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Sr-rich microbial mats at Zhemchug hot springs,
southwest Lake Baikal, Russia
Kazue TAZAKI1, Koushirou MIYATA1, Natalia BELKOVA 2,3 and Ryuji ASADA 2
1. Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma,
Kanazawa, Ishikawa, 920-1192, Japan
2. Graduate School of Natural Science and Technology, Kanazawa University, Kakuma,
Kanazawa, Ishikawa, 920-1192, Japan
3. Limnological Institute of Siberian Division of Russian Academy of Sciences, UlanBatorskaya str., 3, Irkutsk, 664033, Russia
Abstract : At Zhemchug hot springs, southwest Lake Baikal, Russia, the biomats contain high concentration of Ca and Sr, which microbiological, chemical and mineralogicalanalyses were carried. Optical and scanning electron microscopic observation revealed
that carbonate minerals were associated with cyanobacteria. The biomats have colorful
layered structure, such as green, white, brown and black. Chemical analysis of thebiomats revealed high concentration of CaO (85-96 wt%) and SrO (1.1-2.2 wt%) in thefour layers. The mineralogical composition of the layered structure is mainly aragonite
associated with calcite. Elemental layered patterns of the biomats are shown in EPMA
elemental content maps indicating the coincidence of Ca and Sr. By increasing pH andthe oxygen concentration on the surface of biomats, the cyanobacteria could potentially
increase the rate of metal accumulation. The cyanobacteria in hot spring biomats may
carry important role to accumulate Ca with Sr.
Key words : hot springs, biomats, cyanobacteria, Sr, Ca, aragonite

1. Introduction
Hot springs are one of the main important and interesting objects in studying of processes of metal accumulation. In the hot springs, biomineralization has progressed, becauseof high temperature and large amount of nutrients. Biomats in hot springs often havebanded structure and consist of calcium carbonate with high concentration of heavy metals(Yasuda et al., 2000 ; Ohno and Tazaki, 2000 ; Akai, 2000). Chemical and biological diversity of biomats influence water ecosystem (Tazaki, 2000). Microbiological variety in
hot springs also influenced water chemistry and formed different biominerals (Ohno andTazaki, 2000). Microorganisms selectively accumulated heavy metals in mining waste
water system (Tazaki, 2000). In the accumulation processes, various groups of livingorganisms are usually involved. Diatoms can precipitate silicon on their cells walls,
whereas bacteria can accumulate Fe, Mn, Sr, and some other elements in/on the cells

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Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADA(Beveridge, 1989).
In this study, Sr-rich microbial mats with cyanobacteria were found at Zhemchug hot
springs, southwest Lake Baikal, Russia. Observation of the biomats by using optical andscanning electron microscope, ED-XRF, XRD, NCS and EPMA micro techniques clearly
showed layered Ca-Sr biomineral formation.
2. Materials and Methods
Biomats and hot spring water were collected from Zhemchug hot springs in July 2001,
southwest of Irkutsk, Russia (Fig. 1 A, B). The characteristics (pH, Eh, EC, DO and WT)of hot spring water were measured at the field (Table 1) (Eh : Electrode potential versus
the standard hydrogen electrode, EC : Electrical conductivity, DO : Dissolved oxygen,

Fig. 1. Location map of sampling points at Zhemchug hot springs, southwest Lake Baikal, Russia (A).
Field view of biomats at Zhemchug hot springs (B). Cross section of the biomats (C). In biomats
4 layers can be clearly distinguished according to different colors, such as green, white, brownand black layers from the top surface in order.

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Table 1. Water characteristics at Zhemchug hot springs, southwest Lake Baikal, Russia.

