Complete reduction of TCE to ethene by sediment and subsequent enrichments that are not dominated by Dehalococcoides

Trichloroethylene (TCE) bioremediation via reductive dechlorination is one of the most prevalent strategies employed in practice and it is equally as investigated in academia. To date, complete dechlorination (i.e., production of ethene from parent chlorinated solvents) has been attributed to a single microbial genus and species - Dehalococcoides ethenogenes. This is an important microorganism that encompasses many different strains, all of which have disparate metabolic capabilities despite their phylogenetic affiliation. The organism is limited by electron donor. Because hydrogen is considered the sole donor for complete dechlorination, substrates are used that ferment to hydrogen (such as lactate) to stimulate Dehalococcoides in situ. We have been incubating TCE-contaminated sediment incubations with acetate as the sole electron donor added at low, stoichiometric concentration to promote simultaneous dechlorination, Fe(III) reduction, and sulfate reduction without excessive methanogenesis. This unique selective pressure enriched a novel dechlorinating community, and ethene is being produced from TCE and/or VC directly without Dehalococcoides as dominant organisms. While past data suggest that even a small population of DHE-like organisms can drive complete dechlorination, the conditions within these incubations are different than those that normally promote DHE growth. Laboratory incubations were started with TCE-contaminated material obtained from a confidential site in Connecticut. Acetate was added at a low concentration based on the stoichiometry of TCE, Fe(III), and sulfate. TCE was reduced to ethene with simultaneous Fe(III) reduction, which is unique - Fe(III) reduction is often considered a competitive process. Sulfate, Fe(III), and TCE (to ethene) reduction was concurrent in alternate incubations, which was also unique. We extracted DNA from all incubations (nine total - three triplicates) and ran PCR reactions with universal Eubacterial primers versus Dehalococcoides (DHE) specific primers. Dehalococcoides ethenogenes strain FL2 was used as a positive control for the specific primers. DNA from all bottles amplified with universal primers, as did strain FL2. However, DNA was not amplified from the sediment with the DHE-specific primers, although strain FL2 gave a very strong signal. We used nested PCR to amplify the full 16S rRNA genes present (~1500 bases with universal primers) and probed the internal sequence with the DHE-specific primers; again - there was no DHE amplification. Strain FL2 was amplified using the nested approach with an extremely strong signal. 900 clones have been analyzed using amplified ribosomal DNA restriction analysis and unique phylotypes were sequenced. No organisms related to Dehalococcoides have been identified. The dominant clone (comprising 20-40% of the total community depending on the specific incubation) is most closely related to a Desulfosporosinus species. Subsequent liquid enrichment cultures have been developed and reduce TCE or VC to ethene with concurrent Fe(III) reduction. Preliminary data with DHE-specific and universal Eubacterial primers demonstrates that DHE-like organisms are not dominant within these enrichments as well. These data suggest that Dehalococcoides may not be the sole microorganism completely reducing TCE or VC to ethene in situ, which will alter how we approach in situ bioremediation of chlorinated solvents. In addition, the conditions under which complete reduction is taking place (concurrent with Fe(III) reduction with acetate as the sole donor), are unique irrespective of the organisms promoting the reactions.