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Losic, D., Mitchell, J.G., & Voelcker, N.H., “Diatom culture media contain extracellular silica nanoparticles which form opalescent films”. Proceedings of SPIE - The International Society for Optical Engineering, 7267(726712) (2008).

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Diatom culture media contain extracellular silica nanoparticles which form opalescent films

Dusan Losic,*

¹

James G. Mitchell,

²

Nicolas H. Voelcker

³

1

University of South Australia, Ian Wark Research Institute, Mawson Lakes, Adelaide, SA 5095, Australia

2

Flinders University, School of Biological Sciences, Adelaide, SA 5042, Australia.

3

Flinders University, School of Chemistry, Physics and Earth Science, Adelaide, SA 5042, Australia.

ABSTRACT

Diatoms are unicellular photosynthetic algae with enormous diversity of patterns in their silica structures at the nano- to micronscale. In this study, we present results, which support the hypothesis that silica nanoparticles are released into the diatom culture medium. Formation of an opalescent film by the self-assembly of silica nanoparticles produced in the growth medium of diatoms. This film was formed on the filter paper from the culture medium of a Coscinodiscus sp.

culture. A number of diatoms with partially opened valves were observed on the film surface under light microscopy and SEM, which indicates that cell contents inside of diatoms had been released into the culture solution. AFM images of produced an opalescent films show ordered arrays of silica nanoparticles with different diameters depending on the colors observed by light microscopy. The film forming silica nanoparticles are either released by the diatoms during reproduction or after cell death. This approach provides an environmentally friendly means for fabricating silica nanoparticles, decorative coatings and novel optical materials.

Keywords: diatoms, Coscinodiscus sp. silica nanoparticles, biomineralisation, artificial opal films

1. INTRODUCTION

There is a significant interest in the science and technology of gaining inspiration from nature for the production of nanostructured materials [1-2]. Biomaterials made through the process of biomineralisation are highly controlled from the molecular level to the nanoscale and the macroscopic level resulting in complex bioinorganic architectures [3].

Understanding the processes involved in biomineralisation may eventually allow mimicking these structures and producing new materials with advanced mechanical, magnetic, optical, electrical, piezoelectrical, or adhesive properties [3 -5].The exploitation of the biosynthetic activities of microbial cells for the synthesis of nanostructured materials has recently emerged as a novel approach for the fabrication of metal and semiconductor nanoparticles [6].

The unicellular algae known as diatoms are outstanding examples of micro- and nanostructured materials in nature [7].

They produce exceptional three-dimensional silica scaffolds, which have great potential for nanotechnological applications [8-9]. The technological importance of diatoms is the subject of a series of recent reviews on the interface of nano- and biotechnology [5, 812]. Applications in filtration systems, in immunoisolation, biosensing, optoelectronics and

as templates for fabrication on nanomaterials have been demonstrated [7-16].

The diversity of diatom structures is extraordinary and each of the estimated 10⁵ diatom species has a specific shape decorated with a unique pattern of nano-sized features such as pores, ridges, spikes and spines [4]. Furthermore, each diatoms often features several hierarchical layers of porous membrane differing in size and pattern. Their main morphological feature is the frustule, a silica cell wall that consists of two valves, encasing the protoplasm, joined

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together by siliceous girdle bands. The frustule of centric diatoms consists of a honeycomb of hexagonal chambers, called areolae. In general, each chamber has an outer surface, exposed to the external environment and an inner surface.

One of the two surfaces is perforated by large, round holes called foramen, while the other surface contains one or two silica plates (cribellum and cribrum) perforated by a complex and highly symmetrical pore arrangement [17]. An insight into the remarkable diversity of diatom silica architectures can be gained from Figure 1, which shows SEM images obtained from several diatom species.

Figure 1 SEM images of several diatom species.

