Kinetics of PCB Microbial Dechlorination Explained by Freely Dissolved Concentration in Sediment Microcosms.

While microbial dechlorination of polychlorinated biphenyls (PCBs) has been observed in sediments over the last 3 decades, translation to the field has been difficult due to a lack of a clear understanding of the kinetic limitations. To address this issue, the present study used passive dosing/sampling to accurately measure the biological rate of dechlorination of 2,3,4,5-tetrachlorobiphenyl (PCB 61) to 2,3,5-trichlorobiphenyl (PCB 23) by an organohalide-respiring bacterium, Dehalobium chlorocoercia (DF-1). The biological rates were measured over an environmentally relevant concentration range of 1-50 ng/L of freely dissolved concentrations with and without the presence of sediment in bench-scale microcosm studies. The rate of dechlorination was found to be linearly dependent on the freely dissolved concentration of PCB 61 both in sediment and in sediment-free microcosms. The observed rate of dechlorination in sediment microcosms could be predicted within a factor of 2 based on the kinetics measured in sediment-free microcosms. A threshold for dechlorination was not observed down to an aqueous concentration of about 1 ng/L PCB 61. We demonstrate that with the combination of an accurate measurement of the aqueous-phase dechlorination kinetics and an understanding of the site-specific partitioning characteristics, it is possible to predict PCB microbial dechlorination in sediments.

Assessment of PCB contamination, the potential for in situ microbial dechlorination and natural attenuation in an urban watershed at the East Coast of the United States.

Sediment contamination is a major environmental issue in many urban watersheds and coastal areas due to the potential toxic effects of contaminants on biota and human health. Characterizing and delineating areas of sediment contamination and toxicity are important goals of coastal resource management in terms of ecological and economical perspectives. Core and surficial sediment samples were collected from an industrialized urban watershed at the East Coast of the United Stated and analyzed to evaluate the PCB contamination profile and toxicity resulting from dioxin-like PCBs as well as reductive dechlorination potential of indigenous PCB halorespiring bacteria through dechlorination activity assays. To support the experimental results an anaerobic dechlorination model was applied to identify microbial dechlorination pathways. The total PCB concentration in core samples ranged from 3.9 to 225.6 ng/g·dry weight (dw) decreasing with depth compared to 353.2 to 1213.7 ng/g·dw in surficial samples. The results of this study indicated an increase in PCB contamination over the last century as the industrial activity intensified. The toxicity resulting from dioxin-like PCBs was reduced up to 94% in core samples via 21 pathways resulting from the dechlorination model. Dechlorination rates in surficial sediment were between 1.8 and 13.2 ·â€¯10-3 mol% PCB116/day, while lower rates occurred in the core sediment samples. Dechlorination was achieved mainly through meta followed by para dechlorination. However, the rarer ortho dechlorination was also observed. Detection of indigenous PCB dechlorinating bacteria in the sediments and reduction of toxicity indicated potential for natural attenuation when point and nonpoint source PCBs in the urban watershed are controlled and PCB loading reduced.

Colonization and growth of dehalorespiring biofilms on carbonaceous sorptive amendments.

Removal of polychlorinated biphenyls (PCBs) from contaminated sediments is a priority due to accumulation in the food chain. Recent success with reduction of PCB bioavailability due to adsorption onto activated carbon led to the recognition of in situ treatment as a remediation approach. In this study, reduced bioavailability and subsequent break-down of PCBs in dehalorespiring biofilms was investigated using Dehalobium chlorocoercia DF1. DF1 formed a patchy biofilm ranging in thickness from 3.9 to 6.7 µm (average 4.6 ± 0.87 µm), while the biofilm coverage varied from 5.5% (sand) to 20.2% (activated carbon), indicating a preference for sorptive materials. Quantification of DF1 biofilm bacteria showed 1.2-15.3 × 109 bacteria per gram of material. After 22 days, coal activated carbon, bone biochar, polyoxymethylene, and sand microcosms had dechlorinated 73%, 93%, 100%, and 83%, respectively. These results show that a biofilm-based inoculum for bioaugmentation of PCBs in sediment can be an efficient approach.

A Pilot-Scale Field Study: In Situ Treatment of PCB-Impacted Sediments with Bioamended Activated Carbon.

