Modification of the physical and optical properties of the frustule of the diatom Coscinodiscus

wailesii by nickel sulfate

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Nanotechnology 18 (2007) 295101 (5pp) doi:10.1088/0957-4484/18/29/295101

Modification of the physical and opticalproperties of the frustule of the diatomCoscinodiscus wailesii by nickel sulfateHelen E Townley1,4, Kai Lin Woon2,5, Frank P Payne2,Helen White-Cooper1 and Andrew R Parker3

1 Department of Zoology, Oxford University, Tinbergen Building, South Parks Road,Oxford OX1 3PS, UK2 Department of Engineering, Oxford University, Parks Road, Oxford OX1 3PJ, UK3 Department of Zoology, The Natural History Museum, Cromwell Road,London SW7 5BD, UK

E-mail: Helen.Townley@zoo.ox.ac.uk, bomohwk@hotmail.com, Frank.Payne@eng.ox.ac.uk,Helen.White-Cooper@zoo.ox.ac.uk and A.Parker@nhm.ac.uk

Received 23 March 2007, in final form 15 May 2007Published 20 June 2007Online at stacks.iop.org/Nano/18/295101

AbstractIn this paper we demonstrate how the photonic properties of a diatom can bealtered by growth with a metal pollutant. Both the optical and physicalproperties of the silica frustule of the diatom Coscinodiscus wailesii wereaffected by the presence of nickel sulfate in sea water. It was found that asublethal concentration of the metal both significantly modified the size ofthe pores of the valves and quenched the intrinsic PL of the amorphous silica.Since cytoplasmic structures may be involved in determining the frustulearchitecture, we also present TEMs of nickel-grown diatoms and show theaffected organelles. The ability to modify the properties of the frustule showsthat mechanisms exist for the alteration of existing structures in nature tooptimize specific characteristics for exploitation in biotechnologicalapplications.

1. Introduction

Materials science at the nanometre level is being influencedand inspired by the recognition of small-scale biologicalstructures and architectures. Such materials have remarkablycomplex hierarchical structures, and are formed under mildphysiological conditions. Biological organisms can generatelarge amounts of biomaterials simply by replication, andfurthermore there is the opportunity to modify growth throughenvironmental conditions and also to selectively evolveorganisms via mutations.

Naturally derived materials with inherent periodicity areparticularly interesting. Through their photonic crystal-like properties these materials can significantly alter the

4 Author to whom any correspondence should be addressed.5 Present address: OSRAM Opto Semiconductors (Malaysia) Sdn. Bhd.,Bayan Lepas Free Industrial Zone Phase 1, 11900 Bayan Lepas Penang,Malaysia.

propagation of light and the interaction of light and matter.Diatoms are unicellular microalgae which generate a highlyornamented external cell wall, the frustule, composed ofbiogenic amorphous silica. These living cells must constantlyinteract with their environment and as such diatom walls havemyriad openings, such as nanopores and slits, which facilitatesuch exchanges. It is this complex pattern of pores presenton the surface of diatoms which may result in their photonicproperties [1].

The resulting intricate patterns on the surface of thefrustule are both species-specific and partly geneticallydetermined. The precision of this nanoscale architecturefar exceeds the capabilities of present-day materials scienceengineering. Most fabrication techniques in nanotechnologyinvolve planar lithographic approaches [2] in which three-dimensional structures are built up plane by plane, andundercuts and cavities etched away. By contrast, opticalassemblies in nature have evolved over millions of years

0957-4484/07/295101+05$30.00 1 © 2007 IOP Publishing Ltd Printed in the UK

Nanotechnology 18 (2007) 295101 H E Townley et al

to generate intricate nanostructures more complex thananything that could be produced using artificial techniquesand providing materials ideal for biotechnological exploitation.Furthermore, there are over 250 genera of diatoms, withas many as 100 000 species with widely varying frustulearchitecture.

