Arch Microbiol (2003) 180 : 411416 DOI 10.

1007/s00203-003-0607-7

O R I G I N A L PA P E R

Gopalakrishnan Jayachandran Helmut Grisch Lorenz Adrian

Dehalorespiration with hexachlorobenzene and pentachlorobenzene by Dehalococcoides sp. strain CBDB1

Received: 14 August 2003 / Revised: 15 September 2003 / Accepted: 16 September 2003 / Published online: 16 October 2003 Springer-Verlag 2003

Abstract The chlororespiring anaerobe Dehalococcoides sp. strain CBDB1 used hexachlorobenzene and pentachlorobenzene as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with hexachlorobenzene or pentachlorobenzene as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with hexachlorobenzene or pentachlorobenzene when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield (Y) of 2.1 0.24 g protein/mol Cl released with hexachlorobenzene as electron acceptor; with pentachlorobenzene, the growth yield was 2.90.15 g/mol Cl. Hexachlorobenzene was reductively dechlorinated to pentachlorobenzene, which was converted to a mixture of 1,2,3,5- and 1,2,4,5-tetrachlorobenzene. Formation of 1,2,3,4-tetrachlorobenzene was not detected. The final end-products of hexachlorobenzene and pentachlorobenzene dechlorination were 1,3,5-trichlorobenzene, 1,3- and 1,4-dichlorobenzene, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene exclusively to 1,2,4-trichlorobenzene. The organism therefore catalyzes two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-trichlorobenzene, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-dichlorobenzene via 1,2,4-trichlorobenzene singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole

cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive dehalogenases might be induced by the different electron acceptors. To our knowledge, this is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorobenzene via dehalorespiration by a pure bacterial culture. Keywords Dehalococcoides Hexachlorobenzene Pentachlorobenzene Chlorobenzene Reductive dechlorination Dehalorespiration.

Introduction
The use of chlorinated benzenes in a variety of different applications has led to their accumulation in the environment. Hexachlorobenzene was used as a pesticide, fungicide, and seed protectant. In addition, it is released into the environment with process and waste waters and during its pyrotechnical use. Pentachlorobenzene and hexachlorobenzene are very persistent under natural conditions. The United Nations Environment Program identified hexachlorobenzene as one of the twelve most persistent organic pollutants, requiring immediate attention due to its global threat to the health of humans and wildlife (Fisher 1999). The adverse effects of hexachlorobenzene on human health were first noted between 1950 and 1955, when prolonged consumption of hexachlorobenzene-treated seed grain resulted in porphyria turcia, a disease of disturbed heme biosynthesis (Cam 1958). In a systematic survey of the Federal Environmental Agency of Germany, a mean concentration of 0.44 g hexachlorobenzene/l was detected in human blood with a marked increase in hexachlorobenzene concentrations with age (Becker et al. 2002). Although the production of hexachlorobenzene was stopped completely in Germany in 1993, hexachlorobenzene is still ubiquitous in the environment and enters the human body mainly via food (IPCS 1997). Furthermore, pentachlorobenzene and hexachlorobenzene showed hepatocarcinogenic activity (Thomas et al. 1998; Gustafson et al. 2002).

G. Jayachandran H. Grisch L. Adrian () Fachgebiet Technische Biochemie, Institut fr Biotechnologie, Technische Universitt Berlin, Seestrasse 13, Sekr. GG1, 13353 Berlin, Germany Tel.: +49-30-31427509, Fax: +49-30-31427581, e-mail: lorenz.adrian@tu-berlin.de

