Enrichment and characterization of marine organohalide respiring bacteria and of their dehalogenating enzymes

Organohalide pollution of freshwater and marine sediments threatens human and environmental well-being. In freshwater and marine sediments a natural anaerobic microbiological process called reductive dehalogenation (RD) is carried out by organohalide respiring bacteria (OHRB) and reduces toxicity and improves biodegradability of organohalide pollutants. The reaction is catalyzed by enzymes called reductive dehalogenases (rdh). The marine sediment is the final sink for such dangerous and persistent contaminants, which make the very environment noxious, bioaccumulate in living beings and reach human foodstocks. This work explores reductive dehalogenation, OHRB and their rds’s in the marine environment of two sites of the Adriatic Sea: the Venice Lagoon (VL) and Ravenna Harbor (RH). In a microcosm study, primary sediment from the two sites and from an OHRB-enriched PCB-dechlorinating slurry culture were spiked with different organochloride compounds: hexachlorobenzene (HCBe), 1,2,3,5-tetrachlorobenzene (TeCBe), pentachlorophenol (PCP), 2,3,5-trichlorophenol (TCP) and trichloroethylene (TCE), 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) and a commercial mixture of PCBs (Aroclor© 1254). All compounds were dechlorinated to a certain extent in OHRB-enriched cultures, while primary sediments showed MRD of HCBe, TeCBe, TCP, TCE but not of PCP, and TeCDD, while PCBs dechlorination in VL sediments was not observed in RH but only in VL in previous studies. Microbial community analysis revealed the enrichment of bacteria from the Dehalococcoidia class where dechlorination was taking place in most primary cultures. In enriched cultures the increase of previously identified phylotypes from the same taxon, VLD-1 and VLD-2, correlated with dechlorination. A sediment free TCE-dechlorinating consortium was established in a defined mineral medium. PCR degenerate primer pairs screens and Next Generation Sequencing of amplicons revealed 81 novel rdh homologous gene sequences. Gene expression and proteomic mass spectrometry studies on the TCE-dechlorinating cultures revealed the overexpression of a cluster of three rdh genes hinting at a TCE-dechlorinating activity of one or more of them