Biologically, the cell wall has been proposed to have a dualfunction: physical protection and light collection. Mechanicalstrength measurements of Coscinodiscus granii, Thalassiosirapunctigera, and Fragiluriopsis kerguelensis have shown thatthe frustules can withstand mechanical stress correspondingto pressures of 100–700 ton m−2 [3]. Furthermore, the valveof C. granii, which is a quasi-periodic structured materialof limited thickness, has been postulated to act as a planaroptical waveguide supporting several guided modes [1]. Thesewaveguiding properties have the potential to enhance thephotosynthetic capabilities of underlying chloroplasts.

All types of porous silicon show photoluminescence (PL)if the porosity is sufficiently high [4]. Butcher et al [5]investigated whether PL could be seen in the silica frustule ofa freshwater diatom and demonstrated the presence of strongblue photoluminescence when excited by a 325 nm laser, thePL showing a broad peak at 450 nm. There are severalreports which have shown that gases or liquids can reversiblyquench visible PL from porous silicon [6–9] and that artificialdeposition of metal on silica surfaces also affects PL [10].It has previously been demonstrated that the photosyntheticmarine diatom Nitzschia frustulum can assimilate solublegermanium via cell silica transporters and fabricate Si–Geoxide nanostructured composite materials [11]. Furthermore,diatoms are known to be affected by heavy metal pollutionin streams and seas and show a number of cytological effects.Here we investigate the effects of sublethal quantities of nickelon the marine diatom Coscinodiscus wailesii. Nanocompositeswith transition metals could have potentially interesting opticalproperties. In addition, the effects of nickel on diatoms areof biological interest due to its increasing prevalence in theenvironment from its uses in many industrial applications suchas transport, automotive, aerospace, electronics, consumerproducts, chemicals and batteries. The Convention on theProtection of the Marine Environment of the Baltic SeaArea, 1992, recommends that before discharging into sewersor surface waters, treatment should be provided such thatconcentrations of nickel do not exceed 1 mg l−1. We show herethe effects upon the structure of the frustule and organelles ofC. wailesii after growth in 0.5 mg l−1 nickel.

2. Experimental details

2.1. Diatom culture and preparation

The marine diatom C. wailesii was obtained from theProvasoli–Guillard National Centre for Culture of MarinePhytoplankton (Maine, USA; strain B isolated by J Long,1993). The algae were cultured at 22 ◦C, under a regimeof 16 h light, 8 h darkness. Samples were either grown insterilized filtered sea water supplemented with Alga-Gro media(Carolina Biologicals, North Carolina, USA) or on platesadditionally containing 0.8% agar. Nickel sulfate (Sigma,Dorset, UK) was added at concentrations of 5, 1, 0.5 and

0.1 mg l−1. Samples were monitored, and after 1 monthdiatoms grown on 0.5 mg l−1 nickel sulfate were harvested(this was the maximum concentration at which there was nosignificant effect on growth).

Diatoms were filtered using a 63 µm aperture sieve (MeshNo. 240, London, UK) and rinsed with distilled water. Toremove organic matter and clean the frustules, samples weretreated with 50:50 (v/v) water, 30% hydrogen peroxide (BDH,Poole, UK) and incubated at 90 ◦C for 3–4 h, followed bythe addition of hydrochloric acid. Samples were collected bysieving to prevent damage to the frustules, rinsed copiouslywith distilled water and stored in 70% ethanol.

2.2. Scanning electron microscopy

Cleaned samples were dried and mounted on a metal steelsubstrate. They were sputter coated with a 1.5 nm thick layerof platinum and mounted into the JEOL JSM-840F scanningelectron microscope with an acceleration voltage of 5 kV forimage taking.

2.3. Dimensional fast Fourier transform analysis

The spatial frequency spectrum of the frustule pattern ofC. wailesii diatoms was determined by two-dimensional fastFourier transform (2D FFT) of representative scanned SEMimages for both nickel-grown and control samples. Eachpixel of the scanned SEM images corresponds to 0.1 µm, andthe whole circular image of the diatom was extracted usingAdobe Photoshop. The image was imported into ORIGIN7.0 software. By the use of the 2D FFT programme underNumerical Algorithm Group (NAG) within the ORIGIN 7.0software, the 2D Fourier transform diagram correspondingto the amplitude of the periodic distance is obtained. Theresulting two-dimensional spectrum was loaded into a FIT2Dprogram [12] and radially integrated.

