Sci. Rep. Kanazawa Univ.
Vol.49, No.1,2, p.1-24, 2004

Biomineralization of radioactive
sulfide minerals in strong acidic
Tamagawa Hot Springs
Kazue TAZAKI and Hiroaki WATANABE
Department of Earth Sciences, Kanazawa University,
Kanazawa, Ishikawa, 920-1192 JAPAN
Phone/Fax +81-76-264-5736
E-mail : kazuet@kenroku.kanazawa-u.ac.jp
Abstract
Bioaccumulation of radioactive sulfide minerals by bacteria in strong acidic hot
spring water was found at Tamagawa Hot Springs, Akita prefecture in Japan. The hotspring water produces Hokutolite of radioactive minerals containing high radium and radon. The β-ray measurements of sediments and biofilms indicate 1850-2420 and 5700
cpm, respectively, which are 50-100 times higher than that of the water and the air (5090 cpm). The characteristics of hot spring water show pH (1.2), Eh (140 mV), EC (29mS/cm), DO (0.8 mg/l), and water temperature (99.5℃），indicating extremely strong
acidic and reducing conditions. The hot spring water contains mainly HCl associatedwith high concentrations of Ca2+, Al3+, Fe2+, HSO4 - and SO4 2-．SEM-EDX and TEM demonstrate some insight into how microorganisms affect the chemistry and microbiologicalcharacteristics of the strong acidic surroundings with high S, As, Ba, and Ca contents inbiofilms. Especially SEM-EDX, ED-XRF, and STEM-EDX elemental content maps illustrate the distribution of sulfur-bearing compounds of barite (BaSO4), gypsum (CaSO4・2H2O), elemental sulfur (S) and orpiment (As2S3) in the reddish orange biofilms. The
presence of a hydrogen sulfide-rich (H2S) thermal spring and gypsum deposits suggest
the volatilization of H2S from the spring water, oxidation of the H2S gas to sulfuric acid,and reaction of the sulfuric acid. TEM micrographs of bacteria in the biofilms reveal indetail the intimate connections between biological and mineralogical processes that thecells are entirely accumulated with spherical grains, 100〜200 nm in diameter. The relationship among sulfide minerals, such as barite, gypsum, sulfur, orpiment, and Hokutolite, associated with bacteria implies that heavy metals have been transported fromstrong acidic hot spring water to sediments through bacterial metabolism. It is possiblethat the capability of radioactive sulfide biofims for heavy metal immobilization can beused to counteract the disastrous effects of radio nuclide polluted water and radio pharmaceutical medical treatment.
Keywords : Radioactive sulfides, strong acidic hot springs, high β-ray, barite, gypsum,
sulfur, orpiment, Hokutolite.

