Anke Neumann

Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing, strictly anaerobic bacterium

A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H2 as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H2, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H2. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160μM), a dechlorination rate of about 50 nmol min-1 (mg cell protein)-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.

Purification and Characterization of Tetrachloroethene Reductive Dehalogenase from Dehalospirillum multivorans

Tetrachloroethene reductive dehalogenase from the tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, was purified approximately 100-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene (PCE) to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor at a specific activity of 2.6 microkatal/mg. The apparent Km values for tetrachloroethene and trichloroethene were 0.20 and 0.24 mM, respectively. The apparent molecular mass of the native enzyme was determined by gel filtration to be 58 kDa. Sodium dodecyl sulfate-gel electrophoresis revealed a single protein band with a molecular mass of 57 kDa. One mol of dehalogenase contained 1.0 mol of corrinoid, 9.8 mol of iron, and 8.0 mol of acid-labile sulfur. The pH optimum was about 8.0. The enzyme had a temperature optimum of 42°C. It was slightly oxygen-sensitive and was thermolabile above 50°C. The dechlorination of PCE was stimulated by ammonium ions. Chlorinated methanes severely inhibited PCE dehalogenase activity.

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 μM PCE initially, pH 7.5, 25°C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 μM 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 μM) 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 μmol (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.

Properties of tetrachloroethene and trichloroethene dehalogenase of Dehalospirillum multivorans

Some properties of tetrachloroethene and trichloroethene dehalogenase of the recently isolated, tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, were studied with extracts of cells grown on pyruvate plus fumarate. The dehalogenase catalyzed the oxidation of reduced methyl viologen with tetrachloroethene (PCE) or trichloroethene (TCE) as electron acceptor. All other artificial or physiological electron donors tested were ineffective. The PCE and TCE dehalogenase activity was insensitive towards oxygen in crude extracts. When extracts were incubated under anoxic conditions in the presence of titanium citrate as reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (50 μM). Inactivation did not occur in the absence of titanium citrate. The activity of propyl-iodide-treated extracts was restored almost immediately by illumination. The dehalogenase was inhibited by cyanide. The inhibition profile was almost the same under oxic and anoxic conditions independent of the presence or absence of titanium citrate. In addition, N2O, nitrite, and ethylene diamine tetra-acetate (EDTA) were inhibitors of PCE and TCE dehalogenase. Carbon monoxide and azide had no influence on the dehalogenase activity. Trans-1,2-dichloroethene or 1,1-dichloroethene, both of which are isomers of the dechlorination product cis-1,2-dichloroethene, neither inhibited nor inactivated the dehalogenase. PCE and TCE dechlorination appeared to be mediated by the same enzyme since the inhibitors tested had nearly the same effects on the PCE and TCE dehalogenating activity. The data indicated the involvement of a corrinoid and possibly of an additional transition metal in reductive PCE and TCE dechlorination.

Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans: substrate specificity of the native enzyme and its corrinoid cofactor

Abstract. The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans and its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B12) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.

A non-dechlorinating strain of Dehalospirillum multivorans: evidence for a key role of the corrinoid cofactor in the synthesis of an active tetrachloroethene dehalogenase

Abstract. A strain of Dehalosprillum multivorans, designated strain N, was isolated from the same source as the formerly described tetrachloroethene (PCE)-dechlorinating D. multivorans, herein after referred to as strain K. Neither growing cells nor cell extracts of strain N were able to dechlorinate PCE. The pceA and pceB genes encoding for the PCE-reductive dehalogenase were detected in cells of strain N; and they were 100% homologous to the corresponding genes of strain K. Since the PCE dehalogenase of D. multivorans strain K contains a corrinoid cofactor, the corrinoids of strain N cells were extracted. Analysis of the corrinoids revealed the absence of the specific corrinoid, which is the cofactor of the PCE dehalogenase of strain K cells. RT-PCR of mRNA indicated that the pceA gene was transcribed in strain N cells to a far lower extent than the pceA gene of strain K under the same experimental conditions. Western blot analysis of crude extracts of strain N showed that, if at all, an insignificant amount of the apoprotein of the PCE dehalogenase was present. The results indicate that the inability of strain N to dechlorinate is due to the absence of the corrinoid cofactor of the enzyme mediating PCE dechlorination.

Growth-Substrate Dependent Dechlorination of 1,2-Dichloroethane by a Homoacetogenic Bacterium

A rod shaped, gram positive, non sporulating Acetobacterium strain was isolated that dechlorinated 1,2-dichloroethane (1,2-DCA) to ethene at a dechlorination rate of up to 2 nmol Cl- min-1 mg-1 of protein in the exponential growth phase with formate (40 mM) as the substrate. Although with other growth substrates such as pyruvate, lactate, H2/CO2, and ethanol higher biomass productions were obtained,the dechlorination rate with these substrates was more than 10-fold lower compared with formate growing cells. Neither cell extracts nor autoclaved cells of the isolatedAcetobacterium strain mediated the dechlorination of 1,2-DCA at significant rates. The addition of 1,2-DCA to the media did not result in increased cell production. No significant differences in corrinoid concentrations could be measured in cells growing on several growth-substrates. However, these measurements indicated that differences in corrinoid structure might cause the different dechlorination activity. The Acetobacterium sp. strain gradually lost its dechlorination ability during about 10 transfers in pure culture, probably due to undefined nutritional requirements. 16S rDNA analysis of the isolate revealed a 99.7% similarity with Acetobacterium wieringae. However, the type strains of A. wieringae and A. woodii did not dechlorinate 1,2-DCA.

