Influence of Incubation Temperature on the Microbial Reductive Dechlorination of 2,3,4,6-Tetrachlorobiphenyl in Two Freshwater Sediments

We studied the impact of incubation temperatures on the dechlorination of 2,3,4,6-tetrachlorobiphenyl (2346-CB) in two sediments from different climates: polychlorinated biphenyl (PCB)-free sediment from Sandy Creek Nature Center Pond (SCNC) in Athens, Ga., and PCB-contaminated sediment from Woods Pond (WP) in Lenox, Mass. Sediment slurries were incubated anaerobically with 350 (mu)M 2346-CB for 1 year at temperatures ranging from 4 to 66(deg)C. Most of the 2346-CB was dechlorinated between 12 and 34(deg)C in both sediments and, unexpectedly, between 50 and 60(deg)C in WP sediment. This is the first report of PCB dechlorination at thermobiotic temperatures. The data reveal profound differences in dechlorination rate, extent, and products as a function of sediment and temperature. The highest observed rate of dechlorination of 2346-CB to trichlorobiphenyls occurred at 30(deg)C in both sediments, but the rate was higher for WP than for SCNC sediment (46 versus 16 (mu)mol liter(sup-1) day(sup-1)). For SCNC sediment the rate of dechlorination dropped sharply below 30(deg)C, but for WP sediments it was near optimal from 20 to 34(deg)C and then dropped sharply below 20(deg)C. In WP sediment most of the meta chlorines were removed between 8 and 34(deg)C and between 50 and 60(deg)C. para dechlorination was restricted from 18 to 34(deg)C and was optimal at 20(deg)C. ortho dechlorination occurred between 8 and 30(deg)C, with optima around 15 and 27(deg)C, but the extent was highly variable. In SCNC sediment complete meta dechlorination occurred from 12 to 34(deg)C and para dechlorination occurred from 18 to 30(deg)C; both were optimal at 30(deg)C. No ortho dechlorination was observed. Dechlorination products were 246-CB, 236-CB, and 26-CB (both sediments) and 24-CB, 2-CB, and 4-CB (WP sediment). The data suggest that in SCNC sediment similar factors controlled meta and para PCB dechlorination over a broad temperature range (18 to 30(deg)C) but that in WP sediment there were multiple temperature-dependent changes in the factors controlling ortho, meta, and para dechlorination. We attribute the differences observed in the two sediments to differences in their PCB-dechlorinating communities.     

Complete Reductive Dechlorination of 1,2-Dichloropropane by Anaerobic Bacteria

The transformation of 1,2-dichloropropane (1,2-D) was observed in anaerobic microcosms and enrichment cultures derived from Red Cedar Creek sediment. 1-Chloropropane (1-CP) and 2-CP were detected after an incubation period of 4 weeks. After 4 months the initial amount of 1,2-D was stoichiometrically converted to propene, which was not further transformed. Dechlorination of 1,2-D was not inhibited by 2-bromoethanesulfonate. Sequential 5% (vol/vol) transfers from active microcosms yielded a sediment-free, nonmethanogenic culture, which completely dechlorinated 1,2-D to propene at a rate of 5 nmol min(sup-1) mg of protein(sup-1). No intermediate formation of 1-CP or 2-CP was detected in the sediment-free enrichment culture. A variety of electron donors, including hydrogen, supported reductive dechlorination of 1,2-D. The highest dechlorination rates were observed between 20(deg) and 25(deg)C. In the presence of 1,2-D, the hydrogen threshold concentration was below 1 ppm by volume (ppmv). In addition to 1,2-D, the enrichment culture transformed 1,1-D, 2-bromo-1-CP, tetrachloroethene, 1,1,2,2-tetrachloroethane, and 1,2-dichloroethane to less halogenated compounds. These findings extend our knowledge of the reductive dechlorination process and show that halogenated propanes can be completely dechlorinated by anaerobic bacteria.     

