Tetrachloroethene metabolism of Dehalospirillum multivorans

Arch Microbiol (1994) 162:295-301 ORIGINAL

9 Springer-Verlag 1994

PAPER

Anke Neumann 9 Heidrun Scholz-Muramatsu Gabriele Diekert

Tetrachloroethene metabolism of Dehalospirillum multivorans

Received: 6 May 1994 / Accepted: 15 June 1994

A b s t r a c t 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. muItivorans. 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 g M PCE initially, pH 7.5, 25 ~ C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 g 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 gM) 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 gmol (chloride released) rain q (rag protein), q An abiotic reductive dehalogenation could be excluded since cell extracts heated for 10 min at 95~ were inactive. The PCE dehalogenase was recovered in the soluble cell fraction after ultracentrifugation. The enzyme was not inactivated by oxygen. K e y w o r d s DehalospiriIIum multivorans Perchloroethylene 9 Tetrachloroethene - Tetrachloroethene dehalogenase 9 Trichloroethene 9 Dichloroethene Reductive dechlorination

A. Neumann - G. Diekert (N~) Institut ffir Mikrobiologie, Universit/it Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany Tel. + 49 - (0) 711- 68554 83; Fax + 49- (0) 71 l- 6 85 57 25 e-mail: gabriele.diekert @po.uni-stuttgart.de H. Scholz-Muramatsu Institut fiir Siedlungswasserbau, Universit~it Stuttgart, Bandtgle 1, D-70569 Stuttgart, Germany

A b b r e v i a t i o n s PCE Perchloroethylene or tetrachloroethene 9 TCE Trichloroethene 9 DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon MV Methyl viologen

Introduction Tetrachloroethene (perchloroethylene; PCE) is a chlorinated hydrocarbon that has been (and still is) frequently applied in dry cleaning in the textile industry, in the scouring of machines, and in fat extraction. Therefore, it is an abundant pollutant of soil, groundwater, and the atmosphere. Since PCE is not easily combustible, bioremediation of contaminated environments has been suggested. PCE is persistent under aerobic conditions since it cannot be oxidized. Therefore, the complete microbial degradation of PCE has to be initiated by anaerobic bacteria. Anaerobic mixed cultures have been described that perform reductive dechlorination of PCE to ethene (Freedman and Gossett 1989; DiStefano et al. 1992) or ethane (DeBmin et al. 1992) via trichloroethene (TCE), cis-l,2dichloroethene (DCE), and vinylchloride (VC) as intermediates. A highly enriched culture has recently been reported to catalyze the reductive dechlorination of PCE to DCE with H: or formate as electron donor and to couple this reaction to growth (Holliger et al. 1993). Most recently a pure culture of a PCE-dehalogenating anaerobic bacterium was obtained (Scholz-Muramatsu et al., submitted). The organism, Dehalospirillum multivorans, is a gram-negative, anaerobic spirillum able to grow in a defined mineral medium with H2 or formate plus PCE as energy sources and acetate as carbon source. Alternatively, the organism utilized a variety of organic electron donors and fumarate or nitrate as electron acceptor. Growth of the organism was stimulated by yeast extract, resulting in higher growth yields rather than higher growth rates. D. multivorans dechlorinated PCE via TCE to DCE. The present communication describes some features of the dehalogenation reaction in whole cells and in cell extracts of the bacterium.

296

Materials and methods Source of materials All chemicals used were of the highest available purity and purchased from Boehringer (Mannheim, Germany), Fluka (Neu-Ulm, Germany), and Merck (Darmstadt, Germany). Tetrachloroethene was from Ferak (Berlin, Germany), cis-l,2-dichloroethene from Aldrich (Steinheim, Germany). Gases (CO2 grade 4.8 and N2 grade 4.6) were supplied by Messer Griesheim (Dtisseldorf, Germany).

