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In the multibarrier concept for the deep geological disposal of high-level radioactive waste (HLW), bentonite is proposed as a potential barrier and buffer material for sealing the space between the steel canister containing the HLW and the surrounding host rock. In order to broaden the spectra of appropriate bentonites, we investigated the metabolic activity and diversity of naturally occurring microorganisms as well as their time-dependent evolution within the industrial B25 Bavarian bentonite under repository-relevant conditions. We conducted anaerobic microcosm experiments containing the B25 bentonite and a synthetic Opalinus Clay pore water solution, which were incubated for one year at 30 and 60 °C. Metabolic activity was only stimulated by the addition of lactate, acetate, or H2. The majority of lactate- and H2-containing microcosms at 30 °C were dominated by strictly anaerobic, sulfate-reducing, and spore-forming microorganisms. The subsequent generation of hydrogen sulfide led to the formation of iron–sulfur precipitations. Independent from the availability of substrates, thermophilic bacteria dominated microcosms that were incubated at 60 °C. However, in the respective microcosms, no significant metabolic activity occurred, and there was no change in the analyzed biogeochemical parameters. Our findings show that indigenous microorganisms of B25 bentonite evolve in a temperature- and substrate-dependent manner.

Bentonite is currently proposed as a potential backfill material for sealing high-level radioactive waste in underground repositories due to its low hydraulic conductivity, self-sealing ability and high adsorption capability. However, saline pore waters, high temperatures and the influence of microbes may cause mineralogical changes and affect the long-term performance of the bentonite barrier system. In this study, long-term static batch experiments were carried out at 25 °C and 90 °C for one and two years using two different industrial bentonites (SD80 from Greece, B36 from Slovakia) and two types of aqueous solutions, which simulated (a) Opalinus clay pore water with a salinity of 19 g·L−1, and (b) diluted cap rock solution with a salinity of 155 g·L−1. The bentonites were prepared with and without organic substrates to study the microbial community and their potential influence on bentonite mineralogy. Smectite alteration was dominated by metal ion substitutions, changes in layer charge and delamination during water–clay interaction. The degree of smectite alteration and changes in the microbial diversity depended largely on the respective bentonite and the experimental conditions. Thus, the low charged SD80 with 17% tetrahedral charge showed nearly no structural change in either of the aqueous solutions, whereas B36 as a medium charged smectite with 56% tetrahedral charge became more beidellitic with increasing temperature when reacted in the diluted cap rock solution. Based on these experiments, the alteration of the smectite is mainly attributed to the nature of the bentonite, pore water chemistry and temperature. A significant microbial influence on the here analyzed parameters was not observed within the two years of experimentation. However, as the detected genera are known to potentially influence geochemical processes, microbial-driven alteration occurring over longer time periods cannot be ruled out if organic nutrients are available at appropriate concentrations.

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Using two different types of impedance biochips (PS5 and BS5) with ring top electrodes, a distinct change of measured impedance has been detected after adding 1–5 µL (with dead or live Gram-positive Lysinibacillus sphaericus JG-A12 cells to 20 µL DI water inside the ring top electrode. We relate observed change of measured impedance to change of membrane potential of L. sphaericus JG-A12 cells. In contrast to impedance measurements, optical density (OD) measurements cannot be used to distinguish between dead and live cells. Dead L. sphaericus JG-A12 cells have been obtained by adding 0.02 mg/mL of the antibiotics tetracycline and 0.1 mg/mL chloramphenicol to a batch with OD0.5 and by incubation for 24 h, 30 °C, 120 rpm in the dark. For impedance measurements, we have used batches with a cell density of 25.5 × 108 cells/mL (OD8.5) and 270.0 × 108 cells/mL (OD90.0). The impedance biochip PS5 can be used to detect the more resistive and less capacitive live L. sphaericus JG-A12 cells. Also, the impedance biochip BS5 can be used to detect the less resistive and more capacitive dead L. sphaericus JG-A12 cells. An outlook on the application of the impedance biochips for high-throughput drug screening, e.g., against multi-drug-resistant Gram-positive bacteria, is given.

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<p>A multi-barrier system in which the radioactive waste is encapsulated in metal containers surrounded by a geotechnical barrier (e.g. compacted bentonite) deep underground in a stable geological formation is one of the internationally accepted options for the disposal of highly radioactive waste. Bentonites have good properties such as high swelling capacity and low hydraulic conductivity, which makes them favorable as backfilling material. However, indigenous microorganisms may affect these properties. Bentonite samples were collected from the Full-scale Engineered Barrier Experiment (FEBEX) - Dismantling Project [1] at the Grimsel Test Site (Switzerland) to study their microbial diversity. For that, total DNA was extracted directly from the cores and from enrichments of sulfate- and iron-reducing microorganisms as well as microorganisms were isolated from those enrichments. The microbial communities of the bentonites, the enrichments, as well as the isolates were analyzed by 16S rRNA gene sequencing. The results showed that Actinobacteria and Alphaproteobacteria dominated the FEBEX bentonite microbial population, while the dominant phylum in both enrichments was Firmicutes: concretely, Bacilli and Clostridia classes. In addition, bacteria from the genera <em>Desulfitobacterium</em>, <em>Desulfosporosinus</em> and <em>Clostridium</em> were isolated from the enrichments. <em>Desulfosporosinus hippei</em> DSM 8344 as a phylogenetic close relative was selected to study its interactions with uranium and especially its potential to reduce U(VI) to U(IV). This study revealed that microorganisms are present in bentonite samples after a long-term continuous heating. Sulfate- and iron-reducing microbes were enriched by using favorable conditions in specific media and the potential of the sulfate-reducing microorganisms on the reduction of uranium was verified. Therefore, it is important to characterize the microbial population of the bentonite, because microbes might compromise the safety of the deep geological repository of highly radioactive waste.</p><p>[1]http://www.grimsel.com/gts-phase-vi/febex-dp/febex-dp-introduction</p>