Microbial Reductive Dechlorination of Aroclor 1260 in Anaerobic Slurries of Estuarine Sediments

Reductive dechlorination of Aroclor 1260 was investigated in anaerobic slurries of estuarine sediments from Baltimore Harbor (Baltimore, Md.). The sediment slurries were amended with 800 ppm Aroclor 1260 with and without the addition of 350 μM 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) or 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) and incubated in triplicate at 30°C under methanogenic conditions in an artificial estuarine medium. After 6 months, extensive meta dechlorination and moderate ortho dechlorination of Aroclor 1260 occurred in all incubated cultures except for sterilized controls. Overall, total chlorines per biphenyl decreased by up to 34%. meta chlorines per biphenyl decreased by 65, 55, and 45% and ortho chlorines declined by 18, 12, and 9%, respectively, when 2,3,4,5-CB, 2,3,5,6-CB, or no additional congener was supplied. This is the first confirmed report of microbial ortho dechlorination of a commercial polychlorinated biphenyl mixture. In addition, compared with incubated cultures supplied with Aroclor 1260 alone, the dechlorination of Aroclor 1260 plus 2,3,4,5-CB or 2,3,5,6-CB occurred with shorter lag times (31 to 60 days versus 90 days) and was more extensive, indicating that the addition of a single congener stimulated the dechlorination of Aroclor 1260.     

Identification of a bacterium that specifically catalyzes the reductive dechlorination of polychlorinated biphenyls with doubly flanked chlorines

A microorganism whose growth is linked to the dechlorination of polychlorinated biphenyls (PCBs) with doubly flanked chlorines was identified. Identification was made by reductive analysis of community 16S ribosomal DNA (rDNA) sequences from a culture enriched in the presence of 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB), which was dechlorinated at the para position. Denaturing gradient gel electrophoresis (DGGE) analysis of total 16S rDNA extracted from the culture led to identification of three operational taxonomic units (OTUs 1, 2, and 3). OTU 1 was always detected when 2,3,4,5-CB or other congeners with doubly flanked chlorines were present and dechlorinated. Only OTUs 2 and 3 were detected in the absence of PCBs and when other PCBs (i.e., PCBs lacking doubly flanked chlorines) were not dechlorinated. Partial sequences of OTUs 2 and 3 exhibited 98.2% similarity to the sequence of "Desulfovibrio caledoniensis" (accession no. DCU53465). A sulfate-reducing vibrio isolated from the culture generated OTUs 2 and 3. This organism could not dechlorinate 2,3,4,5-CB. From these results we concluded that OTU 1 represents the dechlorinating bacterium growing in a coculture with a Desulfovibrio sp. The 16S rDNA sequence of OTU 1 is most similar to the 16S rDNA sequence of bacterium o-17 (89% similarity), an ortho-PCB-dechlorinating bacterium. The PCB dechlorinator, designated bacterium DF-1, reductively dechlorinates congeners with doubly flanked chlorines when it is supplied with formate or H(2)-CO(2) (80:20).     

The effect of varying levels of sodium bicarbonate on polychlorinated biphenyl dechlorination in Hudson River sediment cultures

The addition of different concentrations of sodium bicarbonate had a profound effect on 2,3,4,5-chlorobiphenyl (2,3,4,5-CB) dechlorination in Hudson River sediment cultures. The most extensive dechlorination was observed in cultures to which 100 mg l(-1) bicarbonate was added. Cultures amended with 1000 mg l(-1) bicarbonate had the least extensive dechlorination, with 2,4-CB and 2,5-CB as predominant end-products. A significant loss of total chlorinated biphenyl mass was observed in cultures to which < or = 500 mg l(-1) bicarbonate was added, suggesting that degradation beyond chlorinated biphenyls occurred. The dynamics of acetate formation were different among the treatments, with high acetate concentrations detected throughout the 303-day experiment in cultures to which 1000 mg l(-1) bicarbonate had been added. Sodium bicarbonate addition also had a significant impact on bacterial community structure as detected by polymerase chain reaction-denaturant gradient gel electrophoresis (PCR-DGGE) of 16S rRNA gene fragments. Three putative polychlorinated biphenyl (PCB) dechlorinators were identified; one Dehalococcoides-like population was detected in all enrichment cultures, whereas two Dehalobacter-like populations were only detected in the enrichment cultures with the most extensive dechlorination. These results suggest that the availability of bicarbonate, and potentially sodium, may affect PCB dechlorination in Hudson River sediment and thus need to be taken into consideration when assessing the fate of PCBs or implementing bioremediation.     