Growth of the bacteria

Dehalospirillum multivorans was isolated with pyruvate plus tetrachloroethene from activated sludge as described elsewhere (Scholz-Muramatsu et al., submitted). The bacteria were routinely grown in anaerobic m e d i u m containing pyruvate (40 rnM) plus fumarate (40 raM) as energy sources. The medium was composed of 1000 ml basal medium, 0.5 ml vitamin solution, 2 ml trace element solution SL10, 1 ml B12 solution (cyanocobalamin, 50 mg/1), 0.2 ml selenite solution (Na2SeO 3 9 5H20, 26 mg/1), 0.1 ml tungstate solution (Na2Wo 4 - 2H20, 33 mg/1), 30 ml NaHCO3 solution (84 gO; autoclaved separately under CO2), and 20 ml yeast extract solution (yeast extract from Oxoid, Wesel, Germany, 100 g/l). Sulfide solution (4 ml; NazS 9 9 H a t , 120 g/l; autoclaved separately under N2) were then added. Pyruvate and fumarate were sterilized by filtration in 1 M solutions and added later to the medium to give a final concentration of 40 m M each. The basal medium contained per liter: 70 mg Na2SO4, 200 mg KH2PO4, 250 mg NH4C1, 1,000 mg NaC1, 400 mg MgC12 - 6H20, 500 m g KC1, 150 mg CaC12 - 2H;O. The trace element solution was composed of 1,000 ml H20, 10 ml HC1 25% (w/v), 1 g FeSO 4 9 7H20, 70 mg ZnClz, 100 mg MnC12 - 4H20, 6 mg H3BO3, 130 mg CaC12 9 6H20, 2 m g CuC12 - 2H20, 24 m g NiC12 9 6H20, and 36 m g N a 2 M o O 4 . 2 H 2 0 . The vitamin solution contained per liter: 80 m g 4-aminobenzoic acid, 20 mg D(+)biotin, 200 mg nicotinic acid, 100 mg Ca-D(+)pantothenate, 300 rag/1 pyridoxamine 9 2 HC1, and 200 mg thiamine - HC1. The final pH of the medium was between 7.2 and 7.4. The bacteria were grown in rubber-stoppered glass bottles. The gas phase was 80% N2/20% CO2 (150 kPa). The medium was inoculated with 10% of a grown culture and incubated at 30~ and 200 rpm in a gyratory water bath shaker. The bacteria grew within less than 24 h to cell densities (AE57s) of near 0.7, corresponding to approximately 175 mg cell protein per liter. Under the conditions chosen, the ability of the organisms to dechlorinate PCE was not lost even after numerous transfers in PCE-free medium.

Experiments with cell suspensions and cell extracts In the late exponential growth phase, the bacteria were harvested anaerobically under a N2 gas phase by centrifngation at 3,300 x g and 4 ~ C for 20 min. For experiments with cell suspensions, bacteria were resuspended in anaerobic assay buffer containing 100 m M 3-morpholinopropane sulfonic acid (Mops), 0.4 m M cysteine, and 16 g M resazurin. Routinely, the pH was adjusted to 7.5. The assays were conducted in 35-ml serum bottles stoppered with Teflon-lined butyl rubber septa. The day before the experiment, the bottles were filled with 20 ml assay buffer (see above) and gassed with N2 (110 kPa). To the bottles, 1 gl tetrachloroethene was added, and the bottles were incubated overnight at 25~ and 300 rpm in a gyratory shaker. Prior to the start of the experiment by the addition of 1 ml freshly prepared anaerobic cell suspension, the assay mixture was supplemented with the components indicated in the Results section. The bottle was then filled with buffer to a volume of 24 ml. The bottles were incubated at 300 rpm and 25~ At the times indicated, samples were taken from the liquid phase and analyzed for tetrachloroethene, trichloroethene and dichloroethene as described below.

The cell suspension activites given in this paper are calculated as amount of CI- released per min. It was determined from the amount of tetrachloroethene (PCE) consumed and trichloroethene (TCE) formed or consumed, respectively, according to the following equation: v = (2 x A[PCE]consumed+ A[TCE])/At (A[PCE] ........ d = [PCE]t = tl - [PCE]t = t2; A[TCE] = [TCEJt = tt - [TCE]~ = t2) Cell extracts were prepared from freshly harvested cells. The cells (1 g wet weight) were resuspended in 1 ml anaerobic 50 m M TrisHC1 (pH 7.5) containing 5 m M dithiothreitol. Lysozyme (10 rag) and DNase I (1 rag) were added to the suspension, which was subsequently incubated for 30 rain at 37 ~ C. Cell debris was removed by eentrifugation at 10,000 x g for 10 rain at 4~ The supernatant was stored under N~ at 0 ~ C in a rubber-stoppered brown serum bottle until used. For investigations on the localization of tetrachloroethene dehalogenase and fumarate reductase, 1.5 g (wet weight) of cells grown with pyruvate plus fumarate plus yeast extract were harvested anaerobically by centrifugation and immediately suspended in 3.5 ml buffer [5 m M Tris-HC1 (pH 7.5) containing 5 m M dithiothreitol and 1 m M EDTA]. To the cell suspension, 10 mg lysozyme was added. After incubation for 1 h at 37~ 10 m M MgC12 and 1 mg DNAse I were added. The suspension was incubated for 10 min at room temperature and centrifuged for 10 rain at 10,000 x g and 4 ~ C. The supernatant (cell extract) was then centrifuged for 60 min at 150,000 x g and 4~ The pellet was resuspended in 3 ml 5 m M Tris-HC1 (pH 7.5) plus 5 m M dithiothreitol. The activities of tetrachloroethene dehalogenase and of fumarate reductase were determined in the cell extract, the supernatant (soluble fraction) and the resuspended pellet (particulate fraction). The procedure was carried out under anaerobic conditions.

