Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by Pseudomonas stutzeri strain JJ.

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.

The potential of anaerobic bacteria to degrade chlorinated compounds.

Chlorinated ethenes and chlorinated aromatics are often found as pollutants in sediments, groundwater, and wastewater. These compounds were long considered to be recalcitrant under anaerobic conditions. In the past years however, dechlorination of these compounds has been found to occur under anaerobic conditions at contaminated sites and in wastewater treatment systems. This dechlorination is mainly attributed to halo-respiring bacteria, which are able to couple this dechlorination to energy conservation via electron transport coupled phosphorylation. The dechlorinating activities of the halo-respiring bacteria seem to be confined to the dechlorination of chloroethenes and chlorinated aromatic compounds. In addition, methanogenic and acetogenic bacteria are also able to reduce the chlorinated ethenes via a-specific cometabolic pathways. Although these latter reactions may not be important in the remediation of contaminated sites, they may be of substantial influence in the start-up of remediation processes and in the application of granular sludge from UASB reactors. Specific halo-respiring bacteria may be used to increase the dechlorination activities via bioaugmentation in the case that the appropriate microorganisms are not present at the contaminated site or in the sludge.

Two distinct enzyme systems are responsible for tetrachloroethene and chlorophenol reductive dehalogenation in Desulfitobacterium strain PCE1.

Desulfitobacterium strain PCE1 is able to use tetrachloroethene and chloroaromatics as terminal electron acceptors for growth. Cell extracts of Desulfitobacterium strain PCE1 grown with tetrachloroethene as electron acceptor showed no dehalogenase activity with 3-chloro-4-hydroxyphenylacetate (Cl-OH-phenylacetate) and other ortho-chlorophenolic compounds in an in vitro assay. Extracts of cells that were grown with Cl-OH-phenylacetate as electron acceptor dechlorinated tetrachloroethene at 10% of the dechlorination rate of Cl-OH-phenylacetate. In both cell extracts dechlorination was inhibited by the addition of 1-iodopropane and dinitrogen oxide, inhibitors of cobalamin-containing enzymes. The enzymes responsible for tetrachloroethene and Cl-OH-phenylacetate dechlorination were partially purified. A 100-fold enriched fraction of chlorophenol reductive dehalogenase was obtained that mainly contained a protein with a subunit size of 48 kDa. The characteristics of this enzyme are similar to that of the chlorophenol reductive dehalogenase of D. dehalogenans. After partial purification of the tetrachloroethene reductive dehalogenase, a fraction was obtained that also contained a 48-kDa protein, but the N-terminal sequence showed no similarity with that of the chlorophenol reductive dehalogenase sequence or with the N-terminal amino acid sequence of tetra- and trichloroethene reductive dehalogenase of Desulfitobacterium strain TCE1. These results provide strong evidence that two different enzymes are responsible for tetrachloroethene and chlorophenol dechlorination in Desulfitobacterium strain PCE1. Furthermore, the characterization of partially purified tetrachloroethene reductive dehalogenase indicated that this enzyme is a novel type of reductive dehalogenase.

Energy yield of respiration on chloroaromatic compounds in Desulfitobacterium dehalogenans.

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with 13C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO2. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.

A Desulfitobacterium strain isolated from human feces that does not dechlorinate chloroethenes or chlorophenols.

An anaerobic bacterium, strain DP7, was isolated from human feces in mineral medium with formate and 0.02% yeast extract as energy and carbon source. This rod-shaped motile bacterium used pyruvate, lactate, formate, hydrogen, butyrate, and ethanol as electron donor for sulfite reduction. Other electron acceptors such as thiosulfate, nitrate and fumarate stimulated growth in the presence of 0.02% yeast extract and formate. Acetate was the only product during fermentative growth on pyruvate. Six mol of pyruvate were fermented to 7 mol of acetate. 13C-NMR labeling experiments showed homoacetogenic 13C-CO2 incorporation into acetate. The pH and temperature optimum of fermentative growth on pyruvate was 7.4 and 37 degrees C, respectively. The growth rate under these conditions was approximately 0.10 h(-1). Strain DP7 was identified as a new strain of Desulfitobacterium frappieri on the basis of 16S rRNA sequence analysis (99% similarity) and DNA-DNA hybridization (reassociation value of 83%) with Desulfitobacterium frappieri TCE1. In contrast to described Desulfitobacterium strains, the newly isolated strain has not been isolated from a polluted environment and did not use chloroethenes or chlorophenols as electron acceptor.