A combined approach involving microbial bioaugmentation and enhanced sorption was demonstrated to be effective for in situ treatment of polychlorinated biphenyls (PCBs). A pilot study was conducted for 409 days on PCB impacted sediments in four 400 m2 plots located in a watershed drainage pond in Quantico, VA. Treatments with activated carbon (AC) agglomerate bioamended with PCB dechlorinating and oxidizing bacteria decreased the PCB concentration in the top 7.5 cm by up to 52% and the aqueous concentrations of tri- to nonachlorobiphenyl PCB congeners by as much as 95%. Coplanar congeners decreased by up to 80% in sediment and were undetectable in the porewater. There was no significant decrease in PCB concentrations in non-bioamended plots with or without AC. All homologue groups decreased in bioamended sediment and porewater, indicating that both anaerobic dechlorination and aerobic degradation occurred concurrently. The titer of the bioamendments based on quantitative PCR of functional marker genes decreased but were still detectable after 409 days, whereas indigenous microbial diversity was not significantly different between sites, time points, or depths, indicating that bioaugmentation and the addition of activated carbon did not significantly alter total microbial diversity. In situ treatment of PCBs using an AC agglomerate as a delivery system for bioamendments is particularly well-suited for environmentally sensitive sites where there is a need to reduce exposure of the aquatic food web to sediment-bound PCBs with minimal disruption to the environment.

A comparative evaluation of anaerobic dechlorination of PCB-118 and Aroclor 1254 in sediment microcosms from three PCB-impacted environments.

Aroclor 1254 (A1254) is the most toxic commercial PCB mixture produced, primarily due to its relatively high concentrations of dioxin-like congeners. This study demonstrates a comparative evaluation of dechlorination of A1254 and PCB-118 by indigenous organohalide respiring bacteria enriched from three PCB impacted sites: Grasse River (GR), NY; Fox River (FR), WI; and Baltimore Harbor (BH), MD. PCB-118 dechlorination rates in GR, BH, and FR was 0.0308, 0.015, and 0.0006 Cl-/biphenyl/day, respectively. A1254 dechlorination rates in GR, FR, and BH were 0.0153, 0.0144, and 0.0048 Cl-/biphenyl/day, respectively. A1254 dechlorination was achieved through the removal of doubly-/singly-flanked chlorines in meta and para positions of mostly penta- followed by hexa- and hepta-chlorinated congeners by 88%, 69%, and 51% in GR, and 88%, 87%, and 83% in FR, respectively, while in BH mostly hepta- (70%) followed by hexa-chlorinated congeners (66%) were dechlorinated. A previously developed Anaerobic Dechlorination Model (ADM) quantified a total of 17 toxicity-related dechlorination pathways in all three sediment microcosms. The toxic equivalency of A1254 based on seven dioxin-like congeners decreased by about 53%, 45% and 21%, in GR, FR and BH microcosms, respectively. The dechlorination products were generally tetra- and tri-chlorinated congeners with unflanked chlorines, all of which is susceptible to further degradation by aerobic bacteria. Concerning the toxic congeners, ADM can be useful to initiate further research focusing on the stimulation of the toxicity reducing pathways for risk assessment and effective remediation strategies.

Mesocosm Studies on the Efficacy of Bioamended Activated Carbon for Treating PCB-Impacted Sediment.

This report describes results of a bench-scale treatability study to evaluate the efficacy of bioaugmentation with bioamended activated carbon (AC) for in situ treatment of polychlorinated biphenyl (PCB) impacted sediments. To this end, the ability of PCB transforming microorganisms to degrade and reduce the overall concentration of PCBs in sediment was determined in 2 L recirculating mesocosms designed to simulate conditions in Abraham's Creek in Quantico, Virginia. Ten sediment mesocosms were tested for the effects of AC alone, AC with slow release electron donor (cellulose) and different concentrations and combinations of PCB dehalogenating and degrading microorganisms added as bioamendments. A 78% reduction of total PCBs was observed using a cell titer of 5 × 105 Dehalobium chlorocoercia and Paraburkholderia xenovorans cells g-1 sediment with 1.5% AC as a delivery system. Levels of both higher and lower chlorinated congeners were reduced throughout the sediment column indicating that both anaerobic reductive dechlorination and aerobic degradation occurred concurrently. Porewater concentrations of all PCB homologues were reduced 94-97% for bioaugmented treatments. Toxicity associated with coplanar PCBs was reduced by 90% after treatment based on toxic equivalency of dioxin-like congeners. These results suggest that an in situ treatment employing the simultaneous application of anaerobic and aerobic microorganisms on AC could be an effective, environmentally sustainable strategy to reduce PCB levels in contaminated sediment.

