Achim Albrecht

Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals

After closure of a waste disposal cell in a repository for radioactive waste, resaturation is likely to cause the release of soluble species contained in cement and bituminous matrices, such as ionic species (nitrates, sulfates, calcium and alkaline ions, etc.), organic matter (mainly organic acids), or gases (from steel containers and reinforced concrete structures as well as from radiolysis within the waste packages). However, in the presence of nitrates in the near-field of waste, the waste cell can initiate oxidative conditions leading to enhanced mobility of redox-sensitive radionuclides (RN). In biotic conditions and in the presence of organic matter and/or hydrogen as electron donors, nitrates may be microbiologically reduced, allowing a return to reducing conditions that promote the safety of storage. Our work aims to analyze the possible microbial reactivity of nitrates at the bitumen - concrete interface in conditions as close as possible to radioactive waste storage conditions in order (i) to evaluate the nitrate reaction kinetics; (ii) to identify the by-products (NO2(-), NH4(+), N2, N2O, etc.); and (iii) to discriminate between the roles of planktonic bacteria and those adhering as a biofilm structure in the denitrifying activity. Leaching experiments on solid matrices (bitumen and cement pastes) were first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface, e.g. highly alkaline pH conditions (10 < pH < 11) imposed by the cement matrix. The screening of a range of anaerobic denitrifying bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these particular conditions of pH. The denitrifying activity of H. desiderata was quantified in a batch bioreactor in the presence of solid matrices and/or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 h) and 2-3 times slower in the presence of bituminous matrix (pH 9.7). The maximal rate of denitrification was approximately 0.063 mM h(-1) and some traces of nitrite were detected for a few hours (<2%). Overall, the presence of solid cement promoted the kinetics of denitrification. The inspection of the solid surfaces at the end of the experiment revealed the presence of a biofilm of H. desiderata on the cement paste surface. These attached bacteria showed a comparable denitrifying activity to planktonic bacterial culture. However, no colonization of bitumen was observed either by SEM or by epifluorescence microscopy.

Microbial Catalysis of Redox Reactions in Concrete Cells of Nuclear Waste Repositories: A Review and Introduction

In order to be able to simulate the behaviour of radionuclides (RN) in waste repositories in space and time it is important to know their chemical speciation. 14C in its reduced form (CH4) does not have the same behaviour as in its oxidised form (CO2, CO 3 − ). Similarly, tritium in the reduced gaseous form, HT, does not at all behave as its oxidised form (liquid water, HTO). For other RN such as U, Se, Tc, Np and Pu the impact is less striking as the change in redox state does not generate a phase change but a change in the sorption behaviour. As a rule of thumb the oxidised form is more mobile than the reduced form. Nuclear waste repositories for both low and intermediate level wastes are characterised by the presence of cementitious phases and zero-valent metals as part of waste, waste containers or engineered materials; organic matter is also likely present in both waste and engineered barrier. Hydrogen gas can be formed either via radiolysis or anaerobic corrosion. We therefore have two main electron donors to participate in redox reactions within an “unnaturally” high pH environment. Oxygen, present during the exploitation phase, is quickly consumed and not considered to diffuse significantly into deep or near-surface repositories. Nitrate, Fe(III) or Mn(IV) are only in specific cases present in significant quantities. Consequently, H+ and C4+ present in water and carbonate will become the main electron acceptors in redox reactions after reduction of sulphates, present in some wastes, concrete and host rocks; H+ and C4+ are likely to control in fine the overall redox potential and the speciation of RN. There is more and more evidence for the microbial control of reactions implying electron transfer within H and C species [Hoehler TM (2005) Biogeochemistry of dihydrogen (H2). In: Sigel A, Sigel H, Sigel RKO (eds) Metal ions in biological systems. Taylor & Francis, Boca Raton, FL, pp 9–48]. Furthermore, the impact of microbial activity on the degradation of complex organic matter (i.e. polymers) adds to the need to evaluate their catalytic impact on waste cell redox potential [Askarieh MM, Chambers AV, Daniel FBD, FitzGerald PL, Holtom GJ, Pilkington NJ, Reesb JH (2000) The chemical and microbial degradation of cellulose in the near field of a repository for radioactive wastes. Waste Manag 20: 93–106]. Quantification of reaction dynamics in alkaline systems involving Fe(0), H2 or organic matter as electron donors and nitrates/sulphates (if present) as well as carbonates or water (and RN in their possibly oxidised form) as electron acceptors will have to consider a microbial catalysis. There are many analogues for testing simulation approaches for microbial catalysis of related redox reactions, but few are in alkaline systems. With H2 almost omnipresent as an energy source, essential and trace nutrients most likely present in the heterogeneous waste cell environment, with space and water available depending on depth, architecture and re-saturation, the high pH may become the most critical parameter controlling microbial activity in space and time. In this chapter, we will review the importance of oxyanions in the nuclear industry and their impact together with concrete, steel and organic matter on the redox state in the near field of a waste storage cell. Particular consideration will be given to the knowledge in relation to alcaliphilic microbial activity in some cases derived from existing natural analogues. Case studies will consider specific redox-sensitive radionuclides in both near surface and deep storage settings. This information will serve as input to two ongoing experimental endeavours dealing with the specific reaction of nitrate reduction by organic matter and/or H2 in the concrete cells for bituminous waste disposal.

Impact of the electron donor on in situ microbial nitrate reduction in Opalinus Clay: results from the Mont Terri rock laboratory (Switzerland)

At the Mont Terri rock laboratory (Switzerland), an in situ experiment is being carried out to examine the fate of nitrate leaching from nitrate-containing bituminized radioactive waste, in a clay host rock for geological disposal. Such a release of nitrate may cause a geochemical perturbation of the clay, possibly affecting some of the favorable characteristics of the host rock. In this in situ experiment, combined transport and reactivity of nitrate is studied inside anoxic and water-saturated chambers in a borehole in the Opalinus Clay. Continuous circulation of the solution from the borehole to the surface equipment allows a regular sampling and online monitoring of its chemical composition. In this paper, in situ microbial nitrate reduction in the Opalinus Clay is discussed, in the presence or absence of additional electron donors relevant for the disposal concept and likely to be released from nitrate-containing bituminized radioactive waste: acetate (simulating bitumen degradation products) and H2 (originating from radiolysis and corrosion in the repository). The results of these tests indicate that—in case microorganisms would be active in the repository or the surrounding clay—microbial nitrate reduction can occur using electron donors naturally present in the clay (e.g. pyrite, dissolved organic matter). Nevertheless, non-reactive transport of nitrate in the clay is expected to be the main process. In contrast, when easily oxidizable electron donors would be available (e.g. acetate and H2), the microbial activity will be strongly stimulated. Both in the presence of H2 and acetate, nitrite and nitrogenous gases are predominantly produced, although some ammonium can also be formed when H2 is present. The reduction of nitrate in the clay could have an impact on the redox conditions in the pore-water and might also lead to a gas-related perturbation of the host rock, depending on the electron donor used during denitrification.