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Introduction
Temperature is one of the most important environmental factors controlling the activities and evolution of organisms, and is one of the easiest variables to measure. Many hot
springs have significant amounts of hydrogen sulfide which thermal environments are mostfit for living organisms. Some hot springs are highly radioactive, whereas others have no
more radioactivity than normal ground waters (Brock, 1967). Radioactive materials are located on the Earth’s surface, which is closely related to the microorganisms in groundwaterand organic materials in soils, and tends to gather at or near lower plants (Markert, 1993 ;Lienert et al., 1994 ; Yoshida et al., 1994). For example, Uranium solute concentration ofriver, lake, tailings, and natural waters at the Elliot Lake district, Ontario, Canada, showshigh Uranium contents. Nordic and Stanrock tailings contain high Ca, Fe, and U, whereas
Euglena algae enriched these elements more than 7-20 times (Mann and Fyfe, 1985). Filamentous fungi, yeasts, algae, Actinomycetes, and bacteria have been evaluated for Uranium
removal biosorption (Lovley, 2001). On the other hand, bacteria live under a wide range ofphysical and chemical conditions and they are quite actively involved in the cycling ofheavy metals. Bacteria can oxidize or reduce various heavy metals, such as Fe, Mn, U, As,
and Cd in order to gain energy or promote to mineralize (Tazaki and Ishida, 1996 ; Tazaki,2003). Bacteria are very small, but have the largest surface area to volume ratio of any life
form. By providing interfaces for sorption of metal cations, bacteria are efficient scavengers of dilute metals and can concentrate them from the surrounding aqueous environment(Waite et al., 1994 ; Tazaki et al., 2002). It is well established that microbes adsorb andconcentrate metals and radionuclide and that the sulfur-cycle impacts radio nuclide solubility. Sulfur-oxidizing bacteria, not aqueous geochemistry, actually control cave growth. The
bacteria oxidize H2S either completely to sulfate for energy or partially to elemental sulfur.
But, very little information of radioactive biofilm had been reported.
There are numerous hot springs in Japan, because Japanese Island has abundant active
volcanoes with built-in hot spring water (Horiuchi, 2001). However, key developmental
processes of radioactive sulfides biomineralization regulating these events are poorly understood. Although the importance of radioactive elements are widely recognized in hydrothermal area and hot springs, there have as yet been remarkably few studies of the interactions by which living microorganisms bind metals. We thus see that bacteria are able togrow in Tamagawa Hot Springs at high temperature and high radioactivity. Unfortunately,
biologists are not concerned with geo-chemical and bio-mineralogical significances, but
the results of the analyses do show that there are many chemical and mineralogical types in
Tamagawa Hot Springs.
In this study, using micro techniques, such as SEM-EDX, TEM, and STEM-EDX,
heavy metal ions with high radioactivity and bacterial interactions were demonstrated instrong acidic hot spring water at Tamagawa Hot Springs, Akita, Japan. The purpose of thispaper is to provide some insight into how microorganisms interact with the radioactive sulfide minerals.

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Area descriptions of Tamagawa Hot Springs
Tamagawa Hot Springs, Akita Prefecture in Japan, is a potentially useful study area ofradioactivity in hydrothermal environments, and is the site of Japan’s highest production of
hot spring water (9,000 l/min) (Fig. 1). The characteristics of hot spring water showed pH(1.2), Eh (140 mV), EC (29 mS/cm), DO (0.8 mg/l), and water temperature (99.5℃）indicating extremely strong acidic and reducing conditions (Table 1). The aqueous chemistryof the hot spring water showed mainly HCl associated with high concentrations of Ca2+,
Al3+, Fe2+, HSO4 -，and SO4 2- (Akita Analytical Center, 2001). Using β-ray survey meter,
sediments and biofilms on the surface of hot spring water indicate 1850-2420 and 5700
cpm, respectively (Fig. 1A, C). The β-ray counts of the biofilms are 50-100 times higher
than those of the hot spring water and the air at Tamagawa Hot Springs (50-90 cpm) (Fig.
1B, Table 2).
Table 1. Characteristics of Ohbuke hot spring water at Tamagawa Hot Springs, Akita Prefecture, Japan.

Table 2. Measurement of β-ray counts of biofilms, sediments, rocks, hokutolite, the air, and the hotspring water surrounding Ohbuke at Tamagawa Hot Springs, Akita Prefecture, Japan.

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Figure 1. Locality map of Tamagawa Hot Springs, Akita Prefecture, Japan. β-ray survey meter (A),strong acidic hot spring water (pH 1.2) at Ohbuke (B), and reddish orange biofilms with thehighest β-ray counts (5700 cpm) (C).

