mass 147 g/mol. With this background, the interference
of glutamine signal was investigated, and it was demonstrated that for equal
concentrations of the two AAs, the interference was less than 2% of the
glutamic acid signal. This is in line with the natural abundance of the 13C and
the different response factors due to ionization and fragmentation of the two
analytes. This interference was taken into account when applying the method
to natural samples, and for the results shown in Paper III, this interference
was statistically insignificant. Leucine and isoleucine could not be resolved
completely either. The fragmentation pattern of both showed a product ion at
m/z 86 corresponding to the neutral loss of H2O + CO. However, only
isoleucine gave the ion m/z 69 corresponding to the neutral loss of H2O + CO
+ NH3. The quantification of isoleucine could thus be done directly from the
m/z 69. In the case of leucine, an interference study demonstrated that along
the entire linear range, the signal obtained for isoleucine at m/z 69
corresponded to 13.5 % of the signal obtained at m/z 86. With this information,
it is possible to calculate the proportion of the signal at m/z 86 corresponding
to isoleucine when both isomers are present in a sample. This interference
should to be taken into account when applying the method to natural samples.
However, this was not necessary for the results shown in Paper III since none
of the isomers was present in quantifiable amounts. See Paper I for more
information about other interferences, especially for cysteine and its dimer
cystine.
Linearity of the method was tested in aqueous solution and in matrix.
Injection volumes of 1 µL resulted in high linearity (r>0.99) for all AAs.
Statistical evaluation showed that the difference in the slopes of the two
regression lines was significant for six of the AAs (see supportive information
39
Paper I). For the extreme cases (isoleucine, proline and ornithine), the ratio
between the slopes was 0.95 (isoleucine) and 1.08 (proline and ornithine).
These values, (corresponding to the effect of matrix), produced a systematic
error less than 10 % and is consistent with the recovery test performed on
matrix at 15 µM and 100 µM. This test showed that the recoveries ranged
between 82-100 % at 100 µM and 89-114 % at 15 µM. Retention times were
highly reproducible, with RSD values from 1.05 % to 3.74 % (n=10). Interday
precision in matrix reported as RSD ranged from 4 % to 16 %. Finally,
repeatability in natural samples (n=5) gave RSD values from 1.6 % to 12.6 %.
See Tables 2 and 3 in Paper I for information on respective LOD and LOQ and
other figures of merit.
A complete comparison about the pros and cons between this method and
several current and newly developed ones is depicted in Paper I. It should be
noted that when methods are evaluated regarding sample throughput, not
only the chromatographic time should be considered. All steps related to the
method, like the mobile phase flow, dimension of column, injection volume
influence the amount of mobile phase used. Additionally, if a system needs to
be equilibrated between runs with a different solvent to reestablish retention
times it will be necessary to have an extra pump to do it automatically
otherwise the method could not be run continuously over weeks. All those
disadvantages are overcome with the method presented here.
The method was successfully applied to the analysis of exudates of
ectomycorrhizal fungi, under the effect of ash treatment; see Paper III.
4.2 Gallium exchange for siderophore screening
(Paper II)
Siderophores are of importance for coping with low concentrations of iron
in nature, so methods to detect the presence of them are of great relevance.
Available screening methods use a competing ligand for iron to obtain a color
40
change that can be monitored visually or with spectroscopic techniques; a
classic example is the chrome azurol S (CAS) assay developed by Schwyn and
Neilands [117]. These methods, however, suffer from high detections limits,
low selectivity and the risk for false positives. More specific and sensitive
methods based on LC-ESI-MS [102, 118-120], LC-ICP-MS [121], LC-ESI-MS
combined with LC-ICP-MS [122] and capillary electrophoresis [123] are now
available, but for screening purposes these methods can actually be too
selective as they are normally limited to known siderophores for which
standards are available.
Paper II presents a straightforward protocol for the universal detection of
potential siderophores in liquid samples. The method is based on a gallium
exchange protocol, which allows determination of siderophore candidates
from MS data. The method utilizes the preference that siderophores have for
chelating iron III rather than iron II, and that their chelating capacity extends
to other ions of similar size such as aluminum III and gallium III [124, 125].
The method also takes advantage of gallium´s only oxidation state, namely III
[124]. The complexing capability of a siderophore obeys to an equilibrium
between the chelated and free forms of iron, respectively. If the free form is
reduced with e.g. ascorbic acid, that is, Fe3+ Fe2+, iron III will be released
from the chelated form to the solution to compensate for the loss of iron III. If
an excess of reducing agent is present, iron III will constantly be reduced to
iron II and the siderophore will thus be depleted of iron III. If a simultaneous
excess of gallium III is added to the system, the gallium will take place in the
siderophore molecule [124, 126, 127], this means that an exchange has
occurred. The method also takes advantage of the isotopic distributions of
iron and gallium. The most stable isotope of iron is 56Fe (91.72 % abundance)
but gallium has two isotopes of considerable stability 69Ga and 71Ga (60.1 %
and 39.9 % abundance, respectively), and both can compete for a siderophore
structure [126].
41
4.2.1 Work flow of the proposed screening method
A suitable background matrix was obtained by pooling growth media
from a separate study on mycorrhizal fungi. Three hydroxamate siderophores
(coprogen, ferricrocin, and ferrioxamine B) were spiked to this matrix at eight
levels ranging from 0 to 500 nM. One of the aims was to establish the
minimum concentration at which the protocol could still work. The protocol
is shown in Fig. 4.4. Briefly, a 12 mL aliquot from each level was treated with
solid phase extraction (SPE), evaporated to dryness and then diluted with a
buffer at pH 4.0. The sample was then split in two portions, one of which was
treated with iron excess and the other subjected to gallium exchange. The two
portions were then analyzed with flow injection ESI-MS. The ESI source was
run in positive mode and scanned for m/z 100-1000 with a step size of 0.1 amu.
Integration was performed over the flow injection peak maxima. The resulting
MS spectra were exported as ASCII files and imported into Matlab via Excel.
In Matlab, two in-house written functions were used to reduce data and select
m/z top candidates of siderophore molecules.
In the first Matlab function, the gallium spectrum was subtracted from the
iron spectrum, and as siderophores are expected to have most of their iron
exchanged by gallium in gallium exchange-treated samples, potential
siderophores should show up as positive peaks in this differential spectrum.
Furthermore, due to the weights of the two stable isotopes of gallium (69Ga
and 71Ga) compared to the most common isotope of iron (56Fe), negative peaks
should show up at m/z=+13 and m/z=+15 from the iron peaks (Figs. 4.5. and
4.6.). All the m/z values presenting this characteristic pattern in the differential
spectrum were listed together and further analyzed with the second Matlab
function. This function selected only the m/z that within an interval of 1 m/z
corresponded to the maximum signal in the Fe-spectrum. Each siderophore
adduct was thereby restricted, giving rise to a single entry in the top candidate
list. Additionally, a ratio between the intensities of the two gallium isotope
peaks 69Ga/71Ga of approximately 1.4 was used to select top candidates.
42
Fig. 4.4. Workflow for a sample treated with the gallium exchange protocol
for the determination of siderophores from mass spectrometry data.
43
Fig. 4.5. Graphic representation of the gallium exchange protocol and
corresponding mass spectra, i.e. the spectra for the sample treated with iron
excess, a sample subjected to gallium exchange, and the differential spectrum
between the two.
4.2.2 Results and discussion
The respective mass spectra corresponding to the sample spiked with the
three siderophores at the concentration 125 nM is depicted in Fig. 4.6. The
entire acquired mass spectra are shown in the left side (up and middle). The
entire differential spectrum is shown in the left side bottom. A close up of the
44
respective spectra in the range 760-780 amu is shown on the right side,
showing the characteristic peaks for ferricrocin and coprogen.
