Development and characterization of stable sediment-free anaerobic bacterial enrichment cultures that dechlorinate aroclor 1260

Abstract

We have developed sediment-free anaerobic enrichment cultures that dechlorinate a broad spectrum of highly chlorinated polychlorinated biphenyls (PCBs). The cultures were developed from Aroclor 1260-contaminated sediment from the Housatonic River in Lenox, MA. Sediment slurries were primed with 2,6-dibromobiphenyl to stimulate Process N dechlorination (primarily meta dechlorination), and sediment was gradually removed by successive transfers (10%) to minimal medium. The cultures grow on pyruvate, butyrate, or acetate plus H(2). Gas chromatography-electron capture detector analysis demonstrated that the cultures extensively dechlorinate 50 to 500 mug/ml of Aroclor 1260 at 22 to 24 degrees C by Dechlorination Process N. Triplicate cultures of the eighth transfer without sediment dechlorinated 76% of the hexa- through nonachlorobiphenyls in Aroclor 1260 (250 mug/ml) to tri- through pentachlorobiphenyls in 110 days. At least 64 PCB congeners, all of which are chlorinated on both rings and 47 of which have six or more chlorines, were substrates for this dechlorination. To characterize the bacterial diversity in the enrichments, we used eubacterial primers to amplify and clone 16S rRNA genes from DNA extracted from cultures grown on acetate plus H(2). Restriction fragment length polymorphism analysis of 107 clones demonstrated the presence of Thauera-like Betaproteobacteria, Geobacter-like Deltaproteobacteria, Pseudomonas species, various Clostridiales, Bacteroidetes, Dehalococcoides of the Chloroflexi group, and unclassified Eubacteria. Our development of highly enriched, robust, stable, sediment-free cultures that extensively dechlorinate a highly chlorinated commercial PCB mixture is a major and unprecedented breakthrough in the field. It will enable intensive study of the organisms and genes responsible for a major PCB dechlorination process that occurs in the environment and could also lead to effective remediation applications.

FIG. 1.

Time course of dechlorination of Aroclor 1260 at various stages of the enrichment process. S3 is the third transfer on autoclaved sediment; SF1, SF2, SF3, SF4, SF5, and SF8 are the first, second, third, fourth, fifth, and eighth transfers without sediment, respectively. The Aroclor 1260 concentration was 5 μg/ml for SF1 to SF4 and 50 μg/ml for SF5 and SF8. For the S3 cultures, the sediment-associated Aroclor 1260 had been partially dechlorinated in situ. The concentration was approximately 45 μg/g (dry weight). Most data are averages for three replicates; for SF4 the data for three individual samples (SF4-A, SF4-B, and SF4-C) are shown. SF4-B showed minimal dechlorination by day 70 and was reinoculated from SF4-A and SF4-C on day 75 (indicated by an arrow). The error bars indicate the standard deviations of the means.

FIG. 2.

Change in PCB congener distribution as a result of dechlorination in enrichment cultures transferred eight times without sediment. The data are the averages for three replicates. The concentration of Aroclor 1260 was 250 μg/ml (675 μM). A complete list of the congener assignments for all DB1 peaks is given in references and .

FIG. 3.

Time course of changes in specific congeners during dechlorination of Aroclor 1260. (A) Dechlorination of five major components of Aroclor 1260. (B) Formation and subsequent dechlorination of intermediates. (C) Accumulation of major dechlorination products of Aroclor 1260. All data are averages for three replicates. The error bars indicate the standard deviations of the means. The concentration of Aroclor 1260 was 250 μg/ml (675 μM).

FIG. 4.

Proposed pathways for dechlorination of six heptachlorobiphenyls to the four major dechlorination products observed in the enrichment cultures. We propose that the first chlorine removed from 2345-chlorophenyl rings may be either the para chlorine (60% of the time) or the meta chlorine in position 3 (40% of the time). The proposed order of removal of the chlorines for each congener is also shown. For congeners with 2345-chlorophenyl rings we indicate the preferred order of attack by 1, 2, and 3 and the less frequent pattern of attack by A, B, and C. Note that the chlorination pattern of both rings affects the order in which chlorines are removed.

FIG. 5.

Bootstrapped neighbor-joining tree of OTUs in sediment-free PCB-dechlorinating enrichment cultures and their closest relatives and type strains. The numbers at the nodes indicate the percentages of times that nodes appeared in 1,000 trials. Nodes that are not labeled appeared 100% of the time. Sequences cloned from DNA extracted from the enrichments after eight transfers without sediment are indicated by boldface type. JN18 refers to the experiment, and the additional letters and numbers indicate the clone name and RFLP designation where applicable. Type strains are indicated (T).