ISSN 0040�5795, Theoretical Foundations of Chemical Engineering, 2013, Vol. 47, No. 1, pp. 14–18. © Pleiades Publishing, Ltd., 2013.Original Russian Text © P.D. Sarkisov, E.G. Vinokurov, N.B. Gradova, E.S. Babusenko, V.V. Bondar’, 2013, published in Teoreticheskie Osnovy Khimicheskoi Tekhnologii, 2013,Vol. 47, No. 1, pp. 18–22.

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INTRODUCTION

Biotechnology is a relevant opportunity for creatingnanomaterials. Thus, for example, the mechanism ofthe biosynthesis of silica nanostructures by diatom algaemakes it possible to develop different nanomaterials.

Diatom algae, the shell material of which is similarto quartz glass [1], are proposed for use as a source ofquartz [2]. Diatom algae are also applied as bioreac�tors to synthesize organic compounds [3] and sorbentsdesigned to separate high�purity components [4] orfilling with catalytically active titanium oxide [5, 6].Silica diatom shells are used to obtain replicas fromgold, carbon, etc. Shell silica is also converted into 2–5�nm silicon nanocrystals, which may be used as gassensors [6]. Diatoms are cultivated under controlledconditions in photobioreactors [7]. The mechanismsof the transportation of silicon by diatom algae areactively studied [8]. Diatoms have skeletons that con�sist of 20–400�nm silicon dioxide nanoparticles,which form in the process of biosilicification. The pro�cess is governed by the special program written in thegenetic code of the proteins responsible for the separa�tion of silicon from seawater, along with dissolvedadmixtures under normal conditions [9]. Silicon isprecipitated with the participation of silaffins that rep�resent peptides catalyzing the polycondensation ofsilicic acid [10]. It is supposed that the distinctions inthe ornamentation of shells and the sizes of silicananostructures are due to the species�specific differ�ence of diatom silaffins [11, 12].

In biosystems, macromolecules strictly control theformation of the crystallization nuclei of inorganic

compounds, the stabilization of phases, and theassemblage and formation of spatial structures [13,14]. Characteristic features of living systems aremolecular selectivity and recognition ability. Biosys�tems can assemble structural blocks several nanome�ters in size into functionally complicated structures.For example, diatom algae proteins can induce theformation of the crystallization nuclei of inorganiccompounds. This process is strictly organized in spaceand time.

The most important moments of studies in the fieldof biological self�organization are the selection of suit�able combinations of biological and inorganic materialsand the determination of their compatibility, the syn�thesis of corresponding structural blocks, and theunderstanding of the mechanism of self�organization ofstructural blocks, as well as the control over this process.

Since the presence of silicon dioxide dissolved inthe surrounding medium is a necessary condition forthe fission of diatom algae cells, it is probable that dia�toms (single or colony) immobilized on the surface ofglass (one of SiO2 sources) can be cultivated in the cor�responding aqueous medium. In this fashion, we canimplement a new scientific field, surface bioengineer�ing, which is environmentally friendly, low�cost tech�nology.

The submitted work is devoted to the experimentalconfirmation of the possibility of the bioengineeringformation of glass surface micro� and nanostructuresusing diatom algae and to the study of the morphologyand some characteristics of a glass surface.

Bioengineering of Glass SurfaceP. D. Sarkisov a†, E. G. Vinokurova,*, N. B. Gradovaa, E. S. Babusenkoa, and V. V. Bondar’b

a Mendeleev Russian University of Chemical Technology, Miusskaya pl. 9, Moscow, 125047 Russiab All�Russia Institute of Scientific and Technical Information, Russian Academy of Sciences,

ul. Usievicha 20, Moscow, 125190 Russia* e�mail: vin@muctr.ruReceived October 1, 2012

Abstract—The possibility of implementing a new scientific field—bioengineering of glass surface—was considered. The growth of different diatom algal cultures and the density of their immobiliza�tion on the surface of glass were studied. The experimental results that confirm the possibility of thebioengineering formation of glass surface micro� and nanostructures using diatom algae were rep�resented.DOI: 10.1134/S0040579513010090

† Deceased.

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BIOENGINEERING OF GLASS SURFACE 15

INVESTIGATION OBJECTS AND METHODS

In our experiments, we used the Achnanthidium sp.,Craticula ef. minusculoides, Diadesmis confervacea,Gomphonema parvulum, Navicula sp., Nitzschia palea,Nitzschia sp., and Sellaphora minima cultures providedby the Department of Algology and Mycology of Mos�cow State University. Diatom algae were grown intightly closed 250�mL flasks containing a Chu�10nutrient medium (100 mL) [15] that was presterilizedin an autoclave at 112°С for 30 min.