Establishment of a Polychlorinated Biphenyl-Dechlorinating Microbial Consortium, Specific for Doubly Flanked Chlorines, in a Defined, Sediment-Free Medium

Estuarine sediment from Charleston Harbor, South Carolina, was used as inoculum for the development of an anaerobic enrichment culture that specifically dechlorinates doubly flanked chlorines (i.e., chlorines bound to carbon that are flanked on both sides by other chlorine-carbon bonds) of polychlorinated biphenyls (PCBs). Dechlorination was restricted to the para chlorine in cultures enriched with 10 mM fumarate, 50 ppm (173 μM) 2,3,4,5-tetrachlorobiphenyl, and no sediment. Initially the rate of dechlorination decreased upon the removal of sediment from the medium. However, the dechlorinating activity was sustainable, and following sequential transfer in a defined, sediment-free estuarine medium, the activity increased to levels near that observed with sediment. The culture was nonmethanogenic, and molybdate, ampicillin, chloramphenicol, neomycin, and streptomycin inhibited dechlorination activity; bromoethanesulfonate and vancomycin did not. Addition of 17 PCB congeners indicated that the culture specifically removes double flanked chlorines, preferably in the para position, and does not attack ortho chlorines. This is the first microbial consortium shown to para or meta dechlorinate a PCB congener in a defined sediment-free medium. It is the second PCB-dechlorinating enrichment culture to be sustained in the absence of sediment, but its dechlorinating capabilities are entirely different from those of the other sediment-free PCB-dechlorinating culture, an ortho-dechlorinating consortium, and do not match any previously published Aroclor-dechlorinating patterns.     

Establishment of a polychlorinated biphenyl-dechlorinating microbial consortium, specific for doubly flanked chlorines, in a defined, sediment-free medium

Estuarine sediment from Charleston Harbor, South Carolina, was used as inoculum for the development of an anaerobic enrichment culture that specifically dechlorinates doubly flanked chlorines (i.e., chlorines bound to carbon that are flanked on both sides by other chlorine-carbon bonds) of polychlorinated biphenyls (PCBs). Dechlorination was restricted to the para chlorine in cultures enriched with 10 mM fumarate, 50 ppm (173 microM) 2,3,4, 5-tetrachlorobiphenyl, and no sediment. Initially the rate of dechlorination decreased upon the removal of sediment from the medium. However, the dechlorinating activity was sustainable, and following sequential transfer in a defined, sediment-free estuarine medium, the activity increased to levels near that observed with sediment. The culture was nonmethanogenic, and molybdate, ampicillin, chloramphenicol, neomycin, and streptomycin inhibited dechlorination activity; bromoethanesulfonate and vancomycin did not. Addition of 17 PCB congeners indicated that the culture specifically removes double flanked chlorines, preferably in the para position, and does not attack ortho chlorines. This is the first microbial consortium shown to para or meta dechlorinate a PCB congener in a defined sediment-free medium. It is the second PCB-dechlorinating enrichment culture to be sustained in the absence of sediment, but its dechlorinating capabilities are entirely different from those of the other sediment-free PCB-dechlorinating culture, an ortho-dechlorinating consortium, and do not match any previously published Aroclor-dechlorinating patterns.     

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.     

Anaerobic dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated microorganisms from sediments.

A polychlorobiphenyl (PCB)-dechlorinating inoculum eluted from upper Hudson River sediments was treated with either heat or ethanol or both. The treated cultures retained the ability to dechlorinate PCBs (Aroclor 1242) under strictly anaerobic conditions. The dechlorination activity was maintained in serial cultures inoculated with transfers of 1% inoculum when the transferred inoculum was treated each time in the same manner. No methane production was detected in any treated culture, although dechlorination of PCBs in the untreated cultures was always accompanied by methane production. All treated cultures preferentially removed meta chlorines, yielding a dechlorination pattern characterized by accumulation of certain ortho- and para-subsituted congeners such as 2-4-chlorobiphenyl (2-4-CB), 2,4-2-CB, and 2,4-4-CB. In contrast, the untreated cultures showed more extensive dechlorination activities, which almost completely removed both meta and para chlorines from Aroclor 1242. These results suggest that microorganisms responsible for the dechlorination of PCBs in the upper Hudson River sediments can be grouped into two populations according to their responses to the heat and ethanol treatments. Microorganisms surviving the heat and ethanol treatments preferentially remove meta chlorines, while microorganisms lost from the enrichment mainly contribute to the para dechlorination activity. These results indicate that anaerobic sporeformers are at least one of the physiological groups responsible for the reductive dechlorination of PCBs. The selection of a dechlorinating population by such treatments may be an important step in isolation of PCB-dechlorinating microorganisms.     

