radiosensitivity of subterranean bacteria in the hungarian upper permian siltstone formation

Journal of

Environmental Radioactivity 61 (2002) 233–239

Technical note

Radiosensitivity of subterranean bacteria in theHungarian upper permian siltstone formation

Gy .oongyi Farkas*, L.G. Gazs !oo, G. Di !oosi

National Research Institute for Radiobiology and Radiohygiene, P.O. Box 101, H-1775 Budapest, Hungary

Received 8 February 2001; received in revised form 12 June 2001; accepted 22 June 2001

Abstract

The main purpose of this work was to study the radioresistance of subterranean aerobic andanaerobic isolates from the Hungarian Upper Permian Siltstone (Aleurolite) Formation, inorder to assess the safety of potential sites of future underground repositories for nuclearwaste. A total of 93 isolates were studied. The radiosensitivities of these aerobic and anaerobic

bacteria isolates were determined: the D10 values (decimal reducing doses) of the aerobicspore-formers lay in the range 0:80–2:44 kGy, and those of the anaerobic spore-formers lay inthe range 1:86–4:93 kGy. The D10 values of the aerobic and anaerobic vegetative isolates weremuch lower, in the ranges 0:11–0:57 and 0:22–0:40 kGy, respectively. The variability inbacterial radioresistance indicates the biodiversity at this potential disposal site. These resultscan affect the construction of a future underground repository, since knowledge of the most

resistant microorganism may be of importance as concerns calculation of the time required toinactivate the bacteria surrounding the containers. r 2002 Elsevier Science Ltd. All rightsreserved.

Keywords: Radiosensitivity; Nuclear waste disposal; Subterranean bacteria

1. Introduction

A number of studies have demonstrated that microbial activity exists in deepgeological formations and rock caverns even under extreme conditions, and variousattempts have been made to evaluate the risks that might result from microbialactivity in proposed nuclear waste disposal (West & McKinley, 1985; Christofi, 1991;

*Corresponding author. Tel.: +36-1-482-2004; fax: +36-1-482-2005.

E-mail address: [email protected] (G. Farkas).

0265-931X/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 2 6 5 - 9 3 1 X ( 0 1 ) 0 0 1 3 0 - 8

Rosevear, 1991). It has been estimated that microbial activity is possible in theplanned repository under the prevailing environmental conditions. The extent ofmicrobial activity will be determined by the composition of the waste and theenvironmental conditions.Containers packed with fuel waste are highly radioactive and thermally hot. In the

immediate vicinity of such fuel waste containers, therefore, most organismsoriginally present at the time of emplacement are likely to be killed in a matter ofhours or days (Stroes-Gascoyne et al., 1995).The sensitivities of microorganisms towards high-energy radiation vary widely,

depending on type, species and strain. Microorganisms in the reactor core after theThree Mile Island disaster grew at a dose rate of 10Gy h�1 (Booth, 1987). SimilarlyMergeay, Bourdos, and Kirchman (1984) found microbes in primary cooling systemsof nuclear reactors that survived neutron fluence rates of up to 5� 1014

neutrons cm�2 s�1. Certain environmental factors also influence the radiationresponse: temperature, oxygen, water, soluble chemical agents, etc. The high-levelnuclear waste in Hungary is currently temporarily stored at a nuclear power plant(Paks) until completion of the final repository site. The preferred site is the PermianSiltstone (Aleurolite) Formation in the area of the Mecsek Hills in southernHungary, close to a former uranium mine. One preliminary and two full studies havebeen undertaken to examine and carry out a phenotypical analysis of the microbespresent in the relevant formations (Farkas et al., 2000).Experiments were carried out on the bacteria isolated from the deep groundwater,

the aleurolite, the surface and the air, in order to obtain information on the bacterialtolerance to extreme conditions of radiation. Thus, the purpose of this study was toassess the ability of native subterranean microorganisms to survive when exposed to*aa-radiation, in an effort to forecast the repository performance and predict thestability of storage canisters to microbially influenced corrosion.

