Factors controlling the rate of DDE dechlorination to DDMU in Palos Verdes margin sediments under anaerobic conditions.

Marine sediments off the coast of the Palos Verdes Peninsula in California have been designated a Superfund site primarily because of the presence of DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethene]. For decades, it was believed that DDE was not microbially transformed, but anaerobic bacteria in the Palos Verdes sediments reductively dechlorinate DDEto DDMU [1-chloro-2,2-bis(p-chlorophenyl)ethene], which is also found in the sediments. The effects of electron donor to sulfate ratio, available carbon, sampling sites, sediment depth, and temperature on the rate and extent of DDE dechlorination in anaerobic Palos Verdes sediment microcosms were investigated. Dechlorination rates varied, depending on the site and depth from which the sediments were collected, but DDE dechlorination occurred with sediments from all locations studied. Sulfate and low temperatures slowed dechlorination, but in the presence of sulfate and at in situ temperature, the dechlorination rates observed in the microcosms agree well with the observed rate of DDE disappearance from the Palos Verdes margin sediments.

Development of a Rhodococcus recombinant strain for degradation of products from anaerobic dechlorination of PCBs.

The Gram-positive bacterium Rhodococcus sp. strain RHA1, naturally containing the biphenyl pathway, was electroporated with a broad host range plasmid containing the 4-chlorobenzoate (4-CBA) degradation operon (fcb) isolated from Arthrobacter globiformis strain KZT1. The recombinant strain grew in medium containing 4-CBA and 4-chlorobiphenyl (4-CB) as the only source of carbon, with stoichiometric release of chloride and a molar growth yield on 4-CB that suggested utilization of both biphenyl rings. In resting cell assays, similar rates of degradation were observed for wild-type and recombinant strains for the most common eight congeners from the anaerobic dechlorination of Aroclor 1242, but the recombinant strain accumulated lower amounts of chlorinated meta-cleavage products and no 4-CBA. Recombinant cells inoculated at 10(4) cells/g into nonsterile soil amended with 4-CB grew to 6-10(5) cells/g, a density consistent with the 4-CB consumed. 4-CB was removed only in the inoculated soil, and the recombinant strain did not grow in the same soil when it was not amended with 4-CB. The fcb operon remained stable in the recombinant strain reisolated from soil after 60 days. This work provides proof of concept that a Rhodococcus strain constructed to grow on a PCB would grow in nonsterile soil if the appropriate chlorobiphenyl is available.

Stimulation of contraction of pregnant rat uterus in vitro by non-dechlorinated and microbially dechlorinated mixtures of polychlorinated biphenyls.

A previous study of six polychlorinated biphenyl (PCB) congeners showed that PCBs with four or fewer chlorines and ortho substitution stimulate uterine contraction frequency in vitro, whereas congeners with a greater number of chlorines or non-ortho substitution are inactive in vitro. We tested the hypothesis that PCB mixtures stimulate uterine contractions in a manner inversely related to the degree of chlorination and the presence of chlorines in the ortho- position of the biphenyl constituents of the mixtures. Uterine strips from pregnant rats were suspended in standard muscle baths and analyzed for changes in isometric contractions in response to in vitro exposure to commercial PCB mixtures (Aroclors) and their dechlorinated products after microbial degradation. The PCB mixtures Aroclor 1242, 1248, and 1254 significantly stimulated uterine contraction frequency, and the least chlorinated mixture, Aroclor 1242, was the most potent stimulant. Microbes from Hudson River sediment dechlorinated Aroclor 1242 and Aroclor 1254 under reducing conditions to produce mixtures with an increased proportion of ortho-substituted congeners with one or two chlorine substitutions. The PCB mixtures that had undergone microbial reductive dechlorination stimulated uterine contraction frequency to a significantly greater extent than the parent mixtures. These results show that increased uterotonic activity was associated with decreased chlorination and increased ortho substitution of the biphenyl constituents of the mixtures.

Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls.

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.

Degradation of anaerobic reductive dechlorination products of Aroclor 1242 by four aerobic bacteria.