Anaerobic dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated microorganisms from sediments.

A polychlorobiphenyl (PCB)-dechlorinating inoculum eluted from upper Hudson River sediments was treated with either heat or ethanol or both. The treated cultures retained the ability to dechlorinate PCBs (Aroclor 1242) under strictly anaerobic conditions. The dechlorination activity was maintained in serial cultures inoculated with transfers of 1% inoculum when the transferred inoculum was treated each time in the same manner. No methane production was detected in any treated culture, although dechlorination of PCBs in the untreated cultures was always accompanied by methane production. All treated cultures preferentially removed meta chlorines, yielding a dechlorination pattern characterized by accumulation of certain ortho- and para-subsituted congeners such as 2-4-chlorobiphenyl (2-4-CB), 2,4-2-CB, and 2,4-4-CB. In contrast, the untreated cultures showed more extensive dechlorination activities, which almost completely removed both meta and para chlorines from Aroclor 1242. These results suggest that microorganisms responsible for the dechlorination of PCBs in the upper Hudson River sediments can be grouped into two populations according to their responses to the heat and ethanol treatments. Microorganisms surviving the heat and ethanol treatments preferentially remove meta chlorines, while microorganisms lost from the enrichment mainly contribute to the para dechlorination activity. These results indicate that anaerobic sporeformers are at least one of the physiological groups responsible for the reductive dechlorination of PCBs. The selection of a dechlorinating population by such treatments may be an important step in isolation of PCB-dechlorinating microorganisms.     

Microbial Dechlorination of 2,3,5,6-Tetrachlorobiphenyl under Anaerobic Conditions in the Absence of Soil or Sediment

Bacterial enrichment cultures developed with Baltimore Harbor (BH) sediments were found to reductively dechlorinate 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) when incubated in a minimal estuarine medium containing short-chain fatty acids under anaerobic conditions with and without the addition of sediment. Primary enrichment cultures formed both meta and ortho dechlorination products from 2,3,5,6-CB. The lag time preceding dechlorination decreased from 30 to less than 20 days as the cultures were sequentially transferred into estuarine medium containing dried, sterile BH sediment. In addition, only ortho dechlorination was observed following transfer of the cultures. Sequential transfer into medium without added sediment also resulted in the development of a strict ortho-dechlorinating culture following a lag of more than 100 days. Upon further transfer into the minimal medium without sediment, the lag time decreased to less than 50 days. At this stage all cultures, regardless of the presence of sediment, would produce 2,3,5-CB and 3,5-CB from 2,3,5,6-CB. The strict ortho-dechlorinating activity in the sediment-free cultures has remained stable for more than 1 year through several transfers. These results reveal that the classical microbial enrichment technique using a minimal medium with a single polychlorinated biphenyl (PCB) congener selected for ortho dechlorination of 2,3,5,6-CB. Furthermore, this is the first report of sustained anaerobic PCB dechlorination in the complete absence of soil or sediment.Anaerobic dechlorination of polychlorinated biphenyls (PCBs) has been demonstrated in situ and with laboratory microcosms containing sediment (reviewed in reference 1a). However, sustained PCB dechlorination has never been shown to occur in the absence of soil or sediments. Morris et al. (6) demonstrated a sediment requirement for the stimulation of PCB dechlorination within freshwater sediment slurries. Wu and Wiegel have recently described PCB-dechlorinating enrichments which required soil for the successful transfer of PCB-dechlorinating activity (9). In addition, no anaerobic microorganisms that dechlorinate PCBs have been isolated or characterized, and this may be due in part to the soil or sediment requirement. The inability to isolate dechlorinating organisms or maintain dechlorination without sediment has limited biogeochemical and physiological investigations into the mechanisms of PCB dechlorination.Dechlorination (ortho, meta, and para) of single PCB congeners has been observed following anaerobic incubation of Baltimore Harbor (BH) sediment under estuarine or marine conditions (2). While sediments from several sites within BH are contaminated with PCBs (1, 5), background contamination of sediment is not necessarily a prerequisite for the development of PCB dechlorination in laboratory microcosms. Wu et al. (8) recently demonstrated meta and ortho dechlorination of Aroclor 1260 when it was added to the same BH sediments. These results showed that more than one dechlorinating activity could be developed with these sediments. It has been proposed that discrete microbial populations are responsible for specific PCB dechlorinations (1a). Consistent with this idea, the ortho dechlorination observed with BH sediments may be catalyzed by discrete microbial populations. In addition, these organisms may be able to couple PCB dechlorination with growth. Therefore we have attempted to select for ortho PCB-dechlorinating organisms by enrichment under minimal conditions with high levels of 2,3,5,6-tetrachlorobiphenyl. We also speculated that given the proper conditions, a PCB-dechlorinating population could be maintained in an actively dechlorinating state in the absence of sediment. Here we report that a distinct PCB-dechlorinating activity, namely, ortho dechlorination, was selected for through sequential transfer initiated with sediments from BH and sustained in the absence of soil or sediment. This is the first report of sustained anaerobic PCB-dechlorinating activity in the total absence of sediment.     