2. Materials and methods

The study site and the sampling and isolating procedures have been described indetail elsewhere (Farkas et al., 2000). A map of the Boda Siltstone formation,including the location of the access tunnel, is shown in Fig. 1. In October 1997 and1998, samples were taken via this access tunnel from the air, groundwater, aleurolitestones and surfaces. The radiation sensitivities of the aerobic and anaerobic, spore-forming and non-spore-forming (vegetative) bacteria were determined. Spores forexamination of the spore-formers were produced as a surface growth (9 days at 351C)on Potato Dextrose Agar (Oxoid CM 139). They were harvested in distilled water,and washed three times by centrifugation. The suspension was then heated at801C for 15min to inactivate any remaining vegetative cells, cooled, washed a furtherthree times and finally resuspended in distilled water to give 107 sporesml�1

(Tallentire & Khan, 1975). The aerobic spore suspensions were irradiated in air,and the anaerobic ones in N2 at room temperature, with exposure to 1; 2; 3; 4 or6 kGy. The irradiation facility was an RH-U-30 60Co apparatus with a dose rate of

G. Farkas et al. / J. Environ. Radioactivity 61 (2002) 233–239234

2:0167–2:0475 kGyh�1. After irradiation, the survivors were detected on NutrientAgar Medium (Oxoid CM 67). The isolated non-spore-forming (vegetative) microbeswere transferred into 5ml Nutrient Broth (Oxoid CM 79) and incubated for 18 h at351C. Cultures in the exponential growth phase were centrifuged, washed twice andresuspended in physiological saline to give 107 viable organisms ml�1. Aerobicsuspensions were irradiated in air, and anaerobic suspensions in N2 at roomtemperature with exposure to 0:2; 0:4; 0:8 or 1:6 kGy. Incubation was performed at351C for 48 h.Dose–response plots were constructed from four experimental points, each point

being determined from at least four replicate plate counts. These plots assumed alinear or near-linear form and were described by the expression

ln S=S0 ¼ ln n� kD;

Fig. 1. Map of the exploratory tunnels, including sampling sites.

G. Farkas et al. / J. Environ. Radioactivity 61 (2002) 233–239 235

where S is the number of microbes surviving a dose D; S0 is the original number ofviable cells, k is the slope of the curve, referred to as the inactivation constant, and nis known as the extrapolation number (the size of the shoulder). The D10 value isdefined as the dose that reduces a given population by a factor of 10: All experimentswere repeated at least twice.

3. Results

Sixty-seven air, groundwater, technical water, rock and surface samples werecollected aseptically from the potential repository site. The number of aerobic andanaerobic isolates was 300: The proportion of spore-forming isolates was 50:6%among the aerobic bacteria and 59:4% among the anaerobic bacteria.The radiosensitivities of 93 isolates were determined. The effect of radiation is

conventionally expressed in terms of the survival curve of the isolates. An example ofthe result of an experiment (isolate W 3=3) is shown in Fig 2. (The deviations of thedata points are not larger than the symbols.)The values of D10 determined for the isolates are listed in Table 1, divided into

four differently radioresistant groups.

Fig. 2. Radiation resistance of spores (isolate W 3/3), D10 ¼ 1:46 kGy.


The D10 values of the aerobic isolates lay in the range 0:8–2:44 kGy, and those ofthe anaerobic isolates lay in the range 1:86–4:93 kGy. The D10 values of thevegetative aerobic and anaerobic isolates were much lower: 0:11–0:57 and 0:22–0:40 kGy, respectively. These results are in the same range as the D10 values cited inthe literature (Urbain, 1986; Jay, 1992; Stroes-Gascoyne & West, 1996) for manyspecies of microorganisms. Among the vegetative bacteria, there were no significantdifferences in radioresistance between the aerobic and the anaerobic isolates, and theminimum and maximum values of D10 did not deviate much from the average. It isalso consistent with the results of other microbiological studies that the anaerobicspores are more resistant than the aerobic ones, and that the differences between theD10 values in both of the spore-forming groups are higher than those for thevegetative cells.

4. Discussion

Since microbes have been found to inhabit almost any subsurface environment,they may play a role in either enhancing or retarding radionuclide transport awayfrom such a disposal vault and this needs to be investigated as well. The speciesdiversity in nuclear waste repositories is quite large, and the different types, speciesand strains among these microorganisms exhibit widely differing sensitivities towardshigh-energy radiation. Certain profound changes in radiation response may berelated to the progress of a cell through the different stages of growth. These may beassociated with changes in the intracellular environment. The literature on theradiation response of bacteria in different growth phases reveals a number ofcontradictory results (Alper, 1980). Unfortunately, a general rule concerning the

Table 1

D10 values of the isolates

Isolates D10 values (kGy)