Constitutive dechlorination of chlorinated ethenes by a methanol degrading methanogenic consortium.

The ability of granular methanogenic sludge to dechlorinate chloroethenes was investigated with unadapted sludge from an upflow anaerobic sludge blanket (UASB) reactor fed with methanol. The sludge degraded chlorinated ethenes, but the degradation rates were low. The addition of primary substrate was necessary to sustain dechlorination. The dechlorinating activity seemed to be constitutively present in the anaerobic bacteria. Usually, one chlorine atom was removed via reductive hydrogenolysis. Only trichloroethene (TCE) was converted to substantial amounts of vinylchloride (VC). 1,1-Dichloroethene (1,1DCE) was observed to be an important intermediate in the dechlorination by unadapted granular sludge, although previously this compound had not been commonly observed. Furthermore, the dechlorination of 1,1DCE was faster than the dechlorination of the other chloroethenes.

Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.

Reductive dechlorination of tetrachloroethene to cis-1, 2-dichloroethene by a thermophilic anaerobic enrichment culture.

Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis-1, 2-dichloroethene at the optimum temperature of 60 to 65 degrees C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H2, formate, or acetate. Fumarate and L-malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H2, CO2, and succinate. With PCE, less H2 was formed, suggesting that PCE competed for the reducing equivalents leading to H2. PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of approximately 60 microM, a high dechlorination rate of 1.1 micromol h-1 mg-1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus, and with the genus Desulfotomaculum (86%).

Degradation and Fate of Carbon Tetrachloride in Unadapted Methanogenic Granular Sludge.

The potential of granular sludge from upflow anaerobic sludge blanket (UASB) reactors for bioremediation of chlorinated pollutants was evaluated by using carbon tetrachloride (CT) as a model compound. Granular sludges cultivated in UASB reactors on methanol, a volatile fatty acid mixture, or sucrose readily degraded CT supplied at a concentration of 1,500 nmol/batch (approximately 10 µM) without any prior exposure to organohalogens. The maximum degradation rate was 1.9 µmol of CT g of volatile suspended solids-1 day-1. The main end products of CT degradation were CO2 and Cl-, and the yields of these end products were 44 and 68%, respectively, of the initial amounts of [14C]CT and CT-Cl. Lower chlorinated methanes accumulated in minor amounts temporarily. Autoclaved (dead) sludges were capable of degrading CT at rates two- to threefold lower than those for living sludges, indicating that abiotic processes (mediated by cofactors or other sludge components) played an important role in the degradation observed. Reduced components in the autoclaved sludge were vital for CT degradation. A major part (51%) of the CT was converted abiotically to CS2. The amount of CO2 produced (23%) was lower and the amount of Cl- produced (86%) was slightly higher with autoclaved sludge than with living sludge. Both living and autoclaved sludges could degrade chloroform. However, only living sludge degraded dichloromethane and methylchloride. These results indicate that reductive dehalogenation, which was mediated better by living sludge than by autoclaved sludge, is only a minor pathway for CT degradation. The main pathway involves substitutive and oxidative dechlorination reactions that lead to the formation of CO2. Granular sludge, therefore, has outstanding potential for gratuitous dechlorination of CT to safe end products.

Enrichment and properties of a 1,2,4-trichlorobenzene-dechlorinating methanogenic microbial consortium.

A methanogenic microbial consortium capable of reductively dechlorinating 1,2,4-trichlorobenzene (1,2,4-TCB) was enriched from a mixture of polluted sediments. 1,2,4-TCB was dechlorinated via 1,4-dichlorobenzene (1,4-DCB) to chlorobenzene (CB). Lactate, which was used as an electron donor during the enrichment, was converted via propionate and acetate to methane. Glucose, ethanol, methanol, propionate, acetate, and hydrogen were also suitable electron donors for dechlorination, whereas formate was not. The addition of 5% (wt/vol) sterile Rhine River sand was necessary to maintain the dechlorinating activity of the consortium. The addition of 2-bromoethanesulfonic acid (BrES) inhibited methanogenesis completely but had no effect on the dechlorination of 1,2,4-TCB. The consortium was also able to dechlorinate other chlorinated benzenes via various simultaneous pathways to 1,3,5-TCB, 1,2-DCB, 1,3-DCB, or CB as an end product. The addition of BrES inhibited several of the simultaneously occurring dechlorination pathways of 1,2,3,4- and 1,2,3,5-tetrachlorobenzene and of pentachlorobenzene, which resulted in the formation of CB as the only final product. Hexachlorobenzene and polychlorinated biphenyls (PCBs) were dechlorinated after a lag phase of ca. 15 days, showing a dechlorination pattern that is different from those observed for lower chlorinated benzenes: only chlorines with two adjacent chlorines were removed. The results show that the consortium possesses at least three distinct dechlorination activities toward chlorinated benzenes and PCBs.

