Elsevier

Geochimica et Cosmochimica Acta

Volume 96, 1 November 2012, Pages 94-104
Geochimica et Cosmochimica Acta

Immobilization of uranium in biofilm microorganisms exposed to groundwater seeps over granitic rock tunnel walls in Olkiluoto, Finland

https://doi.org/10.1016/j.gca.2012.08.012Get rights and content

Abstract

In an underground rock characterization facility, the ONKALO tunnel in Finland, massive 5–10-mm thick biofilms were observed attached to tunnel walls where groundwater was seeping from bedrock fractures at a depth of 70 m. In laboratory experiments performed in a flow cell with detached biofilms to study the effect of uranium on the biofilm, uranium was added to the circulating groundwater (CGW) obtained from the fracture feeding the biofilm. The final uranium concentration in the CGW was adjusted to 4.25 × 10−5 M, in the range expected from a leaking spent nuclear fuel (SNF) canister in a future underground repository. The effects were investigated using microelectrodes to measure pH and Eh, time-resolved laser fluorescence spectroscopy (TRLFS), energy-filtered transmission electron microscopy (EF-TEM), and electron energy-loss spectroscopy (EELS) studies and thermodynamic calculations were utilized as well. The results indicated that the studied biofilms constituted their own microenvironments, which differed significantly from that of the CGW. A pH of 5.37 was recorded inside the biofilm, approximately 3.5 units lower than the pH observed in the CGW, due to sulfide oxidation to sulfuric acid in the biofilm. Similarly, the Eh of +73 mV inside the biofilm was approximately 420 mV lower than the Eh measured in the CGW. Adding uranium increased the pH in the biofilm to 7.27 and reduced the Eh to −164 mV. The changes of Eh and pH influenced the bioavailability of uranium, since microbial metabolic processes are sensitive to metals and their speciation. EF-TEM investigations indicated that uranium in the biofilm was immobilized intracellularly in microorganisms by the formation of metabolically mediated uranyl phosphate, similar to needle-shaped autunite (Ca[UO2]2[PO4]2·2–6H2O) or meta-autunite (Ca[UO2]2[PO4]2·10–12H2O). In contrast, TRLFS studies of the contaminated CGW identified aqueous uranium carbonate species, likely (Ca2UO2[CO3]3), formed due to the high concentration of carbonate in the CGW. The results agreed with thermodynamic calculations of the theoretically predominant field of uranium species, formed in the uranium-contaminated CGW at the measured geochemical parameters.

This investigation clearly demonstrated that biological systems must be considered as a part of natural systems that can significantly influence radionuclide behavior. The results improve our understanding of the mechanisms of biofilm response to radionuclides in relation to safety assessments of SNF repositories.

Introduction

A permanent repository for spent nuclear fuel (SNF) is under construction at an underground rock characterization facility, the ONKALO tunnel, situated near the Olkiluoto nuclear power plants approximately 300 km northwest of Helsinki, Finland. Geological mapping indicates that the tunnel bedrock is mainly composed of veined gneisses with a migmatic appearance, interspersed with numerous fractures (Nordbäck et al., 2008). Groundwater with microorganisms flows through fractures in this bedrock. Analysis of groundwater samples from boreholes indicates an average total number of 3.9 × 105 cells mL−1 in shallow groundwater (at depths of 0–20 m) and 5.7 × 104 cells mL−1 in deep groundwater (at depths of 100–800 m) (Pedersen et al., 2008a). The results show that microbial activity is ongoing in groundwater, down to depths of at least 800 m in Olkiluoto. As described by Stroes-Gascoyne and Sargent (1998), microorganisms can influence the performance of SNF repositories by corroding the nuclear waste containers and mobilizing radionuclides in groundwater in the event of canister failure. Specific interactions between microorganisms and radionuclides are known and can be distinguished as biosorption, bioaccumulation, biotransformation, biomineralization, and bioreduction (Lloyd and Macaskie, 2002), with potential for the substantial retention of radionuclides. The ability to accumulate uranium by biosorption, for example, by Pseudomonas strains (Pons and Fusté, 1993), interactions with S-layers (Merroun et al., 2005), or bioprecipitation (Macaskie et al., 2000), has been found in several bacterial strains. The reduction of uranium(VI) by bioreduction has been studied in microbial communities associated with sediments, soils, groundwater, and biofilms (Abdelouas et al., 1998, Beyenal et al., 2004, Peacock et al., 2004, Geissler and Selenska-Pobell, 2005, Wu et al., 2005).

In nature, microorganisms appear not only as individual, unattached cells but also in multicellular, attached communities called biofilms (Jägevall et al., 2011). These primarily grow attached to aquifer fracture surfaces and consist of microbial cells embedded in extracellular polymeric substances (EPS), for example, polysaccharides, proteins, lipoproteins, and glycoproteins (Flemming, 1991). Interactions of radionuclides with biofilms probably differ from interactions with suspensions of individual cells of single microorganism species. However, relevant studies are rare and little is known of the processes of radionuclide retention in biofilms. Biofilms are known to provide a sink for dissolved heavy metals (Späth et al., 1998) because the EPS, cell walls, cell membranes, and cell cytoplasm can serve as sorption sites (Flemming, 1995). Anderson et al., 2006, Anderson et al., 2007 recently examined the adsorption capacity of subsurface anaerobic biofilms from the Äspö Hard Rock Laboratory tunnel in Sweden using 60Co(II), 147Pm(III), 241Am(III), 234Th(IV), 237Np(V), and 99Mo(VI) as radioactive tracers.

