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.