Because of the chemical similarity of the diatom silica to the silica mineral opal, diatoms are often referred to as jewels of the sea or living opals [18]. Recent studies using high resolution atomic force microscopy imaging revealed that diatom silica structure is build by silica nanoparticles [19-20] The interaction with light produces stunning structural colors with intense diffraction and interference effects when diatoms are observed under a light microscope (Figure 2).

Displaying a play of gleaming colors like those of the rainbow, this phenomenon is caused by multiple reflections from multilayered, semitransparent surfaces in which phase shift and interference of the reflections modulates the incident light by amplifying or attenuating some frequencies more than others. The photonic crystal properties of the diatom’s girdle band structures were recently confirmed suggesting that diatoms are living photonic crystals [21].

Figure 2 a) A photograph of a typical silica mineral, opal. b) Light microscopy images of several diatom species with characteristic colors as result of light interference and diffraction from their silica structure [18] (with permission from S. Ely).

Proc. of SPIE Vol. 7267 726712-2

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The molecular mechanism of the biosilification process is still not well understood. Recent studies have revealed the role of biomolecules including zwitterionic proteins (silaffins) and long chain polyamines in catalysing and controlling silica precipitation and the formation of species-specific nanopatterns [5,17]. Silicic acid Si(OH)4, the precursor of silica in the aqueous environment is supplied to diatoms via transmembrane transporter proteins called silicic acid transporters, The accumulated silicic acid is transported into specialized intracellular vesicles, the silica deposition vesicles (SDVs).

The SDVs are activated after cell division where daughter cells each build one valve and receive one from the mother cell. Two models have been proposed for the rapid two dimensional precipitation of silica nanoparticles and valve morphogenesis that include diffusion limited aggregation and phase separation [18]. In either case, the aqueous interface between vesicles or organic droplets promotes silicification into a honeycomb-like framework. Silica precipitation causes dispersion effects creating new interfaces and continued silica formation [17]. Principles and mechanisms established in recent years have been avidly translated into research on biomimetic synthesis of silica nanostructures, where silaffins, synthetic peptides and polyamines have afforded excellent control over silica morphogenesis [22].

Diatoms can routinely be grown to more than million cells per ml of culture medium, offering the opportunity for large scale and cheap production of nanostructured silica. In this study, we present results that demonstrate the formation of opalescent film of silica nanoparticles from diatom culture medium. We suggest that the film forming silica nanoparticles are either released by the diatoms during reproduction or after cell death. Our observation might lead to an environmentally friendly pathway for fabricating silica nanoparticle films for decorative coatings and novel optical materials.

2. EXPERIMENTAL

Coscinodiscus sp. and Thalassiosira eccentrica were obtained from CSIRO, Marine Research (Hobart, Tasmania, Australia). The cultures were maintained at 20 ^˚C using a 12 hour light / 12 hour dark cycle. GSE medium was used to culture Coscinodiscus sp. and Guillard's medium (f/2) was used for T. eccentrica [23]. The live diatoms were harvested after 1-2 months of culturing and cleaned using a procedure described elsewhere. Aliquots of growth media after the culturing period was filtered through the teflon filter (pore size 1 µm) and examined by light microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The opalescence film on the filter paper is observed through filtration of culture media. In culture media without of diatoms is used as control. To obtain clean diatom structures from diatom culture media, organic material was removed by treatment with concentrated sulphuric acid and surfactant (2 % SDS in 100 mM EDTA). Acid treatment causes the separation of the diatom frustule into the 2 frustule valves and the girdle band. On the other hand, a surfactant treatment preserves the whole diatom structure. Cleaned diatom silica for both procedures was stored in 100% ethanol. The structural characterisation of diatoms was performed by SEM and AFM. One drop of cleaned diatom suspension was deposited onto freshly cleaved mica or silicon wafers previously coated with polylysine (from 0.01% aqueous solution of poly-L-lysine).

SEM studies were performed using a Philips XL 30 field-emission scanning electron microscope in combination with energy-dispersive X ray (EDX) analysis. The samples with diatoms were coated with a thin platinum layer and mounted on microscopy stubs with carbon sticky tape.

