The Dehalococcoides population in sediment-free mixed cultures metabolically dechlorinates the commercial polychlorinated biphenyl mixture aroclor 1260

Abstract

Microbial reductive dechlorination of commercial polychlorinated biphenyl (PCB) mixtures (e.g., Aroclors) in aquatic sediments is crucial to achieve detoxification. Despite extensive efforts over nearly two decades, the microorganisms responsible for Aroclor dechlorination remained elusive. Here we demonstrate that anaerobic bacteria of the Dehalococcoides group derived from sediment of the Housatonic River (Lenox, MA) simultaneously dechlorinate 64 PCB congeners carrying four to nine chlorines in Aroclor 1260 in the sediment-free JN cultures. Quantitative real-time PCR showed that the Dehalococcoides cell titer in JN cultures amended with acetate and hydrogen increased from 7.07 x 10(6) +/- 0.42 x 10(6) to 1.67 x 10(8) +/- 0.04 x 10(8) cells/ml, concomitant with a 64.2% decrease of the PCBs with six or more chlorines in Aroclor 1260. No Dehalococcoides growth occurred in parallel cultures without PCBs. Aroclor 1260 dechlorination supported the growth of 9.25 x 10(8) +/- 0.04 x 10(8) Dehalococcoides cells per mumol of chlorine removed. 16S rRNA gene-targeted PCR analysis of known dechlorinators (i.e., Desulfitobacterium, Dehalobacter, Desulfuromonas, Sulfurospirillum, Anaeromyxobacter, Geobacter, and o-17/DF-1-type Chloroflexi organisms) ruled out any involvement of these bacterial groups in the dechlorination. Our results suggest that the Dehalococcoides population present in the JN cultures also catalyzes in situ dechlorination of Aroclor 1260 in the Housatonic River. The identification of Dehalococcoides organisms as catalysts of extensive Aroclor 1260 dechlorination and our ability to propagate the JN cultures under defined conditions offer opportunities to study the organisms' ecophysiology, elucidate nutritional requirements, identify reductive dehalogenase genes involved in PCB dechlorination, and design molecular tools required for bioremediation applications.

FIG. 1.

Relationship of Dehalococcoides cell titers to Aroclor 1260 dechlorination in JN subcultures. Dechlorination is presented as the percent decrease in PCB congeners with six to nine chlorines, which comprise ∼90% of the PCBs in Aroclor 1260. Data are means ± standard deviations. (a) PCB-dependent growth of Dehalococcoides in JN cultures incubated with Aroclor 1260 (50 μg/ml) and decrease of Dehalococcoides cell titers in cultures transferred four times without PCBs. (b) PCB-dependent growth in subcultures when PCBs were restored following three transfers without PCBs. (c) Correlation between PCB dechlorination and Dehalococcoides (Dhc) cell growth in replicate cultures B and C.

FIG. 2.

Change in Aroclor 1260 congener distribution (Process N dechlorination) in the JN cultures. Data for days 0 and 154 are from the JN cultures depicted in Fig. 1a and represent the averages of triplicate determinations. Parallel cultures transferred nine times in the presence of vancomycin showed the same congener profile in only 54 days. The center profile shows Aroclor 1260 dechlorination in situ in a sediment sample collected from the Housatonic River. A complete list of the congener assignments for all GC peaks is given by Frame et al. (26). Congeners marked with an asterisk are those reported to accumulate to the highest levels in humans (62).

FIG. 3.

Observed Process N dechlorination pathways of an environmentally relevant heptachlorobiphenyl by Dehalococcoides bacteria in the JN cultures. Asterisks denote two of the four PCB congeners that accumulate to the highest levels in humans (62).