Potential risk reduction of Aroclor 1254 by microbial dechlorination in anaerobic Grasse River sediment microcosms.

Aroclor 1254 was the second most produced commercial PCB mixture and is found in soils, sediments and sewage throughout the globe. This commercial PCB mixture is considered particularly toxic because of the relatively high concentrations of congeners with dioxin-like properties. The potential for risk reduction by microbial reductive dechlorination of Aroclor 1254 (A1254) was investigated in sediment microcosms from Grasse River (GR), Massena, NY. The specificity of A1254 dechlorination was doubly- and singly-flanked chlorines in meta positions and to a less extent doubly-flanked para chlorines of 2345-substituted chlorobiphenyl rings. The average dechlorination rate of A1254 was 0.0153 Cl-/biphenyl/day, and dechlorination rates of single congeners ranged between 0.001 and 0.0074 Cl-/biphenyl/day. Potential risk associated with A1254 based on the toxic equivalency factors of the dioxin-like congeners was reduced by 83%. Additional potential risk associated with bioaccumulation in fish was reduced by 35% based on biota-sediment accumulation factor estimates for all detected congeners. Finally, the dechlorination end-products were tri- and tetra-chlorobiphenyls with unflanked chlorines, all of which are susceptible to further degradation by aerobic microorganisms. The combined results indicate that microbial reductive dechlorination has the potential for reducing risk associated with toxicity and bioaccumulation in fish in sites contaminated with A1254.

Evaluation of PCB dechlorination pathways in anaerobic sediment microcosms using an anaerobic dechlorination model.

A detailed quantitative analysis of anaerobic dechlorination (AD) pathways of polychlorinated biphenyls (PCBs) in sediment microcosms was performed by applying an anaerobic dechlorination model (ADM). The purpose of ADM is to systematically analyze changes in a contaminant profile that result from microbial reductive dechlorination according to empirically determined dechlorination pathways. In contrast to prior studies that utilized modeling tools to predict dechlorination pathways, ADM also provides quantification of individual pathways. As only microbial reductive dechlorination of PCBs occurred in the modeled laboratory microcosms, extensive analysis of AD pathways was possible without the complicating effect of concurrent physico-chemical or other weathering mechanisms. The results from this study showed: (1) ninety three AD pathways are active; (2) tetra- to hepta-chlorobiphenyl (CB) congeners were common intermediates in several AD pathways, penta-CBs being the most frequently observed; (3) the highest rates of dechlorination were for penta-CB homologs during the initial 185 days; (4) the dominant terminal products of AD were PCB 32(26-4), 49(24-25), 51(24-26), 52(25-25), 72(25-35), 73(26-35) and 100(246-24), (5) potential toxicity of the sediment was reduced. ADM serves as a powerful tool not only for a thorough analysis of AD pathways, but also for providing necessary input for numerical fate models (as a degradation term) that investigate dechlorination products or outcome of natural attenuation, or bioremediation/bioaugmentation of PCB-impacted sediments.

Kinetics and threshold level of 2,3,4,5-tetrachlorobiphenyl dechlorination by an organohalide respiring bacterium.

The time required for a PCB-contaminated site to recover cannot yet be predicted due in part to lack of quantitative information on rates of PCB dechlorination in the porewater phase. We developed a method to measure rate of dechlorination in the aqueous phase at very low PCB concentrations. This approach utilizes a polymer functioning concurrently as a passive dosing system for maintaining a steady-state PCB substrate concentration in the water phase and as a passive equilibrium sampler to monitor the dechlorination product. Rates of dechlorination of 2,3,4,5-tetrachlorobiphenyl (PCB 61) to 2,3,5-trichlorobiphenyl (PCB 23) by an organohalide respiring bacterium, Dehalobium chlorocoercia DF-1, were measured over an environmentally relevant range of 1 to 500 ng L(-1) in sediment-free medium using a high concentration of cells (>10(6) cells mL(-1)). The results indicate that rate of dechlorination is a linear function of PCB substrate concentration below the maximum aqueous solubility of PCB 61 and occurs at concentrations as low as 1 ng L(-1). Demonstration of PCB 61 dechlorination at environmentally relevant concentrations suggests that low numbers of organohalide respiring bacteria rather than bioavailability accounts for low rates of dechlorination typically observed in sediments. Using passive samplers to measure the concentration of dissolved PCBs in the porewater combined with knowledge of congener-specific rates for organohalide respirer(s), it will be possible to project the in situ rate and final concentration of PCBs for a specific site after treatment by bioaugmentation.

