Sci. Rep. Kanazawa Univ.
Vol.53, p.25-37, 200925

Observation of microbial mats in radioactive hot springs
Kazue TAZAKI
Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa,
Ishikawa 920-1192, Japan
E-mail: Kazue Tazaki: kazuet@kenroku.kanazawa-u.ac.jp
Phone: +81−76−264−6512
Fax: +81−76−264−6545
Abstract : Microbial mats can accumulate heavy metals and radio nuclides through precipitationand complexation on and within the cell surface containing hydroxyl and carboxyl groups.
Processes involving bacterial oxidation-reduction will alter the mobility of the hevy metal
pollutants in water. The microbial immobilization of radio nuclides and other metals wascompared for the high temperature and strongly acidic conditions of Tamagawa Hot Springs in
Akita Prefecture and the low temperature and neutral pH conditions of Misasa Hot Springs in
Tottori Prefecture, Japan. Both hot springs produced microbial mats indicating the Ra and Rn
radioactivity was higher than that of the surrounding water. Here, bacterial biomineralization in
the radioactive hot springs was described by nanometer−resolution observations using electron
microscopic techniques. The analytical results showed the bacterial immobilization of radio
nuclides and other metals. The reddish−brown microbial mats at Tamagawa Hot Springs hadradioactivity of 5700 cps and were composed of Ca2+, Al3+, Fe2+, HSO4− and SO42−, which had
formed sulfur-bearing compounds of barite (BaSO4), elemental sulfur and As−S elements.
Bacterial cells were entirely encrusted with spherical grains having diameters of 100−200 nm,
suggesting that heavy metals were transported from strongly acidic hot spring waters (pH of 1.2)
to sediments. On the other hand, the green microbial mats at Misasa Hot Springs werecharacterized by a neutral pH of 7.2 and a temperature of 42°
C. The radioactive microbial mats
(11.9 μSv/h) were mainly composed of cyanobacteria of Oscillatoria spp. and Phormidium spp.,
which are associated with minerals of ferrihydrite, Mn oxides and calcite. The 226Ra content of
the microbial mats was 6.9 × 104 Bq/kg, which was higher than that of the hot spring water (1.4
× 10−1 Bq/kg). In addition, in reddish-brown microbial mats, coccus and bacillus type bacteria
associated with extracellular polymers around the cell. The results suggest that the
microorganisms in the microbial mats grow and decay with a metabolic reaction, and thenaccumulate heavy metals. It is possible that the capability of radioactive immobilization can beused to counteract the disastrous effects of radionuclide-polluted water and sediments.
Keywords: Microbial immobilization, radionuclide, cyanobacteria, hot springs, Ra, sulfur−bearing
compounds, capsule26

Observation of microbial mats in radioactive hot springs

Introduction
Microbial mats can accumulate heavy metals and radio nuclides through precipitationand complexation on and within the cell surface containing hydroxyl and carboxyl groups.
Processes involving bacterial oxidation-reduction will alter the mobility of the hevy metal
pollutants in water. The interaction between water, microorganisms, and radioactive
materials on the Earth’s surface is of great importance because large amounts ofradioactive material could be released by microbial activity (Macfarlane and Miller 2007;
Stroes-Gascoyne 2007). U-, Ra-, and Rn- biotic interactions could play important roles inradionuclide cycling, involving microorganisms in soils, ground water, and organic matter(Beveridge et al. 1983; Haas et al. 1998; Suzuki et al. 2003; Tazaki et al. 2003).
Filamentous fungi, yeasts, algae, and bacteria have been evaluated for U biosorption(Lovley 1995). Rn converted from Ra is a radioactive gaseous element that mainly emits
α-rays. Although Rn inhalation has been thought to be hazardous in general, springs
containing Rn have been reported to have therapeutic effects on senile brain disorders andhypertension. Another known effect of Rn springs is the promotion of the effects of tissue
perfusion agents such as adrenaline in plasma (Yamaoka et al. 2004).
Tamagawa and Misasa Hot Springs in Japan are well known for producing Ra, Rn,
and radioactive minerals such as hokutolite (plumbian barite). However, only a few studies
have been conducted on Ra- and Rn-bearing microbial mats with nanometer-resolution
observations in the context of environmental remediation. Hokutolite has a chemicalcomposition characterized as (Ba, Pb)SO4 and has high radioactivity owing to the presenceof Ra isotopes and their daughters. Recently, studies on the interaction by which living
microorganisms bind metals have been carried out at Tamagawa Hot Springs (Tazaki and
Watanabe, 2004). It is probable that the presence of Th in hokutolite is due to the coprecipitation with (Ba, Pb)SO4 from the hot spring water. The Ra−Rn hot spring
environments, which support a wide diversity of microbial life, are poorly understood.
In this study, more-detailed studies on the radioactive microbial mats at Tamagawa
and Misasa Hot Springs in Japan using electron microscopic methods are discussed.
Emphasis is given to the radiochemistry of the microbial mats through the determination of
γ-rays. The elemental composition and distribution of radioactive microbial mats are also
examined using scanning electron microscopy (SEM), transmission electron microscopy(TEM), X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction (XRD) analysis,
and Fourier transform infrared (FT-IR) absorbance spectroscopy, and are used to elucidate
nanometer-scale relationships among microorganisms and bio-precipitated materials at thetwo hot springs. Ra and Rn as well as other metals found with minerals on indigenous
bacterial cell walls are described.
Samples and Methods
Measurements of water pH, oxidation-reduction potential (ORP), electricalconductivity (EC), water temperature, and dissolved oxygen (DO) content were carried out