WT : Water temperature). One liter of hot spring water was filtrated through bacterial 0.22
μm filter (Millipore) with peristaltic pump to determine bacteria in hot spring water. The
biomat samples were incubated in hot spring water at room temperature during 4 months atlight conditions. The measurements of water characteristics were also carried after 4
months storage (Table 1).
Biomats have layered structure, which were separated into 4 layers due to differentiation in color (Fig. 1 C). The chemical composition in each layer and mineralogy wereanalyzed. The polished thin section of biomats was prepared for optical microscopic observation and EPMA analysis. Furthermore, optical microscopy and SEM-EDX techniques
were used for observation of green biomats on top surface.
2.1. Optical microscopy observation of biomats and bacteria
Green biomats on top surface and microorganisms collected on the filter (pore size :0.22 μm) were stained with 4’, 6-diamidino-2-phenilindole (DAPI, 50μg/ml) for 3 minutes
to observe under epifluorescent microscope (Nikon EFD 3, Digital camera : COOLPIX
995). For epifluorescent microscopy, a filter UV-1 A (wavelength of exposed light : 360370 nm) was used for DAPI-staining observation and a filter G-2 A (wavelength of exposed light : 510-560 nm) for observation of chlorophyll in cyanobacterial cells. The surface of biomats was observed twice, within 1 week after collection of samples and after 4
months storage. The polished thin section of biomats were examined using a polarizing optical microscope (Nikon UFX-Ⅱ A) and EPMA.
2.2. Energy dispersive X-ray fluorescence spectrometer analysis of biomats
After air-drying, biomat samples were ground to fine powder for ED-XRF analysis.
The powder samples mounted on the Mylar film measured by an energy dispersive X-ray
fluorescence spectrometer (JEOL JSX 3201 using Rh Kα), which operated at an accelerating voltage of 30 kV under a vacuum condition.
2.3. X-ray powder diffraction analysis of biomats
The mineralogical properties of biomats were analyzed by X-ray powder diffractometer (Rigaku RINT 2000) with Cu Kα generated at 40 kV and 30 mA using 2 θ/θ method
and a scan speed 2°/min. The powder of each sample was mounted onto slide glasses to fit
the diffractometer sample-holder.

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2.4. Nitrogen, carbon and sulfur elemental analyses of biomats
By an automatic gas chromatographic elemental analyzer (CE Instruments NA 2500NCS) at 1000°
C with 20 ml oxygen contents of carbon, sulfur and nitrogen of biomats
were determined. All powder samples were analyzed after treatment with HCl.
2.5. Electron probe microanalysis of biomats
Polished thin section of biomats was examined by electron probe microanalysis(EPMA : JEOL JXA 8800 R Super Probe) at an accelerating voltage of 15 kV for elemental content maps and quantitative analysis of major elements.
2.6. Scanning electron microscope (SEM) observation of biomats
Green biomats were micromorphologicaly investigated using a scanning electron microscope (SEM : JEOL-JSM-5200 LV), equipped with an energy dispersive X-ray spectrometer (Philips-EDAX PV 9800 STD).
3. Results
Water chemistry
Field measurements of hot spring water revealed anaerobic condition : pH 7.0, Eh 36
mV, EC 0.67 mS/cm, and DO 3.5 mg/l. After 4 months storage, pH increased till 9.3 andDO also increased twice as (6.8 mg/l) indicating microbial oxygenic activity (Table 1).
3.1. Optical microscopic observation
The observation of green top surface of biomats revealed that different types ofbacteria live in aggregates, and in free-living forms (Fig. 2 A, B). Coccus typed bacterial
cells, 5 μm in size, similar to Anabaena species cells, aggregated together with bacterial
cells and mineral particles were clearly distinguished. In the hot spring water, only small
sized bacterial cells (filtrated through 0.22 μm) aggregates in free-living form were found(Fig. 2 C, D). Numerous community of chains of Anabaena-like cells, filaments, other species of bacteria and mineral particles (Fig. 3 A, B and C) were shown by optical microscopy of green biomats after 4 months storage.
Observation of thin sectioned biomats showed that the layered structures were separated into four sections due to not only by color, but also by structure of minerals (Fig. 4).
The top surface showed colloform structure. White layer was reach in chink, whereasbrown and black layers compacted with radial growth structure were found. Black layer
has well-crystallized banded structure. Underneath the black layer, porous carbonate grains
are present.