MrpA functions in energy conversion during acetate-dependent growth of Methanosarcina acetivorans.

The role of the multisubunit sodium/proton antiporter (Mrp) of Methanosarcina acetivorans was investigated with a mutant deleted for the gene encoding the MrpA subunit. Antiporter activity was 5-fold greater in acetate-grown versus methanol-grown wild-type cells, consistent with the previously published relative levels of mrp transcript. The rate, final optical density, and dry weight/methane ratio decreased for the mutant versus wild type when cultured with a growth-limiting concentration of acetate. All growth parameters of the mutant or wild type were identical when grown with methanol in medium containing a growth-limiting Na(+) concentration of 1.04 M. The lag phase, growth rate, and final optical density for growth of the mutant were suboptimal compared to the wild type when cultured with acetate in medium containing either 0.54 or 1.04 M Na(+). The addition of 25 mM NaCl to resting cell suspensions stimulated ATP synthesis driven by a potassium diffusion potential. ATP synthesis was greater in wild-type than mutant cells grown with acetate, a trend that held for methanol-grown cells, albeit less pronounced. Both sodium and proton ionophores reduced ATP synthesis in the wild type grown with either substrate. The results indicated that the Mrp complex is essential for efficient ATP synthesis and optimal growth at the low concentrations of acetate encountered in the environment.

Remediation of polychlorinated biphenyl impacted sediment by concurrent bioaugmentation with anaerobic halorespiring and aerobic degrading bacteria.

Bioremediation of sediments contaminated with commercial polychlorinated biphenyls (PCBs) is potentially achievable by the sequential activity of anaerobic halorespiration to convert higher chlorinated congeners to less chlorinated congeners that are susceptible to aerobic respiratory degradation. The efficacy of bioaugmentation with anaerobic halorespiring Dehalobium chlorocoercia DF1 and aerobic Burkholderia xenovorans LB400 added concurrently with granulated activated carbon (GAC) as a delivery system was determined in 2 L laboratory mesocosms containing weathered Aroclor-contaminated sediment from Baltimore Harbor, MD, USA. The greatest effect was seen in the mesocosm bioaugmented with both DF1 and LB400 together, which resulted in an 80% decrease by mass of PCBs, from 8 to <2 mg/kg after 120 days. There was no significant increase in lesser-chlorinated congeners, indicating that both anaerobic dechlorination by DF1 and aerobic degradation by LB400 occurred. In contrast, nonbioaugmented controls containing filtered culture supernatant showed only a 25% decrease in total levels of PCBs after 365 days, which was likely due to biostimulation of the indigenous population by the medium. Direct colony counts and molecular analysis targeting a putative reductive dehalogenase gene of D. chlorocoercia or the bphA gene of LB400 showed the presence of viable DF1 and LB400 in bioaugmented mesocosms after 365 days, indicating that both nonindigenous strains were sustainable within the indigenous microbial community. These results suggest that an in situ treatment employing the simultaneous application of anaerobic and aerobic microorganisms could be an effective and environmentally sustainable strategy to reduce PCBs levels in contaminated sediment.

Electrical stimulation of microbial PCB degradation in sediment.

Bioremediation of polychlorinated biphenyls (PCBs) has been precluded in part by the lack of a cost-effective method to stimulate microbial degradation in situ. A common limitation is the lack of an effective method of providing electron donors and acceptors to promote in situ PCB biodegradation. Application of an electric potential to soil/sediment could be an effective means of providing electron-donors/-acceptors to PCB dechlorinating and degrading microorganisms. In this study, electrical stimulation of microbial PCB dechlorination/degradation was examined in sediment maintained under simulated in situ conditions. Voltage was applied to open microcosms filled with PCB-impacted (Aroclor 1242) freshwater sediment from a Superfund site (Fox River, WI). The effect of applied low voltages (1.5-3.0 V) on the microbial transformation of PCBs was determined with: 1) spiked PCBs, and 2) indigenous weathered PCBs. The results indicate that both oxidative and reductive microbial transformation of the spiked PCBs was stimulated but oxidation was dominant and most effective with higher voltage. Chlorobenzoates were produced as oxidation metabolites of the spiked PCBs, but increasing voltage enhanced chlorobenzoate consumption, indicating that overall degradation was enhanced. In the case of weathered PCBs, the total concentration decreased 40-60% in microcosms exposed to electric current while no significant decrease of PCB concentration was observed in control reactors (0 V or sterilized). Single congener analysis of the weathered PCBs showed significant loss of di- to penta-chlorinated congeners, indicating that microbial activity was not limited to anaerobic dechlorination of only higher chlorinated congeners. Degradation was most apparent with the application of only 1.5 V where anodic O(2) was not generated, indicating a mechanism of degradation independent of electrolytic O(2). Low voltage stimulation of the microbial degradation of weathered PCBs observed in this study suggests that this approach could be a cost-effective, environmentally sustainable strategy to remediate PCBs in situ.

