Asada Ryuji

Belkova Natalia

Miyata Koushiro

Tazaki Kazue

田崎 和江

朝田 隆二

Kanazawa University Repository for Academic Resources

Sr-rich microbial mats at Zhemchug hot springs,southwest Lake Baikal, RussiaKazue TAZAKI1, Koushirou MIYATA1, Natalia BELKOVA 2,3 and Ryuji ASADA 21. Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma,Kanazawa, Ishikawa, 920-1192, Japan2. Graduate School of Natural Science and Technology, Kanazawa University, Kakuma,Kanazawa, Ishikawa, 920-1192, Japan3. Limnological Institute of Siberian Division of Russian Academy of Sciences, Ulan-Batorskaya str., 3, Irkutsk, 664033, RussiaAbstract : At Zhemchug hot springs, southwest Lake Baikal, Russia, the biomats con-tain high concentration of Ca and Sr, which microbiological, chemical and mineralogicalanalyses were carried. Optical and scanning electron microscopic observation revealedthat carbonate minerals were associated with cyanobacteria. The biomats have colorfullayered 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 aragoniteassociated with calcite. Elemental layered patterns of the biomats are shown in EPMAelemental content maps indicating the coincidence of Ca and Sr. By increasing pH andthe oxygen concentration on the surface of biomats, the cyanobacteria could potentiallyincrease the rate of metal accumulation. The cyanobacteria in hot spring biomats maycarry important role to accumulate Ca with Sr.Key words : hot springs, biomats, cyanobacteria, Sr, Ca, aragonite1. IntroductionHot springs are one of the main important and interesting objects in studying of proc-esses 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 di-versity of biomats influence water ecosystem (Tazaki, 2000). Microbiological variety inhot springs also influenced water chemistry and formed different biominerals (Ohno andTazaki, 2000). Microorganisms selectively accumulated heavy metals in mining wastewater 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 cells67(Beveridge, 1989).In this study, Sr-rich microbial mats with cyanobacteria were found at Zhemchug hotsprings, southwest Lake Baikal, Russia. Observation of the biomats by using optical andscanning electron microscope, ED-XRF, XRD, NCS and EPMA micro techniques clearlyshowed layered Ca-Sr biomineral formation.2. Materials and MethodsBiomats 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 versusthe 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 biomats4 layers can be clearly distinguished according to different colors, such as green, white, brownand black layers from the top surface in order.68 Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADAWT : Water temperature). One liter of hot spring water was filtrated through bacterial 0.22μm filter (Millipore) with peristaltic pump to determine bacteria in hot spring water. Thebiomat samples were incubated in hot spring water at room temperature during 4 months atlight conditions. The measurements of water characteristics were also carried after 4months storage (Table 1).Biomats have layered structure, which were separated into 4 layers due to differentia-tion 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 obser-vation and EPMA analysis. Furthermore, optical microscopy and SEM-EDX techniqueswere used for observation of green biomats on top surface.2.1. Optical microscopy observation of biomats and bacteriaGreen 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 minutesto observe under epifluorescent microscope (Nikon EFD 3, Digital camera : COOLPIX995). For epifluorescent microscopy, a filter UV-1 A (wavelength of exposed light : 360-370 nm) was used for DAPI-staining observation and a filter G-2 A (wavelength of ex-posed light : 510-560 nm) for observation of chlorophyll in cyanobacterial cells. The sur-face of biomats was observed twice, within 1 week after collection of samples and after 4months storage. The polished thin section of biomats were examined using a polarizing op-tical microscope (Nikon UFX-? A) and EPMA.2.2. Energy dispersive X-ray fluorescence spectrometer analysis of biomatsAfter 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-rayfluorescence spectrometer (JEOL JSX 3201 using Rh Kα), which operated at an accelerat-ing voltage of 30 kV under a vacuum condition.2.3. X-ray powder diffraction analysis of biomatsThe mineralogical properties of biomats were analyzed by X-ray powder diffractome-ter (Rigaku RINT 2000) with Cu Kα generated at 40 kV and 30 mA using 2 θ/θ methodand a scan speed 2?