The three siderophores passed the selection criterion down to 25 nM;
coprogen was selected as low as at 10 nM. The siderophores tested were
selected mainly as adducts of proton [M+H]+, but ferricrocin and coprogen
were also selected as adducts of sodium [M+Na]+, where “M” corresponds to
“siderophore+Fe-3H” for the iron bound state and “siderophore+Ga-3H” for
the gallium bound state (Table 1 in Paper II). Visual inspection of the acquired
spectra showed that the signals of interest were already close to the noise at
25 nM. Despite this fact, the differential spectrum still showed the
characteristic pattern for the three siderophores, and were thus selected with
the Matlab functions.
At 10 nM, the signals corresponding to ferrioxamine B and ferricrocin were
no longer distinguishable from noise. The limits of the developed screening
protocol were therefore established as 10 nM for coprogen and 25 nM for
ferrioxamine B and ferricrocin.
At lower concentrations, the effect of noise became more evident and
limited the performance of the method. For example, for ferricrocin at 10 nM
was observed that the iron signal in the sample treated with gallium exchange
was higher than in the sample treated with iron. Consequently, ferricrocin
was rejected with the first Matlab function. Additionally, if the gallium signals
are too close to the noise, the characteristic ratio between the isotopes can
deviate too much from the parameter established for acceptance and thus
eliminate a true candidate.
The screening method also produced false positives as top candidates,
something that should be considered when applying the method to natural
samples. To eliminate false positives when investigating a real natural sample,
it would be necessary to do replicates of the samples treated with iron excess
and gallium exchange but also replicates of the runs to minimize the influence
of noise. This would eliminate false positive arising by random error. In the
development of this protocol, replicates in the same sense were not necessary
as we used a concentration gradient and for a certain m/z to correspond to a
true siderophore, it should appear in all concentrations levels tested.
45
With a list of top candidates established by the screening protocol, the next
step should be the analysis with MS to optimize ionization parameters in the
ESI source. The sample should then be separated chromatographically to
establish retention times and confirm that at least the two Ga-bound forms of
a specific top candidate co-elute and thus correspond to the same compound.
When Essén et al. [126] separated samples treated with iron excess and
gallium exchange with LC-MS, they found that the iron and gallium bounded
forms of a specific siderophore elute almost at the same time. The next step
should be a fragmentation study to establish with certainty the occurrence of
siderophores in the sample. In first place, the fragmentation patterns of the
respective iron and gallium forms should be compared. It should be necessary
to look for common fragments or common losses. When Essen et al. [126]
performed the screening for siderophores from Pseudomonas stutzeri, they
found similarities in fragmentation patterns between the gallium- and iron
bound ferrioxamines. Furthermore, they found similarities in the
fragmentation of unknown siderophores with the already known structures.
All these steps would allow the elimination of false positives and
determination of siderophores in the sample with high degree of certainty.
The adaptation of this screening method as a semi-targeted metabolomic
approach could definitely enhance resolution and allow the application of the
protocol to very complex samples with a high degree of peak overlap. The
possibility of doing chromatography and scanning at consecutive retention
times enhance resolution but also introduce and extra variable. Mathematical
functions that are more sophisticated would then be necessary to reduce the
raw data to a top candidate list.
46
Fig. 4.6. Mass spectra of sample matrix spiked with 125 nM of ferrioxamine
B, ferricrocin and coprogen, and (a) treated with access FeCl3 or (b) subjected
to gallium exchange. Fig 2c represents the differential spectra between the two.
Fig 2d-f represent the magnification of Fig 2a-c at the interval 760-870 amu for
a closer look at the peaks corresponding to ferricrocin (FCR) and coprogen
(COP).
47
5 Effect of ash on the metabolism of
ectomycorrhizal fungi (Papers III and
IV)
The main purpose of this thesis was to investigate the effect of ash
amendments on ectomycorrhizal fungi metabolism. To accomplish this,
studies were performed using a targeted approach as well as a more general
untargeted approach. The experiments were carried out in vitro to assess the
maximum exudation potential. In the targeted approach, the effects of ash on
growth, external medium pH and exudation of LMMOAs, AAs and HSs by
ECM fungi were investigated. The selection of these metabolites was a natural
decision according to expected production from the tested species. The
untargeted approach investigated the effect of ash on the entire metabolism
of the species in order to establish a list of interesting masses for unknown
compounds for further structural investigation.
5.1 Fungal strains used in the studies
Eight species of ECM fungi were selected for the study. Six of them
belonged to the basidiomycetes phylum: Cortinarius glaucopus (C. glaucopus),
Laccaria bicolor (L. bicolor), Lactarius rufus (L. rufus), Piloderma olivaceum (P.
olivaceum), Rhizopogon roseolus (R. roseolus) and Tomentellopsis submollis (T.
submollis) and two belonged to the ascomycetes phylum: Hymenoscyphus ericae
(H. ericae) and Piceirhiza bicolorata (P. bicolorata). The selection of the species
investigated in the study mirrored those species commonly found in the
rhizosphere of boreal forests.
48
5.2 Ash solution used in the study
The ash solution used in the study was prepared from ash obtained from
a local paper mill (SCA Ortviken, Sundsvall, Sweden). Wood-ash from fir was
collected in January 2015 from the upper part of two boilers corresponding to
“fly ash”. An ash stock solution was prepared by mixing 40 g of ash in 4 L
ultrapure water and stirring for six days before the remaining undissolved
ash was left to settle. After decanting, the supernatant was collected and
filtered twice (the second time with sterile filtration). The element content of
the solution was then analyzed via ICP-MS and FIA. Table 1 in Paper III shows
the element content of the amendment and the nutrients found in greatest
amounts (in decreasing order) were K, Na, Ca and Mg.
5.3 Experimental culture set up
The eight ECM fungal species were grown aseptically in darkness and at
room temperature for up to 28 days. Three levels of ash amendments (low,
medium and high) plus controls were established, and destructive sampling
was conducted after 7, 14 and 28 days. Three replicates were made for every
species, ash treatment and sampling point, yielding a total of 288 samples. See
Table 5.1 for the general set up and Table 1 in Paper III for detailed
information on the medium composition at different ash levels. The entire set
up was used in the targeted study (Paper III), while only the controls and the
high ash treatments samples from the final harvest were included in the
untargeted study (Paper IV).
49
Table 5.1. Composition and pH before inoculation of the four different ash
treatments used in a liquid growth medium experiment for testing
ectomycorrhizal fungal exudation responses.
5.4 Targeted metabolomics (Paper III)
The hypotheses for Paper III [128] were 1) that ash addition will influence
growth of the ECM fungi in a species- and ash concentration-dependent
manner; 2) that the exudation of LMM organic compounds will increase with
increasing ash additions; 3) that ash addition will influence the composition
of the exudates, and 4) that a trade-off in carbon allocation is responsible for
this growth/exudation response to ash, whereby the species either invest
carbon into higher biomass production or into higher exudation.
5.4.1 Parameters included in the study
The parameters included in the study were growth, external pH and the
metabolites LMMOAs, AAs and HSs. The 17 included LMMOAs were:
pyruvic, oxalic, lactic, malonic, fumaric, maleic, succinic, citraconic, glutaric,
malic, α-ketoglutaric, tartaric, shikimic, trans-aconitic, cis-aconitic, isocitric
Control Low Medium High
Basal Norkrans medium (mL) 23 23 23 23
Ash stock solution (mL) 0 1 4 7
Sterilized ultrapure water (mL) 7 6 3 0
Total volume (mL) 30 30 30 30
Dilution factor for ash 0.033 0.13 0.23
pH 4.34 5.11 6.03 6.40
50
and citric acids. The 23 AAs included were: leucine, proline, alanine, valine,
methionine, tryptophan, phenylalanine, isoleucine, glycine, serine,
asparagine, glutamine, threonine, cysteine, tyrosine, aspartic acid, glutamic
acid, lysine, histidine, arginine, ornithine, hydroxyproline and dimmer
cystine. Finally, the 9 HSs included were: triacetylfusarinine C, neocoprogen
I, neocoprogen II, ferricrocin, ferrichrysin, ferrichrome, coprogen, ferrirubin
and ferrirhodin.
5.4.2 Results and discussion
5.4.2.1 Effect of ash treatment on growth
Biomass accumulation varied between species (Figure 2 in Paper III), and
the two ascomycetes (P. bicolorata and H. ericae) produced the largest biomass.