In the present work, biofilm samples were taken from the walls of the ONKALO tunnel and used for experiments in a laboratory on the surface. Laboratory experiments were performed under aerobic conditions thought to represent exceptional conditions over the lifetime of a deep geological repository. Hydrogeochemical results at Olkiluoto indicate that anaerobic conditions will be dominant in groundwater in deep bedrock conditions in the future (Posiva, 2011). Under such conditions, many microorganisms may catalyze the microbial transformation (Lovley et al., 1991, Ganesh et al., 1997) of U(VI) to sparingly soluble and immobile U(IV). In general, there are Fe(III)-reducing bacteria, such as Shewanella spp. and Geobacter spp. (e.g. Lovley et al., 1993), sulfate-reducing bacteria, such as Desulfovibrio spp. (e.g. Tucker et al., 1996, Tucker et al., 1998), and Desulfosporosinus spp. (Suzuki et al., 2002, Suzuki et al., 2003), butyrate-utilizing Desulfotomaculum spp. (Tebo and Obraztsova, 1998), Clostridium spp. (Francis et al., 1994), Salmonella (Shelobolina et al., 2004), Cellulomonas (Sani et al., 2002), and denitrifying Acidovorax spp. (Nyman et al., 2005). But even in the presence of oxic conditions in the bulk solution U may be reduced by microbes as described in Nguyen et al. (2012) assuming anoxic microenvironments inside biofilms. However, the adjustment to anaerobic conditions in the ONKALO tunnel will be a long-lasting process and will be connected to a response in the present microbial diversity.

In our experiments uranium concentrations in the range of those expected from a damaged and leaking waste canister in the far-field were employed to simulate on-site the fate and behavior of this radionuclide in the event of canister failure in a SNF repository. Both biofilm and groundwater were used in studies employing different methods to find evidence of the possibility of uranium retention. Microelectrodes with a few micrometers in diameter allowed measurements at μm scale. Voltage and chemical gradients in the biofilms were helpful in interpreting the in situ microbial metabolic processes as described in general terms in heterogeneous or homogeneous natural environments (e.g., de Beer, 2000, Kühl, 2005, Revsbech, 2005). In the present work, we hypothesized that microbial metabolic processes in biofilms would be sensitive to heavy metals and their speciation and that changes of Eh and pH influence the bioavailability of uranium in natural biofilms. Krawczyk-Bärsch et al., 2008, Krawczyk-Bärsch et al., 2011 have recently described multi-species biofilms, occurring in situ and in the laboratory, from acidic mine drainage waters from a flooded uranium mine.

This study aimed to improve our understanding of the mechanisms by which biofilms respond to exposure to the radionuclide uranium with respect to safety assessments in the far-field of SNF repositories. In this environment, microorganisms must be considered, along with minerals, an important factor influencing radionuclide transport. The results provide new insight, since microelectrodes are rarely used in interpreting radionuclide retention processes in biofilms.

Section snippets

Sampling

In the ONKALO site, massive biofilms approximately 5–10 mm thick were observed attached to the fractured bedrock (Fig. 1) at a depth of 70 m (771 m from the tunnel entrance) during the June 2010 sampling campaign. These biofilms were irregularly distributed in an area approximately 60 cm high and 1 m long, where numerous fractures were distributed over the tunnel wall. The biofilms were removed from the tunnel wall using a knife and collected in sterile boxes. The groundwater seeping over the

Analysis of the groundwater seeps and of the CGW

Minor amounts of 4.2 × 10−9 M uranium were detected in the groundwater seeps (Table 1). This uranium originated from natural leaching from granitic fracture surfaces in the groundwater infiltration area, where oxidizing surface waters recharged the fractures in the granitic rock. The dissolved uranium is transported as mobile U(VI), mainly in the groundwater. In our experiments, uranium was added to the CGW to reach a uranium concentration of 4.25 × 10−5 M. At the end of the experiments, the uranium

Discussion

The combination of microelectrode measurements, EF-TEM/EELS and TRLFS investigations, and theoretical calculation of the predominance fields of aqueous and solid species succeeded in differentiating between the mechanisms proceeding in the biofilm and the CGW. Microelectrodes with a very small tip diameter made it possible to determine the Eh and pH inside the biofilm. Unfortunately, measuring the microprofiles in 50-μm steps, starting from the top of the biofilm and ending at the bottom, was

Conclusions

Improved understanding of the mechanisms of biofilm response to actinides is very important, since microbial metabolic processes are sensitive to metals and their speciation. Consequently, changes of Eh and pH will influence the bioavailability of actinides. In our studies, uranium was removed from solution and immobilized exclusively in biofilm microorganisms in the form of the U-phosphate minerals autunite or meta-autunite. In contrast, aqueous calcium uranyl carbonates species formed in the

Acknowledgments

The research leading to these results has received funding from the European Union’s European Atomic Energy Community’s (Euratom) Seventh Framework Programme FP7/2007-2011 under grant agreement n° 212287 (RECOSYproject). We thank Posiva Oy for their cooperation in biofilm and water sampling. We thank Ursula Schaefer and Carola Eckardt (HZDR Dresden) for analysis. Inge Kristen’s (HZI Braunschweig) skilfull work on sample preparation for analytical EF-TEM is gratefully acknowledged.

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