/min. The powder of each sample was mounted onto slide glasses to fitthe diffractometer sample-holder.Table 1. Water characteristics at Zhemchug hot springs, southwest Lake Baikal, Russia.69Sr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, Russia2.4. Nitrogen, carbon and sulfur elemental analyses of biomatsBy an automatic gas chromatographic elemental analyzer (CE Instruments NA 2500-NCS) at 1000?C with 20 ml oxygen contents of carbon, sulfur and nitrogen of biomatswere determined. All powder samples were analyzed after treatment with HCl.2.5. Electron probe microanalysis of biomatsPolished 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 elemen-tal content maps and quantitative analysis of major elements.2.6. Scanning electron microscope (SEM) observation of biomatsGreen biomats were micromorphologicaly investigated using a scanning electron mi-croscope (SEM : JEOL-JSM-5200 LV), equipped with an energy dispersive X-ray spec-trometer (Philips-EDAX PV 9800 STD).3. ResultsWater chemistryField measurements of hot spring water revealed anaerobic condition : pH 7.0, Eh 36mV, 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 observationThe 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 bacterialcells, 5 μm in size, similar to Anabaena species cells, aggregated together with bacterialcells and mineral particles were clearly distinguished. In the hot spring water, only smallsized 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 spe-cies of bacteria and mineral particles (Fig. 3 A, B and C) were shown by optical micros-copy of green biomats after 4 months storage.Observation of thin sectioned biomats showed that the layered structures were sepa-rated 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 layerhas well-crystallized banded structure. Underneath the black layer, porous carbonate grainsare present.70 Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADA3.2. ED-XRF analyses of biomats and waterThe 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 ofCaO (88.88 wt%) in biomats is very high, especially top surface in green and brown layerscontain more than 90 wt% of CaO. Analysis of hot spring water showed large amount ofCaO (30.42 wt%) and Fe2O3 (23.33 wt%). Such elements as Na2O, MgO and SiO2 in thehot spring water were also found in higher concentration than in biomats. Although the ele-ments 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%) suggestingselective accumulation of SrO in biomats.3.3. XRD analysisThe XRD patterns of all sections in biomats showed the same tendency. The strongaragonite peaks were found at 3.4A, 2.7A, 2.0A in all layers (Fig. 5). The small peaks at3.0A, 2.3A, and 1.9A in white layer of biomats also showed the presence of calcite. AnySrO minerals were not detected at whole biomats.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 micrographswere taken within 1 week after sampling.71Sr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, RussiaFig. 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.72 Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADAFig. 4. Optical micrographs of polished thin section of biomats at Zhemchug hot springs. The layeredstructures were separated into four sections due to not only by color, but also by structure ofminerals.73Sr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, RussiaTable 2. Energy dispersive X-ray fluorescence spectrometer analyses of biomats and water at Zhem-chug hot springs.Fig. 5. X-ray powder diffraction of biomats at Zhemchug hot springs, indicating mainly aragonite withcalcite.74 Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADA3.4. NCS analysisNCS elementary analysis of four layered biomats revealed that the tendency is similarin 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 to0.17 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 analysisEPMA 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 showsFig. 