AFM imaging was performed using a Nanoscope IV Multimode SPM (Veeco Corp., Santa Barbara, USA). Images were acquired in air using contact and tapping modes. Oxide-sharpened silicon nitride probes (NP-S, Veeco Corp.) of spring constant k = 0.15 N/m were used for contact mode experiments. Olympus silicon probes (TESP, Veeco Corp.) were used for tapping mode experiments. These probes were cleaned in water plasma (6 mA source, 0.05 Torr water) for 3 minutes and characterised using a silicon calibrating grating (TGT 01, Silicon-MDT Ltd., Russia). Different parts of the frustule from the central to the peripheral areas were scanned on the inner and outer frustule surface. Image processing was performed using Nanoscope off-line software (Veeco Corp.) and SPIP (Image Metrology, Denmark.

3. RESULTS AND DISCUSSION

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For this research two centric diatom species were cultured, Coscinodiscus species and Thalassiosira eccentrica. Both species are frequently found in marine habitats and are ubiquitous components of diatom blooms. To gain a better understanding of biomineralisation process in diatoms the morphology of their silica structure at both nano- and micronscale is presented in our previous work [19-20]. High resolution images of the frustule prove that the silica wall material is composed of silica nanospheres with size of 80 –150 nm. Light microscopy investigation shows their strong opalescent color from diatom body, (Fig. 2). We assumed this opalescent effect originated from these fused silica nanoparticles. The interference effect depends on the angle at which light strikes the surface, hence the diatom appears to change color as it the observes moves position, as with a thin film of oil on water. Iridescent effects are used widely in color of cosmetics products and personal care packaging, and there is potential for using diatoms in this industry.

Figure 3 Optical microscopy images of colorful biosilica opalescent films formed on filter paper by filtering of the culture medium of Coscinodiscus sp.

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In this work we show that silica nanoparticles films with bright opalescent colors can be obtained from the diatom culture media. Diatoms were grown in culture media for 1-2 moths and the growth media was subsequently filtered

through filter paper. A film with displaying a wide range of colors visible to the naked eye was formed on the filter

surface. Optical microscopy was used for examination of the prepared film and a series of typical images are shown in Figure 3. The opalescent films often show colorful domains of sometime regular geometry (triangular or square). EDX

analysis confirms the silica composition of the film. The obtained films showed different colors from blue to red. In

order to understand the formation of this film we performed investigation of diatoms that were collected on the film. A typical SEM image of an intact diatom Coscinodiscus sp. is shown in Figure 4a. However, light microscopy and SEM images show a numbers of diatoms observed on film surface with fully and partially opened valves (arrow) (Figure 4 b-

d). This indicates that contents inside diatoms have been released into the culture solution. AFM and SEM of cell material escaped from diatom show the presence of spherical nanoparticles (Figure 4 f). It is well known that silica nanoparticles are formed inside of diatoms and SDV during biomineralisation process and the nanoparticles in the film

might originate in the SDV [17]. It was interesting that an opalescent film was formed on the filter from the

Coscinodiscus sp. culture medium (GSE), but not from the culture medium ( Guillard's medium, f/2 ) of another centric

diatom, T. ecentrica’s . These differences are attributed to different chemical composition of culture media and the fact

that valves of T. eccentrica stayed closed under the same condition. The size of T. eccentrica diatoms are about two times smaller than Coscinodiscus sp. Optical microscopy examination has shown significantly lower density of cell

culture grown for the same time and the retaining their compact structure is likely result of their healthier condition. This

suggests that valve opening of diatoms and release of ce ll contents into the culture media is a crucial step for particle film

formation . We propose that diatom death caused by starvation or bacterial attack during longer culturing is responsible for their valve opening.

