Draft Whole-Genome Sequence of the Alkaliphilic Alishewanella aestuarii Strain HH-ZS, Isolated from Historical Lime Kiln WasteContaminated Soil

Here, we present the whole-genome sequence of an environmental Gram-negative Alishewanella aestuarii strain (HH-ZS), isolated from the hyperalkaline contaminated soil of a historical lime kiln in Buxton, United Kingdom

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 (10.513) 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

Microbial Community Evolution Is Significantly Impacted by the Use of Calcium Isosaccharinic Acid as an Analogue for the Products of Alkaline Cellulose Degradation

Diasteriomeric isosaccharinic acid (ISA) is an important consideration within safety assessments for the disposal of the United Kingdoms’ nuclear waste legacy, where it may potentially influence radionuclide migration. Since the intrusion of micro-organisms may occur within a disposal concept, the impact of ISA may be impacted by microbial metabolism. Within the present study we have established two polymicrobial consortia derived from a hyperalkaline soil. Here, α-ISA and a diatereomeric mix of ISAs’ were used as a sole carbon source, reflecting two common substrates appearing within the literature. The metabolism of ISA within these two consortia was similar, where ISA degradation resulted in the acetogenesis and hydrogenotrophic methanogenesis. The chemical data obtained confirm that the diastereomeric nature of ISA is likely to have no impact on its metabolism within alkaline environments. High throughput sequencing of the original soil showed a diverse community which, in the presence of ISA allowed for the dominance the Clostridiales associated taxa with Clostridium clariflavum prevalent. Further taxonomic investigation at the genus level showed that there was in fact a significant difference (p = 0.004) between the two community profiles. Our study demonstrates that the selection of carbon substrate is likely to have a significant impact on microbial community composition estimations, which may have implications with respect to a safety assessment of an ILW-GDF

Whole genome sequence of the anaerobic isosaccharinic acid degrading isolate, Macellibacteroides fermentans strain HH-ZS

The ability of micro-organisms to degrade isosaccharinic acids (ISAs) whilst tolerating hyperalkaline conditions is pivotal to our understanding of the biogeochemistry associated within these environs, but also in scenarios pertaining to the cementitious disposal of radioactive wastes. An alkalitolerant, ISA degrading micro-organism was isolated from the hyperalkaline soils resulting from lime depositions. Here we report the first whole genome sequence, ISA degradation profile and carbohydrate preoteome of a Macellibacteroides fermentans strain HH-ZS, 4.08Mb in size, coding 3,241 proteins, 64 tRNA and one rRNA

Hydrogenotrophic Methanogenesis Under Alkaline Conditions

A cement-based geological disposal facility (GDF) is one potential option for the disposal of intermediate level radioactive wastes. The presence of both organic and metallic materials within a GDF provides the opportunity for both acetoclastic and hydrogenotrophic methanogenesis. However, for these processes to proceed, they need to adapt to the alkaline environment generated by the cementitious materials employed in backfilling and construction. Within the present study, a range of alkaline and neutral pH sediments were investigated to determine the upper pH limit and the preferred route of methane generation. In all cases, the acetoclastic route did not proceed above pH 9.0, and the hydrogenotrophic route dominated methane generation under alkaline conditions. In some alkaline sediments, acetate metabolism was coupled to hydrogenotrophic methanogenesis via syntrophic acetate oxidation, which was confirmed through inhibition studies employing fluoromethane. The absence of acetoclastic methanogenesis at alkaline pH values (>pH 9.0) is attributed to the dominance of the acetate anion over the uncharged, undissociated acid. Under these conditions, acetoclastic methanogens require an active transport system to access their substrate. The data indicate that hydrogenotrophic methanogenesis is the dominant methanogenic pathway under alkaline conditions (>pH 9.0)

The impact of biofilms upon surfaces relevant to an intermediate level radioactive waste geological disposal facility under simulated near field conditions.

The ability of biofilms to form on a range of materials (cementious backfill (Nirex Reference Vault Backfill (NRVB)), graphite and stainless steel) relevant to potential UK intermediate level radioactive waste (ILW) disposal concepts was investigated by exposing these surfaces to alkaliphilic flocs generated by mature biofilm communities. Flocs are aggregates of biofilm material that are able to act as a transport vector for the propagation of biofilms.. In systems where biofilm formation was observed there was also a decrease in the sorption of isosaccharinic acids to the NRVB. The biofilms were composed of cells, extracellular DNA (eDNA), proteins and lipids with a smaller polysaccharide fraction, which was biased towards mannopyranosyl linked carbohydrates. The same trend was seen with the graphite and stainless steel surfaces at these pH values, but in this case the biofilms associated with the stainless steel surfaces had a distinct eDNA basal layer that anchored the biofilm to the surface. At pH 13 no structured biofilm was observed, rather all the surfaces accumulated an indistinct organic layer composed of biofilm materials. This was particularly the case for the stainless steel coupons which accumulated relatively large quantities of eDNA. The results demonstrate that there is the potential for biofilm formation in an ILW-GDF provided an initiation source for the microbial biofilm is present. They also suggest that even when conditions are too harsh for biofilm formation, exposed surfaces may accumulate organic material such as eDNA