2.4. Transmission electron microscopy

Samples were fixed (2.5% glutaraldehyde, 2% formaldehyde in100 mM phosphate buffer (pH 7.2), 0.1% picric acid, sucrose)for 24 h at 4 ◦C. Samples were then washed in 200 mMphosphate buffer and then fixed in 1% osmium tetroxide in100 mM phosphate buffer for 1 h at 4 ◦C. This was followedby washing with 100 mM maleate buffer (pH 5.2). Stainingwas then performed en bloc with 2% uranyl acetate in maleatebuffer for 2 h, at 4 ◦C in darkness. Samples were then washedwith distilled water and dehydrated in acetone, followed byembedding in epoxy resin.

2.5. Fluorescence measurements

Initially, samples were examined under a LSM5 Pascalconfocal fluorescence microscope at an excitation wavelengthof 488 nm. The transmitted light was passed through abandpass filter with transmission between 550 and 625 nm.

Individual diatoms were then excited using the 442 nmline generated by a Kimmon Electric helium–cadmium laserwith a beam power of 20 mW. A fluorescence microscopeset-up as illustrated in figure 1 was used with the opticalfibre acting as a confocal pin-hole. The light collected by

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Nanotechnology 18 (2007) 295101 H E Townley et al

Figure 1. Experimental set-up for the detection ofphotoluminescence from single diatoms.

Figure 2. Scanning electron microscopy images of the silica cellwall of the diatom C. wailesii. Valves grown in sea water ((a), (c)) orsupplemented with 0.5 mg l−1 nickel sulfate ((b), (d)). Magnifiedsections of the valves ((a), (b)) illustrating the difference in pore sizeand shape ((c), (d), respectively).

the fibre optics was detected using an Ocean Optics S2000spectrometer. As a standard, the absorption of nickel sulfateof concentration 10 g l−1 in water was also measured using aPerkin Elmer Lamda 19 UV/vis spectrometer.

3. Experimental results

Frustules of C. wailesii were examined to determine whetherthere were any gross morphological changes when grown inthe presence of nickel sulfate. Representative SEM imagesshow diatoms grown in the presence of 0.5 mg l−1NiSO4

(figures 2(b), (d)) and control samples (figures 2(a), (c)). Thepores of the doped diatoms can be seen to show a moreirregular pattern. The individual pores also appear to belarger and less uniform in shape. The spatial frequencyspectrum of the frustule pattern of doped and undoped diatoms

Figure 3. Fast Fourier transform of valve pores shown in figures 2(a)and (b). Note the curve for undoped sample is offset for clarity. Theintensity is 100 units less than indicated on the graph.

was determined by 2D FFT of the scanned SEM images.The resulting two-dimensional spectrum was then radiallyaveraged using the FIT2D program [12]. The pores in thefrustule surface of the NiSO4-grown diatoms were shown to be2.11 µm in diameter (figure 3, filled circles, peak A), almosttwice as large as the 1.18 µm diameter pores of correspondingcontrol diatoms (figure 3, open circles, peak A). The distancebetween pores increases from 2.05 to 3.87 µm (partly due toincreased pore size) in the presence of nickel sulfate (figure 3,peaks B), resulting in fewer pores over the entire valve surface.

Since diatom frustules are known to photoluminesce,C. wailesii grown in the presence of nickel sulfate wascompared with controls. Confocal images (figure 4) showthat control samples exhibit PL (figure 4(a), (ii)) whereasgrowth in NiSO4 results in quenching of the PL under thesame 442 nm excitation light (figure 4(a), (i)). More detailedanalysis of individual diatoms is shown by a representativetrace (figure 4(b)). The diatoms exhibit a broad PL peakbetween 500 and 650 nm. Growth in NiSO4 shows quenchingof PL, resulting in an almost three-fold decrease in emission.