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Fig. 2. Optical micrographs of green top surface of biomats (A ; light, B ; epifluorescent) and bacteria,collected on 0.22 μm filter (C ; light, D ; epifluorescent) from hot spring water. All micrographs
were taken within 1 week after sampling.

3.2. ED-XRF analyses of biomats and water
The ED-XRF analysis of biomats showed the presence of CaO, Fe2O3, SiO2, and SrOwith the traces of MgO, PO4, SO4, Al2O3, and MnO in biomats (Table 2). The content of
CaO (88.88 wt%) in biomats is very high, especially top surface in green and brown layers
contain more than 90 wt% of CaO. Analysis of hot spring water showed large amount of
CaO (30.42 wt%) and Fe2O3 (23.33 wt%). Such elements as Na2O, MgO and SiO2 in the
hot spring water were also found in higher concentration than in biomats. Although the elements of K2O and SrO were found as trace elements in the water, but other elements of PO4,
Al2O3, SO4, MnO2 and As were not detected. Concentration of SrO on top surface ofgreen biomats (1.11 wt%) was five times as high as in hot spring water (0.27 wt%) suggesting
selective accumulation of SrO in biomats.
3.3. XRD analysis
The XRD patterns of all sections in biomats showed the same tendency. The strong
aragonite peaks were found at 3.4A, 2.7A, 2.0A in all layers (Fig. 5). The small peaks at
3.0A, 2.3A, and 1.9A in white layer of biomats also showed the presence of calcite. Any
SrO minerals were not detected at whole biomats.

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Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADA

Fig. 3. Micrographs of green biomats on top surface (A ; light, B ; epifluorescent with UV-1 A filter set,
C ; epifluorescent with G-2 A filter set) showing cyanobacterial colony with carbonate grains.
The observations were carried after 4 months storage at room temperature.

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Fig. 4. Optical micrographs of polished thin section of biomats at Zhemchug hot springs. The layered
structures were separated into four sections due to not only by color, but also by structure ofminerals.

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Table 2. Energy dispersive X-ray fluorescence spectrometer analyses of biomats and water at Zhemchug hot springs.

Fig. 5. X-ray powder diffraction of biomats at Zhemchug hot springs, indicating mainly aragonite withcalcite.

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3.4. NCS analysis
NCS elementary analysis of four layered biomats revealed that the tendency is similar
in carbon, nitrogen and sulfur contents (Table 3). The concentration of elements ranged ascarbon from 0.55 to 0.75 wt%, nitrogen from 0.06 to 0.14 wt% and sulfur from 0.14 to
0.17 wt%. In white layer, amount of carbon and sulfur were relatively low concentrationsuggesting less organic materials, according to association of calcite with aragonite.
Table 3. Elemental analysis of carbon, nitrogen and sulfur in biomats at Zhemchug hot springs.

3.5. EPMA analysis
EPMA analysis of biomats shows compositional banded structure of Ca, Sr, Fe andMg (Fig. 6). Although Sr and Fe are associated with Ca, the concentration of Mg shows

Fig. 6. Electron microprobe elemental content maps of thin sectioned biomats at Zhemchug hot springs.
Elemental distribution maps of Ca, Sr, Fe and Mg indicated chemical layered structures. The Srrich layers are consistent with Ca-rich layers.

