Survival of sulfate reducing bacteria at different water activities in compacted bentonite

ELSEVIER

FEMS Microbiology Letters 141 (1996) 83-87

Survival of sulfate reducing bacteria at different water activities in compacted bentonite Mehrdad

Motamedi a, Ola Karland b, Karsten Pedersen &I*

a Department of General and Marine Microbiology. The Lundberg Institute, Giiteborg University, Medicinaregatan SC, S-413 90 Gothenburg, Sweden b Clay Technology AB, Ideon Research Center, S-223 70 Lund, Sweden

Received 28 March 1996; accepted 20 May 1996

Long-lived radioactive waste will be buried several hundred meters below ground in metal canisters surrounded by a buffer of compacted bentonite. Sulfate-reducing bacteria present in the bentonite may induce canister corrosion by production of hydrogen sulfide. Here we show that survival of sulfate-reducing bacteria in bentonite depends on the availability of water and that compacting a high quality bentonite to a water activity (uw) of 0.96 was lethal for the species investigated. Keywar&;

Bentonite; Corrosion; Radioactive waste; Sulfate reducing bacteria; Water activity

1. Introduction The concept of deep geological disposal of spent fuel is common to most national nuclear fuel waste programs. Long-lived radioactive waste will be encapsulated in canisters made of corrosion resistant materials (e.g. copper [1,2]) and buried several hundred meters below ground in a geological formation [3]. Different types of compacted bentonite clay, or mixtures with sand, will be placed as a buffer around the waste canisters [4,5]. A common demand to waste disposal concepts developed in countries concerned is that canisters and buffers must remain intact for a very long time. Assessments of the performance of disposal systems are often done covering

* Corresponding author. Tel. : +46 (31) 773 2578; Fax: +46 (31) 773 2599. E-mail: [email protected]

periods of up to 10000 years or more [3,5]. Therefore, bentonite clay has been proposed as buffer material since it reduces the effects on the canister of a possible rock displacement, and it minimizes water flow over the deposition holes [6]. The transport through the buffer will thereby be reduced principally to diffusion both with respect to corrosive components in the ground water and to escaping radionuclides in case of a canister failure. Corrosion is an important process to consider in such an assessment for at least two reasons. The first is obvious: the canisters are an absolute barrier to radionuclide dispersal as long as they remain intact. A second reason is that a separate gas phase may form at a sufhciently high corrosion rate, which may exert a pressure on the system and add to the dispersion of radionuclides by gas bubble transport. Sulfate-reducing bacteria (SRB) may cause corrosion of canister materials due to their dissimilatory reduction of sulfate to hy-

03781097 /96/ $12.00 0 1996 Federation of European Microbiological Societies. All rights reserved PIISO378-1097(96)00213-3

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M. Motunwdi et ul I FEMS Microbiology

drogen sulfide [7-91. Consequently, it is very important to reveal if SRB can survive and produce hydrogen sulfide in bentonite buffers. SRB are obligate anaerobic organisms and they can generally utilize a wide range of carbon substrates. Several species can survive using carbon dioxide and hydrogen as carbon and energy sources [IO]. Bentonite clay contains organic material, and hydrogen and carbon dioxide can also be found at repository depths [11,12]. It has, therefore, been considered plausible that SRB may establish themselves in a repository bentonite environment, as it will be anaerobic, having reducing conditions together with nutrients and energy available for propagation. The temperature in bentonite surrounding the waste canisters will reach 50-80°C during the first 1000 years, but that does not pose any conceptual hindrance for growth of SRB since some are thermophiles that grow at an optimum temperature around 65°C. e.g. Desulfotomaculum and Thermodesulfobacterium [ 131. A corrosive effect on steel from Desulfotomaculum nigr$cans in bentonite at 50°C has recently been demonstrated [8]. Nor will the high pressure at repository depth constitute any limitation for bacteria as many can withstand some of the highest hydrostatic pressures on the planet ~ those in the deepest parts of the ocean [14]. In contrast to nutrient, energy, pressure and temperature constraints, few bacteria can tolerate removal of water from the cell. The term water activthe amount ity, a,, is used to express quantitatively of water available for microorganisms; it is equivalent to the ratio of a solution’s vapor pressure to that of pure water [ 151. Most bacteria grow well at an a,., around 0.98 (the approx. aw for sea water) but relatively few species can grow at an a, of 0.96 or lower. The halophilic bacteria are one exception; several can grow at an a, as low as 0.75 [16]. Sulfate-reducing bacteria are found in natural waters with salinities from near zero to saturation. Certain SRB genera can withstand desiccation by spore formation, e.g. Desulfotomaculum, but spores are inactive and do not produce hydrogen sulfide. They are rarely found at above approximately 2% NaCl, if present they are often not native [17]. Based on the discussion above, it was hypothesized that the only restriction for survival of different SRB in nuclear waste bentonite buffers is a water activity below cer-

