Metagenomic and Cultivation-Based Analysis of Novel Microorganisms and Functions in Metal-Contaminated Environments

Author(s): Yelton, Alexis Pepper | Advisor(s): Banfield, Jillian F | Abstract: Some bacteria and archaea have evolved metabolic strategies that enable them to live in environments contaminated by toxic metals. In fact, many bacteria and archaea take advantage of the redox sensitivity of these very same metals to gain energy via anaerobic respiration. Here, metagenomic techniques were developed and applied along with conventional physiological and ecological methods to elucidate multiple modes of adaptation of bacteria and archaea in metal-contaminated acid mine drainage and aquifer environments contaminated by mine tailings. These approaches provided insight into how these organisms survive and thrive in these environments and how they differentiate themselves from each other.Many of the microbial species in acid mine drainage and mine tailings-contaminated aquifer environments are difficult to culture in the laboratory. Thus, a focus of the research was metabolic analysis of these organisms via analysis of genes and genomes recovered from microbial communities and isolates. Many of the genes are novel, and likely required for specific environmental adaptation, but they are difficult or impossible to functionally characterize based on conventional homology methods. A new method was developed to deal with the challenge of identifying poorly annotated or hypothetical genes of importance in adaptation to metal-contaminated environments. This probabilistic approach is based on conserved gene order between the genomes of interest with distant relatives. The annotation method was used in conjunction with traditional comparative genomics to differentiate a group of co-occurring archaea based on their genetic metabolic potential. These microorganisms grow in biofilm communities in an acid mine drainage system within the Richmond Mine, near Redding in Northern California, USA. Microbial biofilms growing at the air-solution interface were sampled from solutions with pH values of l 1.2, temperatures of up to 48 °C, and mM concentrations of zinc, copper, arsenic, and sub-molar concentrations of dissolved iron. We used a metagenomic approach in which DNA was extracted from biofilm samples, sequenced, and analyzed in order to evaluate differences in the metabolic potential of five closely related Thermoplasmatales archaea and one distant relative. A subset of these organisms appears to be capable of iron oxidation, whereas others appear to live primarily heterotrophically. Another subset is potentially capable of CO oxidation. There are also major differences in motility within the group.A metal-contaminated aquifer adjacent to the Colorado River in Colorado, USA, was studied to investigate microorganisms adapted to high vanadium concentrations. A vanadium-reducing Betaproteobacterium of the genus Simplicispira was isolated (strain BDI). This organism's genome encodes a large number of toxic metal resistance, chemotaxis, motility, and conjugation-related genes that likely allow it to detoxify, avoid contaminants and rapidly adapt in a changing environment. Physiological characterization in the laboratory shows that it is a facultative anaerobic nitrate-reducer capable of reduction of up to 3 mM vanadate.In order to determine the effect of vanadium contamination on the aquifer community structure, soluble vanadium was added to an in-well, flow-through sediment column. Reduction of dissolved vanadate was documented, along with an increase in the number of cells capable of vanadium reduction, and an increase in the relative abundance of strain BDI. An increase in the relative abundance of three families of known vanadium reducing bacteria (Commomonadaceae, Geobacteraceae, and Pseudomonadaceae) was also noted. This experiment confirmed the environmental importance of BDI, and microbial vanadium reduction in response to acetate addition. Following short-term acetate addition to the aquifer, vanadium remained immobile for at least two years. Because the organisms stimulated by amendment were resident in the aquifer and removal of vanadium from solution persists, the acetate addition approach has significant potential for bioremediation of vanadium.In summary, this research used culture-based and culture-independent techniques to elucidate microbial metabolisms that allow organisms to colonize metal-contaminated environments. Vanadium reduction was linked to specific subsurface bacteria, one of which was isolated and characterized. The findings have significance in the fields of genomics, metagenomics, microbial ecology and biogeochemistry, and have potential application for bioremediation.