Enhancing vinyl chloride degradation by bioaugmentation of etheneotrophic bacteria in contaminated groundwater

Vinyl chloride (VC) and dichloro-ethenes (DCE) are often accumulated in chloroethene- contaminated groundwater due to their relatively slow dehalogenation to ethene by organohalide-respiring bacteria. Aerobic bacteria able to use VC and DCE as sole carbon and energy sources (etheneotrophs) can mitigate these contaminations. Etheneotrophs can be stimulated in situ by aerobic treatments installed downstream anaerobic permeable reactive bio-barriers and bioaugmentation with etheneotrophic bacteria can improve and speed up the process of natural attenuation. In this study, two etheneotrophic bacteria belonging to Mycolicibacterium frederiksbergense strain 1.1 and to Ralstonia solanacearum strain M4, were isolated from a highly VC- contaminated aquifer (10,000 μg/L). Kinetic data confirmed their ability to use VC and ethene as growth substrates and genetic characterization highlighted that they carry etnC and etnE genes encoding for alkene monooxygenase (AkMO) and epoxyalkane transferase (EaCoMT), respectively, present in the VC biodegradation pathway. Toxicity tests conducted in the contaminated groundwater revealed that M. frederiksbergense 1.1 and R. solanacearum M4 tolerate high VC (10,000 μg/L) and cis-1,2-DCE (440 μg/L) concentrations. In bioaugmentation experiments with real groundwater, the strains effectively reduced VC and cis-1,2-DCE concentrations to regulatory limits within 5 and 7 days, respectively, compared to 14 days under natural attenuation. Degradation kinetics were correlated with microbial growth. Real Time q-PCR quantification, confirmed that etnC gene was significantly higher in bioaugmented samples than in natural attenuation controls. Illumina sequencing of the bacterial 16S rRNA gene evidenced that etheneotrophic bacteria belonging Mycolicibacterium (2.5%), Simplicispira (1.7%) and Rhodoferax (3.3%), were present in the groundwater. At the present stage, bioaugmentation with autochthonous etheneotrophic bacteria could be tested in pilot scale experiments, as implementation of aerobic reactive biobarriers.