Ash addition did not have any significant effect on the growth of the species,
with the exception for H. ericae for which the observed effect, however, had
no clear trend (Table 4 in Paper III). From these results, the first hypothesis
presented, that ash would have a significant effect on biomass accumulation
was rejected. The results also showed that biomass accumulation mostly
depended on time since most of the species were able to grow and allocate
carbon independently of ash addition (Table 3 in Paper III). The metals (macro
and micronutrients) contained in the ash solution did not promote further
growth compared to the controls. The proportion of e.g. K supplied by the ash
solution (in relation to the K supplied in the growth medium) corresponded
only to 2.9 %, 11.5 % and 20.4 % for low, medium and high treatments,
respectively. Therefore, its contribution was likely insignificant. Furthermore,
the ash amendment did not appear to contribute significantly with heavy
metals that could stunt growth.
51
5.4.2.2 Effect of ash treatment on external pH
The addition of ash to the culturing medium increased the pH as shown in
Table 5.1. This increase in pH is in accordance with the acute effects observed
in nature after the addition of ash [47]. With incubation time, the metabolism
of the species resulted in a significant drop in the external pH (Fig. 3 and Table
3 in Paper III). The exception to this behavior were the two ascomycetes
species (P. bicolorata and H. ericae) for which the trend was not that clear. Ash
addition did have a significant effect on pH drop as shown in Table 4 in Paper
III. With exception of the ascomycetes species, the higher the ash treatment
the greater the pH drop. In falling order the basidiomycetes species R. roseolus,
T. submollis, P. olivaceum and L. bicolor presented the strongest drop in pH at
high ash treatment.
5.4.2.3 General highlights on the exudations patterns
The LMMOAs were detected at the highest concentrations (up to mM),
while AAs were detected in the µM level; only small amounts of the
siderophore ferricrocin were detected.
In general, time had a significant positive effect on the exudation of all
metabolites in all the species. Some exceptions were, for example, P. bicolorata
and C. glaucopus for which a decrease with time was observed for four and
three different LMMOAs, respectively. Ash had a positive effect on the
exudations from most species, but not in P. olivaceum for which only pyruvic
acid increased. Amino acids production was positive affected by ash only for
L. rufus and T. submollis, the largest producers of AAs. Only two species, the
ascomycetes H. ericae and P. bicolorata, exuded measurable amounts of
ferricrocin; these species also had the largest biomass accumulation.
Siderophores act as chelators of ferric iron [34], thereby making iron available
to the fungi, but also improve the resistance against metal toxicity. As ferric
citrate (one of the original ingredients in the Norkrans basal medium) was
omitted, the only iron originated from the ash solution, and possibly from
contamination of the salts used in the preparation of the basal medium. The
52
concentration of iron due to ash amendment corresponded to 11, 42 and 74
nM for the low, medium and high ash treatments, respectively. These
amounts may be insufficient to satisfy the minimum iron requirement for
optimal growth (which is reported to be 10-5-10-7) if no specialized system for
scavenging of iron is present [129]. The ascomycetes may have had an
advantage in comparison to the others species due to the exudation of
ferricrocin, which might explain why these two species could accumulate the
greatest amount of biomass. This may also explain the generally low
exudation of LMMOAs in these two species. The LMMOAs are also known as
chelating agents, but are far less efficient compared to siderophores in this
regard. With a highly specialized iron chelating system, the ascomycetes
species did not need to exude a large amount of LMMOAs. None of the
basidiomycetes were able to produce any of the siderophores examined in the
study, but generally exuded large amounts of LMMOAs. This could have
implications in the species’ carbon economy. The basidiomycetes had to
invest much carbon into the production of LMMOAs, which was detrimental
to biomass accumulation. These results confirmed the fourth hypothesis
presented, namely that the mechanism for the growth/exudation response to
ash is due to a trade-off in carbon allocation, whereby the mycorrhizal fungi
either invest carbon in to higher biomass production or into higher exudation.
A complete description on the exudation patterns of the species studied is
included in the supplementary material of Paper III.
The production of oxalate and other LMMOAs by ECM fungi has been
suggested to play a major role in metal detoxification and nutrient uptake [21].
The exudation response to ash in the present study may be a stress response
to the higher pH and metal exposure in the culture medium, and/or a response
to a greater amount of available nutrients. Metal exposure often causes an
increase in exudation by ECM fungi [76]. The increased starting pH caused by
ash addition, especially in the medium and high ash treatments, approach
neutral values in terms of soil acidity. These values are higher than the
presumed pH optima for the species, originating mainly from acidic podzols
in boreal forest [3]. The exudation of LMMOAs may therefore also have been
a strategy to compensate for the non-optimal pH.
53
Fig. 5.1. Loading and score plot for a PCA exploring the effects of ash
treatment on the growth, external pH and exudation patterns of the eight
ECM fungi.
54
The data obtained in this study was analyzed using PCA to illustrate the
general behavior of the species (Fig. 5.1.). The PC1 and PC2 explained 27 %
and 20 % of the total variation, respectively. The two ascomycetes (score plot
coded by species) diverged in the same direction as growth and ferricrocin
(loading plot). Rhizopogon roseolus diverged in the direction of oxalic acid and
the species T. submollis and L. rufus diverged in the direction of AAs. The
loading plot also showed divergence of pH drop and LMMOAs in the same
direction as P. olivaceum (score plot coded by species). Furthermore, C.
glaucopus diverged to the right in the first component, in the same direction as
the LMMOAs that were exuded in largest amounts. Finally, L. bicolor was
concentrated close to the origo. The score plot coded by ash treatment clearly
depicted the effect of ash amendment, as objects corresponding to high ash
treatments were generally located far from the origo.
Consequently, the species used different strategies in the use of nutrients.
Furthermore, when the species were exposed to ash amendments, they tended
to exude more of the analyzed compounds compared to the controls, with
some exceptions.
5.5 Untargeted metabolomics (Paper IV)
A logical follow-up to the study presented in Paper III was to investigate
the effect of ash amendments on the general metabolism of the eight
ectomycorrhizal fungal species. The aims of the study were: 1) to identify
metabolites exuded by all species that were affected significantly by ash
amendments; 2) to establish if the effect of ash treatment on the exudation of
common metabolites was species dependent or not; 3) investigate the
presence of unique metabolites and possible effects of ash on their exudation;
4) establish how the effects of ash on the exudation of metabolites vary among
the species.
A non-targeted metabolomic assay was chosen to answer these queries
(Paper IV). In total, 48 samples were selected for the assay (8 species x 2
55
treatments x 3 replicates). Additionally, 2x3 replicates of culturing medium
with and without ash were included as controls. All samples and controls
were run in both positive and negative ESI modes. The resulting raw data was
treated as described in section 3.2.4.1.
5.5.1 General results
In general, a significant positive effect of ash amendment was observed in
the exudation patterns of the selected species, meaning that the species tended
to exude greater amounts of the same metabolites compared to the controls.
In some cases, the treatment resulted in new metabolites, not present in the
controls. Negative effects of ash amendments were also observed, but these
were of less magnitude compared to the positive effects. Many of the
metabolites detected were not exuded by all species; indeed, many were
exuded by only one or a few of them.
The number of metabolites detected in all species were in the same order
of magnitude, from 652 in L. rufus to 920 in P. olivaceum, but the two
ascomycetes were the ones exuding the highest percentages of metabolites
that were significantly influenced by ash amendment (t-test at P<0.05 and
P<0.01). Both H. ericae and P. bicolorata diverged from the basidiomycetes in
relation to exudation pattern independently of the ash treatment (Fig. 5.2.),
but also in response to it. The PCA shows that effect of ash was strongest on
H. ericae.
Analysis of the mass distribution of the metabolites exuded by the species
that were affected (positively or negatively) by ash, showed that the
compounds had masses mainly in the 200-300 amu range, allowing
classification of the exuded metabolites as low molecular mass (LMM) organic
compounds.