Biomineralization of radioactive sulfide minerals in strong
acidic Tamagawa Hot Springs

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Tamagawa Hot Springs are famous to produce “Plumbian Barite (Hokutolite)” which
was first found by K. Sakurai (1898). In the following year, Y. Okamoto independently
found similar mineral at the Hokuto Hot Spring in Taiwan. The chemical composition of
Hokutolite is (Ba, Pb)SO4 showed high radioactivity. K. Jimbo(1912) described this mineral as a new radioactive mineral with the composition of (Ba, Pb) SO4 and gave the nameof Hokutolite. The mineral is precipitated in strong acidic hot springs, which was designated as special natural monument of the nation in 1952, and cannot be collected without
the permission of the Government (Sugie, 1986).
In this study, reddish orange biofilms with higher β-ray counts than that of hokutolite
were found in the field at Tamagawa Hot Springs. The presence of H2S thermal springs andHokutolite ((Ba, Pb)SO4) deposits suggested that volatilization of H2S from the springwater contribute to radioactive biominerals.
Methods and materials studied
The β-ray and water characteristics were measured on site on 22nd October 2003 at
Tamagawa Hot Springs, Akita Prefecture, Japan (Fig. 1). The β-ray of hot spring water,
biofilms, sediments, and rocks has been measured by the β-ray GM survey meter (Aloka
Company, Model TGS-136 ; Serial No. R04647) (Fig. 1A). The mineralogical identification of the biofilms was performed by X-ray powder diffraction (XRD) with a Rigaku
RINT2000 carried out with Cu Kα radiation at 40 kV and 30 mA. The samples were also
investigated by using scanning electron microscope (SEM : JEOL JSM-5200 LV)
equipped with an energy dispersive X-ray analyzer (EDX : PHILLIPS EDAX PV 9800
EX) operating at 15 kV.
Microbes in the biofilms were examined with a transmission electron microscope(TEM ; JEOL TEM-2000-EX) using wet samples mounted on a micro grid. After airdrying, the samples were observed without coating at an accelerating voltages of 160 kV.
Complimentary techniques of Energy-Filtering Transmission Electron microscopy (JEM2010FEF) were carried out for the semi-quantitative analyses of the biofilms. Mainly mappings for C, O, Al, Si, S, Cl, Ca, Fe, Cu, and As elements were carried out at the accelerating voltages of 200 kV. Hokutolite sample was cut in half and polished surface for elemental content mapping. The analysis was carried out by an ED-XRF with equipment for Xray probe scanning system (JEOL JSX-3600), using Rh Kα, which operated at an accelerating voltage of 30 kV under the vacuum condition.

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Results
The characteristics of radioactive biofilms and sulfide minerals in strong acidic Tamagawa Hot Springs are described below.
1. β-ray measurements
The β-ray counts were measured in the field for comparison of rocks, sediments, theair, and reddish orange biofilms around the fountainhead at Ohbuke (Fig. 1B). The β-ray of
reddish orange biofilms (Fig. 1C), sediments, rocks, and the air has counted as 5700, 18502420, 350-420, and 80-100 cpm, respectively (Table 2). The β-ray of the biofilms (5700cpm) is the highest on the site, and that of the biofilms are higher than that of Hokutolite(1800-3200 cpm), suggesting high concentration of radioactive components. The sediments around the reddish orange biofilms also have quite high counts as 4700 cpm. Normally the air should be 55 cpm in Morioka City, whereas 80-100 cpm in Tamagawa Hot
Springs area indicates twice much higher than that of normal air value.
2. XRD analysis of biofilms
Wet biofilms were mounted on a glass slide and measured as wet samples. X-ray
powder diffraction analysis of the reddish orange biofilms with the highest β-ray counts
exhibits strong 3.34, 4.06, and 3.85Årefrections identified as sulfur (Fig. 2). However, any
radioactive crystalline phases could not recognized in the XRD pattern. But, some barite,
gypsum, and orpiment can be structurally incorporated in sulfides. The larger arsenate ionsseem to be accommodated in the tetrahedral sites by expansion of the unit cell by deficiencies in adjacent Fe-O (OH) octahedral sites (Paktunc and Dutrizac, 2003).

Figure 2. X-ray powder diffraction pattern of the reddish orange biofilms shows the progressive crystallization of sulfur.

