Growth of Dehalococcoides spp. and increased abundance of reductive dehalogenase genes in anaerobic PCB-contaminated sediment microcosms

Abstract

Polychlorinated biphenyls (PCBs) contaminate 19% of US Superfund sites and represent a serious risk to human and environmental health. One promising strategy to remediate PCB-contaminated sediments utilizes organohalide-respiring bacteria (OHRB) that dechlorinate PCBs.However, functional genes that act as biomarkers for PCB dechlorination processes (i.e., reductive dehalogenase genes) are poorly understood. Here, we developed anaerobic sediment microcosms that harbor an OHRB community dominated by the genus Dehalococcoides. During the 430-day microcosm incubation, Dehalococcoides 16S rRNA sequences increased two orders of magnitude to 10⁷ copies/g of sediment, and at the same time, PCB118 decreased by as much as 70%. In addition, the OHRB community dechlorinated a range of penta- and tetra-chlorinated PCB congeners including PCBs 66, 70 + 74 + 76, 95, 90 + 101, and PCB110 without exogenous electron donor. We quantified candidate reductive dehalogenase (RDase) genes over a 430-day incubation period and found rd14, a reductive dehalogenase that belongs to Dehalococcoides mccartyi strain CG5, was enriched to 10⁷ copies/g of sediment. At the same time, pcbA5 was enriched to only 10⁵ copies/g of sediment. A survey for additional RDase genes revealed sequences similar to strain CG5's rd4 and rd8. In addition to demonstrating the PCB dechlorination potential of native microbial communities in contaminated freshwater sediments, our results suggest candidate functional genes with previously unexplored potential could serve as biomarkers of PCB dechlorination processes.

Keywords: Dehalococcoides mccartyi; PCBs; Polychlorinated biphenyl; Reductive dechlorination; Reductive dehalogenase genes; Sediment microcosms.

Figure 1.

Mass percentage of PCB 118 (245–34-CB) (A) and PCB 66 (24–34-CB) (B) PCB 26/29 (25–3-CB/245-CB) (C) and PCB 25 (24–3-CB) (D) at 0 days incubation compared to 430 days incubation in microcosms with variable sediment origin (A1, C3, D2, E2, F4). Asterisks indicate statistical significance determined by independent two-sample t-tests. Error bars show the standard deviation between replicates.

Figure 2.

Difference between the PCB profile at 430 days and 0 days in microcosms inoculated with A1 and E2 sediments. Only congeners that represent 1% of the total mass fraction at either 0 or 430 days are shown. Error bars show the standard deviation between replicates. Positive values indicate accumulations in the specified congener over the 430 day incubation, and negative values indicate the congener decreased over time.

Figure 3.

Dehalococcoides (top), Dehalobacter (middle), and Dehalogenimonas (bottom) 16S rRNA gene sequence abundance in DNA extracted from spatially variable sediment microcosms (A1, C3, D2, E2, F4) as measured by qPCR. Error bars show the standard deviation between replicates. Each marker represents the average 16S sequence abundance at different lengths of incubation time from 0 to 417 days.

Figure 4.

Phylogenetic tree of Dehalococcoides 16S rRNA nucleotide sequences isolated from microcosms inoculated with A1 and E2 sediments. Evolutionary analysis was conducted in MEGA7 and the tree with the highest log likelihood (−4696.55) supported by a bootstrap analysis (500 replicates) is shown. The Geobacter lovleyi sequence roots the tree. Open diamonds represent bootstrap values greater than 80%, and filled diamonds represent bootstrap values below 80%. Partial Dehalococcoides 16SrRNA gene sequences obtained in this study are displayed in red.

Figure 5.

Quantity of pcbA4 (top), pcbA5 (middle), and CG5 rd14 (bottom) gene sequence abundance in DNA extracted from spatially variable sediment microcosms (A1, C3, D2, E2, F4) as measured by quantitative real time PCR. Error bars show the standard deviation between replicates. Each marker represents the average 16S sequence abundance at different lengths of incubation time from 0 to 417 days.

Figure 6.

Phylogenetic tree of amino acid sequences isolated from microcosms inoculated with A1 sediments. Evolutionary analysis was conducted in MEGA7 and the tree with the highest log likelihood (−482.22) supported by a bootstrap analysis (500 replicates) is shown. The WP_012470907 Geobacter lovleyi sequence roots the tree. Open diamonds represent bootstrap values greater than 80%, and filled diamonds represent bootstrap values below 80%. Partial sequences obtained in this study are displayed in red.