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 Sr-rich layers are consistent with Ca-rich layers.75Sr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, Russiareverse pattern of Ca. Quantitative analysis of the layered biomats produces a profile of ele-mental 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 MgOwas relatively high suggesting mixture of dolomite. The results of EPMA analysis areconsistent with that of ED-XRF (Table 2), showing SrO concentration ranges from 1.11 to2.21 wt%.3.6. SEM observationSEM observation of green biomats on top surface revealed that the biomats were com-posed of Ca grains, Si grains and filamentous bacteria (Fig.7 A, B and C). Ca grains (arrowA in Fig. 7) is about 5 μm in size, Si grains (arrow B in Fig. 7) is about 2 μm in sizewhereas filamentous bacteria are about 1 μm in diameter (arrow C in Fig. 7). The EDXanalysis revealed that bacteria contain high concentration of Si, Cl and Ca with traces of Pand S characteristically (analytical point: arrow C in Fig. 7). The Si grain contains relativelyhigh Fe, whereas the bacteria have no Fe at all. The concentration of Sr was not detectedbecause of detection limits of EDX.4. DiscussionFormation 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). Inthis study, accumulation of SrO and CaO in layered biomats were found in Zhemchug hotsprings, southwest Lake Baikal, Russia, essentially result from bacterial activity. Thebiomats at Zhemchug hot springs composed of mainly aragonite. Observations of Sr-richmicrobial mats described here suggest that cyanobacteria produce structure by periodicbiomineralization of aragonite. Calcite is also formed by fibrous bacterial activity in hotsprings 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 toTable 4. Electron microprobe quantitative analysis of 4 different layers of biomats at Zhemchug hotsprings, southwest Lake Baikal, Russia.76 Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADAform aragonite. Aragonite in hot springs contains higher concentration of Sr in comparisonwith calcite (Ichikuni, 1980). In this study, it was revealed that the Zhemchug hot springbiomats was composed of aragonite with high concentration of Sr. The EPMA contentmaps and electron micrographs of layers suggested that cyanobacteria grew rapidly and ac-cumulated Sr from hot spring water at the initial stages of the microbial process. Bacteriain the biomats absorb abundant Sr in high crystalline aragonite to form layered structurewithin short period. By increasing the pH and the oxygen concentration in the surface sedi-ments, the cyanobacteria could potentially increase the rate of iron oxidation in situ (Pier-son 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 radio-active elements. Furthermore, the biomats can be used for 3 R purposes, as follows ; Re-covery of clean water, Resource of useful bacteria and Recycle of Sr-rich aragonite.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 carbonategrain, (B) : silicate grain, (C) : bacteria).77Sr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, Russia5. ConclusionThe 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.AcknowledgementWe 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 Uni-versity for their cooperation. This study was supported by the grants from the JapaneseMinistry of Education, Science and Culture.ReferencesAkai, J. (2000). Biomineralization at hot springs and mineral springs, and their significance in relation tothe Earth’s history. Biol. Sci. Space, 14, 363-371.Beveridge, T. (1989). Interaction of metal ions with component of bacterial cell walls and their biomin-eralization. In : Poole R., Gadd G (Eds) Metal-Microbe interaction. Spec. Publ. Soc. Gen. Microbiol,pp. 65-83.Fortin, D., Ferris, F.G. and Scott, S.D. (1998). Formation of Fe-silicates and Fe-oxides on acterial sur-faces in samples collected near hydrothermal vents on the Southern Explorer Ridge in the northeastPacific Ocean. American Mineralogist, 83, 1399-1408.Ichikuni, M. (1980). Partition of minor constituents between calcareous sinters and hot-spring waters.Hot spring Scince (Onsenn Kagaku), 30, 168-175.Ohno, M. and Tazaki, K. (2000). Biomineralization in biomats at hirayu hot springs. Earth Science(Chikyu Kagaku), 54, 298-309.Pierson, B.K., Parenteau, M.N. and Griffin, B.M. (1999). Phototrophs in high-iron-concentration micro-bial mats : physiological ecology of phototrophs in an iron-depositing hot spring. Appl.Environ. Mi-crobiol., 65, No. 12, 5474-5483.Tazaki, K. (2000). Formation of banded Iron-Manganese structures by natural microbial communities.Clays and Clay Minerals, 48, No. 5, 511-520.Yasuda, T., Kato, H. and Tazaki, K. (2000). Crystal growth of calcite in microbial mats in hot springs iscontrolled by microoganisms. J.Geol. Soc. Japan, 106, No. 8, 548-559.78 Kazue TAZAKI, Koushirou MIYATA, Natalia BELKOVA and Ryuji ASADA

Sr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, Russia

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Sr-rich microbial mats at Zhemchug hot springs, southwest Lake Baikal, Russia

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