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3. SEM-EDX observations of biofilms
The SEM observation and the EDX analysis of the reddish orange biofilms with thehighest β-ray counts demonstrated the production of sulfides minerals to accumulate highsulfides. In the biofilms, various shapes of sulfur-bearing compounds were found, such as
an ellipse shape which was mainly composed of S and Ca (gypsum) (Fig. 3A, B) as well as
S and Ba (barite) (Fig. 3A-2). The As-Al-Si-S-Cl compounds were found around gypsum(Fig. 3A-1). The high dense of twig shaped crystals rich in S was commonly found, andthat was consistent with mineralogical data by XRD, identified as sulfur (Fig. 2) associatedwith other sulfide minerals. Transparent microorganisms were covered with thin films scattering the spherical grains identified as orpiment (Fig. 4).
EDX analysis of the spherical grains, < 0.3 μm in diameter, attaching to twig
shaped sulfur crystals, exhibited the abundance of S, As, Al, Si, P, Cl, and K (Fig. 4A-1).
Closed-up micrograph of the spherical grins suggested the presence of orpiment (As2S3)(Fig. 4B-2). The biofilms were composed of aggregation of abundant spherical grains associated with bacteria in agreement with TEM micro morphology shown in Figs. 5-10.
4. Elemental content maps of biofilms
The EF-TEM analysis of the reddish orange biofilms provided more detailed elemental distribution and abundant bacteria associated with spherules than that of SEM-EDX
analysis of the sample. The STEM-EDX mapping method had an advantage of distinguishing between sulfide minerals and microorganisms which revealed that abundant bacillus
type bacteria (sulfate-reducing bacteria) coexisted with S-As spherical grains (nano crystalline of orpiment), surrounding the cell (Fig. 5). The STEM-EDX analysis of the reddish orange biofilms with the highest β-ray counts, demonstrated accumulation of high S, Cl, andAs contents on the spherical grains, suggesting the mixture of orpiment and chlorides. In
most cases, these elements (C, O, Si, and N) were detected in the bacterial cell area (Fig. 5).
Especially S and As elements were consistent with the spherical grains, whereas other elements such as C, O, Si and N were consistent with the bacterial cells.
5. TEM observations of biofilms
TEM images of the reddish orange biofilms with the highest β-ray counts clearly
showed abundant sulfur-bearing minerals associated with various shapes of crystalline.
Elongated ellipses were gypsum, 3-5 μm in length and 1-3 μm in wide, with Moire-patternand stacking fault on the surface (Fig. 6). Some of the tips were sharpened as shown in Fig.
6A. On the other hand, disk shaped barite, 1-3 μm in diameter, were commonly foundwhich also showed Moire-pattern and stacking fault on the surface (Fig. 7). The shape andthe size of barite in the natural samples were very similar to the micrographs of BaSO4 particles reported by Yoshiyama (2003). TEM observations clearly showed that some opaquespherical grains were associated with bacterial surface on barite (Fig. 7A, bottom part).
Furthermore, intermediated or combined crystals of gypsum and barite showing elongated/8

Kazue TAZAKI and Hiroaki WATANABE

Figure 3. SEM observations and the EDX analyses of the spherical grains of ellipse shaped gypsum andmixture of sulfur-bearing compounds (A-1) and barite (A-2) in the reddish orange biofilms
showing various kinds of sulfide minerals produced (B-3, 4). The numbers and arrows in the
SEM images coincided with EDX analytical points.

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Kazue TAZAKI and Hiroaki WATANABE

Figure 4. SEM observations and the EDX analyses of spherical grains of barite, gypsum, and orpiment in
the reddish orange biofilms. The numbers and arrows in the SEM images are coincided with
EDX analytical points.

Biomineralization of radioactive sulfide minerals in strong
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BF

O Kα

Cl Kα

C Kα

Si Kα

Ca Kα

N Kα

S Kα

As Lα

Figure 5. EF-TEM micrograph and the elemental content maps of major compositions of the reddish orange biofilms showing high accumulation of S, Cl and As in the spherical grains of orpimentaround bacterial cells.

