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.     

Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate

Abstract A defined 3-chlorobenzoate-degrading methanogenic consortium was constructed by recombining key organisms isolated from a 3-chlorobenzoate-degrading methanogenic sludge enrichment. The organisms comprise a three-tiered food chain which includes: (1) reductive dechlorination of 3-chlorobenzoate; (2) oxidation of benzoate to acetate, H₂ and CO₂; (3) removal of H₂ plus CO₂ by conversion into methane. The defined consortium, consisting of a dechlorinating organism (DCB-1), a benzoate degrader (BZ-1) and a lithotrophic methanogen ( Methanospirillum strain PM-1) grew well in a basal salts medium supplemented with 3-chlorobenzoate (3.2 mM) as the sole energy source. The chlorine released from the aromatic ringe was recovered in stoichiometric amounts as the chloride ion. The reducing power required for reductive dechlorination was obtained from the hydrogen produced in the acetogenic oxidation of benzoate. One-third of the benzoate-derived hydrogen was recycled via the reductive dechlorination of 3-chlorobenzoate, indicating that the consortium operated as a food web rather than a food chain.     

Acetate as a source of reducing equivalents in the reductive dechlorination of 2,5-dichlorobenzoate

Desulfomonile tiedjei is the key dechlorinating organism in a three-tiered bacterial consortium that grows on the methanogenic degradation of 3-chlorobenzoate. 2,5-Dichlorobenzoate, however, is only converted to 2-chlorobenzoate and is not a methanogenic substrate for the consortium. The dechlorinator uses hydrogen produced from benzoate by the benzoate degrading member of consortium as its source of reducing equivalents for the dechlorination reaction. Incubation of 3-chlorobenzoate grown consortium cells with 2,5-dichlorobenzoate resulted in the consumption of acetate concurrent with the formation of 2-chlorobenzoate indicating that acetate can serve as an alternative source of reducing equivalents for reductive dechlorination. This interpretation was confirmed by the finding that the formation of ¹⁴CO₂ from 2-¹⁴C-labeled acetate was stoichiometric. The addition of hydrogen to 2,5-dichlorobenzoate metabolizing cells resulted in (i) an 2.7-fold increase in the rate of dechlorination, and (ii) a drop in the amount of label recovered as CO₂+CH₄ from methyl ¹⁴C-labeled acetate, indicating that hydrogen was the preferred source of reducing equivalents for reductive dechlorination. Benzoate, an indirect source of H₂ in the consortium, also inhibited the oxidation of acetate, while glucose, methanol, and butyrate did not affect labeled gas production and therefore were not suitable electron donors. Concomittant to dechlorination of 2,5-dichlorobenzoate 3- and 4-methoxybenzoate were converted to 3- and 4-hydroxybenzoate respectively. These conversions stimulated the rate of dechlorination 2-fold. Demethylation of 4-methoxybenzoate stimulated, but demethylation of 3-methoxybenzoate inhibited the oxidation of benzoate during the dechlorination of 2,5-dichlorobenzoate, suggesting that these isomers are metabolized through different pathways. Experiments with benzoate, 3-chlorobenzoate and 2,5-dichlorobenzoate metabolizing cells amended with ¹⁴CO₂ showed that actively dechlorinating cells catalyzed an exchange reaction between CO₂ and acetate.     

Strain DCB-1 conserves energy for growth from reductive dechlorination coupled to formate oxidation

Strain DCB-1 is a strict anaerobe capable of the reductive dechlorination of chlorobenzoates. The effect of dechlorination on the yield of pure cultures of DCB-1 was tested. Cultures were incubated with formate or H₂ as electron donors and CO₂ as a putative carbon source. Relative to control cultures with benzoate, cultures which dechlorinated 3-chlorobenzoate and 3,5-dichlorobenzoate had higher yields measured both as protein and cell density. On the media tested the apparent growth yield was 1.7 to 3.4 g cell protein per mole Cl^- removed. Dechlorination also stimulated formate oxidation by growing cultures. Resuspended cells required an electron donor for dechlorination activity, with either formate or elemental iron serving this function. Resuspended cells did not require an electron acceptor for formate consumption, but reductive dechlorination of 3CB to benzoate stoichiometrically stimulated oxidation of formate to CO₂. These results indicate that DCB-1 conserves energy for growth by coupling formate, and probably, H₂ oxidation to reductive dechlorination.Non-standard abbreviations 3CB 3-chlorobenzoate - 35DCB 3,5-dichlorobenzoate - PCF Propionibacterium sp. culture fluid     

Influence of Substituents on Reductive Dehalogenation of 3-Chlorobenzoate Analogs