The retention times recorded in both ESI modes, gave an insight to the
wide range in polarity of the exuded metabolites. Furthermore, H. ericae, C.
glaucopus and P. olivaceum exuded the largest variety of highly retained
56
compounds. This means that (considering the RP chromatography) these
species exuded more compounds of less polarity in comparison to the other
species. Worth to note was the divergence in this regard, between the two
ascomycetes, as P. bicolorata exuded very few compounds with retention times
above 20 min, and then only in ESI negative mode.
Among the basidiomycetes, T. submollis, C. glaucopus and P. olivaceum were
the species with the highest percentages of metabolites that were significantly
affected by ash (Table 1 in Paper IV). This data refers to the number of
metabolites and not specifically, to the magnitude of ash effect. The effect of
ash can be seen in the PCA score plots (Fig. 5.2) where C. glaucopus and P.
olivaceum strongly diverge in the second component followed by T. submollis,
which diverged slightly in the first component. The species R. roseolus, L. rufus
and L. bicolor were less affected by ash, as the respective coordinates, with and
without ash, were concentrated in the PCA.
5.5.1.1 Significance of the findings
The results obtained indicate that there was no metabolite common to all
the species that was affected significantly by ash amendment. Some
metabolites were exuded by up to five species but the effect of ash was highly
varied. Many of the detected metabolites were unique, belonging to
secondary metabolism and thus exuded by only a few or, in some cases, a
single species. Secondary metabolites have properties that ensure adaptation
of an organism to an specific environment [130], thus increase in exudation of
certain compounds may have helped the species to cope with the extra stress
produced by the ash. All species exuded these type of metabolites, but the
effect of ash on their exudation was varied. If only the data that passed the
significance criterion concerning ash effect is considered, H. ericae and P.
olivaceum were the species showing the greatest diversity in unique
metabolites. However, if all data is considered, P. olivaceum was definitively
the species with greatest diversity. In this aspect, the two ascomycetes differ,
as P. bicolorata exuded a rather low amount of metabolites unique to it. Finally,
all species had a more positive response to bio-ash than a negative one, and
the ascomycetes had the largest magnitude of change in either direction.
57
Additionally, it was possible to establish that about 20 % of the exuded
compounds registered belonged to a basal metabolism that is common to all
species, and which was not influenced significantly by ash. Finally, the
answers given to the presented questions point in the direction of exudation
differences due to taxonomy. Complementary studies in the areas of genomics,
transcriptomics and proteomics and a wider variety of species would be
necessary to establish the features behind the responses to ash amendments.
5.5.1.2 Comparison to the prior experiment
The previous study (Paper III) identified a trade-off in the exudation of
LMM organic compounds and accumulation of biomass. The two
ascomycetes exuded lower amounts of LMM organic acids and amino acids
than the basidiomycetes, illustrating the ascomycetes’ preference in allocating
carbon as biomass instead of in the exudation of LMM organic compounds.
Paper IV established that the ascomycetes were more affected by ash in either
a positive or a negative manner. Nevertheless, the findings of both studies are
not directly comparable.
In Paper IV, the metabolites were not quantified; only the relative change
in response to ash treatment was recorded. Comparison of signals areas with
quantified areas is not possible, as different metabolites ionize differently in
the ESI source. For example, a certain metabolite can be present in a sample
in very low amounts, but if it ionizes in the ESI source to a high degree it will
yield a high response, and thus the area will be of high magnitude. Conversely,
another metabolite can be present in the sample in great amounts but if the
molecular structure does not ionize easily, the response will be lower.
Naturally, these differences in ionization behavior make comparison of
results in the present study with the targeted metabolomic approach used in
the previous experiment impossible.
58
Fig. 5.2. Scores plots for a PCA exploring the effects of ash amendments on
the general exudation patterns of the eight ectomycorrhizal fungal species
included in the untargeted metabolomic study.
59
5.6 Conclusions of the ash studies
Bio-ash amendment affected the metabolism of the ectomycorrhizal species
included in the two studies. In both cases, the amendments were added as a
solution of extracted powder ash. This method, along with the experimental
set up employed, simulated the acute effects of ash after rainfall over newly
treated soil. In other words, when the more soluble salts contained in the ash
are dissolved and transferred to the soil solution [47, 131].
Bio-ash had a general positive effect on exudation patterns, meaning that
the species tended to exude more of the same compounds compared to the
controls. In some cases, the ash amendment triggered the exudation of new
metabolites. Negative effects were also observed, but were of lower
magnitude than the positive effects.
Exudation patterns were in general species dependent, as the eight species
included in the study presented different strategies for the utilization of
carbon. Additionally, the ascomycetes H. ericae and P. bicolorata differed from
the basidiomycetes investigated.
Paper III highlighted that ash amendment triggered the exudation of
LMMOAs in C. glaucopus, AAs in L. rufus and ferricrocin in H. ericae. In some
cases though, the exudation pattern changed; P. olivaceum, for example,
showed increases in some organic acids following ash addition, while others
decreased.
In Paper IV, new questions were raised regarding the metabolism of the
species. Why was the exudation from the ascomycetes more positively
affected by bio-ash amendment? What led the strong response to ash
treatment noticed for H. ericae? The species L. bicolor and L. rufus were
generally less affected by ash and furthermore showed low diversity in
relation to unique metabolites. How related are the species that behave
similarly after bio-ash amendment? What features in P. olivaceum lie behind
the great diversity in its exudation? Finally, a list of potentially interesting
unknown compounds that could be subjected to structure elucidation is now
available (Supplementary material Paper IV).
60
In terms of the experimental set up, growing the species in axenic pure
culture had both advantages and disadvantages. One advantage was that it
ensured that the exudates were not mineralized by other microorganisms. Its
disadvantage however was the potential physiological impact of growing the
fungi without their symbiotic plant counterpart, which may reduce the
ecological relevance of the results. This highlights the need for further studies
in order to provide a more complete picture of how ash amendments affect
fungal communities and the chemistry within the rhizosphere of forest soil.
61
6 General conclusions and future
research
This thesis has addressed the effects of ash amendment on the exudation
patterns of eight different ectomycorrhizal fungal species that are normally
encountered in boreal forests.
Both targeted and untargeted metabolomic studies were conducted to
achieve this. Chemical analysis in these studies were based mainly on
established LC-MS methods, but also on a novel method for the analysis of
amino acids developed especially for this thesis. This novel method aimed to
achieve an efficient chromatographic separation that was compatible with MS
detection, but without the need for a derivatization step. Chromatography
and MS parameters were optimized and resulted in a pH gradient using
mixed-mode chromatography that was able to separate amino acids over a
wide range of isoelectric points. The method was shown to be highly stable
and generally unaffected by matrix effects, thus allowing external calibration.
Overall, the method permitted high throughput and detection limits in the
same order of magnitude as other more sophisticated and expensive methods
previously described in the literature. The method development in this thesis
also included the establishment of a screening protocol for the determination
of siderophores in complex samples. This method was based on a gallium
exchange reaction where differential mass spectra obtained via FIA-MS
provided the input for two tailor-made Matlab functions. The method
performance was tested with a suitable matrix spiked with siderophores
representing the most common classes encountered in forest ecosystems, and
it was possible to track the spiked siderophores at least down to 25 nM.
The core of this thesis – the study of effects of ash on the exudation patterns
of ectomycorrhizal fungi- showed that ash treatment generally triggers
exudation. When the species are exposed to ash, they tend to exude larger
amounts of the same metabolites as compared to the controls. In some cases,
the ash treatment also triggered the exudation of new metabolites. Negative
effects were also observed but in general, these effects were of lower
62
magnitude than the positive ones. Both metabolomics studies illustrated a
differentiation between species, where the ascomycetes clearly differed from
the basidiomycetes species; the ascomycetes accumulated more carbon as
biomass compared to the basidiomycetes and presented the greatest number
of different metabolites that were influenced by the ash treatment.
Addition of ash to the culturing medium increased the pH in a manner
that simulated the acute effects of rainfall over a newly treated soil. Over time,
species metabolism counteracted this with a significant drop in external pH,
which correlated with the exudation of LMMOAs. Ash treatment triggered
the exudation of total LMMOAs in five of the eight species studied, and
especially in C. glaucopus. Ash treatment also triggered the exudation of AAs
in T. submollis and ferricrocin in H. ericae.