412

Under natural conditions, hexachlorobenzene and pentachlorobenzene can persist for many decades (Oliver and Nicol 1982). However, some mixed microbial cultures have been reported to reductively dechlorinate these highly chlorinated benzenes under anoxic conditions to less chlorinated benzenes (Fathepure et al. 1988; Holliger et al. 1992; Beurskens et al. 1994; Chang et al. 1998; Yeh and Pavlostathis 2001). We previously reported the isolation of a bacterium, Dehalococcoides sp. strain CBDB1, that is able to reductively dechlorinate all three tetrachlorobenzene isomers, 1,2,3-trichlorobenzene and 1,2,4-trichlorobenzene (Adrian et al. 2000), as well as several polychlorinated dibenzodioxin congeners (Bunge et al. 2003). Strain CBDB1 is phylogenetically closely related to Dehalococcoides ethenogenes strain 195, a bacterium that dechlorinates tetrachloroethene to ethene (Maym-Gatell et al. 1997). Until now, no pure culture that transforms hexachlorobenzene or pentachlorobenzene has been described. Several previous attempts to demonstrate dechlorination of pentachlorobenzene and hexachlorobenzene with strain CBDB1 failed. However, pentachlorobenzene dechlorination was observed in a mixed culture containing strain CBDB1 (Adrian et al. 2000). Here we report the reductive dechlorination of pentachlorobenzene and hexachlorobenzene by strain CBDB1 after adding these compounds as crystals to the growth medium. Furthermore, we demonstrate dehalorespiratory growth of strain CBDB1 with both pentachlorobenzene and hexachlorobenzene.

Materials and methods

Inoculum and culture conditions Dehalococcoides sp. strain CBDB1 growing in a medium containing 15 M 1,2,3-trichlorobenzene and 15 M 1,2,4-trichlorobenzene as electron acceptors was used as initial inoculum. The bacteria were cultured under strictly anaerobic conditions in 120-ml glass flasks containing 60 ml medium and 60 ml gas phase. A synthetic mineral medium with vitamins, trace elements, and 5 mM acetate as carbon source was used and reduced by 0.8 mM titanium (III) citrate as described previously (Adrian et al. 1998). The headspace was flushed with N2/CO2 (4:1, v/v). Hexachlorobenzene and pentachlorobenzene were either added as a 0.2 M solution in hexadecane to a nominal concentration of 10 mM or directly to the cultures as crystals (hexachlorobenzene about 50 g/ml; pentachlorobenzene about 20 g/ml; equivalent to nominal concentrations of 175 and 80 M, respectively) before the flasks were sealed with Teflon-lined butyl-rubber septa (Macherey and Nagel, Dren, Germany) and aluminum crimp caps. Hydrogen was added with a syringe to the headspace of cultures to a nominal concentration of 7.5 mM. The cultures were incubated at 29 C in the dark without agitation. The experiments were set up in duplicate or triplicate. Control experiments were done without inoculum or without electron acceptor. Analytical methods Chlorobenzenes were quantified after extracting 1 ml of bacterial suspension with 1 ml dichloromethane or hexane by capillary gaschromatographic analysis with a flame ionization detector as described previously (Adrian et al. 1998). The use of dichloromethane for extraction resulted in a lower detection limit for hexachlorobenzene and pentachlorobenzene compared to extraction with hexane. The temperature program was adapted to separate tetrachloroben-