The biochemical effects of aryl substituents on the reductive dechlorination of 3-chlorobenzoate analogs were quantified with (i) a stable 3-chlorobenzoate-grown methanogenic sludge enrichment, (ii) Desulfomonile tiedjei DCB-1, isolated from this enrichment and able to catalyze the reductive dechlorination of 3-chlorobenzoate, and (iii) a defined 3-chlorobenzoate-degrading methanogenic consortium with D. tiedjei as the key dechlorinating organism. The addition of hydrogen stimulated the dechlorination rate in the consortium. The extent of this stimulation depended on the substituent. The data were evaluated with various sets of substituent constants compiled for the Hammett equation. None of the sets yielded a satisfactory correlation between experimental values and theoretical constants. This suggests that the microbially catalyzed reductive dechlorination of 3-chlorobenzoate cannot be described simply as either a nucleophilic or an electrophilic substitution reaction. Nevertheless, observations that the presence of a para-amino or -hydroxy group inhibited the rate of dechlorination suggest that the rate-limiting step in the reductive dechlorination of 3-chlorobenzoate is a nucleophilic attack on the negatively charged π electron cloud around the benzene nucleus.     

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.     

Reductive, coenzyme A-mediated pathway for 3-chlorobenzoate degradation in the phototrophic bacterium Rhodopseudomonas palustris

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl-CoA), (ii) reductively dehalogenating 3-chlorobenzoyl-CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.     

Reductive, Coenzyme A-Mediated Pathway for 3-Chlorobenzoate Degradation in the Phototrophic Bacterium Rhodopseudomonas palustris

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl–CoA), (ii) reductively dehalogenating 3-chlorobenzoyl–CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.     

Phenotype Fingerprinting Suggests the Involvement of Single-Genotype Consortia in Degradation of Aromatic Compounds by Rhodopseudomonas palustris

Anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of environmental pollutants. In natural environments, the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. This strategy allows consortium organisms to reduce efforts required for maintenance of the redox homeostasis at each syntrophic level. Cellular mechanisms that maintain the redox homeostasis during the degradation of aromatic compounds by one organism are not fully understood. Here we present a hypothesis that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris forms its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. We have revealed the consortia from large-scale measurements of mRNA and protein expressions under p-coumarate, benzoate and succinate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach, marker genes for known R. palustris phenotypes are employed to determine the relative expression levels of genes and proteins in aromatics versus non-aromatics degrading condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading conditions may lead to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO₂ and H₂. Benzoate degrading conditions may also produce at least three subpopulations utilizing benzoate, CO₂ and H₂, and N₂ and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The N₂-fixing subpopulation in the benzoate degrading consortium has preferential activation of the vanadium nitrogenase over the molybdenum nitrogenase. This subpopulation in the consortium was confirmed in an independent experiment by consumption of dissolved nitrogen gas under the benzoate degrading conditions.     

Kinetics of two complementary hydrogen sink reactions in a defined 3-chlorobenzoate degrading methanogenic co-culture

Abstract The interrelationships between an obligate hydrogen-producing and two different hydrogen-scavenging populations grown as synthrophic members of a 3-chlorobenzoate degrading methanogenic consortium were studied. The hydrogen producer was a benzoate degrader (strain BZ-2), and the hydrogen consumers were a 3-chlorobenzoate dechlorinating bacterium ( Desulfomonile tiedjei ) and a hydrogenotropic methanogen ( Methanospirillum strain PM-1). When a mixture of 3-chlorobenzoate plus benzoate was added to this consortium, the rate of benzoate degradation was 50% higher, at slightly lower H₂ concentrations, than when benzoate alone was added. The enhanced benzoate degradation rate was apparantly triggered by the lower H₂ concentration, as the rate of benzoate degradation was shown to be a function of the H₂ concentration. By offering a hydrogen sink, in addition to methanogenesis, the dechlorinating hydrogen-scavenging population stimulated the rate of benzoate degradation. The lowering of the H₂ concentration was very small, which was in agreement with the observation that the rate of methanogenesis was hardly affected by this lower hydrogen concentration. Thus there was no significant competition for H₂ between the two hydrogen-scavenging populations in the consortium, as they practically complemented each other's hydrogen-scavenging potential at in situ hydrogen concentrations during the degradation of 3-chlorobenzoate. The H₂ concentrations at which hydrogen driven methanogenesis by Methanospirillum occurred in the consortium were well below the threshold concentration extrapolated for this methanogen after growth at high H₂ concentrations.     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

Evidence for chemiosmotic coupling of reductive dechlorination and ATP synthesis in Desulfomonile tiedjei

