Biodegradation of Anaerobic, Alkaline Cellulose Degradation Products

The proposed strategy for the disposal of the United Kingdom’s nuclear waste inventory is placement within a deep geological disposal facility (GDF). The prevailing conditions of a GDF are expected to be anaerobic, with alkaline conditions (10.5 13) over a long timescale. In these anaerobic, alkaline conditions the cellulosic components of intermediate level wastes are expected to degrade, with the major products being the α- and β-forms of isosaccharinic acid (ISA). ISAs have received particular attention because of their ability to form complexes with radionuclides, potentially influencing their migration through the GDF. The potential for microbial colonisation of a GDF means that ISAs present a source of organic carbon for utilisation. The ability of micro-organisms to utilise cellulose degradation products including ISA is poorly understood. The work presented in this thesis has shown that near surface microbial consortia are capable of the degradation of ISA under iron reducing, sulphate reducing and methanogenic conditions at circumneutral pH values expected within geochemical niches of the near field and far field of a facility, with PCR analysis suggesting groups responsible for these metabolic processes were present in each instance. The same near surface consortium studied was capable of ISA degradation up to a pH of 10 within 8 weeks. Degradation rates were retarded by the increase in pH, in particular that of the β- stereoisomer. Clostridia were the likely bacterial Class responsible for fermentation of ISA to acetic acid, carbon dioxide and hydrogen. These secondary metabolites were then used in the generation of methane by methanogenic Archaea, however the acetoclastic methanogen component of the consortium was absent at elevated pH; evidenced by the persistence of acetic acid within the microcosm chemistry. The mesophilic consortium used in these initial investigations was not capable of ISA degradation above pH 10 within the short timescales imposed within the project. As a result, a soil consortium was obtained from a hyper alkaline contaminated site, where waste products from lime burning had occurred between 1883 and 1944. Initial surveying of the site showed that ISA was present and generated through interactions between the hyperalkaline leachate and organic soil matter. Following sub-culture of the soil consortia at pH 11, complete ISA degradation was observed within 14 days where again, fermentation processes followed by methanogenesis occurred. Clone libraries showed that again Clostridia was the dominant phylogenetic Class, represented by species from the genus Alkaliphilus. As observed with the mesophilic microcosms at pH 10, hydrogenotrophic methanogens dominated the Archaeal components of the consortia. The results presented in the following body of work suggest that the microbial colonisation of a GDF is likely within the construction and operational phases of the facility. Carbon dioxide is likely to be the predominant terminal electron acceptor within the facility and here methanogenesis has been observed up to a pH of 11.0. In each case, fermentation is likely to be as a result of alkaliphilic Clostridia, where methanogenesis appears to be limited to the hydrogenotrophic pathway at elevated pH. These findings are likely to inform safety assessments through both the application of rate data and gas generation predictions.