Temperature Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of BASF 13.338 (4-Chloro-5-Dimethylamino-2-Phenyl-3(2H)Pyridazinone)

When Triticum vulgare cv HD 2189 seedlings were grown in the presence of 125 micromolar BASF 13.338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone), the rate of electron transport (H₂O → methyl viologen) in chloroplast thylakoids isolated from the treated seedlings was higher (by 50%) as compared to the control at assay temperatures above 30°C. Below 30°C, however, the rate with the treated seedlings was lower than the control rate. The temperature dependence of the rate of photosystem I electron transport (2-6-dichlorophenol indophenol-reduced → methyl viologen) in the treated system was similar to that in the control. At high temperatures (>30°C), with diphenyl carabazide as electron donor, the rates of electron transfer (diphenyl carbazide → methyl viologen) were similar in the treated and in the control thylakoids. Direct addition of BASF 13.338 to the assay mixture for the measurement of rate of electron transport (H₂O → methyl viologen) in the thylakoids isolated from the control plants did not cause any change in the temperature dependence of photosynthetic electron transport. These results suggested that the donor side of photosystem II became tolerant to heat in the treated plants. Chlorophyll a fluorescence emission was monitored continuously in the leaves of control and BASF 13.338 treated wheat seedlings during continuous increase in temperature (1°C per minute). The fluorescence-temperature profile showed a decrease in the fluorescence yield above 55°C; this decrease was biphasic in the control and monophasic in the treated plants.     

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Demonstration of hydrogenase in extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum.

Cell-free extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum were shown to catalyze the hydrogen-dependent reduction of various artificial electron acceptors. The activity of the hydrogenase was optimal at pH 8.5 to 9 and was extremely sensitive to aeration. EDTA did not significantly reduce the liability of the enzymic activity to oxidation (aeration). At 50 degrees C, when both methyl viologen and hydrogen were at saturating concentrations with respect to hydrogenase, the specific activity of cell-free extracts approximated 4 mumol of H2 oxidized per min per mg of protein; fourfold higher specific activities were obtained when benzyl viologen was utilized as an electron acceptor. Activity stains of polyacrylamide gels demonstrated the presence of a single hydrogenase band, suggesting that the catalytic activity in cell extracts was due to a single enzyme. The activity was stable for at least 32 min at 55 degrees C but was slowly inactivated at 70 degrees C. NAD, NADP, flavin adenine dinucleotide, flavin mononucleotide, and ferredoxin were not significantly reduced, but possible reduction of the particulate b-type cytochrome of C. thermoaceticum was observed. NaCl, sodium dodecyl sulfate, iodoacetamide, and CO were shown to inhibit catalysis. A kinetic study is presented, and the possible physiologic roles for hydrogenase in C. thermoaceticum ar discussed.     

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.     

Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate.

Strain Co23, an anaerobic spore-forming microorganism, was enriched and isolated from a compost soil on the basis of its ability to grow with 2,3-dichlorophenol (DCP) as its electron acceptor, ortho chlorines were removed from polysubstituted phenols but not from monohalophenols. Growth by chlororespiration was indicated by a growth yield of 3.24 g of cells per mol of reducing equivalents (as 2[H]) from lactate oxidation to acetate in the presence of 3-chloro-4-hydroxybenzoate but no growth in the absence of the halogenated electron acceptor. Other indicators of chlororespiration were the fraction of electrons from the electron donor used for dechlorination (0.67) and the H2 threshold concentration of < 1.0 ppm. Additional electron donors utilized for reductive dehalogenation were pyruvate, formate, butyrate, crotonate, and H2. Pyruvate supported homoacetogenic growth in the absence of an electron acceptor. Strain Co23 also used sulfite, thiosulfate, and sulfur as electron acceptors for growth, but it did not use sulfate, nitrate or fumarate. The temperature optimum for growth was 37 degrees C; however, the rates of dechlorination were optimum at 45 degrees C and activity persisted to temperatures as high as 55 degrees C. The 16S rRNA sequence was determined, and strain Co23 was found to be related to Desulfitobacterium dehalogenans JW/IU DC1 and Desulfitobacterium strain PCE1, with sequence similarities of 97.2 and 96.8%, respectively. The phylogenetic and physiological properties exhibited by strain Co23 place it into a new species designated Desulfitobacterium chlororespirans.     

