Sulfate Requirement for the Growth of U(VI)-Reducing Bacteria in an Ethanol-Fed Enrichment

A field experiment at the Oak Ridge Field Research Center has demonstrated the in situ biostimulation of U(VI) reduction with ethanol amendment, but little is known about the stimulated metabolic pathways or composition of the bacterial community mediating the reduction. This work characterized the metabolism and community structure of a sulfate-reducing enrichment developed from sediment from the field site to help address this knowledge gap. Structure was investigated by clone library construction and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA. The enrichment used ethanol concomitantly with sulfate, producing acetate. Hydrogen accumulated intermittently. The clone library contained sequences related to Clostridia, Desulfovibrio, Bacteroides, and Synergistes species. The enrichment reduced U(VI), and the reduction rate was 0.055 L/mg volatile suspended solids (VSS)/day. The enrichment's T-RFLP profile was comprised largely of Desulfovibrio-like fragments, and Desulfovibrio species are known to reduce sulfate and U(VI). A second line of enrichments, inoculated from the sulfate-amended enrichment, was maintained without sulfate. After four transfers of the sulfate-free culture, it was found unable to reduce U(VI). This culture's T-RFLP profile was largely comprised of Clostridia-like fragments, and Clostridia ferment ethanol to acetate. The results indicate a sulfate requirement for the growth of U(VI)-reducing organisms in this community.