Kazue TAZAKI

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in the field. The hot spring water measurements were taken using portable inspection
meters (Horiba and Toa Co.) in the months of December for 2002, 2003, 2004, and 2006.
Radioactivity was measured using a β-ray Geiger−Muller survey meter and a Geiger
counter (α-, β-, and γ-rays) (Aloka Company TGS-136), and the radio activities of air,spring water, microbial mats and rocks were compared.
In this study, reddish-brown microbial mats at Tamagawa Hot Springs and both green
and reddish-brown microbial mats at Misasa Hot Springs were collected for detailed
observation by electron microscopy.
XRD analysis
The mineralogical characteristics of microbial mats collected from Tamagawa andMisasa Hot Springs were analyzed using XRD (Rigaku Rinto 1200) with a Cu-Kα
generator at 40 kV and 30 mA for identification of minerals and the crystalline state of the
products during biomineralization.
Optical and fluorescence microscopy
The microbial mats were observed with an optical microscopy to recognize living
microorganisms and minerals. Wet samples were placed on a glass slide and stained with
4’,6-diamidino-2-phenylindole (DAPI, 50 μg/ml). DAPI-stained DNA in bacteria
fluoresced a blue color that could be seen under ultraviolet rays (wavelength of ~365 nm).
In contrast, minerals with DAPI staining fluoresced yellow.
Energy dispersive X-ray fluorescence (ED-XRF) spectroscopy
Chemical compositions of the microbial mats collected from Tamagawa Hot Springsand Misasa Hot Springs were determined by ED-XRF analysis using a JEOL JSX-3201(Rh-Kα) instrument, which was operated at an accelerating voltage of 30 kV undervacuum conditions.
FT-IR absorbance spectroscopy
The microbial mats at Misasa Hot Springs were examined by FT-IR absorbance
spectroscopy (Jasco FT-IR-610, MICRO-20) to evaluate the chemical bonding of organics
and inorganics. A small quantity of microbial mats was suspended in distilled water. A
drop of the suspension was mounted on a fluorite disk (CaF2, 0.5 mm thick) for FT-IR
analysis. After air-drying, the cells were selected under the IR microscope showing organic
chemical bonding.
Scanning electron microscopy-energy dispersive X-ray (SEM−EDX) spectroscopy
The microbial mats were fixed with glutaraldehyde solution (2.5% final
concentration) for 1 hour. After a sample was washed and substituted with t-butyl alcohol,
it was frozen in liquid nitrogen and dried using a freeze-drying apparatus. The freeze-driedsample was mounted onto copper stubs with double-sided adhesive carbon tape for SEM