Desulfomonile tiedjei (strain DCB-1) was previously shown to conserve energy for growth from reductive dechlorination of 3-chlorobenzoate coupled to formate oxidation. We tested the hypothesis that a chemiosmotic mechanism couples reductive dechlorination and ATP synthesis in D. tiedjei. Dechlorination resulted in an increase in the ATP pool of cells. Uncouplers and ionophores decreased both the dechlorination rate and the ATP pool. However, at low concentrations the inhibitors had relatively greater effects on the ATP pool, and in some cases, even appeared to stimulate dechlorination. Those agents could not completely inhibit ATP synthesis while allowing dechlorination activity. The proton-driven ATPase inhibitor, N,N-dicyclohexylcarbodiimide (DCCD), had similar effects. An imposed pH gradient also resulted in an increase in the ATP pool of cells, and this increase was partially inhibited by DCCD. Addition of 3-chlorobenzoate to cell suspensions caused proton translocation by the cells. Proton translocation was stimulated by the permeant thiocyanate anion and inhibited by uncouplers. A maximum H⁺/3-chlorobenzoate ratio of greater than two was observed. These findings suggest that dechlorination supports formation of a proton-motive force which in turn supports ATP synthesis via a proton-driven ATPase.Abbreviations 3CB 3-chlorobenzoate - CCCP m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dinitrophenol - P proton-motive force - PCP pentachlorophenol     

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.     

Chemotaxis of Pseudomonas putida toward chlorinated benzoates.

The chlorinated aromatic acids 3-chlorobenzoate and 4-chlorobenzoate are chemoattractants for Pseudomonas putida PRS2000. These compounds are detected by a chromosomally encoded chemotactic response to benzoate which is inducible by beta-ketoadipate, an intermediate of benzoate catabolism. Plasmid pAC27, encoding enzymes for 3-chlorobenzoate degradation, does not appear to carry genes for chemotaxis toward chlorinated compounds.     

Chemotaxis of Pseudomonas putida toward chlorinated benzoates

The chlorinated aromatic acids 3-chlorobenzoate and 4-chlorobenzoate are chemoattractants for Pseudomonas putida PRS2000. These compounds are detected by a chromosomally encoded chemotactic response to benzoate which is inducible by beta-ketoadipate, an intermediate of benzoate catabolism. Plasmid pAC27, encoding enzymes for 3-chlorobenzoate degradation, does not appear to carry genes for chemotaxis toward chlorinated compounds.     

Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge

An anaerobic, motile, gram-negative, rod-shaped bacterium is described which degrades benzoate in coculture with an H₂-utilizing organism and in the absence of exogenous electron acceptors such as O₂, SO ₄ ⁼ or NO ₃ ^- . The bacterium was isolated from a municipal primary, anaerobic sewage digestor using anaerobic roll-tube medium with benzoate as the main energy source and in syntrophic association with an H₂-utilizing sulfate-reducing Desulfovibrio sp. which cannot utilize benzoate or fatty acids apart from formate as energy source. The benzoate utilizer produced acetate (3 mol/mol of substrate degraded) and presumably CO₂ and H₂, or formate from benzoate. In media without sulfate and with Methanospirillum hungatei (a methanogen that utilizes only H₂–CO₂ or formate as the energy source) added, 3 mol of acetate and 0.7 mol of methane were produced per mol of benzoate and CO₂ was probably formed. Low numbers of Desulfovibrio sp. were present in the methanogenic coculture and a pure coculture of the benzoate utilizer with M. hungatei was not obtained. The generation times for growth of the sulfate-reducing and methanogenic cocultures were 132 and 166h, respectively. The benzoate utilizer did not utilize other common aromatic compounds, C ₃ ^- –C₇ monocarboxylic acids, or C₄-C₆ dicarboxylic acids for growth, nor did it appear to use SO ₄ ⁼ , NO ₃ ^- or fumarate as alternative electron acceptors. Addition of H₂ inhibited growth and benzoate degradation.     

Isolation and Characterization of Desulfovibrio dechloracetivorans sp. nov., a Marine Dechlorinating Bacterium Growing by Coupling the Oxidation of Acetate to the Reductive Dechlorination of 2-Chlorophenol