12Kazue TAZAKI and Hiroaki WATANABE

500nm

200nm

200nm
Figure 6. TEM observations of elongated ellipses of gypsum produced in the reddish orange biofilms,
showing Moire patterns and stacking faults on the surface of minerals.

Biomineralization of radioactive sulfide minerals in strong
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500nm

200nm
Figure 7. TEM observation of disk shaped barite produced in the reddish orange biofilms, showing
Moire pattern and stacking faults on the surface. Note that bacteria and spherical grains of orpiment attached to barite (A). Barite radically grew during aging time (B).

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Kazue TAZAKI and Hiroaki WATANABE

500nm

200nm

200nm
Figure 8. TEM micrographs of combination of the well-developed gypsum and barite produced in thereddish orange biofilms, showing elongated gypsum / rounded barite (A), twins barite (B) androunded barite / spherical grains of orpiment (C).

Biomineralization of radioactive sulfide minerals in strong
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200nm

100nm
Figure 9. TEM micrographs show spherical grains are stuffed in bacterial cells up. Note that the grain
size is relatively free (A) and uniformed (B) suggesting that formation stages are different. The
grains developed 200 nm in diameter.

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Kazue TAZAKI and Hiroaki WATANABE

rounded/jointed morphologies are shown in Fig. 8. Some of them appear to be a mixture of
gypsum and barite (Fig. 8A), twins of barite (Fig. 8B), and combined crystals of barite andspherical grains of orpiment in the thin films (Fig. 8C). Finally, twins and polytype intergrowths can result if coherencies achieved only in the two dimensions of the interface.
Numerous bacterial cells are entirely coated with high dense of the spherical grains(Fig. 9). The bacterial cells are filled with spherical grains up, <100 nm in diameter, suggesting processes of metabolism. The spherical grains in the bacteria are free sized shownin Fig. 9A, whereas relatively uniform sized grains are shown in Fig. 9B. Few larger sizedgrains stick to the cell wall. The developed grains with high dense, 200 nm in diameter, appear to be pushed from end of the cell out as shown in Fig. 10A, succeed B and C. Some of
spherules are vesicles with cylindrical materials. After all grains were pushed out, the internal cell became empty as shown in Fig. 10-B and -C. It is important to mention that the average size of individual completed grains was approximately 200 nm. Schematic mechanisms of sulfide spherical grains in biofilms with the highest β-ray in strong acidic hot
spring, indicate that the accumulation of HSO4 - or SO4 2- ions in a hot spring water to form
particle are hoard in a cell. The particle size is free at the initial stage. Some larger sizedparticles are jutted from a cell out. The completed particles, approximately 200 nm in diameter, are squeezed from the cell. Note that the bacterium discharge uniform sized particles, < 200 nm in diameter, from the end of the cell. The most of particles are released, andthe cell became empty at the vomited stage (Fig. 11).
The interactions between spherical grains of sulfides and bacterial cells could be seenby using TEM microscopy, but the bacteria could not be seen by the SEM-EDX observation, because of aggregations and covering situations. The STEM-EDX clearly showed bacillus type bacteria and spherical grains in the cell, suggesting radioactive sulfides biomineralization during metal immobilization.
6. Biomineralization of radioactive sulfide minerals
Hydrogen sulfide-rich (H2S) thermal spring and gypsum deposits (CaSO4) were foundin Tamagawa Hot Springs suggesting the volatilization of H2S from the spring water, oxidation of the H2S gas to sulfuric acid. In this study, the results demonstrated that living bacteria in reddish orange biofilms with the highest β-ray (5700 cpm) under pH 1.2 condition
accumulated S, Ba, Ca, and As elements which appeared to be sensitive to the formation of
sulfides of barite, gypsum, sulfur, orpiment, and Hokutolite (Fig. 12). Surprisingly, the βray value of the biofilms was twice higher value than Hokutorite (1800-3200 cpm).
Brown and white layered Hokutolite was found near Tamagawa Hot Springs (Fig.
13). ED-XRF elemental analyses of layered across section of Hokutolite clearly showed
distribution of elements shown in Fig. 14. On the top of surface, Si and Fe were accumulated, whereas Sr, Ba, and Pb were layered in 1-2 mm thick. The layered distributions ofBa and Pb are consistent, but the Sr rich layer is inversely proportional to Ba and Pb richlayers. ED-XRF elemental content maps of layered across section of Hokutolite indicatedBiomineralization of radioactive sulfide minerals in strong
acidic Tamagawa Hot Springs

