Achievement of an enriched anaerobic 1,2-DCA dehalogenating simplified co-culture containing a novel putative 1,2-DCA degrader Geobacter sp. associated to previously identified 1,2-DCA-specific reductive dehalogenase

Chlorinated organic compounds are extensively employed in industry and agriculture. These chemicals are considered among the most severe public health issues, due to their widespread and uncontrolled release in the environment combined to the recalcitrance to degradation and tendency to bio-accumulate. 1,2-dichloroethane (1,2-DCA) is one of the most widespread chlorinated pollutants. This solvent is a key intermediate in the production of PVC. The public health concern and environmental safety issues related to 1,2-DCA, have fueled the scientific interested on the application of feasible and effective bioremediation strategies, on the identification of microorganisms able to efficiently and rapidly degrade it. 1,2-DCA can undergo microbial-mediated degradation through several aerobic or anaerobic metabolic processes. Due to the typical presence of this pollutant in anoxic environments research has been focused on the anaerobic dehalogenation process based on reductive dechlorination in which 1,2-DCA is the final e- acceptor. To date, only relatively few bacterial strains with the ability to reductively dechlorinate 1,2-DCA have been identified. The aims of this work were the evaluation of the 1,2-DCA biodegradative potential of the resident bacterial microbiome of an aquifer polluted with high levels of 1,2-DCA, as well as the enrichment of microbial strains or assemblages directly involved in the dehalorespiration process and the characterization of the functional genes encoding for the putative 1,2-DCA reductive dehalogenases (RDs). We report the characterization of a bacterial community originated from the contaminated aquifer and its development in anaerobic microcosms after biostimulation with different electron donors. 1,2-DCA degradation was monitored by headspace gas-chromatograpic analysis. We investigated the bacterial phylogenetic diversity of the community involved in the degradation process making use of several 16S rRNA-based molecular methods such as PCR-DGGE, clone libraries and high throughput ILLUMINA sequencing, as well as of flow-cytometry. Following the biostimulation treatment the bacterial community underwent a notable change of the main phylotypes. The enrichment culture showed a gradually increasing dechlorinating performance, with up to 50 ppm of 1,2-DCA day-1 degraded. A gradual simplification of the community was observed resulting in the enrichment of few bacterial species. Two populations were highly enriched: a Geobacter sp. (δ-Proteobacteria) and a Pseudomonas sp. (γ-Proteobacteria). These two species were never associated to anaerobic dehalogenation of 1,2-DCA. We were able to assess the inability of the Psedomonas sp. isolated from the dehalogenating consortium, when inoculated in anaerobic as well as aerobic cultures with 1,2-DCA, suggesting Geobacter sp. as the putative dehalogenating bacterium. Through PCR targeting conserved regions within known RD genes novel variants of RDs previously associated to the reductive dechlorination of 1,2-DCA were identified in the metagenome, while no PCR positive product was amplified from the genomic DNA of the Pseudomonas isolate.