Influence of Incubation Temperature on the Microbial Reductive Dechlorination of 2,3,4,6-Tetrachlorobiphenyl in Two Freshwater Sediments

We studied the impact of incubation temperatures on the dechlorination of 2,3,4,6-tetrachlorobiphenyl (2346-CB) in two sediments from different climates: polychlorinated biphenyl (PCB)-free sediment from Sandy Creek Nature Center Pond (SCNC) in Athens, Ga., and PCB-contaminated sediment from Woods Pond (WP) in Lenox, Mass. Sediment slurries were incubated anaerobically with 350 (mu)M 2346-CB for 1 year at temperatures ranging from 4 to 66(deg)C. Most of the 2346-CB was dechlorinated between 12 and 34(deg)C in both sediments and, unexpectedly, between 50 and 60(deg)C in WP sediment. This is the first report of PCB dechlorination at thermobiotic temperatures. The data reveal profound differences in dechlorination rate, extent, and products as a function of sediment and temperature. The highest observed rate of dechlorination of 2346-CB to trichlorobiphenyls occurred at 30(deg)C in both sediments, but the rate was higher for WP than for SCNC sediment (46 versus 16 (mu)mol liter(sup-1) day(sup-1)). For SCNC sediment the rate of dechlorination dropped sharply below 30(deg)C, but for WP sediments it was near optimal from 20 to 34(deg)C and then dropped sharply below 20(deg)C. In WP sediment most of the meta chlorines were removed between 8 and 34(deg)C and between 50 and 60(deg)C. para dechlorination was restricted from 18 to 34(deg)C and was optimal at 20(deg)C. ortho dechlorination occurred between 8 and 30(deg)C, with optima around 15 and 27(deg)C, but the extent was highly variable. In SCNC sediment complete meta dechlorination occurred from 12 to 34(deg)C and para dechlorination occurred from 18 to 30(deg)C; both were optimal at 30(deg)C. No ortho dechlorination was observed. Dechlorination products were 246-CB, 236-CB, and 26-CB (both sediments) and 24-CB, 2-CB, and 4-CB (WP sediment). The data suggest that in SCNC sediment similar factors controlled meta and para PCB dechlorination over a broad temperature range (18 to 30(deg)C) but that in WP sediment there were multiple temperature-dependent changes in the factors controlling ortho, meta, and para dechlorination. We attribute the differences observed in the two sediments to differences in their PCB-dechlorinating communities.     

Phylogenetically Distinct Bacteria Involve Extensive Dechlorination of Aroclor 1260 in Sediment-Free Cultures

