Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of “Desulfomonile tiedjei”

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, “Desulfomonile tiedjei.” We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c₃, or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H₂, but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35°C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in “D. tiedjei” cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in “D. tiedjei” extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.     

Characterization of Chloroethylene Dehalogenation by Cell Extracts of Desulfomonile tiedjei and Its Relationship to Chlorobenzoate Dehalogenation

We characterized the reductive dehalogenation of tetrachloroethylene in cell extracts of Desulfomonile tiedjei and compared it with this organism's 3-chlorobenzoate dehalogenation activity. Tetrachloroethylene was sequentially dehalogenated to trichloro- and dichloroethylene; there was no evidence for dichloroethylene dehalogenation. Like the previously characterized 3-chlorobenzoate dehalogenation activity, tetrachloroethylene dehalogenation was heat sensitive, not oxygen labile, and increased in proportion to the amount of protein in assay mixtures. In addition, both dehalogenation activities were dependent on hydrogen or formate as an electron donor and had an absolute requirement for either methyl viologen or triquat as an electron carrier in vitro. Both activities appear to be catalyzed by integral membrane proteins with similar solubilization characteristics. Dehalogenation of tetrachloroethylene was inhibited by 3-chlorobenzoate but not by the structural isomers 2- and 4-chlorobenzoate. The last two compounds are not substrates for D. tiedjei. These findings lead us to suggest that the dehalogenation of tetrachloroethylene in D. tiedjei is catalyzed by a dehalogenase previously thought to be specific for meta-halobenzoates.     

Influence of sulfur oxyanions on reductive dehalogenation activities in Desulfomonile tiedjei.

The inhibition of aryl reductive dehalogenation reactions by sulfur oxyanions has been demonstrated in environmental samples, dehalogenating enrichments, and the sulfate-reducing bacterium Desulfomonile tiedjei; however, this phenomenon is not well understood. We examined the effects of sulfate, sulfite, and thiosulfate on reductive dehalogenation in the model microorganism D. tiedjei and found separate mechanisms of inhibition due to these oxyanions under growth versus nongrowth conditions. Dehalogenation activity was greatly reduced in extracts of cells grown in the presence of both 3-chlorobenzoate, the substrate or inducer for the aryl dehalogenation activity, and either sulfate, sulfite, or thiosulfate, indicating that sulfur oxyanions repress the requisite enzymes. In extracts of fully induced cells, thiosulfate and sulfite, but not sulfate, were potent inhibitors of aryl dehalogenation activity even in membrane fractions lacking the cytoplasmically located sulfur oxyanion reductase. These results suggest that under growth conditions, sulfur oxyanions serve as preferred electron acceptors and negatively influence dehalogenation activity in D. tiedjei by regulating the amount of active aryl dehalogenase in cells. Additionally, in vitro inhibition by sulfur oxyanions is due to the interaction of the reactive species with enzymes involved in dehalogenation and need not involve competition between two respiratory processes for reducing equivalents. Sulfur oxyanions also inhibited tetrachloroethylene dehalogenation by the same mechanisms, further indicating that chloroethylenes are fortuitously dehalogenated by the aryl dehalogenase. The commonly observed inhibition of reductive dehalogenation reactions under sulfate-reducing conditions may be due to similar regulation mechanisms in other dehalogenating microorganisms that contain multiple respiratory activities.     

Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1.

Although reductive dehalogenation by anaerobic microorganisms offers great potential for the degradation of halocarbons, little is known about the biochemical mechanisms involved. It has previously been demonstrated that the dehalogenase activity involved in 3-chlorobenzoate dehalogenation by Desulfomonile tiedjei DCB-1 is present in the membrane fraction of the cell extracts. We report herein the purification of a 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of D. tiedjei DCB-1. The dehalogenase activity was monitored by the conversion of 3-chlorobenzoate to benzoate with reduced methyl viologen as a reducing agent. The membrane fraction of the cell extracts was obtained by ultracentrifugation, and the membrane proteins were solubilized with either the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) or Triton X-100 in the presence of glycerol. The solubilized dehalogenase was purified by ammonium sulfate fractionation and a combination of anion exchange, hydroxyapatite, and hydrophobic interaction chromatographies. This procedure yielded about 7% of the total dehalogenase activity with a 120-fold increase in specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified dehalogenase consisted of two subunits with molecular weights of 64,000 and 37,000. The enzyme converted 3-chlorobenzoate to benzoate at its highest specific activity in 10 mM potassium phosphate buffer (pH 7.2) at 38 degrees C. The enzyme was yellow and probably a heme protein. The enzyme had an adsorbance peak at 408 nm. The dithionite-reduced enzyme displayed absorbance peaks at 416, 522, and 550 nm. The dithionite-reduced enzyme was able to complex with carbon monoxide. The nature of the heme chromophore is currently unknown.     

