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Si and Fe biomineralization by microorganisms in bottom sediments ofDelta Selenga River, tributary of Lake Baikal, Russia
Natalia BELKOVA1, 2, Valentina PARFENOVA2, Juliya ZAKHAROVA2,and Kazue TAZAKI3
1 Graduate School of Natural Science and Technology, Kanazawa University, Kakuma,Kanazawa, Ishikawa, 920-1192 Japan
2 Limnological Institute of Siberian Division of Russian Academy of Sciences, UlanBatorskaya str., 3, Irkutsk, 664033 Russia
3 Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma,Kanazawa, Ishikawa, 920-1192 Japan
Abstract : Bottom sediment samples, collected from Delta Selenga River, tributary ofLake Baikal, Russia, were analyzed with methods of elemental chemical analysis andoptical microscopy. Chemical composition of sediments was studied with energy dispersive X-ray fluorescence spectrometer, which revealed presence of Si, Fe, Al, Ca and K
as major elements, associated with traces of Mn, Mg, Ti, and Sr in the bottom sediment
samples. Diatoms and bacteria were observed with light and epifluorescence optical
microscopy, showing formation process of organic substances with mineralogical aggregates.
Si and Fe biomineralization on the surface of diatoms has been observed with lightmicroscopy. Fe-oxidizing bacteria with ability to metal accumulation were isolated
on selective medium. The results suggest that Si and Fe biomineralization of diatoms
with Fe-oxidizing bacteria indicate deoxidized ecological environment to form pyrite in/on the cell crust.
Key words : bottom sediments, ED-XRF analysis, diatoms, iron-oxidizing bacteria,
metal accumulation, Si-Fe biomineralization.

1. Introduction
Recently, biomineralization in natural environments is one of the main interesting andkey point in the study of the interactions between microorganisms and minerals. The
process could be visualized not only in the deep core samples, but also in modern habitant,such as hot and mineral springs (Fouke et al., 2000), marine and freshwater sediments(Aguilar and Nealson, 1998). Diatoms, bacteria and fungi usually involved in theseprocesses. Microbial communities are thought to play a significant role in both the oxidationand reduction of iron and manganese in freshwater sediments (Stein et al., 2001). Diatoms
and algae create highly complex and intricate silica structures under physiological conditions, during Si-cycles in marine and freshwater sediments (Cox, 1996 ; Asada and Tazaki,

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Natalia BELKOVA, Valentina PARFENOVA, Juliya ZAKHAROVA, and Kazue TAZAKI