Measurement of minimum substrate concentration (Smin) in a recycling fermentor and its prediction from the kinetic parameters of Pseudomonas strain B13 from batch and chemostat cultures.

The minimum substrate concentration required for growth, Smin, was measured for Pseudomonas sp. strain B13 with 3-chlorobenzoate (3CB) and acetate in a recycling fermentor. The substrates were provided alone or in a mixture. Smin values predicted with kinetic parameters from resting-cell batches and chemostat cultures differed clearly from the values measured in the recycling fermentor. When 3CB and acetate were fed as single substrates, the measured Smin values were higher than the individual Smin values in the mixture. The Smin in the mixture reflected the relative energy contributions of the two substrates in the fermentor feed. The energy-based maintenance coefficients during zero growth in the recycling fermentor were comparable for all influent compositions (mean +/- standard deviation, 0.34 +/- 0.07 J mg [dry weight]-1 h-1). Maintenance coefficient values for acetate were significantly higher in chemostat experiments than in recycling-fermentor experiments. 3CB maintenance coefficients were comparable in both experimental systems. The parameters for 3CB consumption kinetics varied remarkably with the experimental growth conditions in batch, chemostat, and recycling-fermentor environments. The results demonstrate that the determination of kinetic parameters in the laboratory for prediction of microbial activity in complex natural systems should be done under conditions which best mimic the system under consideration.

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations.

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.

Rhodococcus percolatus sp. nov., a bacterium degrading 2,4,6-trichlorophenol.

A bacterial strain that was able to mineralize 2,4,6-trichlorophenol was isolated from a chlorophenol-fed percolator and was identified as a member of the genus Rhodococcus on the basis of chemotaxonomic characteristics and 16S RNA phylogenetic inference data. This organism (strain MBS1T [T = type strain]) exhibited a typical irregular rod-coccus cycle, and the cells had fimbria-like structures on their surfaces. The diagnostic cell wall amino acid was meso-diaminopimelic acid, and the sugars were arabinose and galactose; the mycolic acids contained 46 to 54 carbon atoms. The main menaquinone was MK-8(H2), and MK-9(H2) was a minor component. The cellular phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside, phosphatidylglycerol, and diphosphatidylglycerol. Tuberculostearic acid was present. The whole-cell fatty acids were straight-chain acids with 14 to 18 C atoms. The G+C content of the DNA was 67.4 mol%. This organism grew on sucrose, pyruvate, and 2,4,6-trichlorophenol, and it oxidized a large number of carbon compounds, including catechol, 3-hydroxyphenylacetic acid, and phenol. It also exhibited beta-galactosidase, urease, and 2-acetyl-lactate decarboxylase activities. On a phylogenetic tree that was based on 16S ribosomal DNA gene sequences strain MBS1T was found among the rhodococci on an independent branch. On the basis of the chemotaxonomic and phenotypic characteristics of strain MBS1T and its phylogenetic position we suggest that this bacterium should be placed in a new species, Rhodococcus percolatus; the specific epithet was chosen because the organism was isolated by using an enriched percolator. The type strain is strain MBS1.

Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia.

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm. Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.

Physiological meaning and potential for application of reductive dechlorination by anaerobic bacteria.

The physiological meaning of reductive dechlorination reactions catalyzed by anaerobic bacteria can be explained as a co-metabolic activity or as a novel type of respiration. Co-metabolic activities have been found mainly with alkyl halides. They are non-specific reactions catalyzed by various enzyme systems of facultative as well as obligate anaerobic bacteria. In contrast, the reductive dechlorinations involved in metabolic respiration processes are very specific reactions. Only a limited number of alkyl and aryl chlorinated compounds is presently known to function as a terminal electron acceptor in a few, recently isolated bacteria. Metabolic dechlorination rates are in general several orders of magnitude higher than co-metabolic ones. Both reaction types are suitable for the anaerobic treatment of waste streams.

