Generation of a low pH 1,2-dichloroethane enrichment culture for enhanced bioremediation

The successes of bioremediation field studies with organochlorine respiring bacteria have proved the efficacy of the method to degrade contaminants such as 1,2-dichloroethane (DCA) in situ. The objective of this study was to demonstrate that a DCA degrading consortium, referred to here as AusDCA could be used to bioaugment a DCA contaminated acidic aquifer in situ. Functional characterisation experiments of AusDCA in batch cultures showed that the culture could dechlorinate high concentrations of DCA (6 mM) to ethene anaerobically at pH 5.5 and pH 6.5 and was not inhibited by approximately 15 µM of chloroform (CF). Phylogenetic analysis of an enrichment culture developed after several transfers into fresh medium and two dilution to extinction series of the parent AusDCA culture, identified two closely related Desulfitobacterium clades with sequences 93 and 95 % similarity to Desulfitobacterium metallireducens, a bacterium not known to dechlorinate DCA. Quantitative PCR (qPCR) of both clades demonstrated growth linked with DCA dihaloelimination to ethene at pH 5.5 and ethanol as the sole electron source. Characterisation of the putative DCA reductive dehalogenase, rdhA gene and the putative membrane anchoring protein, rdhB gene showed that the genes and the translated protein sequences were most similar to that of Dehalobacter strain WL, a DCA respiring bacteria from the Firmicutes phylum. This was surprising because not only is Dehalobacter a different genus to Desulfitobacterium, Dehalobacter was not characterised in community analysis of the parent AusDCA culture. A study to characterise the activity and mobility of filtered (planktonic) and unfiltered (planktonic and aggregated) AusDCA culture in aquifer matrix filled columns showed 1) the majority of the inocula had become entrained in the sandy aquifer and 2) aggregated cells are more active than planktonic cells. Application of the parent AusDCA culture in a bioaugmentation field study of the organochlorine contaminated acidic Botany Sands Aquifer, showed evidence of enhanced DCA degradation and longevity of up to 8 months in the bioaugmented well. This is based on in situ field and in vitro microcosm studies established using groundwater extracted 1 and 8 months after bioaugmentation and the detection of Desulfitobacterium specific to the AusDCA culture. This study has implications on bioaugmentation design relating to inoculation, feeding regime and pH buffering in sandy aquifers