In situ treatment of PCBs by anaerobic microbial dechlorination in aquatic sediment: are we there yet?

The remediation of polychlorinated biphenyls (PCBs) in soils and sediments remains a particularly difficult problem to solve. The possibility of in situ degradation by microorganisms has been pursued for many years since this approach has the potential to provide a cost-effective and environmentally sustainable alternative to dredging for treatment of PCB impacted sites. Being hydrophobic, PCBs partition into organic material and accumulate in anoxic environments well poised to support anaerobic dechlorination of highly chlorinated congeners; products of which are susceptible to complete aerobic degradation. Laboratory research over the past 25 years is now leading to new microbial technologies that could soon be tested for treatment of PCB impacted sediments in the field.

Linking energy production and protein synthesis in hydrogenotrophic methanogens.

Hydrogenotrophic methanogens possessing the hydrogen-dependent dehydrogenase Hmd also encode paralogs of this protein whose function is poorly understood. Here we present biochemical evidence that the two inactive Hmd paralogs of Methanocaldococcus jannaschii, HmdII and HmdIII, form binary and ternary complexes with several components of the protein translation apparatus. HmdII and HmdIII, but not the active dehydrogenase Hmd, bind with micromolar binding affinities to a number of tRNAs and form ternary complexes with tRNA(Pro) and prolyl-tRNA synthetase (ProRS). Fluorescence spectroscopy experiments also suggest that binding of HmdII and ProRS involves distinct binding determinants on the tRNA. These biochemical data suggest the possibility of a regulatory link between energy production and protein translation pathways that may allow a rapid cellular response to altered environmental conditions.

Desiccation as a long-term survival mechanism for the archaeon Methanosarcina barkeri.

Viable methanogens have been detected in dry, aerobic environments such as dry reservoir sediment, dry rice paddies and aerobic desert soils, which suggests that methanogens have mechanisms for long-term survival in a desiccated state. In this study, we quantified the survival rates of the methanogenic archaeon Methanosarcina barkeri after desiccation under conditions equivalent to the driest environments on Earth and subsequent exposure to different stress factors. There was no significant loss of viability after desiccation for 28 days for cells grown with either hydrogen or the methylotrophic substrates, but recovery was affected by growth phase, with cells desiccated during the stationary phase of growth having a higher rate of recovery after desiccation. Synthesis of methanosarcinal extracellular polysaccharide (EPS) significantly increased the viability of desiccated cells under both anaerobic and aerobic conditions compared with that of non-EPS-synthesizing cells. Desiccated M. barkeri exposed to air at room temperature did not lose significant viability after 28 days, and exposure of M. barkeri to air after desiccation appeared to improve the recovery of viable cells compared with that of desiccated cells that were never exposed to air. Desiccated M. barkeri was more resistant to higher temperatures, and although resistance to oxidative conditions such as ozone and ionizing radiation was not as robust as in other desiccation-resistant microorganisms, the protection mechanisms are likely adequate to maintain cell viability during periodic exposure events. The results of this study demonstrate that after desiccation M. barkeri has the innate capability to survive extended periods of exposure to air and lethal temperatures.

Enhanced reductive dechlorination of polychlorinated biphenyl impacted sediment by bioaugmentation with a dehalorespiring bacterium.