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200nm

200nm

500nm
Figure 10. TEM micrographs show that abundant spherical grains of orpiment are pushed from the endof bacterium cell out (A), the most spherical grains are discharged (B), and internal bacterium
cell became empty (C). Note that uniform sized spherical grains, approximately 200 nm in diameter, pushed from end of the cell out.

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Kazue TAZAKI and Hiroaki WATANABE

Figure 11. Schematic mechanisms of sulfide spherical grains in biofilms with the highest β-ray in strong
acidic hot spring, indicate that the accumulation of HSO4 - or SO4 2- ions in a hot spring water
to form particle are hoard in a cell. The particle size is free at the initial stage. Some larger
sized particles are jutted from a cell out. The completed particles, approximately 200 nm indiameter, are squeezed from the cell. Note that the bacterium discharge uniform sized particles, < 200 nm in diameter, from the end of the cell. The most of particles are released, andthe cell became empty at the vomited stage.

Biomineralization of radioactive sulfide minerals in strong
acidic Tamagawa Hot Springs

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Figure 12. A schematic diagram of the simple system shows formation of sulfur, gypsum, orpiment, barite, and hokutolite minerals under high β-ray radioactive conditions at Tamagawa Hot Springs.
The biofilms accumulate heavy metals are mainly present as sulfur in hot spring water at pH 1.

Figure 13. Layered Hokutolite was found at Tamagawa Hot Springs.

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Kazue TAZAKI and Hiroaki WATANABE

Figure 14. ED-XRF elemental analyses of layered across section of Hokutolite.

Biomineralization of radioactive sulfide minerals in strong
acidic Tamagawa Hot Springs

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Si rich layer on the top surface, Ba and Pb rich layer in white parts with traces of Mo and
Sr (Fig. 15 middle). The porous area was concentrated with Pb and Ba in bottom of Fig. 15.
In the middle of across section, the white and brown layers appeared to have similar chemistry (Fig. 15), but XRD patterns of white layers and brown layers clearly identified different crystalline as shown in Fig. 16. The white parts contained large amounts of organics oramorphous materials associated with barite, cristobalite, and sulfur minerals. On the other
hand, the brown parts contained only high crystalline of barite. The Si rich layer on the top
surface contained cristobalite in consistent with ED-XRF analytical data. The high background of the XRD result suggested that abundance of microorganisms was presented tocontrol bio-mineral growth.

Figure 15. ED-XRF elemental content maps of layered across section of Hokutolite.

Figure 16. X-ray powder diffraction analysis of the white and brown parts of Hokutolite indicated different mineral formation of crystalline.