Aerobic

vegetative

0.11 0.13 0.15 0.15 0.16 0.16 0.19 0.21 0.26 0.27

0.32 0.38 0.39 0.42 0.57 Average: 0.26

Aerobic

spore-former

0.80 0.90 0.91 0.93 1.00 1.00 1.00 1.10 1.12 1.20

1.20 1.20 1.20 1.24 1.27 1.30 1.32 1.40 1.40 1.40

1.40 1.40 1.40 1.42 1.46 1.47 1.49 1.49 1.50 1.53

1.60 1.60 1.60 1.61 1.62 1.66 1.67 1.67 1.69 1.69

1.72 1.73 1.76 1.80 1.81 1.85 1.85 1.92 2.06 2.07

2.08 2.12 2.44 Average: 1.49

Anaerobic

vegetative

0.22 0.22 0.26 0.26 0.39 0.40 Average: 0.29

Anaerobic

spore-former

1.86 2.03 2.06 2.18 2.18 2.25 2.32 2.33 2.55 2.79

2.79 2.95 2.99 3.10 3.17 3.33 3.37 3.83 4.93

Average: 2.79


growth phase effect is not available. Survival curves are sometimes steeper inexponential than in stationary phases, but the reverse is also sometimes true.As we reported earlier, the minimum and maximum colony-forming unit counts

of aerobic and anaerobic mesophilic microorganisms isolated from groundwaterwere 0:3921:25� 105 and 4� 105 > 106 ml�1, respectively. These data representthe microorganisms naturally present in the groundwater. The proportion of spore-formers was B50% of the aerobic and B60% of the anaerobic isolates.The radiosensitivities of 93 isolates were determined in this study. The D10 valuesof the aerobic spore-formers were 0.8–2.44 kGy, while those of the anaerobic spore-formers were 1.86–4.93 kGy. The D10 values of aerobic spores have been reportedto range generally between 1.2 and 3.3 kGy, while most of their vegetativeforms exhibit lower radiation resistance (Gazs !oo, 1997). The D10 values of theaerobic bacteria isolated from groundwater ranged from 0.90 to 2.44 kGy which wasa larger range of bacterial radioresistance than those for the aerobic isolates from therock and air samples (the D10 values were 0.9–1.76 and 0.8–1.60 kGy, respectively).For the anaerobic isolates, a similar tendency was observed. The range of the D10values of the isolates from the groundwater was 1.86–4.93 kGy, while that forthe isolates from the rock was 2.03–2.99 kGy. The most resistant bacteria wereisolated from the groundwater. The radiosensitivities displayed by the most resistantmicroorganisms appear to be of importance in calculations of the time requiredto inactivate the bacteria in the surroundings of the containers of nuclear fuel waste.At this moment, we do not have any exact information concerning the dose rate atthe waste container surface, because our repository is currently under construction.Stroes-Gascoyne and West (1995) have reported an overview of microbial researchrelating to high-level nuclear waste disposal. They estimated the expected doserate at a titanium fuel waste container surface as 0.052 kGyh�1 for fuel cooled for 10years. If we accept a similar dose rate in our case, an inactivation periodof approximately 24 days is calculated at the surface of the container with regardto the D10 value of the most radioresistant bacteria and the initial count in thegroundwater. Our non-published results revealed corrosion pits (B100 nm deep)after the removal of a 3-week-old biofilm from a stainless steel surface. This meansthat biocorrosion can be initiated within 24 days. Unfortunately, most of theliterature data were obtained under different experimental conditions. As theenvironmental conditions exert a considerable influence on the radiosensitivityobserved, meaningful comparisons are very difficult. A systematic study of theradiosensitivities of soil and subterranean microorganisms of interest as concernsbiocorrosion is still needed. Besides the differences between species, there are anumber of environmental factors (oxygen, water content, chemical radioprotectorsor sensitizers, and temperature) that can greatly affect the radiosensitivity.The different supporting surfaces can also alter the radiosensitivities of bacteria.However, it has been demonstrated that the subterranean conditions andthe overlying uranium-bearing sandstone do not exert significant effects on theradiosensitivities of the bacteria present in the relevant formation. Although ourresults contribute towards an understanding of the impact of microbial activity onthe deep geological disposal of nuclear waste, we do not as yet have sufficient


information to develop a model which will predict the consequences of theseprocesses.

Acknowledgements

This work was supported in part by OTKA grant T20 059.

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radiosensitivity of subterranean bacteria in the hungarian upper permian siltstone formation

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