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Table 4. Electron microprobe quantitative analysis of 4 different layers of biomats at Zhemchug hot
springs, southwest Lake Baikal, Russia.

reverse pattern of Ca. Quantitative analysis of the layered biomats produces a profile of elemental concentrations (Table 4). Layer 1, 2 and 3 of biomats showed the similar chemicalcomposition of SrO, CaO, Fe2O3, MgO, and MnO. The concentration of CaO ranged from52.25 to 52.46 wt%, and SrO from 1.36-1.55 wt%. While, in the layer 4 the concentrationsof SrO, CaO and Fe2O3 were lower than in the other layers. Only the concentration of MgO
was relatively high suggesting mixture of dolomite. The results of EPMA analysis are
consistent with that of ED-XRF (Table 2), showing SrO concentration ranges from 1.11 to
2.21 wt%.
3.6. SEM observation
SEM observation of green biomats on top surface revealed that the biomats were composed of Ca grains, Si grains and filamentous bacteria (Fig.7 A, B and C). Ca grains (arrow
A in Fig. 7) is about 5 μm in size, Si grains (arrow B in Fig. 7) is about 2 μm in size
whereas filamentous bacteria are about 1 μm in diameter (arrow C in Fig. 7). The EDX
analysis revealed that bacteria contain high concentration of Si, Cl and Ca with traces of P
and S characteristically (analytical point: arrow C in Fig. 7). The Si grain contains relatively
high Fe, whereas the bacteria have no Fe at all. The concentration of Sr was not detected
because of detection limits of EDX.
4. Discussion
Formation of periodic alternations of iron, manganese and silica-rich strata are wellknown from the Archean, as represent by Banded Iron Formation (Fortin et al., 1998). In
this study, accumulation of SrO and CaO in layered biomats were found in Zhemchug hot
springs, southwest Lake Baikal, Russia, essentially result from bacterial activity. The
biomats at Zhemchug hot springs composed of mainly aragonite. Observations of Sr-richmicrobial mats described here suggest that cyanobacteria produce structure by periodic
biomineralization of aragonite. Calcite is also formed by fibrous bacterial activity in hot
springs has reported (Yasuda et al., 2000 ; Ohno and Tazaki, 2000). The specially definedpattern of these elements showed layer structure in different color, such as white, brownand black. Various bacteria, including coccoid, rod and filamentous forms, concentrate Si,
Cl and Ca on the cell walls during their lives, and accumulate additional hydroxides toSr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, Russia

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Fig.7. SEM micrograph and the EDX analysis of green biomats on top surface at Zhemchug hot springs.
(arrow A : carbonate grain, B : silicate grain, C : bacteria, (A) : EDX spectrum of carbonate
grain, (B) : silicate grain, (C) : bacteria).

form aragonite. Aragonite in hot springs contains higher concentration of Sr in comparison
with calcite (Ichikuni, 1980). In this study, it was revealed that the Zhemchug hot spring
biomats was composed of aragonite with high concentration of Sr. The EPMA content
maps and electron micrographs of layers suggested that cyanobacteria grew rapidly and accumulated Sr from hot spring water at the initial stages of the microbial process. Bacteria
in the biomats absorb abundant Sr in high crystalline aragonite to form layered structure
within short period. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ (Pierson et al., 1999). The Zhemchug hot spring biomats are suitable models for studying theinteractions among photosynthesis microorganisms, metal accumulation and the cycle ofmicrobial Sr.
The results indicate that biomineralization can be applied to bioremediation of radioactive elements. Furthermore, the biomats can be used for 3 R purposes, as follows ; Recovery of clean water, Resource of useful bacteria and Recycle of Sr-rich aragonite.

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Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADA

5. Conclusion
The biomats at Zhemchug hot springs, southwest Lake Baikal, Russia was composed ofmainly high crystallin aragonite with high concentration of Sr. The EPMA maps show theelemental layered structures. The Sr-rich biomas was formed by cyanobacterial activity.
Acknowledgement
We thank for Dr. Drucker B. and Dr. Parfenova V. of Limnological Institute for ourfield trip. Thanks are also due to the students of the Tazaki’s laboratory at Kanazawa University for their cooperation. This study was supported by the grants from the Japanese
Ministry of Education, Science and Culture.
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