Analytical procedures Protein was determined essentially according to Bradford (1976) using the Bio-Rad reagent (Bio-Rad, Munich, Germany). The reagent was diluted 2 : 5 with quartz-distilled H a t , and 45 gl HNO 3 was added prior to the addition of the sample (see below). For determination of the protein content of cell suspensions, 100 gl 0.2 M NaOH was added to 400 gl samples and the suspensions were incubated at 100~ for 5 min. After cooling at 0 ~ C, the suspensions were centrifuged for 1 rain at 13,000 x g in an Eppendorf centrifuge. Supernatant (455 btl) was added to 500 gl of the modified reagent (see above). The absorption of the solution was immediately measured at 620 nm and 465 nm; the ratio E620/E465 was plotted versus the protein concentration. An albumin solution (concentrate for standard curve preparation with protein assays, Pierce, Rockford, Ill.) was used as a standard. Tetrachloroethene, trichloroethene, and cis-l,2-dichloroethene were determined gas cgromatographicaIIy by flame ionization detection using a 2 m 10% Ucon LB on W A W column ( W G A Analysentechnik, Dtisseldorf, Germany) and N2 as carrier gas. The following temperatures were applied: column, 80~ injector, 150~ detector, 250~ The carrier gas flow was 2 5 - 3 0 ml/min (300 kPa), the gas sample volume was 500 [xl. Under the conditions chosen, the retention times were: PCE, 2.76 min; TCE, 1.76 min; cis-l,2-DCE, 1.2 min; trans-l,2-DCE, 0.8 min; 1,1-DCE, 1.0 min. Standard solutions of PCE, TCE, or DCE were prepared by adding the chlorinated hydrocarbon (CHC) to H20 (10 gl CHC/1 H2 O) and stirring overnight. These standard solutions were then treated as described below for the samples. For the analysis of the chlorinated hydrocarbons in cell suspensions or cell extracts, 1 ml samples were taken from the liquid phase and added with a syringe to 10 ml serum bottles stoppered with Teflon-lined butyl rubber septa and containing 1 g Na2SO4. The serum bottles were incubated for 1 h at 95~ Then 0.5 ml samples were taken from the gas phase, which contained the chlorinated hydrocarbons, and analyzed immediately for tetrachloroethene, trichloroethene, and dichloroethene.

297 Under the conditions chosen for the experiments with cell suspensions (25 ~C, 25 ml liquid phase, 10 ml gas phase, 110 kPa), the amount of the chlorinated hydrocarbons in the liquid phase related to the total amount was 77% for tetrachloroethene, 87% for trichloroethene, and 95% for cis-l,2-dichloroethene. The coefficient strongly depended on the temperature. The dehalogenase activity in cell extracts was determined photometrically in rubber-stoppered 1 ml glass cuvettes by measuring the oxidation of reduced methyl viologen (MV) with tetrachloroethene (PCE) or trichloroethene (TCE) as electron acceptors: 1 PCE + 2 MV+r~d+ 1 H + -+ 1 TCE + 2 MV2+ox+ 1 CI1 TCE + 2 MV+r~d+ 1 H+ --> 1 DCE + 2 MVZ+ox + 1 C1Methyl viologen was either reduced enzymatically with formate dehydrogenase present in the cell extracts using limiting concentrations of formate or chemically with dithionite. Routinely, the assay buffer was 100 mM Tris-HCl (pH 8.0) containing 10 mM methyl viologen, and the reaction was carried out at 25~ Assay buffer (1 ml) was added to anaerobic rubber-stoppered cuvettes (gas phase 100% N2, 120 kPa) with a syringe. Where indicated, formate was added, so that at the end of the methyl viologen reduction in the formate dehydrogenase reaction, the extinction at 578 n m (~578 for MV: 9.7 mM I cm-t) did not exceed 3. The reaction was either started by the addition of the chlorinated hydrocarbon (about 1 gl per cuvette) or of cell extract. The dehalogenase activity is again given as C1- released per rain as calculated here from the rate of methyl viologen oxidation. For the studies on the localization of the PCE dehalogenase and the fumarate reductase, the enzymic activities were determined photometrically at 25~ by the oxidation of reduced methyl viologen with PCE or fumarate, respectively, as electron acceptor. The assay buffer (lml in a rubber-stoppered glass cuvette) was 100 mM Tris-HC1 (pH 7.5) containing 1 mM methyl viologen. To reduce methyl viologen, an anaerobic dithionite solution was added until an absorption at 578 nm of near 3 was measured. Then 20 ~1 extract, soluble fraction, or particulate fraction was added. The reaction was started by the addition of either 10 p,1 of an ethanolic PCE solution (4 gl PCE per ml ethanol) or 10 gl of a 1 M fumarate stock solution to the assay. The final concentrations were 390 gM PCE or 10 mM fumarate.