It is worth noting that none of the metabolites found to be significantly
influenced by ash treatment were common to all investigated species,
indicating that with the culturing conditions used, the ash amendments
mainly affected secondary metabolism. With the experimental set up used,
however, the effect that ash amendment may have had on the primary
metabolism cannot be established with certainty. In addition, it was found
that the ascomycetes H. ericae exuded the greatest number of unique
metabolites affected by ash. On the other hand, P. olivaceum exuded the largest
amount of unique metabolites not influenced by ash.
These studies provided the answer to a number of key research questions,
but other questions nonetheless arise. An investigation of the structure of the
unknown compounds registered in Paper IV would be of particular value.
Furthermore, quantification of the newly identified compounds could reveal
if the trade-off mechanism established in Paper III, which appears to be
related to differences in species metabolism, is maintained across a greater
variety of metabolites. The application of the screening protocol for the
analysis of siderophores to natural samples using a semi-targeted
metabolomic approach, where the retention time variable is included, would
also be useful.
Finally, the results presented here illustrate a diversity of microbial
metabolism in ectomycorrhizal fungi. This is certainly a key factor in the
resilience of the forest ecosystem.
63
7 References
1. Brady, N. and Weil, R., The nature and properties of soils. 13th ed. 2002,
New Jersey: Prentice Hall.
2. Source of the picture: US Department of Agriculture, Soil Profile,
http://soils.usda.gov/education/resources/lessons/profile/. 2006.
3. Smith, S. E. and Read, D. J., Mycorrhizal symbiosis. Third edition ed.
2008, UK: Academic Press.
4. Churchland, C. and Grayston, S. J., Specificity of plant-microbe
interactions in the tree mycorrhizosphere biome and consequences for soil C
cycling. Frontiers in Microbiology, 2014. 5.
5. Rosling, A., Trees, Mycorrhiza and Minerals -Field Relevance of in vitro
Experiments. Geomicrobiology Journal, 2009. 26(6): p. 389-401.
6. Berg, G., Eberl, L. and Hartmann, A., The rhizosphere as a reservoir for
opportunistic human pathogenic bacteria. Environmental Microbiology,
2005. 7(11): p. 1673-1685.
7. Massalha, H., Korenblum, E., Tholl, D. and Aharoni, A., Small
molecules below-ground: the role of specialized metabolites in the rhizosphere.
Plant Journal, 2017. 90(4): p. 788-807.
8. Dinh, Q. T., Li, Z., Tran, T. A. T., Wang, D. and Liang, D. L., Role of
organic acids on the bioavailability of selenium in soil: A review.
Chemosphere, 2017. 184: p. 618-635.
9. Johansson, E. M., Fransson, P. M. A., Finlay, R. D. and van Hees, P. A.
W., Quantitative analysis of exudates from soil-living basidiomycetes in
pure culture as a response to lead, cadmium and arsenic stress. Soil Biology
& Biochemistry, 2008. 40(9): p. 2225-2236.
10. Bergsma-Vlami, M., Prins, M. E., Staats, M. and Raaijmakers, J. M.,
Assessment of genotypic diversity of antibiotic-producing Pseudomonas
species in the rhizosphere by denaturing gradient gel electrophoresis.
Applied and Environmental Microbiology, 2005. 71(2): p. 993-1003.
11. Kinsella, K., Schulthess, C. P., Morris, T. F. and Stuart, J. D., Rapid
quantification of Bacillus subtilis antibiotics in the rhizosphere. Soil
Biology & Biochemistry, 2009. 41(2): p. 374-379.
64
12. Fox, T. R. and Comerford, N. B., Low-molecular-weight organic-acids in
selected forest soils of the southeastern USA. Soil Science Society of
America Journal, 1990. 54(4): p. 1139-1144.
13. Strobel, B. W., Influence of vegetation on low-molecular-weight carboxylic
acids in soil solution - a review. Geoderma, 2001. 99(3-4): p. 169-198.
14. Hubová, P., Tejnecý, V., Ash, C., Borůvka, L. and Drábek, O., Low-
Molecular-Mass Organic Acids in the Forest Soil Environment. Mini-
Reviews in Organic Chemistry, 2017. 14(1): p. 75-84.
15. Olofsson, M. A., On the investigation of chemical parameters reflectimg
microbial activity linked to nutrient availability in forest soil in Faculty of
sciences, technology and media. 2015, Mid sweden University: Sundsvall,
Sweden.
16. van Hees, P. A. W., Jones, D. L. and Godbold, D. L., Biodegradation of
low molecular weight organic acids in coniferous forest podzolic soils. Soil
Biology & Biochemistry, 2002. 34(9): p. 1261-1272.
17. van Hees, P. A. W., Lundström, U. S. and Giesler, R., Low molecular
weight organic acids and their Al-complexes in soil solution - composition,
distribution and seasonal variation in three podzolized soils. Geoderma,
2000. 94(2-4): p. 173-200.
18. Ryan, P. R., Delhaize, E. and Jones, D. L., Function and mechanism of
organic anion exudation from plant roots. Annual Review of Plant
Physiology and Plant Molecular Biology, 2001. 52: p. 527-560.
19. Oburger, E., Leitner, D., Jones, D. L., Zygalakis, K. C., Schnepf, A. and
Roose, T., Adsorption and desorption dynamics of citric acid anions in soil.
European Journal of Soil Science, 2011. 62(5): p. 733-742.
20. Wang, Y. Z., Chen, X., Whalen, J. K., Cao, Y. H., Quan, Z., Lu, C. Y.
and Shi, Y., Kinetics of inorganic and organic phosphorus release influenced
by low molecular weight organic acids in calcareous, neutral and acidic soils.
Journal of Plant Nutrition and Soil Science, 2015. 178(4): p. 555-566.
21. Jones, D. L., Organic acids in the rhizosphere - a critical review. Plant and
Soil, 1998. 205(1): p. 25-44.
22. Blaylock, M. J. and James, B. R., Redox transformations and plant uptake
of selenium resulting from root-soil interactions. Plant and Soil, 1994.
158(1): p. 1-12.
23. Jones, D. L., Dennis, P. G., Owen, A. G. and van Hees, P. A. W.,
Organic acid behavior in soils - misconceptions and knowledge gaps. Plant
and Soil, 2003. 248(1-2): p. 31-41.
65
24. Najeeb, U., Xu, L., Ali, S., Jilani, G., Gong, H. J., Shen, W. Q. and Zhou,
W. J., Citric acid enhances the phytoextraction of manganese and plant
growth by alleviating the ultrastructural damages in Juncus effusus L.
Journal of Hazardous Materials, 2009. 170(2-3): p. 1156-1163.
25. Fox, T., Influence of low-molecular weight organic acids on properties and
processes in forrest soils, in Carbon forms and functions in forest soils,
McFee and Kelly, Editors. 1995, Soils science society of america, Inc.:
Madison, Wisconsin USA. p. 43-62.
26. Hue, N. V., Craddock, G. R. and Adams, F., Effect of organic-acids on
aluminum toxicity in subsoils. Soil Science Society of America Journal,
1986. 50(1): p. 28-34.
27. Campbell, M. and Farrell, S., Amino acids and peptides, in Bioquemistry.
2006, Thomson Brooks/Cole. p. 58-68.
28. Mathews, C. K., van Holde, K. E. and Ahern, G., Introduction to
proteins: The Primary level of Protein Structure, in Biochemistry, B.
Roberts, Editor. 2000, Robyn Heyden: San Francisco, Ca.
29. Kielland, K., McFarland, J. W., Ruess, R. W. and Olson, K., Rapid
cycling of organic nitrogen in taiga forest ecosystems. Ecosystems, 2007.
10(3): p. 360-368.
30. Moe, L. A., Amino acids in the rhizosphere: From plants to microbes.
American Journal of Botany, 2013. 100(9): p. 1692-1705.
