Diversity of planktonic and attached microbial communities in a phenol polluted aquifer.

The sandstone aquifer underlying the Four Ashes industrial site near Wolverhampton, UK, is contaminated with high concentrations of organic pollutants, in particular phenol, cresols and xylenols. Although in the past the geochemistry of the site has been studied extensively, relatively little is known about the in situ microbial communities despite their potential for bioremediation. The aim of this thesis was to investigate the effect of groundwater pollution on the diversity of planktonic and attached microbial communities and to make comparisons between the two. This aim was investigated by sampling planktonic microbial communities at different positions within the fringes of the plume and the planktonic and attached communities at one plume depth (30 metres below ground level in borehole 59). Denaturing Gradient Gel Electrophoresis (DGGE) analysis of peR amplified 16S ribosomal RNA (rRNA) gene fragments indicated that diversity of planktonic microbial communities varied with depth across the steep geochemical gradient of the plume whilst under the same geochemical conditions the planktonic and attached microbial commu~ities differed markedly. The latter result was investigated further by 16S rRNA gene cloning and sequencing. Phylogenetic analysis of the two clone libraries demonstrated that there was limited overlap between the two communities and that the planktonic community was less diverse than the attached community. The 'groundwater' clone library was dominated by four bacterial phylogenetic groups (ex-, (3-Proteobacteria, Firmicutes and Bacteroidetes) whilst the 'sand' clone library was characterised by the presence of a-, {3-, "fProteobacteria, Bacteroidetes as well as a large number of clones (29%) that could not be classified or belonged to minor bacterial phyla. Thirteen percent of the groundwater and 5% of the sand clones had 100% 16S rRNA gene sequence identity to a phenol degrading Azoarcus strain, while 14.7% of the sand clones were closely related (98% sequence identity or more) to members of the Acidovorax genus that have been isolated or detected in phenol contaminated environments. In addition to the in situ studies, laboratory microcosms were inoculated with mixtures of bacteria isolated from the Four Ashes site (with known functional characteristics regarding their abilities to degrade or tolerate phenol and to attach to sand) in order to investigate the influence of different phenol concentrations or changes in phenol concentration on microbial community composition of both planktonic and attached communities. These studies revealed that the relative abundance of microbial isolates within the microcosms altered in response to phenol suggesting that complex metabolic and cell-cell interactions may influence microbial community composition

Domestic shower hose biofilms contain fungal species capable of causing opportunistic infection

The domestic environment can be a source of pathogenic bacteria. We show here that domestic shower hoses may harbour potentially pathogenic bacteria and fungi. Well-developed biofilms were physically removed from the internal surface of shower hoses collected in four locations in England and Scotland. Amplicon pyrosequencing of 16S and 18S rRNA targets revealed the presence of common aquatic and environmental bacteria, including members of the Actinobacteria, Alphaproteobacteria, Bacteroidetes and non-tuberculous Mycobacteria. These bacteria are associated with infections in immunocompromised hosts and are widely reported in shower systems and as causes of water-acquired infection. More importantly, this study represents the first detailed analysis of fungal populations in shower systems and revealed the presence of sequences related to Exophiala mesophila, Fusarium fujikuroi and Malassezia restricta. These organisms can be associated with the environment and healthy skin, but also with infection in compromised and immuno-competent hosts and occurrence of dandruff. Domestic showering may result in exposure to aerosols of bacteria and fungi that are potentially pathogenic and toxigenic. It may be prudent to limit development of these biofilms by the use of disinfectants, or regular replacement of hoses, where immuno-compromised persons are present

Microbial Reduction of U(VI) under Alkaline Conditions: Implications for Radioactive Waste Geodisposal

Although there is consensus that microorganisms significantly influence uranium speciation and mobility in the subsurface under circumneutral conditions, microbiologically mediated U(VI) redox cycling under alkaline conditions relevant to the geological disposal of cementitious intermediate level radioactive waste, remains unexplored. Here, we describe microcosm experiments that investigate the biogeochemical fate of U(VI) at pH 10–10.5, using sediments from a legacy lime working site, stimulated with an added electron donor, and incubated in the presence and absence of added Fe(III) as ferrihydrite. In systems without added Fe(III), partial U(VI) reduction occurred, forming a U(IV)-bearing non-uraninite phase which underwent reoxidation in the presence of air (O2) and to some extent nitrate. By contrast, in the presence of added Fe(III), U(VI) was first removed from solution by sorption to the Fe(III) mineral, followed by bioreduction and (bio)magnetite formation coupled to formation of a complex U(IV)-bearing phase with uraninite present, which also underwent air (O2) and partial nitrate reoxidation. 16S rRNA gene pyrosequencing showed that Gram-positive bacteria affiliated with the Firmicutes and Bacteroidetes dominated in the post-reduction sediments. These data provide the first insights into uranium biogeochemistry at high pH and have significant implications for the long-term fate of uranium in geological disposal in both engineered barrier systems and the alkaline, chemically disturbed geosphere

Bacterial Diversity in the Hyperalkaline Allas Springs (Cyprus), a Natural Analogue for Cementitious Radioactive Waste Repository