Relationship between hydrogen consumption, dehalogenation, and the reduction of sulfur oxyanions by Desulfomonile tiedjei

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate as electron acceptors with rates of 0.50, 0.44, and 0.04 mumol h-1 mg-1, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-1 with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between hydrogen consumption, dehalogenation, and the reduction of sulfur oxyanions by Desulfomonile tiedjei.

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate as electron acceptors with rates of 0.50, 0.44, and 0.04 mumol h-1 mg-1, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-1 with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of the anaerobic dechlorinating microorganism Desulfomonile tiedjei in environmental matrices by its signature lipopolysacchride branched-long-chain hydroxy fatty acids

Abstract Desulfomonile tiedjei is a Gram-negative sulfate-reducing bacterium capable of catalyzing aryl reductive dehalogenation reactions. Since many toxic and persistent contaminants in the subsurface are halogenated aromatic compounds, the detection and enumeration of dehalogenating microorganisms in the environment may be a useful tool for planning and evaluating bioremediation efforts. In this study, we show that D. tiedjei contains unique lipopolysaccharide branched 3-hydroxy fatty acids, unknown as yet in other bacteria, and that it is possible to detect the bacterium in inoculated aquifer sediments based on these signature lipid biomarkers. The detection of D. tiedjei and other dehalogenating microorganisms possessing similar cellular properties in environmental matrices may be possible by this technique. Additionally, the effect of such inoculation on dehalogenation activity is examined.     

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

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

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

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

Microbial reductive dehalogenation.

A wide variety of compounds can be biodegraded via reductive removal of halogen substituents. This process can degrade toxic pollutants, some of which are not known to be biodegraded by any other means. Reductive dehalogenation of aromatic compounds has been found primarily in undefined, syntrophic anaerobic communities. We discuss ecological and physiological principles which appear to be important in these communities and evaluate how widely applicable these principles are. Anaerobic communities that catalyze reductive dehalogenation appear to differ in many respects. A large number of pure cultures which catalyze reductive dehalogenation of aliphatic compounds are known, in contrast to only a few organisms which catalyze reductive dehalogenation of aromatic compounds. Desulfomonile tiedjei DCB-1 is an anaerobe which dehalogenates aromatic compounds and is physiologically and morphologically unusual in a number of respects, including the ability to exploit reductive dehalogenation for energy metabolism. When possible, we use D. tiedjei as a model to understand dehalogenating organisms in the above-mentioned undefined systems. Aerobes use reductive dehalogenation for substrates which are resistant to known mechanisms of oxidative attack. Reductive dehalogenation, especially of aliphatic compounds, has recently been found in cell-free systems. These systems give us an insight into how and why microorganisms catalyze this activity. In some cases transition metal complexes serve as catalysts, whereas in other cases, particularly with aromatic substrates, the catalysts appear to be enzymes.     

Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei DCB-1.

Reductive dehalogenation of chlorophenols has been reported in undefined anaerobic cultures but never before in an anaerobic pure culture. We found that the sulfate-reducing bacterium Desulfomonile tiedjei DCB-1 reductively dehalogenates pentachlorophenol (PCP) and other chlorophenols. The maximum rate of PCP dechlorination observed was 54 mu mol of Cl- h-1 g of protein-1. 3-Chlorobenzoate appeared to serve as a required inducer for PCP dehalogenation; however, neither PCP nor 3-chlorophenol induced dehalogenation. Dehalogenation was catalyzed by living cells, and formate served as a required electron donor. D. tiedjei dehalogenated meta-chlorine substituents of chlorophenols (i.e., PCP was degraded to 2,4,6-trichlorophenol). Generally, more highly chlorinated phenol congeners were more readily dechlorinated, and 3-chlorophenol was not dehalogenated. Growing cultures dehalogenated PCP, but greater than 10 microM PCP (approximately 1.7 mmol g of protein-1) reversibly inhibited growth.     

Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei DCB-1.