Figure 4 a) SEM image of Coscinodiscus sp . with preserved structure. b) Optical microscopy image of opalescent film

with opened valves of several diatoms (a rrows). c-d) SEM images of opened diatom s demonstrating the feasibility of the discharge of the internal components into culture media. e-f) SEM and AFM images of es caped materials that indicate the presence of nanoparticles. Arrows (c-f) show escaped cell material from diatoms.

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AFM characterization of particle size from areas of the opales cent film with different colors were performed and images

reveal that the corresponding co lor of the spot is related to average particle diameter (Figure 5). This result proves that

observed colors in silica film are structural colors. Blue co lored regions feature with sma ller silica spheres (150-250 nm diameter) and red areas larger sized spheres (250-400 nm diameter). The observed colors are generated by Bragg light

diffraction in a similar way to gem opal where the diameter of the spheres controls the color (range) observed. Also the packing of spheres generates voids in the structure of the film which act as an optical diffraction grating for visible light.

Figure 5 a) Optical microscopy image of an opalescent film also showing the opened valve of one diatom (arrow) and

b-c) AFM images of silica nanoparticles from the green/blue and orange area of the film, re spectively, showing different average diameter of silica nanoparticles.

The formation of silica nanoparticles and nanoparticle opalescent f ilm is not clear at this stage. We believe that the silica nanoparticles are either released by the diatoms during reproduction or after cell death. There are two possible options of

their formation. One is that silica nan oparticles were formed inside of cell an d escaped from diatoms after cell death.

These nanoparticles were assembled into opalescent film dur ing filtration process. We obs erved nanoparticles on escaped cell materials that support this hypothesis. A high population of diatoms cells observed in culture media after one month

of culturing was enough to supply silica nanoparticles to form a visible opalescent film. Another possibility is that silica

nanoparticles are formed in the culture medium through the ac tion of biomolecular catalysts such as silaffins or long

chain polyamines released into the growth media during reproduc tion, or after death. The prec ursor of silica, silicic acid Si(OH) 4, exists in culture media and silicic acid can be easily polymerized by these catalysts into small silica aggregates

and form silica nanoparticles [22-23]. In vitro, biomimetic synthesis of silica nanoparticles by silaffins isolated from diatoms has been proved by several groups [22-24]. Current research is in progress to isolate these biomolcules from

culture media and further characteri ze the opalescent biosilica films in or der to resolve this process.

4. CONCLUSION

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The formation of opalescent films by self-assembly of silica nanoparticles produced in the growth medium of marine algae (diatoms) is demonstrated for the first time. AFM images of these films show the presence of ordered arrays of silica nanoparticles. As the color of the film under light microscopy observation changes, so does the average diameter of the nanoparticles in the AFM. A number of diatoms with partially opened valves were observed on the surface of the obtained film which indicates that cell contents inside of diatoms could escape into the culture solution. An opalescent film was formed from Coscinodiscus sp. culture medium but not from T. eccentrica. We believe that the film forming silica nanoparticles are either released by the diatoms during reproduction or after cell death. Alternatively or in addition, silica nanoparticles may be formed through the action of biomolecular catalysts such as silaffins or long chain polyamines released into the growth media. Further research will investigate whether the silica nanoparticles are produced intracellularly and then released or whether synthesis occurs in cell culture medium. This approach provides an environmentally friendly means for fabricating silica nanoparticles, artificial opal films, decorative coatings and novel optical materials. The structural colors made by manipulation on the nanoscale could also provide new and unexplored ways for artistic creation in a new field called NanoArt.

REFERENCES

[1] Sarikaya, M., Biomimetics: Materials fabrication through biology. Proceedings of the National Academy of Sciences of the United States of America, 96 (25), 14183-14185 (1999).

[2] Mann, S., Ozin, G. A., Synthesis of inorganic materials with complex form. Nature, 382(6589), 313-318 (1996).

[3] Wetherbee, R., Craford, S., Mulvaney, P., in Biomineralisation: From Biology to Biotechnology and Medical Application: edited by E. Beurlein, Wiley-VCH, Weinheim, (2000).