We studied the aerobic degradation of eight PCB congeners which comprise from 70 to 85% of the anaerobic dechlorination products from Aroclor 1242, including 2-, 4-, 2,4-, 2,6-, 2,2'-, 2,4'-, 2,2', 4-, and 2,4,4'-chlorobiphenyl (CB), and the biodegradation of their mixtures designed to simulate anaerobic dechlorination profiles M and C. Strains Comamonas testosteroni VP44 and Rhodococcus erythreus NY05 preferentially oxidized a para-substituted ring, while Rhodococcus sp. RHA1, similar to well known strain Burkholderia sp. LB400, preferably attacked an ortho-chlorinated ring. Strains with ortho-directed attack extensively degraded 2,4'- and 2,4,4'-CB into 4-chlorobenzoate, while bacteria with para-directed attack transformed these congeners mostly into potentially problematic meta-cleavage products. The strains that preferentially oxidized an ortho-substituted ring readily degraded seven of the eight congeners supplied individually; only 2,6-CB was poorly degraded. Degradation of 2,2'- and 2,4,4'-CB was reduced when present in mixtures M and C. Higher efficiencies of degradation of the individual congeners and defined PCB mixtures M and C and greater production of chlorobenzoates were observed with bacteria that preferentially attack an ortho-substituted ring. PCB congeners 2,4'-, 2,2',4-, and 2,4,4'-CB can be used to easily identify bacteria with ortho-directed attack which are advantageous for use in the aerobic stage of the two-phase (anaerobic/aerobic) PCB bioremediation scheme.

Altered biologic activities of commercial polychlorinated biphenyl mixtures after microbial reductive dechlorination.

The reductive dechlorination of polychlorinated biphenyls (PCBs) by anaerobic bacteria has recently been established as an important environmental fate of these compounds. This process removes chlorines directly from the biphenyl ring with replacement by hydrogen, resulting in a product mixture in which the average number of chlorines per biphenyl is reduced. In this study, dechlorination of commercial PCB mixtures (Aroclors 1242 and 1254) by microorganisms eluted from PCB-contaminated sediments of the River Raisin (Michigan) and Silver Lake (Massachusetts) caused a depletion in the proportion of highly chlorinated PCB congeners and an accumulation of lesser-chlorinated congeners. Dechlorination occurred primarily at the meta and, to a much lesser extent, para positions of biphenyl. The concentrations of the coplanar congeners including 3,3',4,4',5-pentachlorobiphenyl, the most potent dioxinlike congener, were significantly lowered by reductive dechlorination. Microbial reductive dechlorination of commercial PCB mixtures caused a substantial reduction in biologic activities in several instances. It significantly lowered or eliminated the inhibitory effects of Aroclors on fertilization of mouse gametes in vitro. Similarly, the dechlorinated product mixtures had substantially lower ethoxyresorufin-O-deethylase induction potencies and showed less ability to induce activating protein 1 transcription factor activity as compared to the unaltered Aroclors. In other assays the same dechlorinated product mixtures demonstrated biologic activities similar to the nondechlorinated Aroclors, including the ability of PCB mixtures to stimulate insulin secretion and cause neutrophil activation. The data presented here establish that the biologic activities of commercial PCB mixtures are altered by microbial reductive dechlorination and that an assessment of their toxic potential requires an array of tests that include the different mechanisms associated with PCBs.

Reductive dechlorination of DDE to DDMU in marine sediment microcosms.

DDT is reductively dechlorinated to DDD and dehydrochlorinated to DDE; it has been thought that DDE is not degraded further in the environment. Laboratory experiments with DDE-containing marine sediments showed that DDE is dechlorinated to DDMU in both methanogenic and sulfidogenic microcosms and that DDD is dehydrochlorinated to DDMU three orders of magnitude more slowly. Thus, DDD does not appear to be an important precursor of the DDMU found in these sediments. These results imply that remediation decisions and risk assessments based on the recalcitrance of DDE in marine and estuarine sediments should be reevaluated.

Inhibition of L-aromatic amino acid decarboxylase by polychlorinated biphenyls.

PC12 cells were used to examine the mechanisms by which polychlorinated biphenyls (PCBs) reduce cellular levels of dopamine (DA). In cells treated 3 days with Aroclor 1254, 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB), or 2,2',3,3',4,4'-hexachlorobiphenyl (2,2',3,3',4,4'-HCB), the PCB-mediated reduction in 3H-tyrosine uptake was observed only at high PCB concentrations that produced a reduction in DNA levels. The PCB congener, 2,2',4,4',5,5'-hexachlorobiphenyl (2,2',4,4',5,5'-HCB) did not produce a reduction in 3H-tyrosine uptake at any concentration tested. Thus, there were PCB concentrations at which a reduction in DA levels did not coincide with a decrease in 3H-tyrosine uptake, suggesting that inhibition of tyrosine uptake was not the primary mechanism by which PCBs reduce DA levels. Aroclor 1254-treated cells also exhibited elevated levels of DOPA, further supporting the conclusion that tyrosine levels were not limiting. Incubation of Aroclor 1254-pretreated cells with 3H-tyrosine resulted in a dose-dependent increase in cellular levels of 3H-DOPA and decrease in cellular levels of 3H-DA, suggesting a PCB-mediated inhibition of the conversion of 3H-DOPA to 3H-DA. When the media was supplemented with DOPA, Aroclor 1254-treated cells still exhibited reduced levels of DA, compared to control cells, even though the control and PCB-treated cells had similar cellular levels of DOPA. Thus, one mechanism by which PCBs may reduce cellular levels of DA is by inhibiting L-aromatic amino acid decarboxylase-mediated conversion of DOPA to DA. The PCB congeners, 2,2',4,4'-TCB, 2,2',5,5'-TCB, and 2,2',4,4',5,5'-HCB, also produced dose-dependent increases in DOPA levels. The congener 2,2',3,3',4,4'-HCB did not produce an increase in DOPA levels, although it did mediate reductions in cellular DA levels. However, when PC12 cells were supplemented with DOPA, all four PCB congeners produced a similar reduction in DA levels, suggesting that the conversion of DOPA to DA was inhibited by the PCBs.

Evaluation of strains isolated by growth on naphthalene and biphenyl for hybridization of genes to dioxygenase probes and polychlorinated biphenyl-degrading ability.

Approximately equal numbers of bacteria were isolated from primarily tropical soils by growth on biphenyl and naphthalene to compare their competence in polychlorinated biphenyl (PCB) degradation. The strains isolated by growth on biphenyl catalyzed more extensive PCB degradation than the strains isolated by growth on naphthalene, suggesting that naphthalene cocontamination may be only partially effective in stimulating the cometabolism of lower chlorinated PCBs. Probes were made from the bph, nah, and tod genes encoding the large iron iron sulfur protein of the dioxygenase complex and hybridized to 19 different strains. The hybridization patterns did not correlate well with the substrates of isolation, suggesting that there is considerable diversity in these genes in nature and that probe hybridization is not a reliable indication of catabolic capacity. The strains with the most extensive PCB degradation capacity did strongly hybridize to the bph probe, but a few strains that exhibited strong hybridization had poor PCB-degrading ability. Of the 19 strains studied, 5 hybridized to more than one probe and 2, including one strong PCB degrader, hybridized to all three probes. Southern blots showed that the bph and nah probes hybridized to separate bands, suggesting that multiple dioxygenases were present. Multiple dioxygenases may be an important feature of competitive decomposers in nature and hence may not be rare. Most of the isolates identified were members of the beta subgroup of the Proteobacteria, a few were gram positive, and none were true Pseudomonas species.

Degradation of polychlorinated biphenyl mixtures (Aroclors 1242, 1254, and 1260) by the white rot fungus Phanerochaete chrysosporium as evidenced by congener-specific analysis.

Evidence for substantial degradation of polychlorinated biphenyl mixtures Aroclor 1242, 1254, and 1260 by the white rot fungus Phanerochaete chrysosporium, based on congener-specific gas chromatographic analysis, is presented. Maximal degradation (percent by weight) of Aroclors 1242, 1254, and 1260 was 60.9, 30.5, and 17.6%, respectively. Most of the congeners in Aroclors 1242 and 1254 were degraded extensively both in low-N (ligninolytic) as well as high-N (nonligninolytic) defined media. Even more extensive degradation of the congeners was observed in malt extract medium. Congeners with varying numbers of ortho, meta, and para chlorines were extensively degraded, indicating relative nonspecificity for the position of chlorine substitutions on the biphenyl ring. Aroclor 1260, which has not been conclusively shown to undergo aerobic microbial degradation, was shown to undergo substantial net degradation by P. chrysosporium. Maximal degradation of Aroclor 1260 was observed in malt extract medium (18.4% on a molar basis), in which most of the individual congeners were degraded.

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 micrograms Cl/g sediment x week. PCB dechlorination occurs at 12 degrees 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 Aroclors 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Reductive debromination of the commercial polybrominated biphenyl mixture firemaster BP6 by anaerobic microorganisms from sediments.

Anaerobic microorganisms eluted from three sediments, one contaminated with polybrominated biphenyls (PBBs) and two contaminated with polychlorinated biphenyls, were compared for their ability to debrominate the commercial PBB mixture Firemaster. These microorganisms were incubated with reduced anaerobic mineral medium and noncontaminated sediment amended with Firemaster. Firemaster averages six bromines per biphenyl molecule; four of the bromines are substituted in the meta or para position. The inocula from all three sources were able to debrominate the meta and para positions. Microorganisms from the Pine River (St. Louis, Mich.) contaminated with Firemaster, the Hudson River (Hudson Falls, N.Y.) contaminated with Aroclor 1242, and Silver Lake (Pittsfield, Mass.) contaminated with Aroclor 1260 removed 32, 12, and 3% of the meta plus para bromines, respectively, after 32 weeks of incubation. This suggests that previous environmental exposure to PBBs enhances the debromination capability of the sediment microbial community through selection for different strains of microorganisms. The Pine River inoculum removed an average of 1.25 bromines per biphenyl molecule during a 32-week incubation period, resulting in a mixture potentially more accessible to aerobic degradation processes. No ortho bromine removal was observed. However, when Firemaster was incubated with Hudson River microorganisms that had been repeatedly transferred on a pyruvate medium amended with Aroclor 1242, 17% of the meta and para bromines were removed after 16 weeks of incubation and additional debromination products, including 2-bromobiphenyl and biphenyl, were detected. This suggests the possibility for ortho debromination, since all components of the Firemaster mixture have at least one ortho-substituted bromine.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of polychlorinated biphenyl-degrading enrichment culture with predominantly meta dechlorination.

Enrichment of polychlorinated biphenyl (PCB)-dechlorinating microorganisms from PCB-contaminated sediments from the Upper Hudson River, N.Y., was attempted. The enrichment strategy was to use pyruvate as the electron donor and dechlorination of Aroclor 1242 as the electron acceptor. The enrichment medium also contained non-PCB-contaminated Hudson River sediments, which were required for the PCB-dechlorinating activity. An enrichment culture (that had stable PCBT-dechlorinating activity over nine serial transfers during 1 year) was established under these conditions; however, the rate of dechlorination did not increase after the second serial transfer. Dechlorination occurred primarily from the meta positions of the biphenyl molecule. Hydrogen could be substituted for pyruvate as the electron donor with equal activity, but when acetate was used as the electron donor a delay in dechlorination was observed. Sulfate and bromethane sulfonate inhibited dechlorination activity. The pyruvate-Aroclor 1242 enrichment also dechlorinated Aroclors 1248, 1254, and 1260; the extent of chlorine removed was the greatest for Aroclor 1254. For comparison, nonautoclaved non-PCB-contaminated Hudson River sediments used in the assay also dechlorinated Aroclors, but only after 12 to 16 weeks of incubation. This suggests that PCB-dechlorinating organisms were also present in these sediments but in numbers lower than those in the enrichment culture.

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.

Specific deuteration of dichlorobenzoate during reductive dehalogenation by Desulfomonile tiedjei in D2O.

Desulfomonile tiedjei DCB-1 is a strict anaerobe capable of reductively dechlorinating meta-chlorobenzoates. To probe the mechanism of this aryl dechlorination, we incubated cell suspensions of D. tiedjei in D2O and with 2,5-dichlorobenzoate. The deuterium was incorporated into the dechlorination product exclusively at the position of dehalogenation, as shown by gas chromatography-mass spectrometry and proton magnetic resonance analyses. These results favor a model for dechlorination that should not allow proton exchange at other positions, as would be the case if partial ring reduction occurred.

Reductive dechlorination of polychlorinated biphenyls by anaerobic microorganisms from sediments.

Microorganisms from Hudson River sediments reductively dechlorinated most polychlorinated biphenyls (PCBs) in Aroclor 1242 under anaerobic conditions, thus demonstrating PCB dechlorination by anaerobic bacteria in the laboratory. The most rapid dechlorination was observed at the highest PCB concentration used; at 700 parts per million Aroclor, 53 percent of the total chlorine was removed in 16 weeks, and the proportion of mono- and dichlorobiphenyls increased from 9 to 88 percent. Dechlorination occurred primarily from the meta and para positions; congeners that were substituted only in the ortho position (or positions) accumulated. These dechlorination products are both less toxic and more readily degraded by aerobic bacteria. These results indicate that reductive dechlorination may be an important environmental fate of PCBs, and suggest that a sequential anaerobic-aerobic biological treatment system for PCBs may be feasible.