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tain values; this work was aimed at identifying values.

such

2. Material and methods 2.1. Bacterial strains Two strains of SRB, Desulfomicrobium baculatum and Desulfbvibrio sp., isolated from deep crystalline bedrock groundwater in South-eastern Sweden at the Aspo hard rock laboratory were used [la]. Previous studies have shown that D. baculatum and Desulfovibrio sp. grow in up to 20 and 30 g ll’ NaCl respectively (Motamedi, M. and Pedersen, K. (1996) unpublished results). 2.2. Culture condition The bacteria were cultivated in an anoxic mineral medium (brackish medium containing 7 g 1-l NaCl [19]) enriched with lactate as the substrate, sodium sulfate as the electron acceptor and incubated at 30°C. 2.3. Bentonite composition and characteristics The bentonite material is a natural mixture of smectite and several common minerals like quartz and feldspar. The composition varies considerably depending on the mining site but the smectite component, which normally is montmorillonite, dominates the material [20]. In this study commercial MX-80 Wyoming bentonite clay from American Colloid Co. was used. The bentonite consisted approximately of: sodium montmorillonite clay, 75%; quartz, 15%; feldspars, 7%; carbonates, 1.4%; sulfides, 0.3%; organic carbon, 0.4% and other minerals, 2% [21]. The smectite is characterized by a high water affinity which produces swelling when contacted with water. If swelling is restricted due to mechanical hindrance from the surrounding rock, the smectite will give rise to a swelling pressure and to a reduced water activity (a, < 1). Both effects are sensitive to the ratio between water and smectite (w). This ratio will be controlled in the deposition holes by compacting the bentonite to a relatively high density,

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approaching 2.0 g cme3 (a, = 0.96), either by compaction in situ or by use of pre-compacted bentonite blocks. Bentonite with lower densities will likely be used in other constructions in the repository, e.g. in the tunnel backfill material. The bentonite was heat sterilized at 160°C for 2 h before the start of the experiment. 2.4. Procedure Sodium bentonite was inoculated with two species of SRB and compacted to three different densities using the swelling pressure odometers. All the procedures (except compacting of the samples) were performed under nitrogen atmosphere in a glove box. The parts of the odometers that were in contact with the samples (sample holders and pistons) were heat sterilized at 160°C for 2 h. Two fresh cell suspensions of the strains harvested in late exponential growth phase were used. The initial number of Desulfovibrio sp. and D. baculatum in the clay was adjusted to 1.1 X lo7 and 9.4X 10 7cells g-’ respectively, as counted by acridine orange direct count (AODC)

placed in the central cylindrical sample holder and compacted to densities of 1.5, 1.8 and 2 g cmV3 respectively by forcing the piston down (Fig. 1A). These densities correspond to u,s of 1.0, 0.99 and 0.96 respectively [22]. The nitrogen gas in the sample was evacuated by the confining filters, and pressurized water (2 MPa) was contacted to the sample by the upper three-way stop cock to simulate hydrostatic groundwater pressure (Fig. 1B). All samples were incubated at 30°C for 1 or 60 days. 2.5. Sampling and determination of the number oj viable cells

Sampling of the bentonite was performed after placement of the odometers in an anaerobic box under a mixture of HZ, COs and Ns. The number of viable cells was estimated by a MPN method [23]. The tests were performed in 12 dilution levels with 5 repetitions in each level. The medium used for MPN was brackish medium [19] containing 7 g 1-l NaCl enriched with lactate and sodium sulfate. The results were calculated according to a computer program.