Floc formation reduces the pH stress experienced by microorganisms living in alkaline environments

The survival of microorganisms within a cementitious geological disposal facility for radioactive wastes is heavily dependent on their ability to survive the calcium dominated, hyper-alkaline conditions resulting from the dissolution of the cementitious materials. The present study shows that the formation of flocs, composed of a complex mixture of extracellular polymeric substances (EPS), provides protection against alkaline pH values up to pH 13.0. The flocs were dominated by Alishewanella and Dietzia sp, producing a mannose rich carbohydrate fraction incorporating extracellular DNA, resulting in Ca2+ sequestration. EPS provided a ~10 µm thick layer around the cells within the centre of the flocs, which were capable of growth at pH 11.0 and 11.5, maintaining internal pH values of pH 10.4 and 10.7 respectively. Survival was observed at pH 12.0, where an internal floc pH of 11.6 was observed alongside a reduced associated biomass. Limited floc survival (<2 weeks) was observed at pH 13.0.This study demonstrates that flocs are able to maintain a lower internal pH in response to the hyperalkaline conditions expected to occur within a cementitious, geological disposal facility for radioactive wastes and indicates that floc communities within such a facility would be capable of survival up to a pH of 12.0

Methanogenesis from Mineral Carbonates, a Potential Indicator for Life on Mars

Priorities for the exploration of Mars involve the identification and observation of biosignatures that indicate the existence of life on the planet. The atmosphere and composition of the sediments on Mars suggest suitability for anaerobic chemolithotrophic metabolism. Carbonates are often considered as morphological biosignatures, such as stromatolites, but have not been considered as potential electron acceptors. Within the present study, hydrogenotrophic methanogen enrichments were generated from sediments that had received significant quantities of lime from industrial processes (lime kiln/steel production). These enrichments were then supplemented with calcium carbonate powder or marble chips as a sole source of carbon. These microcosms saw a release of inorganic carbon into the liquid phase, which was subsequently removed, resulting in the generation of methane, with 0.37 ± 0.09 mmoles of methane observed in the steel sediment enrichments supplemented with calcium carbonate powder. The steel sediment microcosms and lime sediments with carbonate powder enrichments were dominated by Methanobacterium sp., whilst the lime/marble enrichments were more diverse, containing varying proportions of Methanomassiliicoccus, Methanoculleus and Methanosarcina sp. In all microcosm experiments, acetic acid was detected in the liquid phase. Our results indicate that chemolithotrophic methanogenesis should be considered when determining biosignatures for life on Mars

Anatomical considerations of cutaneous nerves of scalp for an effective anesthetic blockade for procedures on the scalp.

OBJECTIVE: The anatomy of the scalp nerves varies widely with age, race, and individuals of the same race and even within the same individual and hence need to be studied extensively to avoid complications and improve effectiveness during various surgical and anesthetic procedures of the scalp. MATERIALS AND METHODS: Gross dissection was carried out on 11 cadavers (22 Hemifaces: 11 right and 11 left) with no obvious scalp deformities or surgeries. The distances of the supraorbital nerve (SON), supratrochlear nerve (STN), and greater occipital nerve (GON) from commonly used bony landmarks were measured. The branching pattern and presence of accessory notches/foramina were noted. RESULTS: SON and STN were found almost midway and at the junction between medial and middle one-third of the line joining midline and lateral orbital margin, respectively. The distances of STN and SON from the midline were about ½ and 3/4th of the transverse orbital diameters of the individual. GON was found at the medial 2/5 and lateral 3/5 of the line joining inion to the mastoid. In 40.9% cases, SON gave three branches while STN and GON remained as single trunks in 77.27% and 40.0% cases, respectively. Accessory foramina/notches for SON and STN were found in 36.36% and 4.54% of the specimen, respectively. SON and STN remained lateral in the majority while GON ran medially to corresponding vessels. CONCLUSION: These parameters on the Indian population would give a comprehensive idea of the distribution of these cutaneous scalp nerves and would be beneficial in the targeted and accurate deposition of local anesthetic

Biodegradation of the Alkaline Cellulose Degradation Products Generated during Radioactive Waste Disposal.

The anoxic, alkaline hydrolysis of cellulosic materials generates a range of cellulose degradation products (CDP) including α and β forms of isosaccharinic acid (ISA) and is expected to occur in radioactive waste disposal sites receiving intermediate level radioactive wastes. The generation of ISA's is of particular relevance to the disposal of these wastes since they are able to form complexes with radioelements such as Pu enhancing their migration. This study demonstrates that microbial communities present in near-surface anoxic sediments are able to degrade CDP including both forms of ISA via iron reduction, sulphate reduction and methanogenesis, without any prior exposure to these substrates. No significant difference (n = 6, p = 0.118) in α and β ISA degradation rates were seen under either iron reducing, sulphate reducing or methanogenic conditions, giving an overall mean degradation rate of 4.7×10−2 hr−1 (SE±2.9×10−3). These results suggest that a radioactive waste disposal site is likely to be colonised by organisms able to degrade CDP and associated ISA's during the construction and operational phase of the facility