Results

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Fig. 1 A Kinetics of tetrachloroethene (PCE, 0) dechlorination via trichloroethene (TCE, 4~) to cis-l,2-dichloroethene (DCE, II) and B dependence on the PCE dechlorination rate on the initial PCE concentration in cell suspensions of DehaIospirillum muItivorans. The incubation was carried out at pH 7.5 at 25 ~ C. The cells were grown in the absence of sulfide. The cell concentration corresponded to a cell protein concentration of approximately 0.25 mg/ml. For further experimental details see 'Materials and methods' (CHC Chlorinated hydrocarbon PCE, TCE, or DCE)

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Fig. 2 A Temperature- and B pH-dependence of PCE dechlorination in cell suspensions of Dehalospirillum multivorans. The rates were determined from the initial rates of PCE consumption and TCE formation; the initial PCE concentration was 300 gM. The temperature-dependence assay was carried out at pH 7.5, the pHdependence at 25 ~C. The cell protein concentration corresponded to 0.19 mg cell protein/ml

Dechlorination of tetrachloroethene in whole cells Cell suspensions of Dehalospirillum multivorans were incubated with 20 m M pyruvate as electron donor and about 300 ~tM tetrachloroethene as electron acceptor. The org a n i s m s dechlorinated tetrachloroethene via trichloroethene to cis-1,2-dichloroethene, as s h o w n by the kinetics in Fig. 1 A. The dechlorination rate increased linearly with the cell c o n c e n t r a t i o n (data not shown) and was dependent on the tetrachloroethene concentration (Fig. 1 B). The rate increased c o n t i n u o u s l y up to 300 g M tetrachloroethene, Higher concentrations were inhibitory. Therefore, for most of the experiments with whole ceils, the concentration of tetrachloroethene initially applied did not exceed 300 ~tM. Specific rates b e t w e e n 20 and 150 n m o l (C1- released) rain -1 (mg cell protein) < were usually obtained. The rates often depended on the culture conditions. Sometimes low rates were obtained with cells that had b e e n grown in the presence of NaaS (see below). Tetrachloroethene was also dechlorinated with alternative electron donors such as formate and H2; no dechlori-

nation was observed with succinate or acetate (data not shown). The temperature o p t i m u m of the dechlorination reaction ranged from 25 to 3 7 ~ ( F i g . 2 A ) . Dechlorination was completely inhibited at 42 ~ C. The P C E reduction exhibited a narrow pH o p t i m u m b e t w e e n 7.0 and 7.5 (Fig. 2B). A t p H 6.0 or 8.5, almost no dechlorination was observed.

Influence of potential alternative electron acceptors and of inhibitors

D. multivorans can utilize either tetrachloroethene or fumarate as an electron acceptor for metabolic oxidation reactions. Therefore, the influence of fumarate on the dechlorination reaction was tested in growing cultures of D. multivorans. The bacteria were incubated either with pyruvate (20 raM), with pyruvate plus fumarate (20 raM), pyruvate plus succinate (20 mM), or without any electron donor in the presence of about 150 g M tetrachloroethene.