Bromate Reduction by Denitrifying Bacteria

In the presence of bromide, ozonation as applied in water treatment results in the formation of bromate, an ion with carcinogenic properties. The reduction of bromate by mixed bacterial populations as well as pure cultures was studied under laboratory conditions. Bromate was reduced to bromide by a mixed bacterial population with and without a preceding nitrate reduction step in an anaerobically incubated medium with ethanol as the energy and carbon source at 20 and 25 deg C. The predominating bacteria isolated from the batches showing bromate reduction were identified as Pseudomonas spp. Strains of Pseudomonas fluorescens reduced BrO(inf3)(sup-) to Br(sup-) but at a much lower rate than the mixed bacterial population did. Nitrate is a preferred electron acceptor for the bromate-reducing bacteria. Bromate reduction did not occur in the presence of NO(inf3)(sup-), and the rate of bromate reduction was at least 100 times lower than the rate of nitrate reduction. Bromate was completely converted to Br(sup-), indicating that intermediates, e.g., BrO(inf2)(sup-), did not accumulate during bromate reduction.     

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.     

Photosynthetic properties of permaplasts of anacystis

A treatment procedure using lysozyme and ethylenediaminetetracetic acid gave intact but permeable cells (permeaplasts) of Anacystis nidulans. Rates of electron transport from water to carbon dioxide, ferricyanide, 2,6-dichlorophenol indophenol, benzoquinone, and methyl viologen, and from reduced indophenol to methyl viologen were measured as a function of treatment time. Rates of oxygen evolution in complete photosynthesis and electron flow from water to methyl viologen showed rapid and parallel decline with treatment time. Electron flow from water to ferricyanide and from reduced indophenol to methyl viologen increased during the first half hour of treatment (phase 1) to 60 to 80% of the original photosynthetic rate. Longer treatment (phase 2) resulted in decreased rate of ferricyanide reduction but not in rate of methyl viologen reduction from indophenol. Electron flow from water to quinone was two to three times higher than for complete photosynthesis in intact cells. It remained high during phase 1 and declined during phase 2. Phase 1 permeaplasts apparently retain high activity for photosystems 1 and 2 photoreactions.     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1.

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Lag phase of CO2-dependent O2 evolution by illuminated Anabaena variabilis cells.

The steady-state rate of CO2-dependent O2 evolution by Anabaena variabilis cells in response to illumination was established after a lag phase. The lag phase was shortened (1) in cells incubated with glucose as an oxidizable substrate and (2) upon an increase in light intensity. The lag phase was absent during electron transfer from H2O to p-benzoquinone (in combination with ferricyanide) involving Photosystem II. A lag was observed during electron transfer from H2O to methyl viologen involving Photosystems II and I, but not for electron transfer from N,N,N',N'-tetramethyl-p-phenylenediamine (in combination with ascorbate) to methyl viologen involving only Photosystem I. The lag phases of the light-induced H2O --> CO2 and H2O --> methyl viologen electron transfer reactions showed the same temperature dependences at 10-30 degrees C. The lag was prevented by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations that caused partial inhibition of photosynthetic O2 evolution. Retardation of cell respiration by a combination of CN- and benzylhydroxamate shortened the lag phase of the H2O --> methyl viologen electron transfer. It is concluded that the lag phase is associated with the electron transfer step between Photosystem II and Photosystem I common for the photosynthetic and respiratory chains and is due to the stimulation of cell respiration during the initial period of illumination as a consequence of an increase in the reduced plastoquinone pool and to subsequent retardation of respiration resulting from the transition of the electron transfer chain to the competitive pathway involving Photosystem I.     

Oxidation of protoporphyrinogen in the obligate anaerobe Desulfovibrio gigas.