Diatoms grown under control conditions were crushed toform a powder and re-examined for PL. The crushed sampledid not differ from the intact sample, suggesting that thealterations in quasi-periodic structure of the NiSO4 growndiatoms are not responsible for the changes in PL (data notshown). In addition, diatoms were examined by energydispersive x-ray (EDX) analysis. This showed that if nickelis deposited in the frustule then it is below the detectable limitof 0.1 wt%.

Diatoms were then examined by TEM to assess anyultrastructural changes. Thylakoids of chromophytic plastidsare arranged in stacks of three, appressed along their entirelength and traversing the length of the chloroplast (figure 5(a)).A further ‘girdle’ lamella encircles the rim of the chloroplast.After growth in nickel sulfate there is clear disruption ofthe regular order of the lamellae (figure 5(c)). Figure 5(b)shows Golgi bodies and a number of mitochondria in a controlsample. Comparison with doped samples shows obviousswelling of the mitochondria (figure 5(d)).

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Nanotechnology 18 (2007) 295101 H E Townley et al

(a)

(b)

Figure 4. Diatoms were grown in sea water media, either in thepresence (i) or absence (ii) of 0.5 mg l−1 nickel sulfate. Cleanedfrustules were excited with blue light and photoluminescenceexamined by spectroscopic confocal fluorescence microscopy.(a) Representative confocal images and (b) representative trace fromexcitation of an individual diatom. Control diatom (——), nickelsulfate doped diatom (��), transmission spectrum of nickel sulfate( ).

(This figure is in colour only in the electronic version)

4. Discussion

The present study shows that exposure of C. wailesii tonickel sulfate results in significant modification to the frustule,affecting the size and number of pores, and intrinsic PL.In the presence of nickel sulfate the pores were shown tobe approximately twice as large as those from the controlgroup, and consequently there were fewer pores over the entirefrustule surface. Previously, frustule abnormalities have beenreported in Nitzschia liebethrutti grown in the presence ofmercury and tin [13]. Both metals resulted in a reductionin the length to width ratios of the diatoms, fused poresand a reduction in the number of pores per frustule. Theseabnormalities were thought to arise from enzyme disruptioneither at the silica deposition site or at the nuclear level.Additionally the presence of cations has been shown to givevarious responses on diatoms with respect to silicification.General silica chemistry also dictates that temperature, theconcentration of silicate and the availability of salts and cations(such as Al3+, Ca2+, Fe3+, K+, Na+) and the pH affect thesynthesis of silica and zeolites [14, 15].

Figure 5. TEM images of C. wailesii. Control samples ((a), (b)) andsamples grown in nickel sulfate ((c), (d)). Scale bar is 1 µm.

A previous study [11] has shown that germanium cancompete with silicon to use the same cell transport systemand can be incorporated into the frustule. Similarly, vanBennekom [16, 17] found that soluble aluminium present asAl(OH)3 reacts preferentially with Si(OH)4 and presumedthat Al–silica complexes were formed, finding that Al wasassociated with, most probably, the outer part of diatomfrustules. While we were unable to determine whethernickel was incorporated into the frustule we found thatgrowth in NiSO4-containing media significantly reduced thephotoluminescence of the frustule. This is in accordance withother studies which show that metals can quench the inherentPL of silica. Andsanger et al [10] examined how PL wasaffected by the deposition of metal upon silica. Metals weredistributed at low concentrations over the silica surface to adepth of 100 nm, and copper, silver and gold were found toquench the PL. In addition, Butcher et al [5] examined theluminescence of freshwater diatoms and found that under the325 nm beam of a He–CD laser, the sample exhibited a broadpeak at 450 nm and also showed cathodoluminescence peaksat 640 and 580 nm under exposure to a 25 kV electron beamof a SEM. They also found that diatoms from water coursespresumed to be contaminated with non-specific metals werealso affected in their PL. The PL we detected at 544 nm is mostlikely the result of radiative decay of self-trapped excitons.Nickel sulfate dissolved in water has an absorption curve asshown in figure 4(b). The water molecules act as ligands whichsplit the partially filled d-orbitals into two energy levels. Theabsorption band centred at 700 nm is the result of promotionof electrons from a lower energy level to a higher energylevel in d-orbitals. It is possible that the nickel could haveformed complex ions within the hydrated amorphous silica(SiO2·nH2O)) frustule. The presence of overlapping emissionspectra with the absorption of the nickel ion complex mightallow efficient energy transfer and hence result in quenchingof the photoluminescence. Such energy transfer has beenpreviously demonstrated in studies of Er3+-doped SiO2 filmscontaining Si nanocrystals [18].