Microbial reductive dechlorination of the persistent polychlorinated biphenyls (PCBs) is attracting much attention in cleanup of the contaminated environment. Nevertheless, most PCB dechlorinating cultures require presence of sediment or sediment substitutes to maintain their dechlorination activities which hinders subsequent bacterial enrichment and isolation processes. The information on enriching sediment-free PCB dechlorinating cultures is still limited. In this study, 18 microcosms established with soils and sediments were screened for their dechlorination activities on a PCB mixture – Aroclor 1260. After one year of incubation, 10 out of 18 microcosms showed significant PCB dechlorination with distinct dechlorination patterns (e.g., Process H, N and T classified based on profiles of PCB congeners loss and new congeners formation). Through serial transfers in defined medium, six sediment-free PCB dechlorinating cultures (i.e., CW-4, CG-1, CG-3, CG-4, CG-5 and SG-1) were obtained without amending any sediment or sediment-substitutes. PCB dechlorination Process H was the most frequently observed dechlorination pattern, which was found in four sediment-free cultures (CW-4, CG-3, CG-4 and SG-1). Sediment-free culture CG-5 showed the most extensive PCB dechlorination among the six cultures, which was mediated by Process N, resulting in the accumulation of penta- (e.g., 236-24-CB) and tetra-chlorobiphenyls (tetra-CBs) (e.g., 24-24-CB, 24-25-CB, 24-26-CB and 25-26-CB) via dechlorinating 30.44% hepta-CBs and 59.12% hexa-CBs after three months of incubation. For culture CG-1, dechlorinators mainly attacked double flanked meta-chlorines and partially ortho-chlorines, which might represent a novel dechlorination pattern. Phylogenetic analysis showed distinct affiliation of PCB dechlorinators in the microcosms, including Dehalogenimonas and Dehalococcoides species. This study broadens our knowledge in microbial reductive dechlorination of PCBs, and provides essential information for culturing and stimulating PCB dechlorinators for in situ bioremediation applications.     

Temperature determines the pattern of anaerobic microbial dechlorination of Aroclor 1260 primed by 2,3,4,6-tetrachlorobiphenyl in Woods Pond sediment.

Reductive dechlorination of the Aroclor 1260 residue in Woods Pond (Lenox, Mass.) sediment samples was investigated for a year at incubation temperatures from 4 to 66 degrees C. Sediment slurries were incubated anaerobically with and without 2,3,4,6-tetrachlorobiphenyl (2346-CB; 350 microM) as a primer for dechlorination of the Aroclor 1260 residue. Dechlorination of the Aroclor residue occurred only in live samples primed with 2346-CB and only at 8 to 34 degrees C and 50 to 60 degrees C. The extent and pattern of polychlorinated biphenyl (PCB) dechlorination were temperature dependent. At 8 to 34 degrees C, the dechlorination resulted in 28 to 65% decreases of the hexathrough nonachlorobiphenyls and corresponding increases in the tri- and tetrachlorobiphenyls. At 12 to 30 degrees C, 30 to 40% of the hexa- through nonachlorobiphenyls were dechlorinated in just 3 months. The optimal temperature for overall chlorine removal was 20 to 27 degrees C. We observed four different microbial dechlorination processes with different but partially overlapping temperature ranges, i.e., Process N (flanked meta dechlorination) at 8 to 30 degrees C, Process P (flanked para dechlorination) at 12 to 34 degrees C, Process LP (unflanked para dechlorination) at 18 to 30 degrees C, and Process T (a very restricted meta dechlorination of specific hepta- and octachlorobiphenyls) at 50 to 60 degrees C. These temperature ranges should aid in the development of strategies for the enrichment and isolation of the microorganisms responsible for each dechlorination process. The incubation temperature determined the relative dominance of the four PCB dechlorination processes and the extent and products of dechlorination. Hence, understanding the effects of temperature on PCB dechlorination at contaminated sites should assist in predicting the environmental fate of PCBs or planning bioremediation strategies at those sites.     

Effect of Incubation Temperature on the Route of Microbial Reductive Dechlorination of 2,3,4,6-Tetrachlorobiphenyl in Polychlorinated Biphenyl (PCB)-Contaminated and PCB-Free Freshwater Sediments