The anaerobic oxidation of protoporphyrinogen to protoporphyrin was demonstrated in extracts of Desulfovibrio gigas. Protoporphyrin formation occurred in the presence of nitrite, hydroxylamine, sulfite, thiosulfate, ATP plus sulfate, NAD+, NADP+, flavin adenine dinucleotide, flavin mononucleotide, fumarate, 2,6-dichlorophenol-indophenol, methyl viologen, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. With dialyzed cell extracts, highest activities were observed with sulfite, NAD+, and NADP+ as electron acceptors. The enzyme for protoporphyrinogen oxidation was localized in the membrane of D. gigas and displayed optimal activity at pH 7.3 and 28 degrees C.     

Comparative studies on tetrachloroethene reductive dechlorination mediated by Desulfitobacterium sp. strain PCE-S

Tetrachloroethene reductive dechlorination was studied with cell extracts of a newly isolated, tetrachloroethene-utilizing bacterium, Desulfitobacterium sp. strain PCE-S. Tetrachloroethene dehalogenase mediated the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with artificial electron donors such as methyl viologen. The chlorinated aromatic compounds tested so far were not reduced. A low-potential electron donor (E ₀′ < –0.4 V) was required for tetrachloroethene reduction. The enzyme in its reduced state was inactivated by propyl iodide and reactivated by light, indicating the involvement of a corrinoid in reductive tetrachloroethene dechlorination. Received: 28 April 1997 / Accepted: 11 July 1997     

Fraction of electrons consumed in electron acceptor reduction and hydrogen thresholds as indicators of halorespiratory physiology.

Measurements of the hydrogen consumption threshold and the tracking of electrons transferred to the chlorinated electron acceptor (f(e)) reliably detected chlororespiratory physiology in both mixed cultures and pure cultures capable of using tetrachloroethene, cis-1, 2-dichloroethene, vinyl chloride, 2-chlorophenol, 3-chlorobenzoate, 3-chloro-4-hydroxybenzoate, or 1,2-dichloropropane as an electron acceptor. Hydrogen was consumed to significantly lower threshold concentrations of less than 0.4 ppmv compared with the values obtained for the same cultures without a chlorinated compound as an electron acceptor. The f(e) values ranged from 0.63 to 0.7, values which are in good agreement with theoretical calculations based on the thermodynamics of reductive dechlorination as the terminal electron-accepting process. In contrast, a mixed methanogenic culture that cometabolized 3-chlorophenol exhibited a significantly lower f(e) value, 0.012.     

Reduction of azo dyes by intestinal anaerobes.

Reduction of seven azo dyes (amaranth, Ponceau SX, Allura Red, Sunset Yellow, tartrazine, Orange II, and methyl orange) was carried out by cell suspensions of predominant intestinal anaerobes. It was optimal at pH 7.4 in 0.4 M phosphate buffer and inhibited by glucose. Flavin mononucleotide caused a marked enhancement of azo reduction by Bacteroides thetaiotaomicron. Other electron carriers, e.g., methyl viologen, benzyl viologen, phenosafranin, neutral red, crystal violet, flavin adenine dinucleotide, menadione, and Janus Green B can replace flavin mononucleotide. These data suggest that an extracellular shuttle is required for azo reduction.     

Reduction of azo dyes by intestinal anaerobes.

Reduction of seven azo dyes (amaranth, Ponceau SX, Allura Red, Sunset Yellow, tartrazine, Orange II, and methyl orange) was carried out by cell suspensions of predominant intestinal anaerobes. It was optimal at pH 7.4 in 0.4 M phosphate buffer and inhibited by glucose. Flavin mononucleotide caused a marked enhancement of azo reduction by Bacteroides thetaiotaomicron. Other electron carriers, e.g., methyl viologen, benzyl viologen, phenosafranin, neutral red, crystal violet, flavin adenine dinucleotide, menadione, and Janus Green B can replace flavin mononucleotide. These data suggest that an extracellular shuttle is required for azo reduction.