2001). Microbial reduced silicon is removed from the sediments to the water column.
Delta Selenga River has been actively sedimented to form extensive delta (Votintsevet al., 1963). Chemistry and microbiology of bottom sediments of Selenga River have beenreported (Goldurev et al., 1971 ; Mladova, 1971). Their results show Si, Fe, Mn, and Al
were major elements in the river sediments (Votintsev et al., 1963 ; Goldurev et al., 1971).
River sediments counting of total number of bacteria revealed a large number of microorganisms to compare with sediments of Lake Baikal (Mladova, 1971). Cultivation ofheterothrophic bacteria was carried, and microorganisms belong to Pseudomonas, Arthrobacter, Flexibacter, Bacillus, Micrococcus, Desulfotomaculum, Cytophaga, and Clostridium genera were identified in Selenga River sediments (Namsarayev et al., 1994). Diatoms,
collected from Selenga River and Lake Baikal, were identified, as follows, Aulacoseiraspp., Synedra spp., Stephanodiscus spp., Cyclotella spp., Melosira spp., and Novicula spp.
(Likhoshway et al., 1996).
In this study the bottom sediment of Delta Selenga River were microbiologically andchemically discribed. The role of microorganisms in silicon cycles is discussed.
2. Materials and Methods
Bottom sediment samples were collected from 13 points of 10 cross-sections, as in theaquatory of open Baikal (cross-section 1 p.106, cross-section 10 p.102), as in the delta ofSelenga River (points of cross-sections 2-9) (Fig. 1). Upper layer of sediments werefreezed immediately after collecting samples and stored at temperature -20°
C before analysis.
Energy dispersive X-ray fluorescent analysis was carried on JEOL Element Analyzer
JSX-3201 with Rh Kα, at 30 kV in vacuum condition. Sediment samples were airdried andgrounded to fine powder. Optical micrographs were taken with Nikon EFD 3 microscope.
Samples were also stained with 4’, 6-diamidino-2-phenilindole solution (DAPI, 50 μg/ml)
during 3 min and visualized with UV-1 A filter set for epifluorescent microscopy. Fe-oxidizing
bacteria were cultivated on selective medium with agar at 25°
C during 2 - 8 weeks.
Medium contained, as follows, (NH4)2SO4 (0.5 g/l), NaNO3 (0.5 g/l), K2HPO4 (0.5 g/l),MgSO4 (0.5 g/l), FeSO4 (5.9 g/l), triptone (1 g/l), agar (15-20 g/l), pH 6.8.
3. Results
Chemical analysis of bottom sediments
Chemical composition of the bottom sediments of Delta Selenga River is shown inTable 1. The results revealed presence of Si, Fe, Al, Ca and K as major elements, associated with traces of Mn, Mg, Ti, and Sr. In Selenga River sediments Si was found in
higher concentration than in lake sediments. Si concentration ranges from 53.61 to 66.07
wt % in river sediments, whereas from 39.05 - 46.44 wt % of Si are in lake sediments. Fe
concentration ranges from 3.85 to 12.90 wt % in river sediments, whereas 25.87 - 29.25 wt %of Fe are in lake sediments. Sr concentration varies in from 0.17 to 0.33 wt % without any
dependence to sampling points. Rb was detected only in few river samples. Cr was detectedSi and Fe biomineralization by microorganisms in bottom sediments ofDelta Selenga River, tributary of Lake Baikal, Russia

41Fig. 1. Location maps of the study area of Delta Selenga River and Lake Baikal. The numbers indicate
sampling points.

in one sample and the concentration was 0.10 wt % in the region of open Baikal−c-s 10p.102.

42Natalia BELKOVA, Valentina PARFENOVA, Juliya ZAKHAROVA, and Kazue TAZAKI

Table 1. Energy dispersive X-ray fluorescent analysis of bottom sediment samples from Delta Selenga
River and Lake Baikal, Russia.

Optical microscopy of bottom sediment samples
With optical microscopy of bottom sediments large amount of diatoms with variety ofspecies were revealed. Fig. 2 A shows common view of the bottom sediment samples.
More than 4 or 5 different species of diatoms were visualized in each sampling points. The
diatoms belong to Aulacosira, Cyclotella, Cymbella, and Novicula genera were foundcommonly in sediments (Fig. 2 A, B, C). Single diatom cells with clearly distinguished internal structure were visualized in bottom samples, showing attached bacterial cells (Fig. 2D, E). The diatom cell was covered on whole surface by mineral particles (Fig. 2 F).
Spherical aggregates formed with mineral particles, diatoms and bacterial cells (Fig. 2 G).
Chemical composition of bottom sediments
High concentration of silicon was found in the samples c-s 9, p.52 - c-s 2, p.3. In theregions of open Baikal c-s 1, p.106 ; c-s 10, p.102, Si content is quite low (Fig.3 A).
Cultivation and isolation of Fe-oxidizing bacteria
Fe-oxidizing bacteria were isolated on selective medium. Mixture of microorganisms
was initially cultivated at 25°
C during 3-8 weeks (Fig. 4 A). Colonies with different colorand growth ability were observed. Microorganisms were purified and grown on the same
medium after 3 or 4 passages (Fig. 4 B, C). The size of single colony was varied from 1 to
10 mm in diameter. Colonies of microorganisms have metallic color, white and transparent(Fig. 4 B, C). The colonies have various shapes. Rounded shape in metallic color (B) andstar-typed shape colony (C) grow through agar.
4. Discussion
According to the previously investigations, the total amount of diatoms in the upper
layer of Selenga River water is higher than in Lake Baikal water (Likhoshway et al., 1996).
The sedimentation progress actively in river sediments than in lake sediments (Votintsev etal., 1963). Two processes of Si biomineralization were observed in the sediments by optical microscopy, as follows ; one is concerning to accumulation of silicon by diatoms, andother one is by microorganisms on the cell walls (Fig. 2 F, G). Digestion of diatom cells bymicroorganisms are shown in Fig. 2D, E. Because of extreme conditions in Lake Baikal

