Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Characterization of Thermophilic Consortia from Two Souring Oil Reservoirs

The microbial consortia from produced water at two different oil fields in Alaska (Kuparuk) and the North Sea (Ninian) were investigated for sulfate-reducing and methanogenic activity over a range of temperatures and for a variety of substrates. The consortia were sampled on site, and samples were either incubated on site at 60(deg)C with various substrates or frozen for later incubation and analyses. Temperature influenced the rates of sulfate reduction, hydrogen sulfide production, and substrate oxidation, as well as the cell morphology. The highest rates of sulfate reduction and substrate oxidation were found between 50 and 60(deg)C. Formate and n-butyrate were the most favorable electron donors at any tested temperature. Acetate was utilized at 35(deg)C but not at 50 or 70(deg)C and was produced at 60(deg)C. This indicates that the high levels of acetate found in produced water from souring oil formations are due mainly to an incomplete oxidation of volatile fatty acids to acetate. The cell size distribution of the microbial consortium indicated a nonuniform microbial composition in the original sample from the Kuparuk field. At different temperatures, different microbial morphologies and physiologies were observed. Methane-producing activity at thermophilic temperatures (60(deg)C) was found only for the Kuparuk consortium when hydrogen and carbon dioxide were present. No methane production from acetate was observed. Suppression of methanogenic activity in the presence of sulfate indicated a competition with sulfate-reducing bacteria for hydrogen.     

微生物纳米导线的导电机制及功能

微生物种间直接电子传递是指在厌氧条件下,一种微生物将电子直接传递给另外一种微生物,将两种不同微生物的代谢途径耦合在一起,以达到互养共生的目的。细菌-古菌之间的直接电子传递是其物质转换与能量代谢的新途径和新调控机制,直接参与甲烷的合成以及与硫酸盐还原耦合的厌氧甲烷氧化,在驱动碳和硫的地球化学转化与循环中起着十分重要的作用。目前研究结果认为细菌-古菌之间的直接电子传递主要是由含多个血红素的C型细胞色素介导的,这些细胞色素能形成不间断的胞外电子传递途径,以电子多步跃迁机制在细菌和古菌的细胞质膜之间传递电子。     

Microbiological and biogeochemical processes in a pockmark of the Gdansk depression,Baltic Sea

Comprehensive microbiological and biogeochemical investigation of a pockmark within one of the sites of gas-saturated sediments in the Gdansk depression, Baltic Sea was carried out during the 87th voyage of the Professor Shtokman research vessel. Methane content in the near-bottom water and in the underlying sediments indicates stable methane flow from the sediment into the water. In the 10-m water layer above the pockmark, apart from methane anomalies, elevated numbers of microorganisms and enhanced rates of dark CO₂ fixation (up to 1.15 µmol C/(l day)) and methane oxidation (up to 2.14 nmol CH₄/(l day)) were revealed. Lightened isotopic composition of suspended organic matter also indicates high activity of the near-bottom microbial community. Compared to the background stations, methane content in pockmark sediments increased sharply from the surface to 40–60 ml/dm³ in the 20–30 cm horizon. High rates of bacterial sulfate reduction (SR) were detected throughout the core (0–40 cm); the maximum of 74 µmol S/(dm³ day) was located in subsurface horizons (15–20 cm). The highest rates of anaerobic methane oxidation (AMO), up to 80 µmol/dm³ day), were detected in the same horizon. Good coincidence of the AMO and SR profiles with stoichiometry close to 1: 1 is evidence in favor of a close relation between these processes performed by a consortium of methanotrophic archaea and sulfate-reducing bacteria. Methane isotopic composition in subsurface sediments of the pockmark (from ?53.0 to ?56.5‰) does not rule out the presence of methane other than the biogenic methane from the deep horizons of the sedimentary cover.     