[la The quantities of dry bentonite and SRB suspensions used in the mixtures were: 31.2 g bentonite and 27.8 g SRB suspension, 50.0 g bentonite and 20.8 g SRB suspension, and 62.4 g bentonite and 16.2 g cell suspension, corresponding to water ratios of 89%, 42% and 26% respectively. The SRB inoculated bentonite-water mixtures at ratios 89, 42 and 26% were

3. Results and discussion The amount of water available in the bentonite significantly influenced the survival of the studied SRB. Both strains were 100% non-viable after 1 day at the lowest a, studied, 0.96. The dry condi-

Table 1 The number of viable cells of Desulfomicrobium baculatum and Desulfovibrio sp. in bcntonite tivities (a,) for 1 and 60 davs incubation at 30°C Sample density

Water activity

(S cmm3)

(aw)

Hydrostatic pressure (MPa)

samples with different densities and water ac-

No. of viable bacteria Incubation

per g clay

time (days)

0"

lb

60b

D. baculatum

1.5 1.8 2.0

1.0 0.99 0.96

2.0 2.0 2.0

9.4 x 10’ 9.4x 107 9.4x 107

1.6x lo7 1.2x 10s 0.0

1.8xlos 0.0 0.0

Desuljovibrio sp.

1.5 1.8 2.0

1.0 0.99 0.96

2.0 2.0 2.0

1.1x107 1.1x10’ 1.1x10’

4.2~ lo4 1.4x 10s 0.0

0.0 0.0 0.0

“Total number of bacteria as counted by AODC. bDetermined by MPN (most probable number), SD=0.26

M. Motamedi et al. I FEMS Microbiology

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Sample compaction

A

Expelled N, Piston Cylindrical sample holder Bentonite - SRB mixture Filter Expelled N, 0 t

1Ocm

I

B

Test conditions

Water filled pipe to water pressurizing system 12 MPal Piston Cylindrical sample holder Bentonite - SRB sample Filter Water filled pipe closed stop-cock

Fig. 1. Schematic drawing

Letters 141 (1996)

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buffer from groundwater, improbable. The only way in which bacteria can be seeded in nuclear waste buffers is during mixing of the bentonite and water (sometimes groundwater) before compaction (Fig, 1A). A similar inoculation process has been suggested for viable bacteria that are found in subsurface confined clay layers. These bacteria were probably mixed into the clay when it was laid down during sedimentation and they have remained viable since then [l 11. Here, we used a compaction technique similar to what will be used for production of buffer on an industrial scale, and we deliberately introduced very high levels of viable SRB to simulate a ‘worst case scenario’ in such a production. The results show that survival of these SRB depended on the amount of water that was available (a,). When a, approached 0.96 in the bentonite, they were presumably killed by desiccation. In conclusion, the mechanism of microbiologically induced sulfide corrosion inside a nuclear waste bentonite buffer will probably be restrained if an a, of 0.96, or lower, is maintained. Extreme halophilism in Desulfovibrio exists but has not been studied extensively [17]. Indeed, the types of sulfate-reducing bacteria which colonize highly saline soils and water need to be studied, preferably in full scale experiments at actual deposition depths.

Acknowledgments

of swelling pressure odometer.

tions at this density of 2 g cmP3 effectively killed more than lo7 SRB per g bentonite in less than 24 h. The best survival was observed in the bentonite with an a, of 1.0, but the survival differed markedly between the species. About 10% of the initial population of D. baculatum survived for 60 days, but DesuJhovibrio sp. did not survive at all after this time (Table 1). Limitation in nutrients and energy sources, accumulation of hydrogen sulfide and interference of the redox potential may add constraints to a closed batch system like the one used here (Fig. 1). A better survival may be expected in an open system at non-limiting a, values, i.e. at a, close to 1. The pore size of highly compacted bentonite is in the nanometer range [6], which makes contamination of a compacted buffer with SRB, migrating into the

This research was supported by the Swedish Nuclear Fuel and Waste Management Co. We are grateful to Fred Karlsson, Virginia Oversby and Lars Werme for technical and editorial comments on the manuscript.