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Observation of microbial mats in radioactive hot springs(JEOL JSM-5200 LV) at an accelerating voltage of 15 kV. The scanning electron
microscope was equipped with an EDX spectrometer (Philips EDAX PV 9800) and thechemical composition of the microbial mats was analyzed. Bacteria (Oscillatoria spp. andPhormidium spp.) were identified using Bergey’s Manual of Determinative Bacteriology(Ninth Edition).
Electron microscopic techniques
Wet samples of microbial mats mounted on a micro-grid were observed with atransmission electron microscope (JEOL TEM- 2000-EX). After air-drying, the samples
were observed without coating at an accelerating voltage of 160 kV in the bright-fieldmode. Energy-filtering transmission electron microscopy (EF-TEM) and scanningtransmission electron microscopy-energy dispersive X-ray (STEM-EDX) spectroscopy
were used for nm scale observations. Complimentary techniques of EF-TEM (JEOL JEM2010 FEF) using a field emission gun, an in-column energy filter, an EDX spectrometer(JEOL JED-2300), and the scanning transmission mode of an advanced scanning imaging
device were carried out for semi-quantitative analyses of the microbial mats at anaccelerating voltage of 200 kV. STEM-EDX was also used for the point analysis ofchemical compositions of nanometer-order.
Results
The study areas of Tamagawa Hot Springs in Akita Prefecture and Misasa Hot
Springs in Tottori Prefecture are on the island of Honshu in Japan (Fig. 1).
Tamagawa Hot Springs
Tamagawa Hot Springs is a potentially useful area for the study of radioactivity inhydrothermal environments, and the site has the highest production of hot spring water(9,000 L/min) in Japan. The characteristics of the hot spring water were strong acidity witha pH of 1.2, high temperature of 99.5°
C, ORP of 140 mV, EC of 29 mS/cm, a DO
concentration of 0.8 mg/L, and high β-ray counts. The β-ray counts of reddish-orange
microbial mats (5700 cps) were 2−100 times those of the hot spring water (50−90 cps),
sediments (1850−2420 cps), hokutolite (1800−3200 cps) and air (80−100 cpm) on 22
October 2003.
The aqueous chemistry of hot spring water was mainly HCl associated with highconcentrations of Ca2+, Al3+, Fe2+, HSO4−, and SO42− (Tazaki and Watanabe 2004). Using theGeiger−Muller counter, the reddish-brown microbial mats were found to have radioactivityof 5000 cpm whereas the background in the air had radioactivity of 100 cpm (Fig. 2 A, B).
The β-ray counts of the microbial mats were 50−100 times those of the hot spring waterand the air. The area used to produce the radioactive mineral hokutolite.
Small cubic hokutolite, 10 μm in diameter, was found in the reddish-brown microbial
mats under SEM−EDX observation. The hokutolite was rich in S and Ba and had a small
amount of Pb (as indicated in Fig. 2 C). The XRD analysis of the microbial mats identified

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Fig. 1. Locality map of sampling places at Tamagawa and Misasa Hot Springs in Japan.

Fig. 2. Reddish brown microbial mats (A, B), the SEM-EDX micrograph of Hokutolite (C) and the
XRD pattern (D) in the microbial mats collected from Tamagawa Hot Springs.

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Observation of microbial mats in radioactive hot springs

low quantities of crystalline sulfur and tridymite. Hokutolite was not identified because its
small amounts and quantity were too small for this method (Fig. 2 D). In addition, the EDXRF analysis of the microbial mats clearly indicated granular Hokutolite (Fig. 3 C),
associated with As element (Figs. 3 A and B). The STEM−EDX micrograph of thebacterial cells showed a thick cell wall encrusted with Fe component associated with P, Ca,
and As elements. The result suggests that the existence of these materials around them
defend from radioactivity (Fig. 4).
Misasa Hot Springs
The characteristics of the hot spring water at Misasa Hot Springs were a neutral pH of
7.2, low temperature of 42.0°
C, ORP of 226 mV, EC of 1.3 mS/cm, and DO concentrationof 1.5 mg/L, which were associated with radioactive microbial mats. The microbial matsaccumulated elements such as 238U (6.4 Bq/kg), 226Ra (6.9 × 104 Bq/kg), 228Ra (8.3 × 104
Bq/kg), 228Th (8.8 × 103 Bq/kg), Fe (total of 2.2 × 105 ppm) and Mn (total of 6.2 × 104 ppm).
The counts for the microbial mats were several hundred times those for the water (Fujisawa
and Tazaki 2007).

Fig. 3. ED-XRF analyses of the microbial mats at Tamagawa Hot Springs, showing Hokutolite small
grains (arrows), and analytical points, showing the presence of As element and Hokutolite (C).

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Fig. 4. STEM-EDX micrograph of unstained thin-section of Fe-rich coatings around the cells at
Tamagawa Hot Springs. The area contains small amount of P, Ca, and As (Cu peak is due tosupporting grid)(an arrow).