31. Näsholm, T., Ekblad, A., Nordin, A., Giesler, R., Högberg, M. and
Högberg, P., Boreal forest plants take up organic nitrogen. Nature, 1998.
392(6679): p. 914-916.
32. Owen, A. G. and Jones, D. L., Competition for amino acids between wheat
roots and rhizosphere microorganisms and the role of amino acids in plant N
acquisition. Soil Biology & Biochemistry, 2001. 33(4-5): p. 651-657.
33. Matzanke, B., Structures, coordination chemistry and functions of
microbial iron chelates, in Handbook of microbial iron chelates, G.
Winkelmann, Editor. 1991, CRC Press: Florida, United States.
34. Neilands, J., Siderophores: structure and function of microbial iron
transport compounds. Journal of Biological Chemistry, 1995. 270(45): p.
26723-26726.
35. Holmström, s., Organic acids and siderophores in forest soil solution, in
Department od Natural and Environmental Sciences. 2003, Mid Sweden
University: Sundsvall, Sweden.
66
36. Luján, A. M., Gómez, P. and Buckling, A., Siderophore cooperation of the
bacterium Pseudomonas fluorescens in soil. Biology Letters, 2015. 11(2):
p. 4.
37. Nilsson, S. I., Miller, H. G. and Miller, J. D., Forest growth as a possible
cause of soil and water acidification - an examination of the concepts. Oikos,
1982. 39(1): p. 40-49.
38. Olsson, B. A., Bengtsson, J. and Lundkvist, H., Effects of different forest
harvest intensities on the pools of exchangeable cations in coniferous forest
soils. Forest Ecology and Management, 1996. 84(1-3): p. 135-147.
39. Norström, S. H., Bylund, D., Vestin, J. L. K. and Lundström, U. S.,
Initial effects of wood ash application to soil and soil solution chemistry in a
small, boreal catchment. Geoderma, 2012. 187: p. 85-93.
40. Rolff, C. and Ågren, G. I., Predicting effects of different harvesting
intensities with a model of nitrogen limited forest growth. Ecological
Modelling, 1999. 118(2-3): p. 193-211.
41. Demirbas, M. F., Balat, M. and Balat, H., Potential contribution of
biomass to the sustainable energy development. Energy Conversion and
Management, 2009. 50(7): p. 1746-1760.
42. Hagerberg, D. and Wallander, H., The impact of forest residue removal
and wood ash amendment on the growth of the ectomycorrhizal external
mycelium. Fems Microbiology Ecology, 2002. 39(2): p. 139-146.
43. Egnell, G., A review of Nordic trials studying effects of biomass harvest
intensity on subsequent forest production. Forest Ecology and
Management, 2017. 383: p. 27-36.
44. Iwald, J., Löfgren, S., Stendahl, J. and Karltun, E., Acidifying effect of
removal of tree stumps and logging residues as compared to atmospheric
deposition. Forest Ecology and Management, 2013. 290: p. 49-58.
45. Nykvist, N. and Rosén, K., Effect of clear-felling and slash removal on the
acidity of Northern coniferous soils. Forest Ecology and Management,
1985. 11(3): p. 157-169.
46. Thiffault, E., Hannam, K. D., Paré, D., Titus, B. D., Hazlett, P. W.,
Maynard, D. G. and Brais, S., Effects of forest biomass harvesting on soil
productivity in boreal and temperate forests - A review. Environmental
Reviews, 2011. 19: p. 278-309.
47. Augusto, L., Bakker, M. R. and Meredieu, C., Wood ash applications to
temperate forest ecosystems - potential benefits and drawbacks. Plant and
Soil, 2008. 306(1-2): p. 181-198.
67
48. Huotari, N., Tillman-Sutela, E., Moilanen, M. and Laiho, R., Recycling
of ash - For the good of the environment? Forest Ecology and
Management, 2015. 348: p. 226-240.
49. Ludwig, B., Rumpf, S., Mindrup, M., Meiwes, K. J. and Khanna, P. K.,
Effects of lime and wood ash on soil-solution chemistry, soil chemistry and
nutritional status of a pine stand in Northern Germany. Scandinavian
Journal of Forest Research, 2002. 17(3): p. 225-237.
50. Meiwes, K. J., Application of lime and wood ash to decrease acidification of
forest soils. Water Air and Soil Pollution, 1995. 85(1): p. 143-152.
51. Saarsalmi, A., Mälkonen, E. and Piirainen, S., Effects of wood ash
fertilization on forest soil chemical properties. Silva Fennica, 2001. 35(3):
p. 355-368.
52. Saarsalmi, A., Smolander, A., Kukkola, M., Moilanen, M. and
Saramäki, J., 30-Year effects of wood ash and nitrogen fertilization on soil
chemical properties, soil microbial processes and stand growth in a Scots pine
stand. Forest Ecology and Management, 2012. 278: p. 63-70.
53. Saarsalmi, A., Smolander, A., Moilanen, M. and Kukkola, M., Wood
ash in boreal, low-productive pine stands on upland and peatland sites:
Long-term effects on stand growth and soil properties. Forest Ecology and
Management, 2014. 327: p. 86-95.
54. Khan, A. A., de Jong, W., Jansens, P. J. and Spliethoff, H., Biomass
combustion in fluidized bed boilers: Potential problems and remedies. Fuel
Processing Technology, 2009. 90(1): p. 21-50.
55. James, A. K., Thring, R. W., Helle, S. and Ghuman, H. S., Ash
Management Review-Applications of Biomass Bottom Ash. Energies, 2012.
5(10): p. 3856-3873.
56. Nurmesniemi, H., Manskinen, K., Pöykiö, R. and Dahl, O., Forest
fertilizer properties of the bottom ash and fly ash from a large-sized (115 mw)
industrial power plant incinerating wood-based biomass residues. Journal
of Chemical Technology and Metallurgy, 2012. 47(1).
57. Swedish Forest Board, Recommendations on harvesting of residues and
ash recycling. Report 2. 2008: Sweden.
58. Demeyer, A., Nkana, J. C. V. and Verloo, M. G., Characteristics of wood
ash and influence on soil properties and nutrient uptake: an overview.
Bioresource Technology, 2001. 77(3): p. 287-295.
59. Perkiomäki, J., Tom-Petersen, A., Nybroe, O. and Fritze, H., Boreal
forest microbial community after long-term field exposure to acid and metal
68
pollution and its potential remediation by using wood ash. Soil Biology &
Biochemistry, 2003. 35(11): p. 1517-1526.
60. Nieminen, M., Piirainen, S. and Moilanen, M., Release of mineral
nutrients and heavy metals from wood and peat ash fertilizers: Field studies
in Finnish forest soils. Scandinavian Journal of Forest Research, 2005.
20(2): p. 146-153.
61. Ozolinčius, R., Varnagirytė-Kabašinskienė, I., Armolaitis, K.,
Gaitnieks, T., Buožytė, R., Raguotis, A., Skuodienė, L., Aleinikovienė,
J. and Stakėnas, V., Short term effects of compensatory wood ash
fertilization on soil, ground vegetation and tree foliage in Scots pine stands.
Baltic Forestry, 2007. 13(2): p. 158-168.
62. Eriksson, H. M., Short-term effects of granulated wood ash on forest soil
chemistry in SW and NE Sweden. Scandinavian Journal of Forest
Research, 1998: p. 43-55.
63. Perkiömäki, J. and Fritze, H., Short and long-term effects of wood ash on
the boreal forest humus microbial community. Soil Biology &
Biochemistry, 2002. 34(9): p. 1343-1353.
64. Karltun, E., Saarsalmi, A., Ingerslev, M., Mandre, M., Andersson, S.,
Gaitnieks, T., Ozolinčius, R. and varnagiryte-Kabasinskiene, I., Wood
ash recicling- possibilities and risks, in Sustainable use of forest biomass for
energy: A synthesis with focus on the baltic and nordic region, D. Röser,
Asikainen, A., Raulund-Rasmussen, K., Stupak, I., Editor. 2008,
Springer. p. 79-108.