References [l] Marsh, G.P., Bland, I.W., Desport, J.A., Naish, C., Westcott, C. and Taylor, K.J. (1983) Corrosion assessment of metal overpacks for radioactive waste disposal. Eur. Appl. Res. Rept-Nucl. Sci. Technol. 5, 2233252. [2] McCright, R.D. (1994) Metal container materials for nuclear waste. MRS Bull. 14, 3942. [3] Pedersen, K. and Karlsson, F. (1995) SKB Technical Report 95-10, 222 pp. Swedish Nuclear Fuel and Waste Management Co., Stockholm.

M. Motamedi et al. IFEMS Microbiology Letters 141 (1996) 83-87 141 Atabek, R., Jorda, M. and Andre-Jehan, R. (1985) Programs and means developed by C.E.A. for clay characterization. Eng. Geol. 21, 209-213. PI Werme, L., Kjellbert, N. and Sellin, P. (1992) SKB Technical Report 92-26, 25 pp. Swedish Nuclear Fuel and Waste Management Co., Stockholm. VI Puch, R. (1983) SKB Technical Rapport 83-46, 79 pp. Swedish Nuclear Fuel and Waste Management Co., Stockholm. 171 Hamilton, W.A. (1985) Sulfate-reducing bacteria and anaerobic corrosion. Ann”. Rev. Microbial. 39, 195-217. PI Philp, J.C., Taylor, H.J. and Christofi, N. (1991) Consequences of sulfate-reducing bacteria1 growth in a lab-simulated waste disposal regime. Experientia 47, 553-559. 191 Pedersen, K. (1996) Investigations of subterranean bacteria in deep crystalline bedrock and their importance for the disposal of nuclear waste. Can. J. Microbial. 42, in press. [lOI Widdel, F. (1988) Microbiology and ecology of sulfate- and sulfur-reducing bacteria. In: Biology of Anaerobic Microorganisms (Zender, A.J.B., Ed.) pp. 469-585. John Wiley and Sons, New York. u11 Pedersen, K. (1993) Bacteria1 process in nuclear waste disposal. Microbial. Eur. 18-23. v21 Stevens, T.O. and McKinley, J.P. (1995) Lithoautotrophic microbial ecosystem in deep basaltic aquifers. Science 270, 45&454. 1131 Widdel, F. and Hansen, T.A. (1992) The dissimilatory sulfateand sulfur-reducing bacteria. In: The Prokaryotes (Balows, A., Triiper, G.H., Dworkin, M., Harder, W. and Schleifer, K.H., Eds.) Vol. 2, pp. 582624. Springer-Verlag, New York.

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[14] Kato, C., Hata, S., Suzuki, S., Smorawinska, M. and Horikoshi, M. (1994) The isolation and properties of deep-sea bacteria at high hydrostatic pressure. High Press. Biosci. 4, 152-163. [15] Potts, M. (1994) Desiccation tolerance of prokaryotes. Microbiol. Rev. 58, 755-805. [16] Kushner, D.J. (1978) Microbial Life in Extreme Environments, Academic Press, London. [17] Postgate, J.R. (1984) The Sulphate-Reducing Bacteria, Cambridge University Press, Cambridge, 208 pp. [IS] Pedersen, K. and Ekendahl, S. (1990) Distribution and activity of bacteria in deep granitic groundwaters of southeastern Sweden. Microbial. Ecol. 30, 37-52. [19] Widdel, F. and Bak, F. (1992) Gram-negative mesophilic sulfate-reducing bacteria. In: The Prokaryotes (Balows, A., Triiper, H.G., Dworkin, M., Harder, W. and Schleifer, K.H., Eds.) Vol. 4, pp. 3352-3378. Springer-Verlag, New York. [20] Miiller-Vonmoos, M. and Kahr, G. (1983) Technischer Bericht 83-12, NAGRA, Boden. [21] Grim, R.E. and Guven, N. (1978) Developments in Sedimentology, Elsevier, Amsterdam. [22] Kahr, G., Kraehenbuehl, F., Miiller-Vonmoos. M. and Stoeckli, H.F. (1986) Technischer Bericht 86-14, NAGRA, Baden. [23] Koch, A.L. (1994) Growth measurement. In: Methods for General and Molecular Bacteriology (Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R., Eds.) pp. 249-296. American Society for Microbiology, Washington, DC,