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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. Voelcker3
1
3
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.
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
Smart Materials V, edited by Nicolas H. Voelcker, Helmut W. Thissen,
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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).
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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
Proc. of SPIE Vol. 7267 726712-3
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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.
Proc. of SPIE Vol. 7267 726712-4
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In this work we show that silca na oparticles films with
culture media. Diatoms wer grown in culture media for 1-2 moths and the growth media was ubsequently filter d
through filter pa er. A film with displaying a wide range of col rs visble to the naked ey was formed on the filter
surface. Optical microscopy was used for
Figure 3. The opalescent films often show col rful domains of sometime regular geometry (triangular or square). EDX
an lysi confirms the silca compositon of the film. The obtained films howed if er nt col rs from blue to red. In
order to understand the formation of this filmwe performed
typical SEM image of an intact diatom
images howanumbersofdiatomsobservedonfilmsurfacewithfulyandpartialyopen dvalves(ar ow)(Figure4bd). This indicates that contents inside diatoms have be n
material escaped from diatom show the pres nce of spherical
na oparticles are formed inside of diatoms and SDV during biomineralisation proces and the na oparticles in the film
might orignate in the SDV [17]. It was inter sting that
Coscinodiscus p.
culture medium (GSE), but not from the culture medium (
diatom,
T. ec ntrica’s
. Thes dif er nces are atributed to dif er nt chemical compositon of culture media nd the fact
that valves of
T. ec entrica
stayed closed under the same conditon. The size of
times maler than
Coscinodiscus p.
culturegrownforthesametimeandthe
sug est hatvalveopeni gofdiatomsandrel aseofce
formation
. We prop se that diatom death caused by starvation or bacterial at ck during longer culturing is responsible
fortheirvalveopeni g.
Acc.V Spot Magn
100 kV 3.0 3482x
bright opalescent col rs can be obtained from the diatom
examination of the prepared film and a
Coscinodiscus
series of typical images are shown in
sp.
investigation of diatoms that wer colected on the film. A
is hown in Figure 4a. Howev r, light microscopy and SEM
rel ased into the culture solution. AFM and SEM of cel
na oparticles (Figure 4 f). It is wel known that silca
an opalescent film was formed on the filter from the
Guilard's medium, f/2
) of another centric
T. ec entrica
diatoms are about wo
Optical microscopy examination has hown signifcantly lower density of cel
retain gtheircompactsructureislikelyresultoftheirhealthierconditon.This
l contentsintotheculturemedia sacrucialstepfor
particlefilm
Dot WD I
SE
101 nanogold2Osl
.'N
ApnfPnDnfWDI
Figure 4 a) SEM image of
withopen dvalvesofsev raldiatoms(a
discharge of the internal compone ts into culture media.
thepres nceofna oparticles.Ar ows(c-f)
Coscinodiscus p
.withpres rved structure.
b)Optical microscopy image of opalescentfilm
r ows).c-d)SEMimagesofopen d iatom
sdemonstratingthefeasiblityofthe
e-f) SEM and AFM images of es
showescapedcel materialfromdiatoms.
Proc. of SPIE Vol. 7267 726712-5
caped materials that indicate
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AFMchar cterizationofparticlesizefromareasoftheopales
rev al that he cor esponding co
observedcol rsinsilcafilmarestructuralcol rs. Blueco
diamet r) and red areas larger sized spher s (250-40 nm diamet r). The observed col rs are gen rated by Brag light
dif raction inasimilarwaytogemopalwher thediamet rof
packingofspher sgen ratesvoidsinthestructureofthefilmwhichactasanopticaldif ractiongratingforvisble ight.
centfilmwithdif er ntcol rswer performedandimages
lor of the spot is related to average particle
diamet r (Figure 5). This result proves that
loredregionsfeaturewithsma
lersilcaspher s(150-250nm
thespher scontrolsthecol r ( ange)observed.Also the
\
Figure5 a)Optical microscopyimage of anopalescentfilmalso showingtheopen dvalveofonediatom (ar ow)and
b-c)AFMimagesofsilcana oparticlesfromthegre n/blue
averagediamet rofsilcana oparticles.
Theformationofsilcana oparticlesandna oparticleopalescentf
na oparticlesare ither el ased
their formation. One is that silca na
Thes na oparticleswer as embledinto palescentfilmdur
cel materialsthatsup ort hishypothesi .Ahighpo ulationofdiatomscelsobservedinculturemedia fteronemonth
ofculturingwasenoughtosup lysilcana oparticlestoformavisbleopalescentfilm.Anotherpos iblityisthatsilca
na oparticles are formed in the culture medium through the ac
chainpolyaminesrel asedintothegrowthmediaduringreproduc
Si(OH)
4 , exist in culture media nd sil ci acid can be asily polymerized by thes cat lyst into smal sil ca g regates
and form silca na oparticles [2 -23]. In vitro, biomimetic synthesi of silca na oparticles by silaf ins isolated from
diatoms has be n proved by sev ral groups [2 -24]. Cur ent res arch is in progres to isolate thes biomolcules from
culturemedia ndfurtherchar cteri
andorangeareaofthefilm,re
spectively,showingdif er nt
ilmisnotclearat his tage.Webeli vethat hesilca
bythediatomsduringreproductionoraftercel death.Ther aretwopos ibleoptionsof
oparticles wer formed inside of cel an
d escaped from diatoms after cel death.
ingfiltrationproces .Weobs
ervedna oparticlesonescaped
tion of biomolecular cat lyst such as ilaf ins or long
tion,orafterdeath.Theprec
zetheopalescentbiosilcafilmsinor
ursorofsilca,silci acid
dertoresolvethisproces .
4.
CONCLUSION
Proc. of SPIE Vol. 7267 726712-6
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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.
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