We studied the influence of temperature (4 to 66(deg)C) on the microbial dechlorination of 2,3,4,6-tetrachlorobiphenyl (2,3,4,6-CB) incubated for 1 year in anaerobic sediments from Woods Pond in Lenox, Mass., and Sandy Creek Nature Center Pond (SCNC) in Athens, Ga. Seven discrete dechlorination reactions were observed, four of which occurred in both sediments. These were 2,3,4,6-CB (symbl) 2,4,6-CB, 2,3,4,6-CB (symbl) 2,3,6-CB, 2,4,6-CB (symbl) 2,6-CB, and 2,3,6-CB (symbl) 2,6-CB. Three additional reactions occurred only in Woods Pond sediment. These were 2,4,6-CB (symbl) 2,4-CB, 2,4-CB (symbl) 2-CB, and 2,4-CB (symbl) 4-CB. The dechlorination reactions exhibited at least four different temperature dependencies in SCNC sediment and at least six in Woods Pond sediment. We attribute the discrete dechlorination reactions to different polychlorinated biphenyl (PCB)-dechlorinating microorganisms with distinct specificities. Temperature influenced the timing and the relative predominance of parallel pathways of dechlorination, i.e., meta versus para dechlorination of 2,3,4,6-CB and ortho versus para dechlorination of 2,4,6-CB and 2,4-CB. meta dechlorination of 2,3,4,6-CB to 2,4,6-CB dominated at all tested temperatures except at 18 and 34(deg)C, where para dechlorination to 2,3,6-CB dominated in some replicates. The dechlorination of 2,4,6-CB was restricted to (symbl)15 to 30(deg)C in both sediments. Temperature affected the lag time preceding the dechlorination of 2,4,6-CB in both sediments and affected the preferred route of its dechlorination in Woods Pond sediment. para dechlorination dominated at 20(deg)C, and ortho dechlorination dominated at 15(deg)C, but at 18 and 22 to 30(deg)C the relative dominance of ortho versus para dechlorination of 2,4,6-CB varied. These data indicate that field temperatures play a significant role in controlling the nature and the extent of the PCB dechlorination that occurs at a given site.     

Two anaerobic polychlorinated biphenyl-dehalogenating enrichments that exhibit different para-dechlorination specificities.

Two anaerobic polychlorinated biphenyl (PCB)-dechlorinating enrichments with distinct substrate specificities were obtained: a 2,3,4,6-tetrachlorobiphenyl (2346-CB) para-dechlorinating enrichment derived from Aroclor 1260-contaminated Woods Pond (Lenox, Mass.) sediment and a 2,4,6-trichlorobiphenyl (246-CB) unflanked para-dechlorinating enrichment derived from PCB-free Sandy Creek Nature Center (Athens, Ga.) sediment. The enrichments have been successfully transferred to autoclaved soil slurries over 20 times by using 300 to 350 microM 2346-CB or 246-CB. Both enrichments required soil for successful transfer of dechlorination activity. The 2346-CB enrichment para dehalogenated, in the absence or presence of 2346-CB, only 4 of 25 tested para halogen-containing congeners: 234-CB, 2345-CB, 2346-CB, and 2,4,6-tribromobiphenyl (246-BrB). In the presence of 246-CB, the 246-CB enrichment para dehalogenated 23 of the 25 tested congeners. However, only three congeners (34-CB, 2346-CB, and 246-BrB) were dehalogenated in the absence of 246-CB, indicating that these specific congeners initiate dehalogenation in this enrichment culture. The addition of the 2346-CB (para)-dechlorinating enrichment did not further stimulate the 2346-CB-primed dechlorination of the Aroclor 1260 residue in Woods Pond sediment samples. Compared to the addition of the primer 246-CB or the 246-CB unflanked para-dechlorinating enrichment alone, the addition of both 246-CB (300 microM) and the 246-CB enrichment stimulated the unflanked para dechlorination of the Aroclor 1260 residue in Woods Pond sediments. These results indicate that the two enrichments contain different PCB-dechlorinating organisms, each with high substrate specificities. Furthermore, bioaugmentation with the enrichment alone did not stimulate the desired dechlorination in PCB-contaminated Woods Pond sediment.     

Effects of Polychlorinated Biphenyl Congener Concentration and Sediment Supplementation on Rates of Methanogenesis and 2,3,6-Trichlorobiphenyl Dechlorination in an Anaerobic Enrichment

We have employed a method of enrichment that allows us to significantly increase the rate of reductive polychlorinated biphenyl (PCB) dechlorination. This method shortens the time required to investigate the effects that culture conditions have on dechlorination and provides an estimate of the potential activity of the PCB-dechlorinating anaerobes. The periodic supplementation of sterile sediment and PCB produced an enhanced, measurable, and sustained rate of dechlorination. We observed volumetric rates of the dechlorination of 2,3,6-trichlorobiphenyl (2,3,6-CB) to 2,6-dichlorobiphenyl (2,6-CB) of more than 300 μmol liter^-1 day^-1 when the cultures were supplemented daily. A calculation of this activity that is based on an estimate of the number of dechlorinating anaerobes present indicates that 1.13 pmol of 2,3,6-CB was dechlorinated to 2,6-CB day^-1 bacterial cell^-1. This rate is similar to that of the reductive dechlorination of 3-chlorobenzoate by Desulfomonile tiedjei. Methanogenesis declined from 585.3 to 125.9 μmol of CH₄ liter^-1 day^-1, while dechlorination increased from 8.2 to 346.0 μmol of 2,3,6-CB dechlorinated to 2,6-CB liter^-1 day^-1.     

