Dehalorespiration with polychlorinated biphenyls by an anaerobic ultramicrobacterium

Anaerobic microbial dechlorination is an important step in the detoxification and elimination of polychlorinated biphenyls (PCBs), but a microorganism capable of coupling its growth to PCB dechlorination has not been isolated. Here we describe the isolation from sediment of an ultramicrobacterium, strain DF-1, which is capable of dechlorinating PCBs containing double-flanked chlorines added as single congeners or as Aroclor 1260 in contaminated soil. The isolate requires Desulfovibrio spp. in coculture or cell extract for growth on hydrogen and PCB in mineral medium. This is the first microorganism in pure culture demonstrated to grow by dehalorespiration with PCBs and the first isolate shown to dechlorinate weathered commercial mixtures of PCBs in historically contaminated sediments. The ability of this isolate to grow on PCBs in contaminated sediments represents a significant breakthrough for the development of in situ treatment strategies for this class of persistent organic pollutants.     

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.     

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.     

Reductive dechlorination of low concentration polychlorinated biphenyls as affected by a rhamnolipid biosurfactant

We investigated whether the threshold concentration for polychlorinated biphenyl (PCB) dechlorination may be lower in biosurfactant-amended sediments compared with biosurfactant-free samples. At PCB concentrations of 40, 60, and 120 ppm, the surfactant amendment enhanced the PCB dechlorination rate at all concentrations and the rate was also faster at higher concentrations. On a congener group basis, dechlorination proceeded largely with group A (congeners with low threshold) in both surfactant-free and -amended sediments, accumulating mainly group C (residual products of dechlorination) congeners, and surfactant enhanced the dechlorination rate of group A congeners. Since the PCB threshold concentration for the inoculum in the experiment was lower than 40 ppm, we carried out another experiment using sediments with lower PCB concentrations, 10, 20, and 30 ppm. Sediments with 100 ppm were also performed to measure dechlorination at a PCB saturation concentration. Comparison between the plateaus exhibited that the extent of dechlorination below 40 ppm PCBs was much lower than that at a saturation concentration of 100 ppm. There was no significant difference in the extent of dechlorination between surfactant-free and -amended sediments. Moreover, surfactant did not change the congener specificity or broaden the congener spectrum for dechlorination at PCB concentrations below 40 ppm. Taken together, it seems that at a given PCB concentration, dechlorination characteristics of dechlorinating populations may be determined by not only the congener specificity of the microorganisms but also the affinity of dechlorinating enzyme(s) to individual PCB congeners.     

Microbial reductive dechlorination of PCBs

Reductive dechlorination is an advantageous process to microorganisms under anaerobic conditions because it is an electron sink, thereby allowing reoxidation of metabolic intermediates. In some organisms this has been demonstrated to support growth. Many chlorinated compounds have now been shown to be reductively dechlorinated under anaerobic conditions, including many of the congeners in commercial PCB mixtures. Anaerobic microbial communities in sediments dechlorinate Aroclor at rates of 3 µg Cl/g sediment × week. PCB dechlorination occurs at 12° C, a temperature relevant for remediation at temperate sites, and at concentrations of 100 to 1000 ppm. The positions dechlorinated are usually meta > para > ortho. The biphenyl rings, and the mono-ortho- and diorthochlorobiphenyls were not degraded after a one year incubation. Hence subsequent aerobic treatment may be necessary to meet regulatory standards. Reductive dechlorination of Arochlors does reduce their dioxin-like toxicity as measured by bioassay and by analysis of the co-planar congeners. The most important limitation to using PCB dechlorination as a remediation technology is the slower than desired dechlorination rates and no means yet discovered to substantially enhance these rates. Long term enrichments using PCBs as the only electron acceptor resulted in an initial enhancement in dechlorination rate. This rate was sustained but did not increase in serial transfers. Bioremediation of soil contaminated with Aroclor 1254 from a transformer spill was dechlorinated by greater than 50% following mixing of the soil with dechlorinating organisms and river sediment. It is now reasonable to field test reductive dechlorination of PCBs in cases where the PCB concentration is in the range where regulatory standards may be directly achieved by dechlorination, where a subsequent aerobic treatment is feasible, where any co-contaminants do not pose an inhibitory problem, and where anaerobic conditions can be established.This paper was presented at the Pacific Basin Conference on Hazardous Waste, April, 1992, Bangkok, Thailand. Published by permission of the Pacific Basin Consortium for Hazardous Waste Research, East-West Center, Honolulu, HI     

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.     