The Misasa Hot Springs water had aqueous chemistry of Fe (0.10 ppm), Mn (0.12ppm), and 226Ra (0.14 Bq/kg), whereas the microbial mats had quite high concentrations ofFe (2.2 × 105 ppm), Mn (6.2 × 104 ppm), and 226Ra (6.9 × 104 Bq/kg), indicating highradioactivity conditions. Using the Geiger counter, the radio activities of the background(0.18 μSv/h), surface of granite (0.30 μSv/h), hot spring water (0.17 μSv/h), and microbial
mats (11.9 μSv/h) were measured in the field (Fig. 5). The green and reddish-brown partsof the microbial mats were collected from the spring source and areas of water overflow
for this study (Fig. 5, top). The ED-XRF chemical analysis showed the microbial matscontained high concentrations of Mn and Fe components associated with Si, S, and Ca andAs (Fig. 5, middle left). The mineralogical composition determined from the XRD analysisof the microbial mats was mainly ferrihydrite and calcite (Fig. 5, middle right). The FT-IR
analysis indicated abundant organic materials, showing chemical bonds of C−H, C=O, and
CNH in the organics, suggests that microbial mats can accumulate heavy metals and radio
nuclides through precipitation and complexation on and within the cell surface containinghydroxyl and carboxyl groups (Fig. 5, bottom).

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Observation of microbial mats in radioactive hot springs

Fig. 5. Sampling points at Misasa Hot Springs in Tottori Prefecture in Japan, showing higher
radioactivity of the green and reddish brown microbial mats (11.9 μSv/h; an arrow in centre) thanthat of hot spring water (0.17 μSv/h; an arrow in bottom), granite (0.30 μSv/h), and back groundin the air (0.18 μS/h; BG). Energy dispersive X-ray fluorescence analysis (ED-XRF) of the
microbial mats showed Mn and Fe- rich compositions (center left). X-ray powder diffraction
analysis (XRD) of the microbial mats shows mainly ferrihydrite and calcite minerals (center right).
Fourier transform-infrared absorbance spectroscopy (FT-TR) of the microbial mats showed richin organics (circles)(bottom).

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The SEM−EDX analyses showed abundant cyanobacteria of Oscillatoria spp. andPhormidium spp. composed of Si, P, S, Ca and Fe other than C, whereas the mineralfragments of ferrihydrite (Fig. 6 A). The filmy materials were composed of Si, Ca, Mn, Fe
and organic thin films (Fig. 6 B). TEM showed the bacterial cells encrusted with fibrous
Mn minerals on cell walls (Fig. 6 C). Thin-section samples of Mn-rich microbial matsrevealed a cell structure having thick capsules around the cell. Most of the bacteria were
encrusted with filamentous Mn minerals. Fine extracellular polymers extending away fromthe cell were covered with Mn minerals, and a detailed cross section of cell structures of acytoplasm membrane, peptidoglycan layer, outer membrane, plasma membrane, and thickcapsule could be observed (Fig. 6 C). The capsule of the slime layer with Fe and Mn
contained organics in the forms of protein molecules, enzymatic formations andphospholipids, as shown in the FT-IR analysis (Fig. 5, bottom).
Discussion
The bacterial biomineralization of sulfur, tridymite, and Hokutolite at Tamagawa Hot
Springs under strong acidic and high temperature conditions, and ferrihydrite, calcite, andMn minerals at Misasa Hot Springs under neutral pH and low temperature conditions aredescribed in this study. At both hot springs, Ra−Rn concentrations were quite high, but thebacterial cells with capsules had different structures. The results provide a basis for thedesign of an in situ bioremediation process using indigenous bacteria. The microbial
immobilization of radionuclide and other metals in the two Ra−Rn hot springs play
important roles in biomineralization, bioremediation, and the ecosystem.
The microbial mats in the hot springs had higher radioactivity than the surrounding
water did as shown in Figs. 2 and 5. Most of the radionuclide was present in cell walls asshown in Figs. 4 and 6 C. The result was a gram-positive cell envelope that highlyinteracted with dissolved metal ions, was extremely robust, and was resistant to harshenvironmental conditions (Beveridge and Fyfe 1985). Usually, gram-positive cell walls are
exemplified by those of Bacillus subtilis and are linear polymers constructed of
peptidoglycan, rich in carboxylate groups and covalently link together as they assemble
around the cell (Beveridge et al. 1983). The U-loaded B. subtilis cell is entirely coated withextracellular precipitates, having the EDX spectrum of U (Suzuki et al. 2003).
Microorganisms use a wide variety of minerals for their skeletons, including silica, apatite,
Fe, Mn, several polymorphs of carbonate, uranium phosphate, and in particular, aragonite
and calcite (Beveridge et al. 1983; Banfield and Nealson 1997; Tazaki 2000, 2005, 2006).
TEM-EDX analysis has confirmed that most U removed from solution with kaolinite clay
is associated with B . subtilis (Ohnuki et al. 2007). For living bio-films to consist of avariable distribution of cells and cellular aggregates, their extracellular polymers and void
spaces or water channels are continuously grown, as shown by TEM in this study andreported by Costerton et al. (1995).
Bacteria in nature often exist as sessile communities called microbial films. The