65. Reid, C. and Watmough, S. A., Evaluating the effects of liming and wood-
ash treatment on forest ecosystems through systematic meta-analysis.
Canadian Journal of Forest Research, 2014. 44(8): p. 867-885.
66. Bååth, E. and Arnebrant, K., Growth rate and response of bacterial
communities to pH in limed and ash treated forest soils. Soil Biology &
Biochemistry, 1994. 26(8): p. 995-1001.
67. Mahmood, S., Finlay, R. D., Fransson, A. M. and Wallander, H., Effects
of hardened wood ash on microbial activity, plant growth and nutrient
uptake by ectomycorrhizal spruce seedlings. Fems Microbiology Ecology,
2003. 43(1): p. 121-131.
68. Zimmermann, S. and Frey, B., Soil respiration and microbial properties in
an acid forest soil: effects of wood ash. Soil Biology and Biochemistry,
2002. 34(11): p. 1727-1737.
69
69. Bååth, E. and Arnebrant, K., Microfungi in coniferous forest soils treated
with lime or wood ash. Biology and Fertility of Soils, 1993. 15(2): p. 91-
95.
70. García-Sánchez, M., Siles, J. A., Cajthaml, T., García-Romera, I.,
Tlustoš, P. and Száková, J., Effect of digestate and fly ash applications on
soil functional properties and microbial communities. European Journal of
Soil Biology, 2015. 71: p. 1-12.
71. Noyce, G. L., Fulthorpe, R., Gorgolewski, A., Hazlett, P., Tran, H. and
Basiliko, N., Soil microbial responses to wood ash addition and forest fire in
managed Ontario forests. Applied Soil Ecology, 2016. 107: p. 368-380.
72. Fransson, P., Andersson, A., Norström, S., Bylund, D. and Bent, E.,
Ectomycorrhizal exudates and pre-exposure to elevated CO2 affects soil
bacterial growth and community structure. Fungal Ecology, 2016. 20: p.
211-224.
73. Dennis, P. G., Miller, A. J. and Hirsch, P. R., Are root exudates more
important than other sources of rhizodeposits in structuring rhizosphere
bacterial communities? Fems Microbiology Ecology, 2010. 72(3): p. 313-
327.
74. Moore, J. A. M., Jiang, J., Patterson, C. M., Mayes, M. A., Wang, G. S.
and Classen, A. T., Interactions among roots, mycorrhizas and free-living
microbial communities differentially impact soil carbon processes. Journal
of Ecology, 2015. 103(6): p. 1442-1453.
75. Johansson, E. M., Fransson, P. M. A., Finlay, R. D. and van Hees, P. A.
W., Quantitative analysis of soluble exudates produced by ectomycorrhizal
roots as a response to ambient and elevated CO2. Soil Biology &
Biochemistry, 2009. 41(6): p. 1111-1116.
76. Plassard, C. and Fransson, P., Regulation of low-molecular weight organic
acid production in fungi. Fungal Biology Reviews, 2009. 23(1–2): p. 30-
39.
77. Ray, P. and Adholeya, A., Correlation between organic acid exudation and
metal uptake by ectomycorrhizal fungi grown on pond ash in vitro.
Biometals, 2009. 22(2): p. 275-281.
78. Blaudez, D., Jacob, C., Turnau, K., Colpaert, J. V., Ahonen-Jonnarth,
U., Finlay, R., Botton, B. and Chalot, M., Differential responses of
ectomycorrhizal fungi to heavy metals in vitro. Mycological Research,
2000. 104: p. 1366-1371.
70
79. Johansson, E. M., Fransson, P. M. A., Finlay, R. D. and van Hees, P. A.
W., Quantitative analysis of root and ectomycorrhizal exudates as a response
to Pb, Cd and As stress. Plant and Soil, 2008. 313(1-2): p. 39-54.
80. Kim, C. G., Power, S. A. and Bell, J. N. B., Effects of cadmium on growth
and glucose utilisation of ectomycorrhizal fungi in vitro. Mycorrhiza, 2003.
13(4): p. 223-226.
81. Holler, F. J., Skoog, D. A. and Crouch, S. R., Principles of intrumental
analysis. Sixth editioon ed, ed. R. broyer. 2007, USA: Thomson,
Brooks/Cole.
82. Chesnut, S. M. and Salisbury, J. J., The role of UHPLC in pharmaceutical
development. Journal of Separation Science, 2007. 30(8): p. 1183-1190.
83. Dong, M. W. and Guillarme, D., Perspectives in modern hplc uhplc, part
ii: Benefits. Lc Gc North America, 2017. 35(8): p. 486-495.
84. Stevenson, R. L., Transitioning From HPLC to UHPLC of Proteins.
American Laboratory, 2016. 48(2): p. 48-50.
85. Fritz, J. S., Principles and applications of ion-exclusion chromatography.
Journal of Chromatography, 1991. 546(1-2): p. 111-118.
86. Dejaegher, B., Mangelings, D. and Heyden, Y. V., Method development
for HILIC assays. Journal of Separation Science, 2008. 31(9): p. 1438-
1448.
87. Greco, G. and Letzel, T., Main Interactions and Influences of the
Chromatographic Parameters in HILIC Separations. Journal of
Chromatographic Science, 2013. 51(7): p. 684-693.
88. Apfelthaler, E., Bicker, W., Lämmerhofer, M., Sulyok, M., Krska, R.,
Lindner, W. and Schuhmacher, R., Retention pattern profiling of fungal
metabolites on mixed-mode reversed-phase/weak anion exchange stationary
phases in comparison to reversed-phase and weak anion exchange separation
materials by liquid chromatography–electrospray ionisation-tandem mass
spectrometry. Journal of Chromatography A, 2008. 1191(1-2): p. 171-
181.
89. Hsieh, Y., Duncan, C. J. G. and Liu, M., A mixed-mode liquid
chromatography-tandem mass spectrometric method for the determination of
cytarabine in mouse plasma. Journal of Chromatography B, 2007. 854(1–
2): p. 8-12.
90. Lamouroux, C., Foglia, G. and Le Rouzo, G., How to separate ionic
liquids: Use of Hydrophilic Interaction Liquid Chromatography and mixed
71
mode phases. Journal of Chromatography A, 2011. 1218(20): p. 3022-
3028.
91. Nogueira, R., Lämmerhofer, M. and Lindner, W., Alternative high-
performance liquid chromatographic peptide separation and purification
concept using a new mixed-mode reversed-phase/weak anion-exchange type
stationary phase. Journal of Chromatography A, 2005. 1089(1–2): p.
158-169.
92. Zhang, K., Dai, L. and Chetwyn, N. P., Simultaneous determination of
positive and negative pharmaceutical counterions using mixed-mode
chromatography coupled with charged aerosol detector. Journal of
Chromatography A, 2010. 1217(37): p. 5776-5784.
93. Arpino, P., Combined liquid-chromatography mass-spectrometry .3.
Applications of thermospray. Mass Spectrometry Reviews, 1992. 11(1): p.
3-40.
94. Pitt, J. J., Principles and Applications of Liquid Chromatography-Mass
Spectrometry in Clinical Biochemistry. The Clinical Biochemist Reviews,
2009. 30(1): p. 19-34.
95. Van Berkel, G., The electrolitic nature of the electrospray, in Electrospray
ionization mass spectrometry. Fundamentals, instrumentation and
applications., R. Cole, Editor. 1997, John wiley & Sons, Inc: New York.
96. Kruve, A., Kaupmees, K., Liigand, J., Oss, M. and Leito, I., Sodium
adduct formation efficiency in ESI source. Journal of Mass Spectrometry,
2013. 48(6): p. 695-702.
97. Banerjee, S. and Mazumdar, S., Electrospray Ionization Mass
Spectrometry: A Technique to Access the Information beyond the Molecular
Weight of the Analyte. International Journal of Analytical Chemistry,
2012.
98. Kebarle, P. and Ho, Y., On the mechanism of electrospray mass
spectrometry, in Electrspray ionization mass spectrometry. Fundamentals,
instrumentation and applications, R. Cole, Editor. 1997, john Wiley &
Sons, Inc: New York.