Inocula were placed into flasks filled with a nutri�ent medium and incubated at 20°С and an illumi�nance of 3000 lx for 14 days. Algae samples for micro�scopic monitoring were taken in each 5 days. Afterincubating these cultures for 14 days, we placed three20 × 20�mm glass plates into each flask, after whichtheir incubation was continued under the same condi�tions.

In 2 (first sampling) and 6 (second sampling)weeks, one plate was retrieved from each flask,washed, dried, and thermally treated to removeorganic products (heating rate, 10°C/min; exposuretime, 0.5 h; temperature, 500°С, inertial cooling in aclosed furnace). The microscopic studies of theobtained samples and the calculation of the density ofcells immobilized on the glass surface were performedon an optical microscope at a ×300 and ×500 magnifi�cation. The hypothesis on the significant distinction ofsamplings was verified as described in [16] by themethod of nonparametric statistics using the Mann–Whitney test.

RESULTS AND DISCUSSION

In the course of the experiment, we have revealedthat the Craticula ef. minusculoides and Diadesmis con�fervacea diatoms in submerged culture grow slowlyand their cells are not almost immobilized on the wallsof a flask. The Navicula sp. diatom alga demonstrates

intensive growth, and its cells are immobilized on thewalls of a flask; however, upon shaking, they form afilm conglomerates that settle to the bottom of theflask. The Nitzschia palea and Nitzschia sp. culturesare also actively reproduced and immobilized on thewalls of a flask; however, upon shaking, they formcloddy conglomerates that settle to the bottom. TheSellaphora minima, Achnanthidium sp. and Gompho�nema parvulum cultures are intensively reproduced,and their cells are immobilized on the walls and bot�tom of a flask, which remain immobilized upon shak�ing. The further studies on the possibility of the forma�tion of glass surface micro� and nanostructures werepreformed with Achnanthidium sp., Gomphonema par�vulum, Navicula sp., Nitzschia palea, Nitzschia sp., andSellaphora minima.

To a considerable degree, the species of a culturegoverns the occupancy of the glass surface, which isdecreased in the order of Nitzschia palea > Navicu�la sp. > Achnanthidium sp. > Nitzschia sp. > Sellaphoraminima > Gomphonema parvulum (see table, glassexposure time is 2 weeks).

A surface of glass with immobilized shells of Ach�nanthidium sp. and Nitzschia sp. diatom algae after 6weeks of incubation in a nutrient medium is shown inFig. 1 as an example.

Based on the data of Table 1, it follows that, underexperimental conditions, the most active growth andoccupation of the surface of glass by diatoms wasobserved for up to two weeks. Under further incuba�tion, the surface occupancy remained almostunchanged (the experimental Mann–Whitney test Ue

exceeded its critical value Ucr for Gomphonema parvu�lum, Nitzschia palea, Nitzschia sp., Sellaphora minima)or decreased (Ue < Ucr for Achnanthidium sp. and Nav�icula sp.). In the latter case, the presence of diatomalgae shells destroyed probably due to their reuse bygrowing microorganisms was observed in photos of thesamples. For samples with Nitzschia palea, we

Average density of different diatom algae cultures immobilized on glass surface in different time periods

Glass exposure time, weeks

Density of different diatom algae shells immobilized on the surface of glass, cells/mm2

Achnanthidium sp.

Gomphonema parvulum Navicula sp. Nitzschia palea Nitzschia sp. Sellaphora

minima

2 800 (n = 2) 39 (n = 3) 998 (n = 2) 2064 (n = 4) 503 (n = 2) 487 (n = 6)

6 151 (n = 14) 43 (n = 13) 16 (n = 8) 1350 (n = 9) 273 (n = 8) 486 (n = 5)

Ue 1 13.5 0 8.5 4 15

Ucr 3 6 1 7 1 5

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SARKISOV et al.

(а)

(b)

100 μm

20 μm

Fig. 1. Glass surface regions with immobilized shells of (a) Achnanthidium sp. and (b) Nitzschia sp. diatoms after 6 weeks of incu�bation in nutrient medium.

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BIOENGINEERING OF GLASS SURFACE 17

observed the presence of surface regions with a highdensity of occupancy in the form of linear surfacestructures (Fig. 2). However, the role of factors thatgovern the uniformity and value of the density ofimmobilized diatom algae shells remains unclarifiedand requires further study.

CONCLUSIONS

In the present work, the possibility of the bioengi�neering formation of micro� and nanostructures onglass surfaces using diatom algae has been experimen�tally confirmed. In future, it is necessary to study thebehavior of diatoms in stress situations (for example,by changing the concentration of silicon and the illu�minance), the conditions that govern the uniformityand value of the density of immobilized shells, and thedirections of the growth of diatom algae colonies, aswell as to develop scientific foundations for obtaininghigh�purity micro� and nanostructured quartz surface.