Biogeochemistry and microbial ecology of methane oxidation in anoxic environments: a review

Evidence supporting a key role for anaerobic methane oxidation in the global methane cycle is reviewed. Emphasis is on recent microbiological advances. The driving force for research on this process continues to be the fact that microbial communities intercept and consume methane from anoxic environments, methane that would otherwise enter the atmosphere. Anaerobic methane oxidation is biogeochemically important because methane is a potent greenhouse gas in the atmosphere and is abundant in anoxic environments. Geochemical evidence for this process has been observed in numerous marine sediments along the continental margins, in methane seeps and vents, around methane hydrate deposits, and in anoxic waters. The anaerobic oxidation of methane is performed by at least two phylogenetically distinct groups of archaea, the ANME-1 and ANME-2. These archaea are frequently observed as consortia with sulfate-reducing bacteria, and the metabolism of these consortia presumably involves a syntrophic association based on interspecies electron transfer. The archaeal member of a consortium apparently oxidizes methane and shuttles reduced compounds to the sulfate-reducing bacteria. Despite recent advances in understanding anaerobic methane oxidation, uncertainties still remain regarding the nature and necessity of the syntrophic association, the biochemical pathway of methane oxidation, and the interaction of the process with the local chemical and physical environment. This review will consider the microbial ecology and biogeochemistry of anaerobic methane oxidation with a special emphasis on the interactions between the responsible organisms and their environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane

Anaerobic oxidation of methane (AOM) with sulfate is catalysed by microbial consortia of archaea and bacteria affiliating with methanogens and sulfate-reducing Deltaproteobacteria respectively. There is evidence that methane oxidation is catalysed by enzymes related to those in methanogenesis, but the enzymes for sulfate reduction coupled to AOM have not been examined. We collected microbial mats with high AOM activity from a methane seep in the Black Sea. The mats consisted mainly of archaea of the ANME-2 group and bacteria of the Desulfosarcina-Desulfococcus group. Cell-free mat extract contained activities of enzymes involved in sulfate reduction to sulfide: ATP sulfurylase (adenylyl : sulfate transferase; Sat), APS reductase (Apr) and dissimilatory sulfite reductase (Dsr). We partially purified the enzymes by anion-exchange chromatography. The amounts obtained indicated that the enzymes are abundant in the mat, with Sat accounting for 2% of the soluble mat protein. N-terminal amino acid sequences of purified proteins suggested similarities to the corresponding enzymes of known species of sulfate-reducing bacteria. The deduced amino acid sequence of PCR-amplified genes of the Apr subunits is similar to that of Apr of the Desulfosarcina/Desulfococcus group. These results indicate that the major enzymes involved in sulfate reduction in the Back Sea microbial mats are of bacterial origin, most likely originating from the bacterial partner in the consortium.     

Inhibition Experiments on Anaerobic Methane Oxidation

Anaerobic methane oxidation is a general process important in controlling fluxes of methane from anoxic marine sediments. The responsible organism has not been isolated, and little is known about the electron acceptors and substrates involved in the process. Laboratory evidence indicates that sulfate reducers and methanogens are able to oxidize small quantities of methane. Field evidence suggests anaerobic methane oxidation may be linked to sulfate reduction. Experiments with specific inhibitors for sulfate reduction (molybdate), methanogenesis (2-bromoethanesulfonic acid), and acetate utilization (fluoroacetate) were performed on marine sediments from the zone of methane oxidation to determine whether sulfate-reducing bacteria or methanogenic bacteria are responsible for methane oxidation. The inhibition experiment results suggest that methane oxidation in anoxic marine sediments is not directly mediated by sulfate-reducing bacteria or methanogenic bacteria. Our results are consistent with two possibilities: anaerobic methane oxidation may be mediated by an unknown organism or a consortium involving an unknown methane oxidizer and sulfate-reducing bacteria.     

Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME‐1 group

Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate‐reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME‐1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82–90% of a composite ANME‐1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME‐1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron‐accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]‐hydrogenases are lacking in the dataset, genes for an [FeFe]‐hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c‐type cytochromes were identified, which have not yet been correlated with methanogenesis‐related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME‐1 to the bacterial sulfate‐reducing partner.     

Simultaneous removal of phenol, ortho- and para-cresol by mixed anaerobic consortia.

Two different anerobic consortia, one removing phenol and ortho (o-) cresol and other removing para(p-) cresol, were cultivated in serum bottles using whey as cosubstrate substitute for proteose peptone. Phenol and p-cresol removal with the phenol-removing consortium were the same with 0.0125% (w/v) whey as with 0.05% proteose peptone. For the other consortium, 8 days were required to decrease the p-cresol concentration from 35 to 2 mg/L with 0.025% whey, while 35 days were required to achieve a similar removal with 0.5% proteose peptone. The two consortia were mixed and cultivated with 0.025% whey. Phenolic compound removal with the mixed consortia was as good as that achieved by each of the two initial consortia against their respective substrates. This removal activity was maintained after several transfers. In a continuous upflow fixed-film reactor, the mixed consortia removed over 98% of 150 mg/L of phenol and 35 mg/L of each o- and p-cresol in the influent at 29 degrees C, with 0.025% whey as cosubstrate. The hydraulic retention time (HRT) was 0.25 day, corresponding to a phenolic compound volumic loading rate of 880 mg/(L of reactor x day). Once the continuous flow reactor achieved constant phenolic compound removal, no intermediates were found in the effluent, while in serum bottles, m-toluic acid, an o-cresol intermediate, accumulated. Measurements of the specific activity for the uptake of different substrates demonstrated the presence of all trophic groups involved in methanogenic fermentation. These activities were, in mg of substrate/(g of volatile suspended solids x day), as follows: 849 +/- 25 for the acidogens; 554 +/- 15 for the acetogens; 934 +/- 37 for the aceticlastic methanogens; and 135 +/- 15 for the hydrogenophilic methanogens. Electron micrographs of the mixed consortia showed seven different morphological bacterial types, including Methanotrix-like bacteria.     

Gene expression and ultrastructure of meso‐ and thermophilic methanotrophic consortia

The sulfate‐dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon‐rich environments under different temperature regimes: ANME‐1a/HotSeep‐1 (60°C), ANME‐1a/Seep‐SRB2 (37°C) and ANME‐2c/Seep‐SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME‐2c encodes all enzymes, while ANME‐1a lacks the gene for N5,N10‐methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c‐type cytochromes, some predicted to be extracellular. ANME‐2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME‐1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire‐like structures and heme‐rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate‐dependent AOM, and that both partners synthesize molecules to enable it.     

Exocellular electron transfer in anaerobic microbial communities

Exocellular electron transfer plays an important role in anaerobic microbial communities that degrade organic matter. Interspecies hydrogen transfer between microorganisms is the driving force for complete biodegradation in methanogenic environments. Many organic compounds are degraded by obligatory syntrophic consortia of proton-reducing acetogenic bacteria and hydrogen-consuming methanogenic archaea. Anaerobic microorganisms that use insoluble electron acceptors for growth, such as iron- and manganese-oxide as well as inert graphite electrodes in microbial fuel cells, also transfer electrons exocellularly. Soluble compounds, like humic substances, quinones, phenazines and riboflavin, can function as exocellular electron mediators enhancing this type of anaerobic respiration. However, direct electron transfer by cell-cell contact is important as well. This review addresses the mechanisms of exocellular electron transfer in anaerobic microbial communities. There are fundamental differences but also similarities between electron transfer to another microorganism or to an insoluble electron acceptor. The physical separation of the electron donor and electron acceptor metabolism allows energy conservation in compounds as methane and hydrogen or as electricity. Furthermore, this separation is essential in the donation or acceptance of electrons in some environmental technological processes, e.g. soil remediation, wastewater purification and corrosion.     