Strain SF3, a gram-negative, anaerobic, motile, short curved rod that grows by coupling the reductive dechlorination of 2-chlorophenol (2-CP) to the oxidation of acetate, was isolated from San Francisco Bay sediment. Strain SF3 grew at concentrations of NaCl ranging from 0.16 to 2.5%, but concentrations of KCl above 0.32% inhibited growth. The isolate used acetate, fumarate, lactate, propionate, pyruvate, alanine, and ethanol as electron donors for growth coupled to reductive dechlorination. Among the halogenated aromatic compounds tested, only the ortho position of chlorophenols was reductively dechlorinated, and additional chlorines at other positions blocked ortho dechlorination. Sulfate, sulfite, thiosulfate, and nitrate were also used as electron acceptors for growth. The optimal temperature for growth was 30°C, and no growth or dechlorination activity was observed at 37°C. Growth by reductive dechlorination was revealed by a growth yield of about 1 g of protein per mol of 2-CP dechlorinated, and about 2.7 g of protein per mole of 2,6-dichlorophenol dechlorinated. The physiological features and 16S ribosomal DNA sequence suggest that the organism is a novel species of the genus Desulfovibrio and which we have designated Desulfovibrio dechloracetivorans. The unusual physiological feature of this strain is that it uses acetate as an electron donor and carbon source for growth with 2-CP but not with sulfate.     

Isolation and characterization of Desulfovibrio dechloracetivorans sp. nov., a marine dechlorinating bacterium growing by coupling the oxidation of acetate to the reductive dechlorination of 2-chlorophenol

Strain SF3, a gram-negative, anaerobic, motile, short curved rod that grows by coupling the reductive dechlorination of 2-chlorophenol (2-CP) to the oxidation of acetate, was isolated from San Francisco Bay sediment. Strain SF3 grew at concentrations of NaCl ranging from 0.16 to 2.5%, but concentrations of KCl above 0. 32% inhibited growth. The isolate used acetate, fumarate, lactate, propionate, pyruvate, alanine, and ethanol as electron donors for growth coupled to reductive dechlorination. Among the halogenated aromatic compounds tested, only the ortho position of chlorophenols was reductively dechlorinated, and additional chlorines at other positions blocked ortho dechlorination. Sulfate, sulfite, thiosulfate, and nitrate were also used as electron acceptors for growth. The optimal temperature for growth was 30 degrees C, and no growth or dechlorination activity was observed at 37 degrees C. Growth by reductive dechlorination was revealed by a growth yield of about 1 g of protein per mol of 2-CP dechlorinated, and about 2.7 g of protein per mole of 2,6-dichlorophenol dechlorinated. The physiological features and 16S ribosomal DNA sequence suggest that the organism is a novel species of the genus Desulfovibrio and which we have designated Desulfovibrio dechloracetivorans. The unusual physiological feature of this strain is that it uses acetate as an electron donor and carbon source for growth with 2-CP but not with sulfate.     

Biodegradation of 1,1,1-trichloroethane by a carbon tetrachloride-degrading denitrifying consortium

A denitrifying consortium capable of degrading carbon tetrachloride (CT) was shown to also degrade 1,1,1-trichloroethane (TCA). Fed-batch experiments demonstrated that the specific rate of TCA degradation by the consortium was comparable to the specific rate of CT degradation (approximately 0.01 L/gmol/min) and was independent of the limiting nutrient. Although previous work demonstrated that 4-50% of CT transformed by the consortium was converted to chloroform (CF), no reductive dechlorination products were detected during TCA degradation, regardless of the limiting nutrient. The lack of chlorinated TCA degradation products implies that the denitrifying consortium possesses an alternate pathway for the degradation of chlorinated solvents which does not involve reductive dechlorination. Copyright 1998 John Wiley & Sons, Inc.     

Degradation of 4-Chlorobenzoic Acid by Arthrobacter sp

A mixed population, enriched and established in a defined medium, from a sewage sludge inoculum was capable of complete mineralization of 4-chlorobenzoate. An organism, identified as Arthrobacter sp., was isolated from the consortium and shown to be capable of utilizing 4-chlorobenzoate as the sole carbon and energy source in pure culture. This organism (strain TM-1), dehalogenated 4-chlorobenzoate as the initial step in the degradative pathway. The product, 4-hydroxybenzoate, was further metabolized via protocatechuate. The ability of strain TM-1 to degrade 4-chlorobenzoate in liquid medium at 25°C was improved by the use of continuous culture and repeated sequential subculturing. Other chlorinated benzoates and the parent compound benzoate did not support growth of strain TM-1. An active cell extract was prepared and shown to dehalogenate 4-chloro-, 4-fluoro-, and 4-bromobenzoate. Dehalogenase activity had an optimum pH of 6.8 and an optimum temperature of 20°C and was inhibited by dissolved oxygen and stimulated by manganese (Mn²⁺). Strain improvement resulted in an increase in the specific activity of the cell extract from 0.09 to 0.85 nmol of 4-hydroxybenzoate per min per mg of protein and a decrease in the doubling time of the organism from 50 to 1.6 h.