Microbial reductive dehalogenation of polychlorinated biphenyls

Under anaerobic conditions, microbial reductive dechlorination of polychlorinated biphenyls (PCBs) occurs in soils and aquatic sediments. In contrast to dechlorination of supplemented single congeners for which frequently ortho dechlorination has been observed, reductive dechlorination mainly attacks meta and/or para chlorines of PCB mixtures in contaminated sediments, although in a few instances ortho dechlorination of PCBs has been observed. Different microorganisms appear to be responsible for different dechlorination activities and the occurrence of various dehalogenation routes. No axenic cultures of an anaerobic microorganism have been obtained so far. Most probable number determinations indicate that the addition of PCB congeners, as potential electron acceptors, stimulates the growth of PCB-dechlorinating microorganisms. A few PCB-dechlorinating enrichment cultures have been obtained and partially characterized. Temperature, pH, availability of naturally occurring or of supplemented carbon sources, and the presence or absence of H(2) or other electron donors and competing electron acceptors influence the dechlorination rate, extent and route of PCB dechlorination. We conclude from the sum of the experimental data that these factors influence apparently the composition of the active microbial community and thus the routes, the rates and the extent of the dehalogenation. The observed effects are due to the specificity of the dehalogenating bacteria which become active as well as changing interactions between the dehalogenating and non-dehalogenating bacteria. Important interactions include the induced changes in the formation and utilization of H(2) by non-dechlorinating and dechlorinating bacteria, competition for substrates and other electron donors and acceptors, and changes in the formation of acidic fermentation products by heterotrophic and autotrophic acidogenic bacteria leading to changes in the pH of the sediments.     

Biotransformations of Aroclor 1242 in Hudson River test tube microcosms.

A microcosm system to physically model the fate of Aroclor 1242 in Hudson River sediment was developed. In the dark at 22 to 25 degrees C with no amendments (nutrients, organisms, or mixing) and with overlying water being the only source of oxygen, the microcosms developed visibly distinct aerobic and anaerobic compartments in 2 to 4 weeks. Extensive polychlorinated biphenyl (PCB) biodegradation was observed in 140 days. Autoclaved controls were unchanged throughout the experiments. In the surface sediments of these microcosms, the PCBs were biologically altered by both aerobic biodegrading and reductive dechlorinating microorganisms, decreasing the total concentration from 64.8 to 18.0 micromol/kg of sediment in 1140 days. This is the first laboratory demonstration of meta dechlorination plus aerobic biodegradation in stationary sediments. In contrast, the primary mechanism of microbiological attack on PCBs in aerobic subsurface sediments was reductive dechlorination. The concentration of PCBs remained constant at 64.8 micromol/kg of sediment, but the average number of chlorines per biphenyl decreased from 3.11 to 1.84 in 140 days. The selectivities of microorganisms in these sediments were characterized by meta and para dechlorination. Our results provide persuasive evidence that naturally occurring microorganisms in the Hudson River have the potential to attack the PCBs from Aroclor 1242 releases both aerobically and anaerobically at rapid rates. These unamended microcosms represent a unique method for determining the fate of released PCBs in river sediments.     

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.     

Reductive dechlorination of PCB-contaminated raisin river sediments by anaerobic microbial granules

A polychlorinated biphenyl (PCB)-dechlorinating anaerobic microbial consortium, developed in a granular form, demonstrated extensive dechlorination of PCBs present in Raisin River sediments at room (20 degrees to 22 degrees C) and at a relatively low (12 degrees C) temperature. Highly chlorinated PCB congeners were dechlorinated and less chlorinated compounds were produced. The homolog comparison showed that tri-, tetra-, penta-, hexa-, and heptachlorobiphenyl compounds decreased significantly, and mono- and dichlorobiphenyl compounds increased. After 32 weeks of incubation at 12 degrees C, the predominant less chlorinated products included 2-, 4-, 2-2/26-, 24-, 2-4-, 24-2-, 26-2-, and 26-4-CB. Among these, 24- and 24-2-CB did not accumulate at room temperature, suggesting a further dechlorination of these congeners. Predominantly meta dechlorination (i.e., pattern M) was catalyzed by the microbial consortium in the granules. Dechlorination in the control studies without granules was not extensive. This study is the first demonstration of enhanced reductive dechlorination of sediment PCBs by an exogenous anaerobic microbial consortium. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 182-190, 1997.     

Brominated Biphenyls Prime Extensive Microbial Reductive Dehalogenation of Aroclor 1260 in Housatonic River Sediment

The upper Housatonic River and Woods Pond (Lenox, Mass.), a shallow impoundment on the river, are contaminated with polychlorinated biphenyls (PCBs), the residue of partially dechlorinated Aroclor 1260. Certain PCB congeners have the ability to activate or “prime” anaerobic microorganisms in Woods Pond sediment to reductively dehalogenate the Aroclor 1260 residue. We proposed that brominated biphenyls might have the same effect and tested the priming activities of 14 mono-, di-, and tribrominated biphenyls (350 μM) in anaerobic microcosms of sediment from Woods Pond. All of the brominated biphenyls were completely dehalogenated to biphenyl, and 13 of them primed PCB dechlorination. Measured in terms of chlorine removal and decrease in the proportion of hexa- through nonachlorobiphenyls, the microbial PCB dechlorination primed by several brominated biphenyls was nearly twice as effective as that primed by chlorinated biphenyls. Congeners containing a meta bromine primed Dechlorination Process N (flanked meta dechlorination), and congeners containing an unflanked para bromine primed Dechlorination Process P (flanked para dechlorination). Two ortho-substituted congeners, 2-bromobiphenyl and 2,6-dibromobiphenyl (2-BB and 26-BB), also primed Process N dechlorination. The most effective primers were 26-BB, 245-BB, 25-3-BB, and 25-4-BB. The microbial dechlorination primed by 26-BB converted ~75% of the hexa- through nonachlorobiphenyls to tri- and tetrachlorobiphenyls in 100 days and removed ~75% of the PCBs that are most persistent in humans. These results represent a major step toward identifying an effective method for accelerating PCB dechlorination in situ. The challenge now is to identify naturally occurring compounds that are safe and effective primers.     