[4] Round, F.E., Crawford, R.M., Mann, D.G., “The Diatoms, Biology & Morphology of the Genera.” Cambridge, Cambridge University Press (1990).

[5] Sumper, M., Brunner, E., Learning from diatoms: Nature's tools for the production of nanostructured silica.

Advanced Functional Materials, 16(1), 17-26 (2006).

[6] Mandal, D. et al, The use of micorganisms for the formation of metal nanoparticles and their applications. Applied Microbiology and Biotechnology, 69, 485–492 (2006).

[7] Parkinson, J., Gordon, R., Beyond micromachining: the potential of diatoms, Trends in Biotechnology 17, 190-196 (1999)

[8] Drum, R. W., Gordon, R., Star Trek replicators and diatom nanotechnology, Trends in Biotechnology 21, 325-328 (2003).

[9] Kroth, P., Molecular biology and the biotechnological potential of diatoms, Advances in Experimental Medicine &

Biology 616, 23-33 (2008).

[10] Gordon, R., et al. A Special Issue on Diatom Nanotechnology, Journal of Nanoscience and Nanotechnology 5, 1-4 (2005)

[11] Lopez J. L, et al. Prospects in diatom research, Current opinion in Biotechnology, 16, 180-186 (2005).

[12] Losic, D., Mitchell, J. G., Voelcker, N. H., Fabrication of gold nanostructures by templating from porous diatom frustules, New Journal of Chemistry, 30(6), 908-914 (2006).

[13] Losic, D., Mitchell, J. G., Voelcker, N. H., Complex gold nanostructures derived by templating from diatom frustules, Chemical Communications, 39, 4905-4907 (2005).

[14] Losic, D., Mitchell, J. G., Lal, R., Voelcker, N. H., Rapid fabrication of micro- and nanoscale patterns by replica molding from diatom biosilica. Advanced Functional Materials, 17(14), 2439-2446 (2007).

[15] Losic, D., Triani, G., Evans, P. J., Atanacio, A., Mitchell, J. G., Voelcker, N. H. Controlled pore structure modification of diatoms by atomic layer deposition of TiO2, Journal of Materials Chemistry, 16(41), 4029-4034 (2006).

[16] Losic, D., et al. AFM nanoindentation of diatom biosilica surface, Langmuir, 23, 5014-5021 (2007)

[17] Sumper, M. A, phase separation model for the nanopatterning of diatom biosilica. Science, 295(5564), 2430-2433 (2002).

[18] Gordon, R., Losic, D., et al. The Glass Menagerie: diatoms for novel applications in nanotechnology, Trends in Biotechnology, 27, 2 (2009) (in press)

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[19] Losic, D., et al. Atomic force microscopy (AFM) characterisation of the porous silica nanostructure of two centric diatoms. J. Porous Mater. 14, 61-69(2007)

[20] Losic, D., Rosengarten, G., Mitchell, J. G., & Voelcker, N. H., Pore architecture of diatom frustules: Potential nanostructured membranes for molecular and particle separations. Journal of Nanoscience and Nanotechnology, 6(4), 982-989 (2006).

[21] Fuhrmann, T., et al. Diatoms as living photonic crystals, Applied Physics B., Lasers and Optics, 78, 257-260 (2004)

[22] Bauer, C. A., Robinson, D. B., Simmons, B. A. Silica particle formation in confined environments via bioinspired polyamine catalysis at near-neutral pH, Small, 3(1), 58-62 (2007).

[23] Warren, S. C., Disalvo, F. J., Wiesner, U., Nanoparticle-tuned assembly and disassembly of mesostructured silica hybrids. Nature Materials, 6 (2), 156-U123 (2007).

[24] Sumper, M., Lehmann G., Silica pattern formation in diatoms: species-specific polyamine biosynthesis.

Chembiochem 7(9), 1419-1427 (2006).