The biogeochemical gradients that will develop across the interface between a highly alkaline cementitious geological disposal facility for intermediate level radioactive waste and the geosphere are poorly understood. In addition, there is a paucity of information about the microorganisms that may populate these environments and their role in biomineralization, gas consumption and generation, metal cycling, and on radionuclide speciation and solubility. In this study, we investigated the phylogenetic diversity of indigenous microbial communities and their potential for alkaline metal reduction in samples collected from a natural analogue for cementitious radioactive waste repositories, the hyperalkaline Allas Springs (pH up to 11.9), Troodos Mountains, Cyprus. The site is situated within an ophiolitic complex of ultrabasic rocks that are undergoing active low-temperature serpentinization, which results in hyperalkaline conditions. 16S rRNA cloning and sequencing showed that phylogenetically diverse microbial communities exist in this natural high pH environment, including Hydrogenophaga species. This indicates that alkali-tolerant hydrogen-oxidizing microorganisms could potentially colonize an alkaline geological repository, which is predicted to be rich in molecular H2, as a result of processes including steel corrosion and cellulose biodegradation within the wastes. Moreover, microbial metal reduction was confirmed at alkaline pH in this study by enrichment microcosms and by pure cultures of bacterial isolates affiliated to the Paenibacillus and Alkaliphilus genera. Overall, these data show that a diverse range of microbiological processes can occur in high pH environments, consistent with those expected during the geodisposal of intermediate level waste. Many of these, including gas metabolism and metal reduction, have clear implications for the long-term geological disposal of radioactive waste

Influence of riboflavin on the reduction of radionuclides by Shewanella oneidenis MR-1

Uranium (as UO22+), technetium (as TcO4−) and neptunium (as NpO2+) are highly mobile radionuclides that can be reduced enzymatically by a range of anaerobic and facultatively anaerobic microorganisms, including Shewanella oneidensis MR-1, to poorly soluble species. The redox chemistry of Pu is more complicated, but the dominant oxidation state in most environments is highly insoluble Pu(IV), which can be reduced to Pu(III) which has a potentially increased solubility which could enhance migration of Pu in the environment. Recently it was shown that flavins (riboflavin and flavin mononucleotide (FMN)) secreted by Shewanella oneidensis MR-1 can act as electron shuttles, promoting anoxic growth coupled to the accelerated reduction of poorly-crystalline Fe(III) oxides. Here, we studied the role of riboflavin in mediating the reduction of radionuclides in cultures of Shewanella oneidensis MR-1. Our results demonstrate that the addition of 10 μM riboflavin enhances the reduction rate of Tc(VII) to Tc(IV), Pu(IV) to Pu(III) and to a lesser extent, Np(V) to Np(IV), but has no significant influence on the reduction rate of U(VI) by Shewanella oneidensis MR-1. Thus riboflavin can act as an extracellular electron shuttle to enhance rates of Tc(VII), Np(V) and Pu(IV) reduction, and may therefore play a role in controlling the oxidation state of key redox active actinides and fission products in natural and engineered environments. These results also suggest that the addition of riboflavin could be used to accelerate the bioremediation of radionuclide-contaminated environments

Microbially mediated reduction of FeIII and AsV in Cambodian sediments amended with 13C-labelled hexadecane and kerogen

Microbial activity is generally accepted to play a critical role, with the aid of suitable organic carbon substrates, in the mobilisation of arsenic from sediments into shallow reducing groundwaters. The nature of the organic matter in natural aquifers driving the reduction of AsV to AsIII is of particular importance but is poorly understood. In this study, sediments from an arsenic rich aquifer in Cambodia were amended with two 13C-labelled organic substrates. 13C-hexadecane was used as a model for potentially bioavailable long chain n-alkanes and a 13C-kerogen analogue as a proxy for non-extractable organic matter. During anaerobic incubation for 8 weeks, significant FeIII reduction and AsIII mobilisation were observed in the biotic microcosms only, suggesting that these processes were microbially driven. Microcosms amended with 13C-hexadecane exhibited a similar extent of FeIII reduction to the non-amended microcosms, but marginally higher AsIII release. Moreover, gas chromatography–mass spectrometry analysis showed that 65 % of the added 13C-hexadecane was degraded during the 8-week incubation. The degradation of 13C-hexadecane was microbially driven, as confirmed by DNA stable isotope probing (DNA-SIP). Amendment with 13C-kerogen did not enhance FeIII reduction or AsIII mobilisation, and microbial degradation of kerogen could not be confirmed conclusively by DNA-SIP fractionation or 13C incorporation in the phospholipid fatty acids. These data are, therefore, consistent with the utilisation of long chain n-alkanes (but not kerogen) as electron donors for anaerobic processes, potentially including FeIII and AsV reduction in the subsurface

Nitrate and nitrite reduction at high pH in a cementitious environment by a microbial microcosm

The possible release of oxyanions, such as nitrate, from radioactive waste repositories may influence redox-conditions of the near field environment and thus promote mobility of some redox sensitive radionuclides. The fate of dissolved oxyanions will be significantly conditioned by microbial activities, if present in the aqueous interstitial phase of a waste cell. This study investigates microbial nitrate reduction in a cementitious environment. A consortium of microorganisms was used, an inoculum prepared with sediments collected from a former lime works site, characterized by a pH of pore water of 11–12. The biomass was acclimated to cement leachate supplemented with nitrate, acetate and yeast extract. According to experiments performed in closed and in dynamic systems, the microbial consortium was adapted to reduce nitrate and nitrite in a cementitious, anaerobic environment (pH 11, with and without hardened cement paste and leachate). Although, nitrite accumulation was observed in close system and temporally in dynamic system. The rate of nitrate reduction was between 0.12 and 0.75 mM/h with incoming nitrate concentrations between 6 and 48 mM, respectively. The microorganism diversity and the biofilm present on the hardened cement paste helped maintain microbial activity in all of the conditions simulating cementitious environments