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Fig. 3 Effect of fumarate (Fum) on PCE dechlorinadon with pyrurate (Pyr) as electron donor in growing cultures (+Pyr +Fum, ~). The cells were incubated at pH 7.3 and 25 ~ C. In control experiments, incubations were performed with succinate (+Pyr +Succ, i ) as the product of fumarate reduction, in the absence of fumarate and succinate (+Pyr, 0) or in the absence of pyruvate, fumarate, and succinate (-Pyr, A), under which conditions no growth was observed. The initial concentrations of pyruvate, fumm'ate, and succinate were 20 raM. The inoculum (10%) was grown with pyruvate as sole energy source

The kinetics of tetrachloroethene consumption are shown in Fig. 3. Obviously, fumarate inhibited the dechlorination almost completely under the conditions applied. Only very low amounts of trichloroethene and almost no dichloroethene were formed. Succinate, which was the product of fumarate reduction (data not shown), did not have an inhibitory effect (Fig. 3). In the absence of pyruvate no dechlorination occurred. The inhibitory effect of fumarate could be reproduced in cell suspensions, although the effect was less pronounced (about 50% inhibition at a fumarate concentration of 40 mM). Until now, only fumarate, tetrachloroethene (or trichloroethene), and nitrate have been definitely known to serve as electron acceptors for D. multivorans (H. ScholzMuramatsu et al., submitted). However, the presence of elemental sulfur as an electron acceptor in growing cultures often resulted in decreased turbidity of the culture medium, indicating that sulfur might be reduced with pyruvate as electron donor. Moreover, it was sometimes observed that in cells grown in the presence of sulfide, the dechlorination activity was very low when sulfur was formed in the medium. Therefore, the influence of polysulfide (to replace the sulfur, which is almost insoluble in HaO) on the dechlorination activity was studied. Polysulfide significantly inhibited the dechlorination, depending on the concentration applied. In the presence of 5 m M polysulfide, almost no dechlorination was observed; with 1 m M polysulfide, the activity was approximately 15% of that of the control. Sulfate, which was not utilized by the organism, had not significant influence. Corrinoids and corrinoid proteins have been demonstrated to mediate the abiotic dechlorination of chlorinated ethenes at very low rates (Gantzer and Wackett 1991; Jablonski and Ferry 1992). It is therefore conceiv-

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Fig.4 Effect of propyl iodide on the reductive dechlorination of PCE in cell suspensions of Dehalospirillum multivorans. The cell concentration corresponded to a protein concentration of 0.22 mg protein/ml. For further experimental detail, see 'Materials and methods'. No propyl iodide; ~ plus 10 HM propyl iodide; 9 plus 50 g M propyl iodide

able that the dehalogenating enzyme system might contain a corrinoid as cofactor. Since many corrinoid enzymes are known to be inhibited or inactivated by alkylation with propyl iodide, the influence of this alkylating agent on the dechlorinadon of tetrachloroethene was tested. The inhibitor was dissolved in 2-propanol. Propyl iodide (_> 50 gM) almost completely inhibited dechlorination of tetrachloroethene, whereas 10 ~tM had no significant effect (Fig. 4). 2-Propanol had no effect. Other potential inhibitors tested were cyanide (as complex ligand for transition metal centers in proteins), thiocyanate (as an inhibitor of the anaerobic dechlorination of methyl chloride; Megmer et al. 1993), and malonate (as a competitive inhibitor of succinate dehydrogenase). None of these agents had a significant effect on the dechlorination in cell suspensions of D. multivorans (data not shown).

Dechlorination of tetrachloroethene in cell extracts Reductive dechlorination of PCE or TCE was determined in cell extracts by the oxidation of reduced methyl viologen with tetrachloroethene or trichloroethene, respectively, as described in Materials and methods. This enzymic activity is referred to as "PCE dehalogenase" or simply "dehalogenase" in this communication. In the experiment shown in Fig. 5 A, methyl viologen was reduced with dithionite. Tetrachloroethene ( - l gl) was added after the reduction of methyl viologen. The reaction was started by the addition of extract (E), The oxidation of methyl viologen started immediately (Fig. 5 A). In a control experiment, the cell extract was incubated for 10 min at 95~ prior to the addition to the cuvette. With heated cell extract (HE), no reaction was observed, independent of whether the reaction was started with PCE (Fig. 5 B) or with heated extract (data not shown). This indicated that no abiotic reductive dehalogenation of tetra-

299 tein) -1, depending on the experimental conditions, for example, the P C E concentration applied (see above). It was nearly the same with tetrachloroethene and trichloroethene as substrates. The tetrachloroethene dehalogenase was stable after freezing the cell extract for 12 h at - 2 0 ~ and also after stirring in the presence o f air for 5 min at 0 ~ C, whereas the hydrogenase lost about 50% of the initial activity after the same treatment (data not shown).

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