A highly purified enrichment culture couples the reductive dechlorination of tetrachloroethene to growth.

A microscopically pure enrichment culture of a gram-negative anaerobic bacterium, in the present article referred to as PER-K23, was isolated from an anaerobic packed-bed column in which tetrachloroethene (PCE) was reductively transformed to ethane via trichloroethene (TCE), cis-1,2-dichloroethene (cis-1,2-DCE), chloroethene, and ethene. PER-K23 catalyzes the dechlorination of PCE via TCE to cis-1,2-DCE and couples this reductive dechlorination to growth. H2 and formate were the only electron donors that supported growth with PCE or TCE as an electron acceptor. The culture did not grow in the absence of PCE or TCE. Neither O2, NO3-, NO2-, SO4(2-), SO3(2-), S2O3(2-), S, nor CO2 could replace PCE or TCE as an electron acceptor with H2 as an electron donor. Also, organic electron acceptors such as acetoin, acetol, dimethyl sulfoxide, fumarate, and trimethylamine N-oxide and chlorinated ethanes, DCEs, and chloroethene were not utilized. PER-K23 was not able to grow fermentatively on any of the organic compounds tested. Transferring the culture to a rich medium revealed that a contaminant was still present. Dechlorination was optimal between pH 6.8 and 7.6 and a temperature of 25 to 35 degrees C. H2 consumption was paralleled by chloride production, PCE degradation, cis-1,2-DCE formation, and growth of PER-K23. Electron balances showed that all electrons derived from H2 or formate consumption were recovered in dechlorination products and biomass. Exponential growth could be achieved only in gently shaken cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum delta H catalyzes the reductive dechlorination of 1,2-dichloroethane to ethylene and chloroethane.

Reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene and chloroethane (CA) by crude cell extracts of Methanobacterium thermoautotrophicum delta H with H2 as the electron donor was stimulated by Mg-ATP. The heterodisulfide of coenzyme M (CoM) and 7-mercaptoheptanoylthreonine phosphate together with Mg-ATP partially inhibited ethylene production but stimulated CA production compared Mg-ATP alone. The pH optimum for the dechlorination was 6.8 (at 60 degrees C). Michaelis-Menten kinetics for initial product formation rates with different 1,2-DCA concentrations indicated the enzymatic character of the dechlorination. Apparent Kms for 1,2-DCA of 89 and 119 microM and Vmaxs of 34 and 20 pmol/min/mg of protein were estimated for ethylene and CA production, respectively. 3-Bromopropanesulfonate, a specific inhibitor for methyl-CoM reductase, completely inhibited dechlorination of 1,2-DCA. Purified methyl-CoM reductase, together with flavin adenine dinucleotide and a crude component A fraction which reduced the nickel of factor F430 in methyl-CoM reductase, converted 1,2-DCA to ethylene and CA with H2 as the electron donor. In this system, methyl-CoM reductase was also able to transform its own inhibitor 2-bromoethanesulfonate to ethylene.

Evidence for the involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri.

Cobalamin and the native and diepimeric forms of factor F430 catalyzed the reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene or chloroethane (CA) in a buffer with Ti(III) citrate as the electron donor. Ethylene was the major product in the cobalamin-catalyzed transformation, and the ratio of ethylene to CA formed was 25:1. Native F430 and 12,13-di-epi-F430 produced ethylene and CA in ratios of about 2:1 and 1:1, respectively. Cobalamin dechlorinated 1,2-DCA much faster than did factor F430. Dechlorination rates by all three catalysts showed a distinct pH dependence, correlated in a linear manner with the catalyst concentration and doubled with a temperature increase of 10 degrees C. Crude and boiled cell extracts of Methanosarcina barkeri also dechlorinated 1,2-DCA to ethylene and CA with Ti(III) citrate as the reductant. The catalytic components in boiled extracts were heat and oxygen stable and had low molecular masses. Fractionation of boiled extracts by a hydrophobic interaction column revealed that part of the dechlorinating components had a hydrophilic and part had a hydrophobic character. These chemical properties of the dechlorinating components and spectral analysis of boiled extracts indicated that corrinoids or factor F430 was responsible for the dechlorinations. The ratios of 3:1 to 7:1 of ethylene and CA formed by cell extracts suggested that both cofactors were concomitantly active.