Consortia of cyanobacteria/microalgae and bacteria: biotechnological potential

Microbial metabolites are of huge biotechnological potential and their production can be coupled with detoxification of environmental pollutants and wastewater treatment mediated by the versatile microorganisms. The consortia of cyanobacteria/microalgae and bacteria can be efficient in detoxification of organic and inorganic pollutants, and removal of nutrients from wastewaters, compared to the individual microorganisms. Cyanobacterial/algal photosynthesis provides oxygen, a key electron acceptor to the pollutant-degrading heterotrophic bacteria. In turn, bacteria support photoautotrophic growth of the partners by providing carbon dioxide and other stimulatory means. Competition for resources and cooperation for pollutant abatement between these two guilds of microorganisms will determine the success of consortium engineering while harnessing the biotechnological potential of the partners. Relative to the introduction of gene(s) in a single organism wherein the genes depend on the regulatory- and metabolic network for proper expression, microbial consortium engineering is easier and achievable. The currently available biotechnological tools such as metabolic profiling and functional genomics can aid in the consortium engineering. The present review examines the current status of research on the consortia, and emphasizes the construction of consortia with desired partners to serve a dual mission of pollutant removal and commercial production of microbial metabolites.     

Biomarker Evidence for Widespread Anaerobic Methane Oxidation in Mediterranean Sediments by a Consortium of Methanogenic Archaea and Bacteria

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in ¹³C (δ¹³C values are as low as −95‰). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of ¹³C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

Characterization of Anaerobic Dechlorinating Consortia Derived from Aquatic Sediments

Four methanogenic consortia which degraded 2-chlorophenol, 3-chlorophenol, 2-chlorobenzoate, and 3-chlorobenzoate, respectively, and one nitrate-reducing consortium which degraded 3-chlorobenzoate were characterized. Degradative activity in these consortia was maintained by laboratory transfer for over 2 years. In the methanogenic consortia, the aromatic ring was dechlorinated before mineralization to methane and carbon dioxide. After dechlorination, the chlorophenol consortia converted phenol to benzoate before mineralization. All methanogenic consortia degraded both phenol and benzoate. The 3-chlorophenol and 3-chlorobenzoate consortia also degraded 2-chlorophenol. No other cross-acclimation to monochlorophenols or monochlorobenzoates was detected in the methanogenic consortia. The consortium which required nitrate for the degradation of 3-chlorobenzoate degraded benzoate and 4-chlorobenzoate anaerobically in the presence of KNO₃, but not in its absence. This consortium also degraded benzoate, but not 3-chlorobenzoate, aerobically.     

Removal of sulphate, COD and Cr(VI) in simulated and real wastewater by sulphate reducing bacteria enrichment in small bioreactor and FTIR study

The present study was conducted to investigate the chromium(VI), COD and sulphate removal efficiency from aqueous solution and treatment of real effluent (CETP) in a small scale bioreactor using sulphate reducing bacteria consortium. Effect of different hydraulic retention times (HRTs), initial metal concentrations, various carbon sources and temperatures were studied on removal of chromium(VI), COD and sulphate. Maximum chromium(VI) and sulphate removal was found to be 96.0% and 82.0%, respectively, at initial concentration of 50 mg l⁻¹ using lactate as carbon source. However, highest COD removal was 36.2% in medium containing fructose as the carbon source and electron donor. NADH dependent chromate reductase activity was not observed which indicated the anaerobic consortium. Initially consortium medium with a strong negative oxidation reduction potential indicated the reducing activity. The FTIR spectrum of the sulphate reducing bacteria consortium clearly shows the existence of the sulphate ions and signifies that sulfate reducing bacteria have used sulfate during the growth phase.     