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Nanotechnology 18 (2007) 295101 H E Townley et al

An examination of the cytoplasmic morphology ofC. wailesii grown in the presence of nickel sulfate showeddisruption of the thylakoid stacks, and swelling of themitochondria. A previous study of Skeletonema costatum [19]examined growth in the presence of mercury (6.5 µg l−1),cadmium (50 µg l−1) or zinc (265 µg l−1). Alterations incytoplasmic morphology were observed in cells treated withsublethal concentrations of these metals. Swollen organelles,dilated membranes and vacuolated cytoplasm indicated plasmamembrane damage and subsequent osmotic disorganization.Electron-dense inclusions in vesicles, multivesiculate bodiesand cytoplasmic ‘tubules’ appear to be derived from the Golgibody and were interpreted as mechanisms of sequesteringthe metals. Mercury and zinc had a marked effecton the fine structure whereas treatment with cadmiumresulted in little or no cytoplasmic damage. Subsequentstudies [20] have, however, demonstrated that diatoms havea unique metalloenzyme that specifically requires cadmiumfor biological function, which could explain the lack ofphysiological effects. The effects shown by the cytotoxicmetals which result in cytological and frustule abnormalitiescould be linked by the mechanism of silica deposition. Silicais concentrated in a silica deposition vesicle (SDV) wheremacromorphogenesis determines the pattern formation. In thisprocess the silica is moulded into a pattern by the presence oforganelles, such as mitochondria, spaced at regular intervalsalong the cytoplasmic side of the SDV [21]. Consequentlyabnormalities arising in the internal structures would affect theeventual frustule pattern.

It therefore appears that the effects of NiSO4 are two-fold: firstly, the frustule patterning is altered most likely dueto modification of internal structures used to mould the silica,and secondly the incorporation of very low levels of nickel intothe frustule alters its optical properties.

The aberration of the frustule may have implications forthe photosynthetic capacity of the organism. This is animportant point, since globally diatoms are more effectiveat removing atmospheric CO2 than all the world’s rainforests [22]. The role of the frustule in light collection wasreinforced by Fuhrmann et al [1] who indicated the possiblerole of waveguiding in the visible range for valves of C. granii.Furthermore, diatoms are equipped with several hundredchloroplasts which are located close to the cell walls allowingthem to effectively make use of the confined light. Thesechloroplasts are reported to be redistributed and to migrateaway from the cell walls at high light intensities [23]. Anincrease in pore size would be predicted to reduce the effectivewaveguide refractive index of the diatom valve. This couldeventually decrease the ability of the valves to support higherorder optical modes and consequently their ability to confinelight, potentially reducing the photosynthetic competency ofthese vital primary producers.

The ability to alter the frustule by changing environmentalconditions may be useful for biotechnological applications.Already the photoluminescent properties of diatoms havefound applications in sensing devices: the PL emission fromthe silica frustule of the marine diatom Thalassiosira rotulahas been shown to be strongly dependent on the surroundingenvironment [24]. It was shown that both the optical intensity

and the position of the peaks were affected by gases andorganic vapours with electrophilic substances quenching PLwhilst nucleophilic substances had the opposite effect [24].

5. Conclusions

In summary, we have shown that a metal pollutant can alterthe optical, physical and cytological properties of the marinediatom C. wailesii. Photoluminescence of the silica frustulewas quenched as a result of growth in nickel sulfate. Inaddition, the pores were increased in size, and organelles wereshown to have aberrant structures. Given these biologicaleffects we propose that diatom frustules could have potentialas the active component in an aquatic pollution sensor.

Acknowledgments

The authors thank Dr Alison Crossley and Dr Chris Salterfor EDX analysis. We are grateful to Dr M Shaw for experttechnical assistance with TEM. This work was supported bythe EPSRC.

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