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Observation of microbial mats in radioactive hot springs

Fig. 6. Scanning electron micrographs of reflected image show cyanobacteria attaching to detrial silicate
and carbonate fragments (A), thin film materials with organics contain large amount of Si, Ca, Mn
and Fe (B), and unstained thin-section of Mn-rich coatings collected from microbial mats. The
living bacteria are encrusted with filamentous Mn minerals. Fine extracellular polymers extending
away from the cell are covered with Mn minerals (C; arrows).

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communities of Pseudomonas aeruginosa in the microbial films develop structures that aremorphologically and physiologically differentiated from those of free-living bacteria(Davies et al. 1998). A repeatable pattern of cell death and lysis occurs in bio-films of
Pseudomonas aeruginosa during the normal course of development (Webb et al. 2003).
Effective biological remediation and biological treatments of radioactive materials
from localized sources have also received much attention. There has been increased
interest in the microbial uptake of radioactive materials as one of the primary ways bywhich Ra and Rn are eliminated from hot spring hydrothermal sites. Active resistance tothe radioactive materials is a widely distributed trait among bacteria under different
conditions (Beveridge and Fyfe 1985; Yoshida 1994; Costerton et al. 1995; Abdelouas etal. 1998; Davies et al. 1998; Suzuki et al. 2003; Tazaki and Watanabe 2004). Bacterial
cells can accumulate heavy metals through ion exchange, precipitation, and complexation
on and within a cell surface containing hydroxyl, carboxyl, and phosphate groups.
Processes involving bacterial oxidation-reduction will occur after heavy-metal pollutants
become mobile in soil (Yong et al. 2006). The bacterial surface might enhance the heavy
metal adsorption of Mn, Fe, As, Ba, and Pb, which are associated with radioactive
elements (Ra and Rn), at a pH of 1 or 7 under high temperature (98°C) or low temperature(42°C) conditions at Tamagawa and Misasa Hot Springs, respectively.
Conclusion
Microbial immobilizations of radionuclides at Tamagawa Hot Springs in Akita
Prefecture and Misasa Hot Springs in Tottori, Japan, were studied. The hot springs are apotentially useful area for the study of high radioactivity in environments rich in Ra−Rn.
The water of Tamagawa Hot Springs had a high temperature of 98°
C and was strongly
acidic with a pH of 1.2, whereas the water of Misasa Hot Springs had a temperature of 42°
C and neutral pH of 7.2. Both waters had high EC values owing to the reduced microbial
mats having high radioactivity; the microbial mats were 100 times more radioactive than
the surrounding groundwater. Barite and elemental sulfur were found to be associated with
abundant bacteria in the reddish-brown microbial mats at Tamagawa Hot Springs. The
bacterial cells were entirely encrusted with spherical grains with diameters of 100−200 nm.
The heavy metals have been transported from the strongly acidic hot spring water tosediments. On the other hand, the green microbial mats at Misasa Hot Springs contained ahigh concentration of 226Ra (6.9 × 104 Bq/kg), which was present in living cyanobacteria(Oscillatoria spp. and Phormidium spp.) with ferrihydrite, Mn oxides and calcite. Thissuggests that 226Ra is adsorbed to the capsular surfaces of cyanobacteria and coccus andbacillus bacteria in microbial mats, thus producing capsules and slime layers of
extracellular polymers around cells that protect against radionuclides and other metals in
hot springs. It is possible that the capability that specific bacteria for immobilizing heavy
metals can be used to counteract the disastrous effects of radionuclide pollution.

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Observation of microbial mats in radioactive hot springs

Acknowledgments
I would like to thank Akiko Fujisawa and Tatsuya Shirotori for assistance with theexperiments, and Prof. Masashi Nakano for valuable comments on the progress of theresearch.
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