Microbial Community Changes in Response to Ethanol or Methanol Amendments for U(VI) Reduction

Microbial community responses to ethanol, methanol, and methanol plus humics amendments in relationship to U(VI) bioreduction were studied in laboratory microcosm experiments using sediments and ground water from a uranium-contaminated site in Oak Ridge, TN. The type of carbon source added, the duration of incubation, and the sampling site influenced the bacterial community structure upon incubation. Analysis of 16S rRNA gene clone libraries indicated that (i) bacterial communities found in ethanol- and methanol-amended samples with U(VI) reduction were similar due to the presence of Deltaproteobacteria and Betaproteobacteria (members of the families Burkholderiaceae, Comamonadaceae, Oxalobacteraceae, and Rhodocyclaceae); (ii) methanol-amended samples without U(VI) reduction exhibited the lowest diversity and the bacterial community contained 69.2 to 92.8% of the family Methylophilaceae; and (iii) the addition of humics resulted in an increase of phylogenetic diversity of Betaproteobacteria (Rodoferax, Polaromonas, Janthinobacterium, Methylophilales, and unclassified) and Firmicutes (Desulfosporosinus and Clostridium).The use of uranium in nuclear research, fuel production, and weapons manufacturing has resulted in environmental contamination at production, manufacturing, and storage sites throughout the United States. Although all of the common isotopes of uranium (²³⁸U 99.27%], ²³⁵U 0.72%], and ²³⁴U 0.005%]) are radioactive, it is the chemical toxicity of uranium that is usually of greatest concern when it is present as a contaminant.The U.S. Department of Energy (DOE) has ongoing efforts to identify and remediate contaminated areas under its control. Stimulating the in situ metabolism of microorganisms capable of reduction of U(VI) to U(IV), producing the insoluble mineral uraninite which precipitates and renders uranium immobile in ground water, has been proposed as an environmentally safe and a potentially cost-effective remediation method (37). Typically, an organic substrate is added to stimulate microbial growth and promote the development of anaerobic conditions, under which the reduction of U(VI) is favored (67). Various substrates (e.g., acetate, ethanol, glucose, and methanol) have been used either in the field or in microcosm studies, and most were capable of stimulating microbial U(VI) reduction (1, 8, 42, 43, 47, 60); however, the addition of methanol did not always result in U(VI) reduction (49). Many microorganisms are known to reduce U(VI) in pure culture, including a hyperthermophilic archaeon (28), a thermophilic bacterium Thermoterrabacterium ferrireducens (29), the mesophilic dissimilatory metal-reducing bacteria Geobacter and Shewanella (67) and Anaeromyxobacter dehalogenans (71), the sulfate-reducing bacterium Desulfovibrio sp. (61), and fermentative bacteria such as Clostridium spp. (20). These data suggest that U(VI) can be reduced by many microorganisms once suitable electron donors are available.The purpose of this study was to analyze the ability of various amendments to stimulate the reduction of U(VI) by the indigenous microbial communities found in subsurface sediments collected from a uranium-contaminated site. A previous publication from this project (42) gave a very limited analysis of the microbial community. Here we present a detailed phylogenetic analysis of the bacterial community structure and link community structure to capability of U(VI) reduction in sediments stimulated with ethanol and methanol. This study was designed to explore whether microbial communities that demonstrate U(VI) reduction after stimulation with different alcohols show a similar structure. Also, it was designed to detect differences between the methanol-stimulated communities that were capable of U(VI) reduction and those that were not capable of U(VI) reduction. Since humic substances have been reported to promote U(VI) reduction (10, 34), we also examined the effects of humics on the community structure and reduction of U(VI).