ACKNOWLEDGMENTS

This work was supported by the Ministry of Scienceand Education of the Russian Federation (projectno. 11G34.31.0027).

NOTATION

n—sampling number;

U—statistical Mann–Whitney test.

SUBSCRIPTS AND SUPERSCRIPTS

cr—critical;

e—experimental.

REFERENCES

1. Hamm, C.H., Merkel, R., Springer, O., et al., Architec�ture and Material Properties of Diatom Shells ProvideEffective Mechanical Protection, Nature, 2003, vol. 421,no. 2, p. 841.

100 μm

10 μm

Fig. 2. Glass surface region with high density of immobilized Nitzschia palea diatom algae shells in the form of a linear structureafter 6 weeks of incubation in a nutrient medium.

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2. Grachev, M.A., Annenkov, V.V., and Vereshchagin, A.L.,RF Patent 2319672, 2008.

3. Kalinovskii, A.I., Gorshkov, A.G., Ponomarenko, L.P.,et al., Preparation of 13C�24�Methylcholesta�5,24(28)�Dien�2β�ol by Cultivation of the Baikal Diatom Synedraacus in NaH13CO3, Russ. Chem. Bull., 2010, vol. 59,no. 1, p. 236.

4. Annenkov, V.V., Gorshkov, A.G., Zelinskii, S.N., andDanilovtseva, E.N., Macroporous Liquid�Chromatog�raphy Matrices Based on Siliceous Valves of Diatoms,Dokl. Chem., 2010, vol. 432, part 2, p. 175.

5. Gordon, R., Sterrenburg, F.A.S., and Sandhage, K.H.,A Special Issue on Diatom Nanotechnology, J. Nanosci.Nanotechnol., 2005, vol. 5, no. 1, p. 1.

6. Milovzorov, D., Thin Film Nanocrystalline (111) Siliconfor Solar Energy and Electronics, Nanoindustriya, 2010,no. 3, p. 52.

7. Vereshchagin, A.L., Glyzina, O.Yu., Basharina, T.N.,Safonova, T.A., Latyshev, N.A., Lyubochko, S.A.,Korneva, E.S., Petrova, D.P., Annenkov, V.V., Danilovt�seva, E.N., Chebykin, E.P., Volokitina, N.A., andGrachev, M.A., Culturing of a Fresh�Water DiatomicAlga Synedra acus in One�Hundred�Liter Photobioreac�tor and Analysis of Biomass Composition, Biotekhnol.Russ., 2008, no. 4, p. 77.

8. Safonova, T.A., Annenkov, V.V., Chebykin, E.P., et al.,Aberration of Morphogenesis of Siliceous Frustule Ele�ments of the Diatom Synedra acus in the Presence ofGermanic Acid, Biochemistry (Moscow), 2007, vol. 72,no. 11, p. 1261.

9. Voznesenskiy, S.S., Galkina, A.N., and Kulchin, Yu.N.,The Features of Nanostructured Biosilica, Proc. 16th Int.Symp. “Nanostructures: Physics and Technology,” Vladi�vostok, 2008, p. 51.

10. Kroger, N., Deutzmann, R., and Sumper, M., Polyca�tionic Peptides from Diatom Biosilica That Direct SilicaNanosphere Formation, Science, 1999, vol. 286, no. 5,p. 1129.

11. Lee, R.E., Phycology, Cambridge: Cambridge Univ.Press, 2008, 4th ed.

12. Poulsen, N., Sumper, M., and Kroger, N., Biosilica For�mation in Diatoms: Characterization of Native Silaffin�2and Its Role in Silica Morphogenesis, Proc. Natl. Acad.Sci. U. S. A, 2003, vol. 100, no. 21, p. 12075.

13. Belcher, A.M., Wu, X.H., Christensen, R.J., Hans�ma, P.K., and Stucky, G.D., Control of Crystal PhaseSwitching and Orientation by Soluble Mollusc�ShellProteins, Nature, 1996, vol. 381, p. 56.

14. Falini, G., Albeck, S., Weiner, S., and Addadi, L., Con�trol of Aragonite or Calcite Polymorphism by MolluskShell Macromolecules, Science, 1996, vol. 271, p. 67.

15. Gaisina, L.A., Fazlutdinova, A.I., and Kabirov, R.R.,Sovremennye metody vydeleniya i kul’tivirovaniya vodor�oslei: Uchebnoe posobie (Contemporary Methods of Iso�lation and Cultivating of Algae: A Textbook), Ufa:BGTU, 2008.

16. Vinokurov, E.G., Burukhina, T.F., Kolesnikov, V.A., andFadina, S.V., Concentration Criterion for ClassifyingResource�Saving Compositions of Solutions for MetalElectroplating, Theor. Found. Chem. Eng., 2012, vol. 46,no. 5, p. 486.