Si and Fe biomineralization by microorganisms in bottom sediments ofDelta Selenga River, tributary of Lake Baikal, Russia

43Fig. 2. Optical micrographs of diatoms in the bottom sediments collected from Selenga River. Common
view of the sample : diatoms are marked with arrows (A). Diatoms commonly visualized in river
sediment samples : Cyclotella sp. (B) and Navicula radiosa (C). Light (D) and epifluorescence(E) micrographs of Cymbella sp. with attached bacteria cells. Diatoms cell covered with attachedbacteria and mineral particles (F). Aggregates formed with diatoms, microorganisms and mineralparticles (G).

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Natalia BELKOVA, Valentina PARFENOVA, Juliya ZAKHAROVA, and Kazue TAZAKI

SiFeAlTiMg

MnSrFig. 3. Chemical composition of bottom sediments of Delta Selenga River and Lake Baikal. Si and Fe
concentrations (A) ; Al, Ti and Mg concentrations (B) ; Mn and Sr concentrations (C) showing
distribution tendency between delta and lake sediments.

Si and Fe biomineralization by microorganisms in bottom sediments ofDelta Selenga River, tributary of Lake Baikal, Russia

45Fig. 4. Optical photographs of colonies of Fe-oxidizing microorganisms, isolated from bottom sedimentsof Delta Selenga River. A, mixture of microorganisms cultivated on selective medium ; B, C,
colonies of isolated strains with different capacity to growth.

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Natalia BELKOVA, Valentina PARFENOVA, Juliya ZAKHAROVA, and Kazue TAZAKI

water and sediments, such as low mineral content and low concentration of nutrients,
microorganisms in natural environments have grown with high enzymatic activities (Parfenova, 1985). Microorganisms, such as Si-utilized bacteria, utilized silica from diatoms
cell walls during precipitation from the upper layer of water to bottom sediments. Fig. 2 (D,E) shows this process on the cell walls of Cymbella sp., found in the sediments from Lake
Baikal. On epifluorescence micrographs (Fig. 2 E), large amount of bacterial cells werefound. The process of accumulation and precipitation of Si is more dependent on diatoms(Fig. 2 H, G). According to chemical analysis of sediments (Fig. 3 A, B, C), weight percent
concentrations of Si tend to curve similar to Al and Sr. Diatoms might be responsible for
Al and Sr accumulation in river sediments.
The attempt to isolate pure strains of microorganisms associated with metal accumulation was carried out. Diversity of Fe-oxidizing bacteria, isolated from sediments, shows
variety of existing strains : ones have metal color of the colony (Fig. 4 B), while others
have grown and change the medium color to neutral (Fig. 4 C). These strains have different
mechanisms of metal accumulation deals with either the capacity to adsorption, accumulation
or transportation of metal on/in the cells (Ledin, 2000). The tendency weight percent concentration was found on Mn, Mg, and Ti similar to curve of Fe (Fig. 3 A, B, C). These metals
could be involved in the processes of biomineralization corresponding with microorganisms.
5. Conclusion
According to chemical analysis of bottom sediments, optical microscopical observationand isolation of Fe-oxidizing bacteria, we can suggest that Si, Al and Sr biomineralization
process is corresponding with diatoms which metal accumulation of Fe, Mn, Mg and Ti isdeals with bacteria. In further perspectives, pure strains of microorganisms with metal
accumulation abilities would be cultivated, and mechanisms of biomineralization process
would be studied in laboratory experiments.
Acknowledgement
We are thankful to Dr. R. Asada for help in chemical analysis of the samples, to colleagues from laboratory of Water Microbiology, Limnological Institute SD RAS for help
in isolation of microorganisms and fruitful discussion.
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