99. deHoffmann, E., Tandem mass spectrometry: A primer. Journal of Mass
Spectrometry, 1996. 31(2): p. 129-137.
100. Kondrat, R. W., McClusky, G. A. and Cooks, R. G., Multiple reaction
monitoring in mass spectrometry mass spectrometry for direct analysis of
complex-mixtures. Analytical Chemistry, 1978. 50(14): p. 2017-2021.
72
101. Bylund, D., Norström, S. H., Essén, S. A. and Lundström, U. S.,
Analysis of low molecular mass organic acids in natural waters by ion
exclusion chromatography tandem mass spectrometry. Journal of
Chromatography A, 2007. 1176(1-2): p. 89-93.
102. Olofsson, M. A. and Bylund, D., Analysis of hydroxamate siderophores in
soil solution using liquid chromatography with mass spectrometry and
tandem mass spectrometry with on-line sample preconcentration. Journal
of Separation Science, 2015. 38(19): p. 3305-3312.
103. Thomas, R., Prcatical guide to ICP-MS. 2004, United States: Marcel
Dekker, Inc.
104. Xu, W. H., Sandford, R. C., Worsfold, P. J., Carlton, A. and Hanrahan,
G., Flow injection techniques in aquatic environmental analysis: Recent
applications and technological advances. Critical Reviews in Analytical
Chemistry, 2005. 35(3): p. 237-246.
105. Swedish Standards, Digestion for total phosphorus, SS- 02 81 27, 2003.
106. Swedish Standards, Digestion for total nitrogen, EN ISO 11905-1, Editor.
2003.
107. Environmental Stress Screening, ESS Method 220.3: ammonia, nitrogen
and nitrate + nitrite by automated flow injection analysis method, 1991.
108. Tecator, F., Application note: detrmination of the sum of nitrate and nitrite
in water by FIASTAR 5000 F.T. AB, Editor. 2000.
109. Tecator, F., Application Note: Determination of total phosphorus in water
by FIASTAR 5000, F.T. AB., Editor. 2000.
110. Xiao, J. F., Zhou, B. and Ressom, H. W., Metabolite identification and
quantitation in LC-MS/MS-based metabolomics. Trac-Trends in
Analytical Chemistry, 2012. 32: p. 1-14.
111. Patti, G. J., Yanes, O. and Siuzdak, G., Metabolomics: the apogee of the
omics trilogy. Nature Reviews Molecular Cell Biology, 2012. 13(4): p.
263-269.
112. Viant, M. R. and Sommer, U., Mass spectrometry based environmental
metabolomics: a primer and review. Metabolomics, 2013. 9(1): p. S144-
S158.
113. Piraud, M., Vianey-Saban, C., Petritis, K., Elfakir, C., Steghens, J. P.,
Morla, A. and Bouchu, D., ESI-MS/MS analysis of underivatised amino
acids: a new tool for the diagnosis of inherited disorders of amino acid
metabolism. Fragmentation study of 79 molecules of biological interest in
73
positive and negative ionisation mode. Rapid Communications in Mass
Spectrometry, 2003. 17(12): p. 1297-1311.
114. Piraud, M., Vianey-Saban, C., Petritis, K., Elfakir, C., Steghens, J. P.
and Bouchu, D., Ion-pairing reversed-phase liquid
chromatography/electrospray ionization mass spectrometric analysis of 76
underivatized amino acids of biological interest: a new tool for the diagnosis
of inherited disorders of amino acid metabolism. Rapid Communications
in Mass Spectrometry, 2005. 19(12): p. 1587-1602.
115. Thiele, B., Fullner, K., Stein, N., Oldiges, M., Kuhn, A. J. and Hofmann,
D., Analysis of amino acids without derivatization in barley extracts by LC-
MS-MS. Analytical and Bioanalytical Chemistry, 2008. 391(7): p. 2663-
2672.
116. Vilches, A. P., Norström, S. H. and Bylund, D., Direct analysis of free
amino acids by mixed-mode chromatography with tandem mass
spectrometry. Journal of Separation Science, 2017: p. n/a-n/a.
117. Schwyn, B. and Neilands, J. B., Universal chemical-assay for the detection
and determination of siderophores. Analytical Biochemistry, 1987. 160(1):
p. 47-56.
118. Carroll, C. S., Amankwa, L. N., Pinto, L. J., Fuller, J. D. and Moore, M.
M., Detection of a Serum Siderophore by LC-MS/MS as a Potential
Biomarker of Invasive Aspergillosis. Plos One, 2016. 11(3): p. 16.
119. Sidebottom, A. M., Karty, J. A. and Carlson, E. E., Accurate Mass
MS/MS/MS Analysis of Siderophores Ferrioxamine B and E1 by Collision-
Induced Dissociation Electrospray Mass Spectrometry. Journal of the
American Society for Mass Spectrometry, 2015. 26(11): p. 1899-1902.
120. Zhang, L., Yuan, D. X., Fang, K. and Liu, B. M., Determination of
Siderophores in Seawater by High Performance Liquid Chromatography-
Tandem Mass Spectrometry Coupled with Solid Phase Extraction. Chinese
Journal of Analytical Chemistry, 2015. 43(9): p. 1285-1290.
121. Boiteau, R. M., Fitzsimmons, J. N., Repeta, D. J. and Boyle, E. A.,
Detection of Iron Ligands in Seawater and Marine Cyanobacteria Cultures
by High-Performance Liquid Chromatography-Inductively Coupled Plasma-
Mass Spectrometry. Analytical Chemistry, 2013. 85(9): p. 4357-4362.
122. Boiteau, R. M. and Repeta, D. J., An extended siderophore suite from
Synechococcus sp PCC 7002 revealed by LC-ICPMS-ESIMS. Metallomics,
2015. 7(5): p. 877-884.
74
123. Mucha, P., Rekowski, P., Kosakowska, A. and Kupryszewski, G.,
Separation of siderophores by capillary electrophoresis. Journal of
Chromatography A, 1999. 830(1): p. 183-189.
124. Emery, T., Exchange of iron by gallium in siderophores. Biochemistry,
1986. 25(16): p. 4629-4633.
125. Neilands, J. B., Siderophores - structure and function of microbial iron
transport compounds. Journal of Biological Chemistry, 1995. 270(45): p.
26723-26726.
126. Essén, S. A., Johnsson, A., Bylund, D., Pedersen, K. and Lundström,
U. S., Siderophore production by Pseudomonas stutzeri under aerobic and
anaerobic conditions. Applied and Environmental Microbiology, 2007.
73(18): p. 5857-5864.
127. Moberg, M., Nilsson, E. M., Holmstrom, S. J. M., Lundstrom, U. S.,
Pettersson, J. and Markides, K. E., Fingerprinting metal-containing
biomolecules after reductive displacement of iron by gallium and subsequent
column-switched LC-ICPMS analysis applied on siderophores. Analytical
Chemistry, 2004. 76(9): p. 2618-2622.
128. Vilches, A. P., Norström, S. H., Olofsson, M. A., Fransson, P. and
Bylund, D., Biofuel ash addition increases ectomycorrhizal fungal
exudation in pure culture. Environmental Chemistry, 2018: p. -.
129. Lehner, S. M., Atanasova, L., Neumann, N. K. N., Krska, R., Lemmens,
M., Druzhinina, I. S. and Schuhmacher, R., Isotope-Assisted Screening
for Iron-Containing Metabolites Reveals a High Degree of Diversity among
Known and Unknown Siderophores Produced by Trichoderma spp. Applied
and Environmental Microbiology, 2013. 79(1): p. 18-31.
130. Hautbergue, T., Jamin, E. L., Debrauwer, L., Puel, O. and Oswald, I.
P., From genomics to metabolomics, moving toward an integrated strategy
for the discovery of fungal secondary metabolites. Natural Product Reports,
2018. 35(2): p. 147-173.
131. Brännvall, E., Wolters, M., Sjöblom, R. and Kumpiene, J., Elements
availability in soil fertilized with pelletized fly ash and biosolids. Journal of
Environmental Management, 2015. 159: p. 27-36.