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.     

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.     

Population Dynamics of Polychlorinated Biphenyl-Dechlorinating Microorganisms in Contaminated Sediments

The growth dynamics of polychlorinated biphenyl (PCB)-dechlorinating microorganisms were determined for the first time, along with those of sulfate reducers and methanogens, by using the most-probable-number technique. The time course of Aroclor 1248 dechlorination mirrored the growth of dechlorinators; dechlorination ensued when the dechlorinating population increased by 2 orders of magnitude from 2.5 x 10(sup5) to 4.6 x 10(sup7) cells g of sediment(sup-1), at a specific growth rate of 6.7 day(sup-1) between 2 and 6 weeks. During this period, PCB-dechlorinating microorganisms dechlorinated Aroclor 1248 at a rate of 3.9 x 10(sup-8) mol of Cl g of sediment(sup-1) day(sup-1), reducing the average number of Cl molecules per biphenyl from 3.9 to 2.8. The growth yield was 4.2 x 10(sup13) cells mol of Cl dechlorinated(sup-1). Once dechlorination reached a plateau, after 6 weeks, the number of dechlorinators began to decrease. On the other hand, dechlorinators inoculated into PCB-free sediments decreased over time from their initial level, suggesting that PCBs are required for their selective enrichment. The numbers of sulfate reducers and methanogens increased in both PCB-free and contaminated sediments, showing little difference between them. The maximum population size of sulfate reducers was about an order of magnitude higher than that of dechlorinators, whereas that of methanogens was slightly less. Unlike those of dechlorinators, however, numbers of both sulfate reducers and methanogens remained high even when dechlorination ceased. The results of this study imply that PCB concentrations may have to exceed a certain threshold to maintain the growth of PCB dechlorinators.     

The Impact of Sediment Characteristics on PCB-dechlorinating Cultures: Implications for Bioaugmentation

Polychlorinated Biphenyl (PCB)-dechlorinating cultures with complimentary activities, previously derived from estuarine Baltimore Harbor (B), marine Palos Verdes (P), and riverine Hudson River (H) sediments, were mixed and then inoculated into sterile sediments from the same sources. In the treatments containing sterile B sediment, the different inocula had limited impact on the bacterial community development and on dechlorination patterns, all of which were similar. In treatments containing sterile P or H sediment, however, different inocula resulted in significantly different PCB dechlorination patterns and bacterial communities. The B sediment appeared to support not only the most extensive and rapid dechlorination of the three sediments, but also supported a more diverse bacterial community. This was thought to be a result of nutritional richness, as it was high in organic carbon and micronutrients such as zinc and cobalt. Although mixing three PCB-dechlorinating cultures was able to produce a culture capable of enhanced PCB-dechlorinating activity as compared to single cultures, some activities were lost upon culture transfer. This indicates that care must be taken to establish robust PCB-dechlorinating cultures capable of extensive dechlorination prior to pursuing bioaugmentation. In addition, our results indicate that the concentration and availability of macro-and micronutrients could have a significant impact on the microbial community structure, and thus a thorough characterization of the sediment at contaminated sites is essential for implementing bioaugmentation for PCB bioremediation.     

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.     

Evidence for para Dechlorination of Polychlorobiphenyls by Methanogenic Bacteria

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.     

Molecular characterization of polychlorinated biphenyl-dechlorinating populations in contaminated sediments

Polychlorinated biphenyl (PCB)-dechlorinating microorganisms were characterized in PCB-contaminated sediments using amplified ribosomal DNA restriction analysis (ARDRA). The sediments were prepared by spiking Aroclor 1248 into PCB-free sediments, and were inoculated with microorganisms eluted from St. Lawrence River sediments. PCB-free sediments inoculated with the same inoculum served as the control. Four restriction fragment length polymorphism (RFLP) groups in the eubacterial and two in the archaeal domain were found exclusively in PCB-spiked sediment clone libraries. Sequence analysis of the four eubacterial clones showed homology to Escherichia coli, Lactosphaera pasteurii, Clostridium thermocellum, and Dehalobacter restrictus. The predominant archaeal sequence in the PCB-spiked sediment clone library was closely related to Methanosarcina barkeri, which appear to support earlier findings that methanogens are involved in PCB dechlorination. When the dot-blot hybridization was performed between the sediment DNA extract and the probes designed with eubacterial RFLP groups, the intensity of two of eubacterial RFLP groups, which showed high sequence homology to C. pascui and D. restrictus, was highly correlated with the number of dechlorinating microorganisms suggesting these two members intend to contribute to PCB dechlorination.