Impacts of Co-Solvent Flushing on Microbial Populations Capable of Degrading Trichloroethylene

With increased application of co-solvent flushing technologies for removal of nonaqueous phase liquids from groundwater aquifers, concern over the effects of the solvent on native microorganisms and their ability to degrade residual contaminant has also arisen. This study assessed the impact of ethanol flushing on the numbers and activity potentials of trichloroethylene (TCE)-degrading microbial populations present in aquifer soils taken immediately after and 2 years after ethanol flushing of a former dry cleaners site. Polymerase chain reaction analysis revealed soluble methane monooxygenase genes in methanotrophic enrichments, and 16S rRNA analysis identified Methylocystis parvus with 98% similarity, further indicating the presence of a type II methanotroph. Dissimilatory sulfite reductase genes in sulfate-reducing enrichments prepared were also observed. Ethanol flushing was simulated in columns packed with uncontaminated soils from the dry cleaners site that were dosed with TCE at concentrations observed in the field; after flushing, the columns were subjected to a continuous flow of 500 pore volumes of groundwater per week. Total acridine orange direct cell counts of the flushed and nonflushed soils decreased over the 15-week testing period, but after 5 weeks, the flushed soils maintained higher cell counts than the nonflushed soils. Inhibition of methanogenesis by sulfate reduction was observed in all column soils, as was increasing removal of total methane by soils incubated under methanotrophic conditions. These results showed that impacts of ethanol were not as severe as anticipated and imply that ethanol may mitigate the toxicity of TCE to the microorganisms

Role of the geosphere in deep nuclear waste disposal – An England and Wales perspective

To dispose permanently of its higher activity nuclear waste England and Wales have chosen deep geological disposal as the most appropriate solution currently available. The purpose of this paper is to describe the main geological features, events and processes relevant to England and Wales that will need to be considered to demonstrate that a site is suitable for a geological disposal facility (GDF). England and Wales are in the early stages of a GDF siting process in which areas of interest are being evaluated using mainly existing data from surface mapping and hydrocarbon exploration and production. Sites are evaluated consistently under six overarching headings, three of which are impacted by their geological setting – safety, engineering feasibility and value for money. “Suitable” geology is that which is safe during the operational and long-term post-closure period, which could have a GDF and its accessways constructed within it, and which delivers value for money. A GDF needs to fulfil dual safety functions wherever it is located: long-term containment of radionuclides, and isolation of the waste from human actions and from natural processes such as glaciations and earthquakes. The role of the geosphere in delivering these safety functions is to provide a low-flux groundwater environment with geochemical conditions that minimise degradation of the engineered components of the GDF, to promote retention of mobilised radionuclides, and to protect the waste from the impacts of humans and natural processes. The containment function of a GDF is provided by a combination of rock and engineering generally referred to as the multibarrier system. It comprises the engineered barriers – solid wasteforms, canisters, buffers, backfill materials, plugs and seals – that work together with the rock to ensure long-term containment. The GDF Programme in England and Wales seeks to identify suitable geological environments for which bespoke engineered barriers can be tailored to optimize the performance of the multibarrier system. The post-closure period over which independent regulators will require a safety case to demonstrate the long-term containment and isolation capabilities of a GDF is up to 1 million years. The long timescales make post-closure safety assessments a unique feature of deep geological disposal programmes. A comprehensive site characterization programme will use information mostly from seismic surveying and deep investigation boreholes to establish adequate rock availability (host rock depth, thickness, areal extent and compartmentalisation), suitable properties and behaviour of the deep geological environment, and the constructability and operability of a potential GDF site including its surface to subsurface access ways. Nuclear Waste Services, the organisation tasked with developing a GDF in England and Wales, is currently engaged with four Community Partnerships through a volunteer siting process: three in west Cumbria, and one on the English east coast in Theddlethorpe, Lincolnshire. In all of these areas Mesozoic claystones have been provisionally identified as potentially suitable GDF host rocks and are being investigated further, with a dedicated 3D seismic survey acquired off the coast of Cumbria in 2022. The main conclusion to be drawn from this paper is that a GDF could be sited in a large number of geological settings in England and Wales, and that the success of the current siting process will largely depend on engaging effectively with willing communities and building enduring relationships with them

Benchmarking DNA extraction methods for phylogenomic analysis of sub-Antarctic Rhodococcus and Williamsia species

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii