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.     

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.     

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.     

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.     

Reductive dechlorination of chlorophenols by a pentachlorophenol- acclimated methanogenic consortium.

Anaerobic digester sludge fed 5,300 mg of acetate per liter, 3.4 microM pentachlorophenol, and nutrients for 10 days biotransformed pentachlorophenol by sequential ortho dechlorinations to produce 2,3,4,5-tetrachlorophenol and 3,4,5-trichlorophenol. Upon acclimation to 3.4 microM pentachlorophenol for 6 months, the methanogenic consortium removed chlorines from the ortho, meta, and para positions of pentachlorophenol and its reductive dechlorination products. Pentachlorophenol was degraded to produce 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, and 2,3,5,6-tetrachlorophenol. Dechlorination of 2,3,4,5-tetrachlorophenol produced 3,4,5-trichlorophenol, which was subsequently degraded to produce 3,4-dichlorophenol and 3,5-dichlorophenol. 2,3,4,6-Tetrachlorophenol was dechlorinated at the ortho and meta positions to produce 2,4,6-trichlorophenol and 2,4,5-trichlorophenol. 2,3,5,6-Tetrachlorophenol yielded 2,3,5-trichlorophenol, followed by production of 3,5-dichlorophenol. 2,4,6-Trichlorophenol was degraded to form 2,4-dichlorophenol, and 2,4,5-trichlorophenol was dechlorinated at two positions to form 2,4-dichlorophenol and 3,4-dichlorophenol. Of the three dichlorophenols produced (2,4-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol), only 2,4-dichlorophenol was degraded significantly within 3 weeks, to produce 4-chlorophenol.     

Reductive dechlorination of chlorophenols by a pentachlorophenol- acclimated methanogenic consortium.

Anaerobic digester sludge fed 5,300 mg of acetate per liter, 3.4 microM pentachlorophenol, and nutrients for 10 days biotransformed pentachlorophenol by sequential ortho dechlorinations to produce 2,3,4,5-tetrachlorophenol and 3,4,5-trichlorophenol. Upon acclimation to 3.4 microM pentachlorophenol for 6 months, the methanogenic consortium removed chlorines from the ortho, meta, and para positions of pentachlorophenol and its reductive dechlorination products. Pentachlorophenol was degraded to produce 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, and 2,3,5,6-tetrachlorophenol. Dechlorination of 2,3,4,5-tetrachlorophenol produced 3,4,5-trichlorophenol, which was subsequently degraded to produce 3,4-dichlorophenol and 3,5-dichlorophenol. 2,3,4,6-Tetrachlorophenol was dechlorinated at the ortho and meta positions to produce 2,4,6-trichlorophenol and 2,4,5-trichlorophenol. 2,3,5,6-Tetrachlorophenol yielded 2,3,5-trichlorophenol, followed by production of 3,5-dichlorophenol. 2,4,6-Trichlorophenol was degraded to form 2,4-dichlorophenol, and 2,4,5-trichlorophenol was dechlorinated at two positions to form 2,4-dichlorophenol and 3,4-dichlorophenol. Of the three dichlorophenols produced (2,4-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol), only 2,4-dichlorophenol was degraded significantly within 3 weeks, to produce 4-chlorophenol.     

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium. Received: 30 April 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

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.     

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.     

Synthesis of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->3)]-beta-D-GlcpOLauryl, an oligosaccharide with anti-tumor activity

A concise and effective synthesis of lauryl heptasaccharide 17 was achieved from the key intermediates lauryl 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4-di-O-benzoyl-beta-D-glucopyranoside (10) and isopropyl 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-1-thio-beta-D-glucopyranoside (15). The key trisaccharide glycosyl acceptor 10 was constructed by coupling 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) with lauryl 6-O-acetyl-2,4-di-O-benzoyl-beta-D-glucopyranoside (9), followed by deacetylation. The thioglycoside donor 15 was obtained by condensation of 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (12), followed by debenzylidenation and acetylation. A bioassay of the inhibition of S180 noumenal tumors showed that lauryl heptasaccharide 17 could be employed as a potential agent for cancer treatment.     

Reductive dechlorination of dichlorophenols by nonadapted and adapted microbial communities in pond sediments

Fresh and dichlorophenol (DCP)-adapted sediments from two ponds near Athens, Georgia exhibited distinctly different dechlorinating activities. These differences centered on the relative rates of reductive dehlorination in both fresh and adapted sediments and on the substrate specificity of the adapted sediments. Fresh Cherokee Trailer Park Pond sediment dechlorinated 2,3-, 2,4-, and 2,6-DCP to monochlorophenols at a faster rate and after a shorter lag period than fresh Bolton's Pond sediment. Lag periods were not observed in either Cherokee or Bolton's sediments that had been adapted to dechlorinate either 2,3-, 2,4-or 2,6-DCP. Adapted Cherokee sediments exhibited faster dechlorinating rates and a broader substrate specificity than the adapted Bolton's sediments. The broad substrate specificity of each of the adapted Cherokee sediments contrasted sharply with the narrow specificity of the 2,6-DCP-adapted Bolton's sediment. The preference for reductive dechlorination wasortho>meta orpara in sediments from both ponds.     

Regiospecific dechlorination of pentachlorophenol by dichlorophenol-adapted microorganisms in freshwater, anaerobic sediment slurries.

The reductive dechlorination of pentachlorophenol (PCP) was investigated in anaerobic sediments that contained nonadapted or 2,4- or 3,4-dichlorophenol (DCP)-adapted microbial communities. Adaptation of sediment communities increased the rate of conversion of 2,4- or 3,4-DCP to monochlorophenols (CPs) and eliminated the lag phase before dechlorination was observed. Both 2,4- and 3,4-DCP-adapted sediment communities dechlorinated the six DCP isomers to CPs. The specificity of chlorine removal from the DCP isomers indicated a preference for ortho-chlorine removal by 2,4-DCP-adapted sediment communities and for para-chlorine removal by 3,4-DCP-adapted sediment communities. Sediment slurries containing nonadapted microbial communities either did not dechlorinate PCP or did so following a lag phase of at least 40 days. Sediment communities adapted to dechlorinate 2,4- or 3,4-DCP dechlorinated PCP without an initial lag phase. The 2,4-DCP-adapted communities initially removed the ortho-chlorine from PCP, whereas the 3,4-DCP-adapted communities initially removed the para-chlorine from PCP. A 1:1 mixture of the adapted sediment communities also dechlorinated PCP without a lag phase. Dechlorination by the mixture was regiospecific, following a para greater than ortho greater than meta order of chlorine removal. Intermediate products of degradation, 2,3,5,6-tetrachlorophenol, 2,3,5-trichlorophenol, 3,5-DCP, 3-CP, and phenol, were identified by a combination of cochromatography (high-pressure liquid chromatography) with standards and gas chromatography-mass spectrometry.     

Synthesis of trisaccharide methyl glycosides related to fragments of the capsular polysaccharide of Streptococcus pneumoniae type 18C.

The synthesis is reported of methyl 3-O-(4-O-beta-D-galactopyranosyl-alpha-D- glucopyranosyl)-alpha-L-rhamnopyranoside (1), methyl 2-O-alpha-D-glucopyranosyl-4-O-beta-D-glucopyranosyl-beta-D- galactopyranoside (3), methyl 3-O-(4-O-beta-D-galactopyranosyl-alpha-D-glucopyranosyl)-alpha-L- rhamnopyranoside 3"-(sn-glycer-3-yl sodium phosphate) (2), and methyl 2-O-alpha-D-glucopyranosyl-4-O-beta-D- glucopyranosyl-beta-D-galactopyranoside 3-(sn-glycer-3-yl sodium phosphate) (4), which are trisaccharide methyl glycosides related to fragments of the capsular polysaccharide of Streptococcus pneumoniae type 18C ([----4)-beta-D- Glcp-(1----4)-[alpha-D-Glcp-(1----2)]-[Glycerol-(1-P----3)]-beta-D-Galp - (1----4)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-(1----]n). Ethyl 4-O-acetyl-2,3,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (10) was coupled with benzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (6). Deacetylation of the product, followed by condensation with 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-galactopyranosyl trichloroacetimidate (18), gave benzyl 2,4-di-O-benzyl-3-O-[2,3,6-tri-O- benzyl-4-O-(2,4,6-tri-O-acetyl-3-O-allyl-beta-D-galactopyranosyl)-alpha- D- glucopyranosyl]-alpha-L-rhamnopyranoside (19). Acetolysis of 19, followed by methylation, deallylation (----22), and further deprotection afforded 1. Condensation of methyl 2,4-di-O-benzyl-3-O-[2,3,6-tri-O-benzyl-4-O-(2,4,6-tri- O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L- rhamnopyranoside (22) with 1,2-di-O-benzyl-sn-glycerol 3-(triethyl-ammonium phosphonate) (24), followed by oxidation and deprotection, yielded 2. Condensation of ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (27) with methyl 3-O-allyl-4,6-O-benzylidene-beta-D-galactopyranoside (28), selective benzylidene ring-opening of the product, coupling with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (31), and deallylation afforded methyl 6-O-benzyl-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-2-O- (2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-beta-D-galactopyranoside (33). Deprotection of 33 gave 3, and condensation of 33 with 24, followed by oxidation and deprotection, gave 4.     

The reductive dechlorination of 2,3,4,5-tetrachlorobiphenyl in three different sediment cultures: evidence for the involvement of phylogenetically similar Dehalococcoides-like bacterial populations

Anaerobic cultures capable of reductively dechlorinating 2,3,4,5-tetrachlorobiphenyl (CB) were enriched from three different sediments, one estuarine, one marine and one riverine. Two different electron donors were used in enrichments with the estuarine sediment (elemental iron or a mixture of fatty acids). The removal of doubly flanked meta and para chlorines to form 2,3,5-CB and 2,4,5-CB was observed in all cultures. Bacterial community analysis of PCR-amplified 16S rRNA gene fragments revealed different communities in these cultures, with the exception of one common population that showed a high phylogentic relatedness to Dehalococcoides species. No Dehalococcoides-like populations were ever detected in control cultures to which no PCBs were added. In addition, the dynamics of this Dehalococcoides-like population were strongly correlated with dechlorination. Subcultures of the estuarine sediment culture demonstrated that the Dehalococcoides-like population disappeared when dechlorination was inhibited with 2-bromoethanesulfonate or when 2,3,4,5-CB had been consumed. These results provide evidence that Dehalococcoides-like populations were involved in the removal of doubly flanked chlorines from 2,3,4,5-CB. Furthermore, the successful enrichment of these populations from geographically distant and geochemically distinct environments indicates the widespread presence of these PCB-dechlorinating, Dehalococcoides-like organisms.     

The anaerobic degradation of 3-chloro-4-hydroxybenzoate in freshwater sediment proceeds via either chlorophenol or hydroxybenzoate to phenol and subsequently to benzoate.

To study the anaerobic degradation of the chimera 3-chloro-4-hydroxybenzoate (3-Cl,4-OHB), anaerobic freshwater sediment samples from the vicinity of Athens, Ga., were adapted for the transformation of 4-hydroxybenzoate (4-OHB), 3-chlorobenzoate (3-CB), 2-chlorophenol (2-CP), and 2,4-dichlorophenol (2,4-DCP). In nonadapted samples, both 4-OHB (product of aryl dechlorination) and 2-CP (product of aryl decarboxylation) were observed as intermediates in the transformation of 3-Cl,4-OHB to phenol. The accumulated phenol was subsequently transformed to benzoate, an intermediate in the conversion to methane and CO2. In 4-OHB-adapted samples (i.e., samples adapted for aryl decarboxylation), 2-CP was the first intermediate which was subsequently dechlorinated to phenol. In 3-CB-adapted samples (i.e., samples adapted for meta-chlorobenzoate dehalogenation), 3-Cl,4-OHB was stoichiometrically dechlorinated to 4-OHB. In 2-CP-adapted samples (i.e., samples adapted for ortho-chlorophenol dehalogenation), 4-OHB was the first major intermediate. Furthermore, 3-CB was not dechlorinated in 2-CP-adapted sediment samples, suggesting the possibility that different 3-Cl,4-OHB dechlorinating systems were induced in the 2-CP- and 3-CB-adapted sediments. Adaptation of sediment samples for dechlorination of 2,4-DCP did not lead to adaptation for dechlorination of 3-Cl,4-OHB. However, 3-Cl,4-OHB was dechlorinated to 4-OHB in our stable, sediment-free 2,4-DCP-dechlorinating enrichment, isolated previously from the same environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The anaerobic degradation of 3-chloro-4-hydroxybenzoate in freshwater sediment proceeds via either chlorophenol or hydroxybenzoate to phenol and subsequently to benzoate.

To study the anaerobic degradation of the chimera 3-chloro-4-hydroxybenzoate (3-Cl,4-OHB), anaerobic freshwater sediment samples from the vicinity of Athens, Ga., were adapted for the transformation of 4-hydroxybenzoate (4-OHB), 3-chlorobenzoate (3-CB), 2-chlorophenol (2-CP), and 2,4-dichlorophenol (2,4-DCP). In nonadapted samples, both 4-OHB (product of aryl dechlorination) and 2-CP (product of aryl decarboxylation) were observed as intermediates in the transformation of 3-Cl,4-OHB to phenol. The accumulated phenol was subsequently transformed to benzoate, an intermediate in the conversion to methane and CO2. In 4-OHB-adapted samples (i.e., samples adapted for aryl decarboxylation), 2-CP was the first intermediate which was subsequently dechlorinated to phenol. In 3-CB-adapted samples (i.e., samples adapted for meta-chlorobenzoate dehalogenation), 3-Cl,4-OHB was stoichiometrically dechlorinated to 4-OHB. In 2-CP-adapted samples (i.e., samples adapted for ortho-chlorophenol dehalogenation), 4-OHB was the first major intermediate. Furthermore, 3-CB was not dechlorinated in 2-CP-adapted sediment samples, suggesting the possibility that different 3-Cl,4-OHB dechlorinating systems were induced in the 2-CP- and 3-CB-adapted sediments. Adaptation of sediment samples for dechlorination of 2,4-DCP did not lead to adaptation for dechlorination of 3-Cl,4-OHB. However, 3-Cl,4-OHB was dechlorinated to 4-OHB in our stable, sediment-free 2,4-DCP-dechlorinating enrichment, isolated previously from the same environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of reductive dehalogenation of substituted ortho-chlorophenols by Desulfitobacterium dehalogenans JW/IU-DC1.

Resting cells of Desulfitobacterium dehalogenans JW/IU-DC1 growth with pyruvate and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor and inducer of dehalogenation reductively ortho-dehalogenate pentachlorophenol (PCP); tetrachlorophenols (TeCPs); the trichlorophenols 2,3,4-TCP, 2,3,6-TCP, and 2,4,6-TCP; the dichlorophenols 2,3-DCP, 2,4-DCP, and 2,6-DCP; 2,6-dichloro-4-R-phenols (2,6-DCl-4-RPs, where R is -H, -F, -Cl, -NO2, -CO2, or -COOCH3; 2-chloro-4-R-phenols (2-Cl-4-RPs, where R is -H, -F, -Cl, -Br, -NO2, -CO2-, -CH2CO2, or -COOCH3); and the bromophenols 2-BrP, 2,6-DBrP, and 2-Br-4ClP [corrected]. Monochlorophenols, the dichlorophenols 2,5-DCP, 3,4-DCP, and 3,5-DCP, the trichlorophenols 2,3,5-TCP, 2,4,5-TCP, and 3,4,5-TCP, and the fluorinated analog of 3-Cl-4-OHPA, 3-F-4-OHPA ("2-F-4-CH2CO2- P"), are not dehalogenated. A chlorine substituent in position 3 (meta), 4 (para), or 6 (second ortho) of the phenolic moiety facilitates ortho dehalogenation in position 2. Chlorine in the 5 (second meta) position has a negative effect on the dehalogenation rate or even prevents dechlorination in the 2 position. In general, 2,6-DCl-4-RPs are dechlorinated faster than the corresponding 2-Cl-4-RPs with the same substituent R in the 4 position. The highest dechlorination rate, however, was found for dechlorination of 2,3-DCP, with a maximal observed first-order rate constant of 19.4 h-1 g (dry weight) of biomass-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.