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

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.     

Dechlorination of Four Commercial Polychlorinated Biphenyl Mixtures (Aroclors) by Anaerobic Microorganisms from Sediments

The rate, extent, and pattern of dechlorination of four Aroclors by inocula prepared from two polychlorinated biphenyl (PCB)-contaminated sediments were compared. The four mixtures used, Aroclors 1242, 1248, 1254, and 1260, average approximately three, four, five, and six chlorines, respectively, per biphenyl molecule. All four Aroclors were dechlorinated with the loss of meta plus para chlorines ranging from 15 to 85%. Microorganisms from an Aroclor 1242-contaminated site in the upper Hudson River dechlorinated Aroclor 1242 to a greater extent than did microorganisms from Aroclor 1260-contaminated sediments from Silver Lake, Mass. The Silver Lake inoculum dechlorinated Aroclor 1260 more rapidly than the Hudson River inoculum did and showed a preferential removal of meta chlorines. For each inoculum the rate and extent of dechlorination tended to decrease as the degree of chlorination of the Aroclor increased, especially for Aroclor 1260. The maximal observed dechlorination rates were 0.3, 0.3, and 0.2 μg-atoms of Cl removed per g of sediment per week for Aroclors 1242, 1248, and 1254, respectively. The maximal observed dechlorination rates for Hudson River and Silver Lake organisms for Aroclor 1260 were 0.04 and 0.21 μg-atoms of Cl removed per g of sediment per week, respectively. The dechlorination patterns obtained suggested that the Hudson River microorganisms were more capable than the Silver Lake organisms of removing the last para chlorine. These results suggest that there are different PCB-dechlorinating microorganisms at different sites, with characteristic specificities for PCB dechlorination.     

Microbial reductive dechlorination of aroclor 1260 in Baltimore harbor sediment microcosms is catalyzed by three phylotypes within the phylum Chloroflexi

The specific dechlorination pathways for Aroclor 1260 were determined in Baltimore Harbor sediment microcosms developed with the 11 most predominant congeners from this commercial mixture and their resulting dechlorination intermediates. Most of the polychlorinated biphenyl (PCB) congeners were dechlorinated in the meta position, and the major products were tetrachlorobiphenyls with unflanked chlorines. Using PCR primers specific for the 16S rRNA genes of known PCB-dehalogenating bacteria, we detected three phylotypes within the microbial community that had the capability to dechlorinate PCB congeners present in Aroclor 1260 and identified their selective activities. Phylotype DEH10, which has a high level of sequence identity to Dehalococcoides spp., removed the double-flanked chlorine in 234-substituted congeners and exhibited a preference for para-flanked meta-chlorines when no double-flanked chlorines were available. Phylotype SF1 had similarity to the o-17/DF-1 group of PCB-dechlorinating bacteria. Phylotype SF1 dechlorinated all of the 2345-substituted congeners, mostly in the double-flanked meta position and 2356-, 236-, and 235-substituted congeners in the ortho-flanked meta position, with a few exceptions. A phylotype with 100% sequence identity to PCB-dechlorinating bacterium o-17 was responsible for an ortho and a double-flanked meta dechlorination reaction. Most of the dechlorination pathways supported the growth of all three phylotypes based on competitive PCR enumeration assays, which indicates that PCB-impacted environments have the potential to sustain populations of these PCB-dechlorinating microorganisms. The results demonstrate that the variation in dechlorination patterns of congener mixtures typically observed at different PCB impacted sites can potentially be mediated by the synergistic activities of relatively few dechlorinating species.     

Sequential Reductive Dechlorination of meta-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different Chloroflexi Phylotypes

Three species within a deeply branching cluster of the Chloroflexi are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the Chloroflexi including Dehalococcoides spp. and the o-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the para-flanked meta position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the Dehalococcoides group. This same species reductively dechlorinated the para- and ortho-flanked meta-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the o-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the Chloroflexi exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of Chloroflexi with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.