Anaerobic reductive dehalogenation of commercial PCBs such as Aroclor 1260 has a critical role of transforming highly chlorinated congeners to less chlorinated congeners that are then susceptible to aerobic degradation. The efficacy of bioaugmentation with the dehalorespiring bacterium Dehalobium chlorocoercia DF1 was tested in 2-L laboratory mesocosms containing sediment contaminated with weathered Aroclor 1260 (1.3 ppm) from Baltimore Harbor, MD. Total penta- and higher chlorinated PCBs decreased by approximately 56% (by mass) in bioaugmented mesocosms after 120 days compared with no activity observed in unamended controls. Bioaugmentation with DF-1 enhanced the dechlorination of doubly flanked chlorines and stimulated the dechlorination of single flanked chlorines as a result of an apparent synergistic effect on the indigenous population. Addition of granulated activated carbon had a slight stimulatory effect indicating that anaerobic reductive dechlorination of PCBs at low concentrations was not inhibited by a high background of inorganic carbon that could affect bioavailability. The total number of dehalorespiring bacteria was reduced by approximately half after 60 days. However, a steady state level was maintained that was greater than the indigenous population of putative dehalorespiring bacteria in untreated sediments and DF1 was maintained within the indigenous population after 120 days. The results of this study demonstrate that bioaugmentation with dehalorespiring bacteria has a stimulatory effect on the dechlorination of weathered PCBs and supports the feasibility of using in situ bioaugmentation as an environmentally less invasive and lower cost alternate to dredging for treatment of PCB impacted sediments.

Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14.

The modified nucleosides N(2)-methylguanosine and N(2)(2)-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m(2)G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m(2)G at position 6 in tRNA(Cys). The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m(2)G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20-30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m(2)G6 tRNA modification.

Effects of bioaugmentation on indigenous PCB dechlorinating activity in sediment microcosms.

Bioaugmentation is an attractive mechanism for reducing recalcitrant pollutants in sediments, especially if this technology could be applied in situ. To examine the potential effectiveness of a bioaugmentation strategy for PCB contamination, PCB dehalorespiring populations were inoculated into Baltimore Harbor sediment microcosms. A culture containing the two most predominant indigenous PCB dehalorespiring microorganisms and a culture containing a strain with a rare ortho dechlorination activity and a non-indigenous strain that attacks double-flanked chlorines, were inoculated into sediment microcosms amended with 2,2',3,5,5',6-hexachlorobiphenyl (PCB 151) and Aroclor 1260. Although we observed a similar reduction in the concentration of PCB 151 in all microcosms at day 300, a reduced lag time for dechlorination activity was observed only in the bioaugmented microcosms and the pattern of dechlorination was altered depending on the initial combination of microorganisms added. Dechlorination of Aroclor 1260 was most extensive when dehalorespiring microorganisms were added to sediment. Overall numbers of dehalorespiring microorganisms in both bioaugmented and non-bioaugmented microcosms increased 100- and 1000-fold with PCB 151 and Aroclor 1260, respectively, and they were sustained for the full 300 days of the experiments. The ability of bioaugmentation to redirect dechlorination reactions in the sediment microcosms indicates that the inoculated PCB dehalorespiring microorganisms effectively competed with the indigenous microbial populations and cooperatively enhanced or altered the specific pathways of PCB dechlorination. These observations indicate that bioaugmentation with PCB dehalorespiring microorganisms is a potentially tractable approach for in situ treatment of PCB impacted sites.

The effect of co-substrate activation on indigenous and bioaugmented PCB dechlorinating bacterial communities in sediment microcosms.

Microbial reductive dechlorination by members of the phylum Chloroflexi, including the genus Dehalococcoides, may play an important role in natural detoxification of highly chlorinated environmental pollutants, such as polychlorinated biphenyls (PCBs). Previously, we showed the increase of an indigenous bacterial population belonging to the Pinellas subgroup of Dehalococcoides spp. in Anacostia River sediment (Washington DC, USA) microcosms treated with halogenated co-substrates ("haloprimers"), tetrachlorobenzene (TeCB), or pentachloronitrobenzene (PCNB). The PCNB-amended microcosms exhibited enhanced dechlorination of weathered PCBs, while TeCB-amended microcosms did not. We therefore developed and used different phylogenetic approaches to discriminate the effect of the two different haloprimers. We also developed complementary approaches to monitor the effects of haloprimer treatments on 12 putative reductive dehalogenase (rdh) genes common to Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1. Our results indicate that 16S rRNA gene-based phylogenetic analyses have a limit in their ability to distinguish the effects of two haloprimer treatments and that two of rdh genes were present in high abundance when microcosms were amended with PCNB, but not TeCB. rdh gene-based phylogenetic analysis supports that these two rdh genes originated from the Pinellas subgroup of Dehalococcoides spp., which corresponds to the 16S rRNA gene-based phylogenetic analysis.