The influence of different nitrogen and carbon sources on mycotoxin production in Alternaria alternata.

The aim of this study was to determine the influence of different carbon and nitrogen sources on the production of the mycotoxins alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by Alternaria alternata at 28°C using a semi-synthetic medium (modified Czapek-Dox broth) supplemented with nitrogen and carbon sources. Additionally the effect of shaken and static cultivation on mycotoxin production was tested. Initial experiments showed a clear dependency between nitrogen depletion and mycotoxin production. To assess whether nitrogen limitation in general or the type of nitrogen source triggers the production, various nitrogen sources including several ammonium/nitrate salts and amino acids were tested. In static culture the production of AOH/AME can be enhanced greatly with phenylalanine whereas some nitrogen sources seem to inhibit the AOH/AME production completely. TA was not significantly affected by the choice of nitrogen source. In shaken culture the overall production of all mycotoxins was lower compared to static cultivation. Furthermore tests with a wide variety of carbon sources including monosaccharides, disaccharides, complex saccharides such as starch as well as glycerol and acetate were performed. In shaken culture AOH was produced when glucose, fructose, sucrose, acetate or mixtures of glucose/sucrose and glucose/acetate were used as carbon sources. AME production was not detected. The use of sodium acetate resulted in the highest AOH production. In static culture AOH production was also stimulated by acetate and the amount is comparable to shaken conditions. Under static conditions production of AOH was lower except when cultivated with acetate. In static cultivation 9 of 14 tested carbon sources induced mycotoxin production compared to 4 in shaken culture. This is the first study which analyses the influence of carbon and nitrogen sources in a semi-synthetic medium and assesses the effects of culture conditions on mycotoxin production by A. alternata.

Anaerobic transformation of compounds of technical toxaphene. I. Regiospecific reaction of chlorobornanes with geminal chlorine atoms

Technical toxaphene (Melipax) and the single compounds of technical toxaphene (CTTs) 2,2,5-endo,6-exo,8,8,9,10-octachlorobornane (B8-806), 2,2,5-endo,6-exo, 8,9,9,10-octachlorobornane (B8-809), 2,2,5,5,8,9,9,10,10-nonachlorobornane (B9-1025), 2-endo,3-exo,5-endo,6-exo,8,8,9,10,10-nonochlorobornane (B9-1679), 2-endo,3-exo,5-endo,6-exo,8,9,10,10-octachlorobornane (B8-1414), 2-endo,3-exo,5-endo,6-exo,8,8,9,10-octachlorobornane (B8-1412), and 2-exo,3-endo,5-exo,9,9,10,10-heptachlorobornane (B7-1453) were treated with suspensions of the anaerobic bacterium Dehalospirillum multivorans. After 7 d, more than 50% of technical toxaphene was transformed, and the relative amount of early eluting CTTs increased. After 16 d, only 2-exo,3-endo,6-exo, 8,9,10-hexachlorobornane (B6-923), 2-endo,3-exo,5-endo,6-exo, 8,9,10-heptachlorobornane (B7-1001), and a few minor penta- and hexachloro-CTTs were detected in the samples. The result of the transformation was comparable with observations in naturally contaminated sediments and soil. However, the performance with D. multivorans was more simple and reproducible, as well as faster, than use of soil, sediment, or anaerobic sewage sludge. In agreement with reports in the literature, reductive dechlorination at geminal chlorine atoms (gem-C1s) was found to be the major CTT transformation pathway. Experiments conducted with CTTs and gem-C1s at both primary and secondary carbons clarified that the initial C1 --> H substitution takes place at the secondary carbon C2. Furthermore, the 2-endo-C1 position was preferably substituted with hydrogen. In the case of B8-806, the dechlorination at the secondary carbon C2 was approximately 20-fold faster than the subsequent, slow reduction at the primary carbon C8. The three different formerly unknown heptachloro-CTTs, 2-exo,3-endo,6-exo,8,9,9,10-heptachlorobornane (B7-1473), 2-exo, 3-endo,6-endo,8,9,9,10-hepatchlorobornane (B7-1461), and 2-exo, 3-endo,6-exo,8,8,9,10-heptachlorobornane (B7-1470) were found as intermediates of the B8-806/809 transformation. Treatment of B9-1679 with D. multivorans indicated that gem-C1s on the bridge (C8 and C9) are dechlorinated faster than gem-C1s on the bridgehead (C10).

Non-porous magnetic micro-particles: Comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion

In this study the kinetics of conversion of a low-soluble substrate by an immobilized enzyme was investigated with respect to the diffusion limitation within porous and non-porous carriers. Non-porous micro-magnetic beads in comparison to conventional porous supports like Eupergit and Sepharose were tested. Due to their small diameters and their magnetic properties, micro-magnetic beads are especially applicable in diffusion rate-controlled processes in biological suspensions. The enzymatic reaction studied was the conversion of emulsified dirhamnolipid by immobilized Naringinase from Penicillium decumbens to monorhamnolipid and L-rhamnose. Taking into account mass transfer phenomena, the variation of the reaction effectiveness factor with increasing enzyme loading was estimated and compared with experimental efficiencies utilizing different enzyme loaded immobilized preparations. For comparison, carrier activities were also determined with the model substrate p-nitro-phenyl-rhamnoside. Intrinsic enzyme activities were thereby evaluated for porous supports. Highest specific activities were obtained with the micro-magnetic beads. These non-porous micro-beads demonstrated to be the most suitable carrier for bioconversion of a low-soluble substrate like rhamnolipids, where mass diffusional resistances in the three-phase reaction process are completely overcome. However, the smaller particle surface available limited the specific activity obtained at high protein loadings.