Dynamics of Methane Production, Sulfate Reduction, and Denitrification in a Permanently Waterlogged Alder Swamp

The dynamics of sulfate reduction, methane production, and denitrification were investigated in a permanently waterlogged alder swamp. Molybdate, an inhibitor of sulfate reduction, stimulated methane production in soil slurries, thus suggesting competition for common substrates between sulfate-reducing and methane-producing bacteria. Acetate, hydrogen, and methanol were found to stimulate both sulfate reduction and methane production, while trimethylamine mainly stimulated methane production. Nitrate addition reduced both methane production and sulfate reduction, either as a consequence of competition or poisoning of the bacteria. Sulfate-reducing bacteria were only slightly limited by the availability of electron acceptors, while denitrifying bacteria were seriously limited by low nitrate concentrations. Arrhenius plots of the three processes revealed different responses to temperature changes in the slurries. Methane production was most sensitive to temperature changes, followed by denitrification and sulfate reduction. No significant differences between slope patterns were observed when comparing summer and winter measurements, indicating similar populations regarding temperature responses.     

Contribution of quinone-reducing microorganisms to the anaerobic biodegradation of organic compounds under different redox conditions

The capacity of two anaerobic consortia to oxidize different organic compounds, including acetate, propionate, lactate, phenol and p-cresol, in the presence of nitrate, sulfate and the humic model compound, anthraquinone-2,6-disulfonate (AQDS) as terminal electron acceptors, was evaluated. Denitrification showed the highest respiratory rates in both consortia studied and occurred exclusively during the first hours of incubation for most organic substrates degraded. Reduction of AQDS and sulfate generally started after complete denitrification, or even occurred at the same time during the biodegradation of p-cresol, in anaerobic sludge incubations; whereas methanogenesis did not significantly occur during the reduction of nitrate, sulfate, and AQDS. AQDS reduction was the preferred respiratory pathway over sulfate reduction and methanogenesis during the anaerobic oxidation of most organic substrates by the anaerobic sludge studied. In contrast, sulfate reduction out-competed AQDS reduction during incubations performed with anaerobic wetland sediment, which did not achieve any methanogenic activity. Propionate was a poor electron donor to achieve AQDS reduction; however, denitrifying and sulfate-reducing activities carried out by both consortia promoted the reduction of AQDS via acetate accumulated from propionate oxidation. Our results suggest that microbial reduction of humic substances (HS) may play an important role during the anaerobic oxidation of organic pollutants in anaerobic environments despite the presence of alternative electron acceptors, such as sulfate and nitrate. Methane inhibition, imposed by the inclusion of AQDS as terminal electron acceptor, suggests that microbial reduction of HS may also have important implications on the global climate preservation, considering the green-house effects of methane.     

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.     

Pentachlorophenol Dechlorination in Fluidized-Bed Reactors under Methanogenic Conditions

The reductive dechlorination of chlorophenols was studied in three fluidized-bed reactors (FBRs) with respect to enrichment, pathways, complete dechlorination, and overall performance. The methanogenic consortia, developed by previous researchers in our laboratory, have been further enriched by reducing the ratio of substrate to pentachlorophenol (PCP) and increasing the PCP loading. The performance of the consortia was improved, and complete dechlorination at high PCP loading rates was observed, reaching a PCP loading of 1227 µmol/L d with 99% chlorophenol removal. The dechlorination rates in the reactors for chlorophenol (CP) congeners were obtained and were used to evaluate the performance of the three consortia and to quantitatively estimate the fates of these chlorophenols in the reactors. The consortium with the best performance was further investigated in bottle tests by treatment with heat and metabolic inhibitors to examine chlorophenol degradation and to characterize the CP degraders. The degradation of all monochlorophenols was completely inhibited after heat treatment, but the degradation of all other tested chlorophenols was hardly affected by heat treatment, indicating that spore-forming bacteria likely were involved in dechlorination. Addition of sulfate negatively affected CP degradation, but addition of molybdate reduced the effect of sulfate. Tests with 2-bromoethanesulfonic acid and vancomycin indicated that bacteria were responsible for chlorophenol degradation in the consortium.