Reductive dehalogenation of chlorophenols has been reported in undefined anaerobic cultures but never before in an anaerobic pure culture. We found that the sulfate-reducing bacterium Desulfomonile tiedjei DCB-1 reductively dehalogenates pentachlorophenol (PCP) and other chlorophenols. The maximum rate of PCP dechlorination observed was 54 mu mol of Cl- h-1 g of protein-1. 3-Chlorobenzoate appeared to serve as a required inducer for PCP dehalogenation; however, neither PCP nor 3-chlorophenol induced dehalogenation. Dehalogenation was catalyzed by living cells, and formate served as a required electron donor. D. tiedjei dehalogenated meta-chlorine substituents of chlorophenols (i.e., PCP was degraded to 2,4,6-trichlorophenol). Generally, more highly chlorinated phenol congeners were more readily dechlorinated, and 3-chlorophenol was not dehalogenated. Growing cultures dehalogenated PCP, but greater than 10 microM PCP (approximately 1.7 mmol g of protein-1) reversibly inhibited growth.     

Introduction of anaerobic dechlorinating bacteria into soil slurry microcosms and nested-PCR monitoring.

Desulfomonile tiedjei and Desulfitobacterium dehalogenans were chosen as model bacteria to demonstrate the introduction of an anaerobic microbia reductive dechlorination activity into nonsterile soil slurry microcosms by inoculation. De novo 3-chlorobenzoate dechlorination activity was established with the bacterium D. tiedjei in microcosms normally devoid of this dechlorination capacity. The addition of D. tiedjei to microcosms supplemented with 20 mM pyruvate as the cosubstrate resulted in total biotransformation of 1.5 mM 3-chlorobenzoate within 7 days. The introduction of the bacterium Desulfitobacterium dehalogenans into nonsterile microcosms resulted in a shortening of the period required for dechlorination activity to be established. In microcosms inoculated with Desulfitobacterium dehalogenans, total degradation of 6 mM 3-chloro-4-hydroxy phenoxyacetic acid (3-Cl-4-OHPA) was observed after 4 days in contrast to the result in noninoculated microcosms, where the total degradation of 3-Cl-4-OHPA by indigenous microorganisms was observed after 11 days. Both externally introduced bacterial strains were detected in soil slurry microcosms by a nested-PCR methodology.     

Evidence for a Chemiosmotic Model of Dehalorespiration in Desulfomonile tiedjei DCB-1

Desulfomonile tiedjei DCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized chemiosmotic process. Respiratory electron transport components were examined in D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation, and sulfate reduction. Reductive dehalogenation was inhibited by the respiratory quinone inhibitor 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that a respiratory quinoid is a component of the electron transport chain coupled to reductive dehalogenation. Moreover, reductive dehalogenation activity was dependent on 1,4-naphthoquinone, a possible precursor for a respiratory quinoid. However, no ubiquinone or menaquinone could be extracted from D. tiedjei. Rather, a UV-absorbing quinoid which is different from common respiratory quinones in chemical structure according to mass spectrometric and UV absorption spectroscopic analyses was extracted. ATP sulfurylase, adenosine phosphosulfate reductase, and desulfoviridin sulfite reductase, enzymes involved in sulfate reduction, were constitutively expressed in the cytoplasm of D. tiedjei cells grown under all three metabolic conditions. A periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. A membrane-bound, periplasm-oriented formate dehydrogenase was detected only in cells grown with formate as electron donor, while a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with D. tiedjei whole-cell suspensions and cell extracts suggest that the membrane-bound reductive dehalogenase is cytoplasm oriented. The data clearly demonstrate an enzyme topology in D. tiedjei which produces protons directly in the periplasm, generating a proton motive force by a scalar mechanism.     

Tetrachloroethene metabolism of Dehalospirillum multivorans

Dehalospirillum multivorans is a strictly anaerobic bacterium that is able to dechlorinate tetrachloroethene (perchloroethylene; PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) as part of its energy metabolism. The present communication describes some features of the dechlorination reaction in growing cultures, cell suspensions, and cell extracts of D. multivorans. Cell suspensions catalyzed the reductive dechlorination of PCE with pyruvate as electron donor at specific rates of up to 150 nmol (chloride released) min^-1 (mg cell protein)^-1 (300 M PCE initially, pH 7.5, 25°C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 M inhibited dehalogenation. The temperature optimum was between 25 and 30°C; the pH optimum at about 7.5. Dehalogenation was sensitive to potential alternative electron acceptors such as fumarate or sulfur; nitrate or sulfate had no significant effect on PCE reduction. Propyl iodide (50 M) almost completely inhibited the dehalogenation of PCE in cell suspensions. Cell extracts mediated the dehalogenation of PCE and of TCE with reduced methyl viologen as the electron donor at specific rates of up to 0.5 mol (chloride released) min^-1 (mg protein).^-1 An abiotic reductive dehalogenation could be excluded since cell extracts heated for 10 min at 95°C were inactive. The PCE dehalogenase was recovered in the soluble cell fraction after ultracentrifugation. The enzyme was not inactivated by oxygen.Abbreviations PCE Perchloroethylene or tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon - MV Methyl viologen     

