Molecular analysis of diversity, gene expression and activity of mineral-associated bacteria

Abstract

This dissertation investigated the diversity and hydrogenase activity and gene expression of mineral-associated microorganisms. Surface-associated microbes have been shown to dominate diversity and activity in the environment, however molecular analysis of sediment-associated communities is hindered by both inaccessibility to the subsurface and co-extraction of inhibitory compounds. In order to analyze microbial communities in which the environmental conditions previously had precluded the use of traditional sediment extraction techniques, biofilm coupons (metal, mesh cylinders containing surrogate substrata) were used to recover microorganisms able to attach and compete in a biofilm. The community recovered from the contaminated site using hematite as a surrogate substratum was dominated by microbes most closely related to Alcaligenes sp. (metal-tolerant), Frateuria sp. (acidophilic), and Methylobacterium radiotolerans (radionuclide-tolerant) which together reflect the acidic, metal-, and radionuclide-contaminated environment. Hematite, as compared to other substrata, was shown to recover communities most closely representative of sediment communities inhabiting the saturated zone. Surface-associated cells have been shown to express greater activity than suspended populations and mineral-associated sulfate-reducing bacteria (SRB) mediate the formation of different secondary minerals as compared to suspended cells. In order to investigate the affect of surface-association on enzyme activity, hydrogenase enzyme activity was compared in hematite-associated and suspended populations of the SRB Desulfovibrio desulfuricans Essex 6. Hydrogenase activity of surface-associated populations was higher than that displayed by suspended cells. Hydrogenase likely affects the pH and pE of the conditions immediately surrounding the cell. The greater rate of activity may be one factor which contributes to the formation of a mineral phase not observed in the presence of suspended populations of this bacterium. In order to determine the portion of cells expressing hydrogenase in the surface-associated populations, in situ reverse-transcription PCR was applied to the hematite-associated cells and all cells were expressing the [NiFe] hydrogenase gene. This thesis demonstrates that environmental conditions of contaminated subsurface environments select for microorganisms able to tolerate or utilize the contaminants. Also, the hydrogenase activity of surface-associated populations is not representative of the suspended cells thus indicating the importance of studying attached populations where enzyme activity likely influences the conditions at the mineral-microbe interface.