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Kazue TAZAKI and Hiroaki WATANABE

Discussions
The metal-loaded cells nucleate the formation of a mixed assemblage of crystalline
metal phosphates and metal sulfides (Beveridge et al., 1983). There are many potential intermediate sulfur and sulfoxy compounds generated in the process, including elemental
sulfur, thiosulfate, tetrathionate, and sulfite. In addition to water-soluble compounds, special microorganisms are also involved in radioactive mineralization processes (Suzuki et al.,2003). Thus, certain microorganisms can utilize oxidation of some sulfur-bearing compounds (by oxygen) to generate metabolic energy (Banfield and Welch, 2000). The bacteria oxidize H2S either complexly to sulfate for energy or partially to elemental sulfur that isstored intra cellular for later use. Elemental sulfur interacted with the metal-loaded cells,
and formed a variety of crystalline metal sulfides. Several sulfate-reducing microorganisms
are able to reduce U (VI). The U (VI) reduction is the likely explanation for the concentration of Uranium in the reduction spot of microbial mats. The ability of bottom sediments ofwaste treatment ponds to remove dissolved Uranium from Uranium mine wastewater isprobably due to reductive precipitation of Uranium (Lovley et al., 1991).
The sulfide spherules were of uniform size and some of them were aggregated, showing a matrix of colonies. The uniformed particles (200 nm in diameter) passed easily
through holes at the end of the cell, but larger particles up to 200 nm in diameter failed togo out of an opening. This may lead to preservation of cell-related morphology, which issignificance to fossilization and bio-signature preservation (Fig. 11). Interestingly, uniformed particles were able to traverse the blockade by squeezing cells. Leaving the particles emerges out at the only one side of the end. This kind of device might offer a rapid
screen for agents that inhibited or reversed the biomechanical effects. A fundamental property of many products of biomineralization reactions, is very small particle size, resultsfrom rapid nucleation due to super-saturation following iron oxidation (Banfield andWelch, 2000). Small nucleus size is preserved due to low solubility of ions, which limits
diffusion-based crystal growth. All microstructures that arise from nm-crystal aggregation
have the potential to subsequently modify the reactivity, and thus the form and fate, of
biomineralization products.
In this study, the correlation between the results of β-ray measurements and the microscopic observations of natural evidence of radioactive biominerals are considered to be
helpful for further discussion of the nuclide medical treatments. This organism clearly has
the potential for use in bioremediation of radioactive areas under strong acidic conditions.
Conclusions
TEM micrographs clearly showed the biological formation of radioactive sulfide minerals in strong acidic hot springs. Bacteria in reddish orange biofilms were able to accumulate S, Ba, Ca, and As elements from strong acidic hot spring water (pH 1.2), indicating theBiomineralization of radioactive sulfide minerals in strong
acidic Tamagawa Hot Springs

23

highest β-ray counts (5700 cpm) at Tamagawa Hot Springs, Akita, Japan. Bacterial
biomineralization processes were revealed by direct observation of analytical electron microscopy. SEM-EDX and TEM observations provided some insight into how microorganisms affected the chemistry and microbiological characteristics of the strong acidic surroundings. The chemical analysis of the biofilms showed S, Ba, Ca, and As, as the major
elemental composition. Especially SEM-EDX, ED-XRF, and STEM-EDX elemental content maps of the reddish orange biofilms illustrated the distribution of sulfur-bearing compounds of barite (BaSO4）, gypsum (CaSO4・2H2O), sulfur (S), orpiment (As2S3), and Hokutolite ((Ba, Pb)SO4)．These minerals were associated with living bacteria. TEM micrographs of bacteria in the biofilms revealed in detail the intimate connections between
biological and mineralogical processes that the cells were entirely accumulated withspherical grains, 100〜200 nm in diameter. This shows that microbial activity may play asignificant role in the radioactive environments, and that this may be a common occurrence.
The relationship among radioactive sulfide minerals and bacteria implies that heavy metals
had been transported from strong acidic hot spring water to minerals through bacterial metabolism. Sulfide minerals formation in hot spring water may be substantially enhanced by
the presence of bacteria. This bacterium clearly has the potential for use in bioremediationof radioactive areas.
Acknowledgements
We thank to Dr. O. Nishikawa, Dr. K. Shiraki and Mr. T. Morikawa of the Department of Earth Sciences, Kanazawa University for their technical help. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science,and Culture, Japan, awarded to Kazue Tazaki.
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