The coexistence in rat muscle cells of two distinct classes of Ca2+-dependent K+ channels with different pharmacological properties and different physiological functions

An Arthrobacter sp. has been shown to dehalogenate 4-chlorobenzoate yielding 4-hydroxybenzoate. Experiments with 18O indicate that, in the presence of cell-free extracts, the hydroxyl group which is substituted onto the aromatic nucleus during dehalogenation is derived from water and not from molecular oxygen. Dehalogenation therefore is not catalysed by a mixed-function oxidase; instead a novel aromatic hydroxylase is implicated in the reaction.     

Hydrogenase Activity and the H2-Fumarate Electron Transport System in Bacteroides fragilis

Hydrogenase activity and the H₂-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H₂. Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H₂ was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H₂, but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H₂ when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H₂ to fumarate in B. fragilis is proposed.     

Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium,strain DCB-1

Thermodynamic data that the reductive dechlorination of 3-chlorobenzoate is exergonic have led to the hypothesis that this reaction yields biologically useful energy. This hypothesis was tested with strain DCB-1, a dehalogenating bacterium. The organism was grown under strictly anaerobic conditions in vitamin-amended mineral medium with formate plus acetate as electron donor and 3-chlorobenzoate as electron acceptor. The cell yield increased stoichiometrically to the amount of 3-chlorobenzoate dechlorinated. No growth was observed in the absence of 3-chlorobenzoate, or when 3-chlorobenzoate was replaced by benzoate. To obtain further evidence on that energy is derived from dechlorination, 3-chlorobenzoate was added to starved cells. This amendment resulted in an increase in the ATP level of the cells at 10 nmol per mg protein versus 3 nmol per mg protein in non-amended controls. These data indicate that the reductive dehalogenation of chlorinated aromatic compounds can be coupled to a novel type of chemotrophy.     

Reductive dehalogenation of chlorobenzene congeners in cell extracts of Dehalococcoides sp. strain CBDB1

Enzymatic reductive dehalogenation of tri-, tetra-, penta-, and hexachlorobenzenes was demonstrated in cell extracts with low protein concentration (0.5 to 1 micro g of protein/ml) derived from the chlorobenzene-respiring anaerobe Dehalococcoides sp. strain CBDB1. 1,2,3-trichlorobenzene dehalogenase activity was associated with the membrane fraction. Light-reversible inhibition by alkyl iodides indicated the presence of a corrinoid cofactor.     

Introduction of a de novo bioremediation ability, aryl reductive dechlorination, into anaerobic granular sludge by inoculation of sludge with Desulfomonile tiedjei.

Methanogenic upflow anaerobic granular-sludge blanket (UASB) reactors treat wastewaters at a high rate while simultaneously producing a useful product, methane; however, recalcitrant environmental pollutants may not be degraded. To impart 3-chlorobenzoate (3-CB)-dechlorinating ability to UASB reactors, we inoculated granular sludge in UASB reactors with either a pure culture of Desulfomonile tiedjei (a 3-CB-dechlorinating anaerobe) or a three-member consortium consisting of D. tiejei, a benzoate degrader, and an H2-utilizing methanogen. No degradation occurred in an uninoculated control reactor which was started with the same granular sludge, but inoculated reactors and granules from the inoculated UASB systems rapidly transformed 3-CB (54 mumol/day/g of granule biomass). After several months at a hydraulic retention time of 0.5 day, much shorter than the generation time of D. tiedjei, the reactors still dechlorinated 3-CB. This indicated that the bacteria were immobilized in the reactor granules, and by using an antibody probe for D. tiedjei, we demonstrated that this microorganism had colonized the sludge granules. These results represent the first addition of a pure culture or a defined microbial mixture to a viable waste treatment process to introduce a specific de novo degradative pathway into a granular-sludge consortium.