zenes, pentachlorobenzene, and hexachlorobenzene. Splitless injection was done at a column temperature of 55 C. After 1 min, the split was opened and the column temperature was steadily increased to 225 C at a rate of 6 C/min. Chlorobenzene congeners were identified and quantified by injecting single compounds as standards. Dehalogenase activity was determined with whole cells of strain CBDB1 using reduced methyl viologen as an artificial electron donor. The assay solution contained 100 mM Tris-HCl, pH 7.5, 1 mM methyl viologen, 2 mM titanium(III) citrate and nominal concentrations of 50 M 1,2,3-trichlorobenzene, 50 M 1,2,3,4-tetrachlorobenzene, 15 M pentachlorobenzene or 15 M hexachlorobenzene (Hlscher et al. 2003). The chlorobenzenes were added as 5 mM solutions in acetone. Assay solution (800 l) was filled in a 2-ml auto-sampler vial in an anaerobic chamber and tightly closed with a Teflon-lined butyl septum (Macherey and Nagel) and an aluminum crimp cap. Two-hundred l of bacterial cultures pregrown with trichlorobenzene, pentachlorobenzene, or hexachlorobenzene as electron acceptor and containing between 2107 and 5107 cells/ml were added with a syringe and the vials were incubated at 25 C. The reaction was stopped after 2 h when trichlorobenzene, tetrachlorobenzene, or pentachlorobenzene was used as an electron acceptor or after 4 h when hexachlorobenzene was used by extracting the reaction mixture with 1 ml hexane or dichloromethane. Concentrations of the produced chlorobenzenes were determined by gas chromatography as described above. Reductive dechlorination of chlorinated benzenes results in the stoichiometric formation of lesser chlorinated benzenes as end products. Therefore, enzymatic activities were calculated from the sum of all formed products and are expressed in nkat (nmol products per second at 25 C). Direct counting of cells in a counting chamber was not possible because cells of Dehalococcoides sp. strain CBDB1 are very small (1 m) and diffuse rapidly out of focus. Therefore, for estimating cell numbers 10 l of a culture was placed on a microscopic slide and covered with a 2020 mm cover glass. This procedure led to an even distribution of the culture volume underneath the total area of the cover glass, forming a thin film with a thickness of 25 m such that diffusion of cells out of focus was reduced compared to a counting chamber with a film thickness of 100 m. The average number of cells observed in 20 different microscopic fields was used to calculate the number of cells per ml. Counting was done on a phasecontrast microscope (Axioskop 2 plus, Zeiss, Jena, Germany). Total protein was quantified by a fluorescent test using whole cells (NanoOrange-kit, Molecular Probes, Leiden, The Netherlands). According to the instructions of the manufacturer, excitation/emission wavelengths of 485/590 nm were used. Fluorescence measurements were carried out on a RF-5001PC-fluorometer (Shimadzu, Tokyo, Japan). Chemicals All chlorobenzenes were purchased from Aldrich (Steinheim, Germany), Merck-Schuchardt (Hohenbrunn,Germany), Acros (New Jersey, USA) or Fluka (Buchs, Switzerland). Titanium chloride (synthesis-grade solution) was obtained from Merck-Schuchardt. Titanium(III) citrate (0.1 M with respect to Ti(III)) was prepared as described by Zehnder and Wuhrmann (1976). All other chemicals used were at least of analytical grade and were purchased from Sigma (Deisenhofen, Germany) or Merck-Schuchardt. N2 and H2 were obtained in 99.999% (v/v) and CO2 in 99.8% (v/v) quality from Messer Griesheim (Berlin, Germany). Traces of oxygen were removed by a reduction column (Ochs, Gttingen, Germany).

Results
Dechlorination of pentachlorobenzene and hexachlorobenzene Pure cultures of Dehalococcoides sp. strain CBDB1 grown with 15 M 1,2,3- and 15 M 1,2,4-trichlorobenzene as

413

electron acceptor were used as an inoculum to assess the dechlorination potential towards highly chlorinated benzenes. Addition of pentachlorobenzene or hexachlorobenzene to a nominal concentration of 10 mM as a solution in hexadecane did not consistently result in the production of less chlorinated benzenes. Most parallel cultures remained inactive even after 35 months of incubation and only trace concentrations (below 2 M) of dechlorination products could be detected. However, when pentachlorobenzene or hexachlorobenzene was directly added as crystals, significant concentrations of less chlorinated products (>3 M) could be detected within 4 days of incubation (Fig. 1). The cultures were subcultured eight times within 1 year using 0.2% inocula and medium containing acetate, hydrogen, and crystals of pentachlorobenzene or hexachlorobenzene. Reproducible growth of strain CBDB1 was observed, and both hexachlorobenzene and pentachlorobenzene were dechlorinated to 1,3,5-trichlorobenzene, 1,3-dichlorobenzene, and 1,4-dichlorobenzene in a ratio of about 3:2:5 (Fig. 1). These products were not further dechlorinated. Product formation was much faster in cultures with pen-

tachlorobenzene crystals than in cultures with hexachlorobenzene crystals (Fig. 1). Only trace concentrations (below 2 M) of pentachlorobenzene were detected in cultures incubated with hexachlorobenzene during the first few days of incubation. Intermediates detected both in cultures with hexachlorobenzene and in cultures with pentachlorobenzene were 1,2,4,5-tetrachlorobenzene, 1,2,3,5-tetrachlorobenzene, and 1,2,4-trichlorobenzene. Tetrachlorobenzenes were detected only during the first few days of incubation at concentrations below 2 M. As reported previously, strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene and 1,2,4,5-tetrachlorobenzene to 1,2,4-trichlorobenzene (Adrian et al. 2000). Formation of 1,2,3,4-tetrachlorobenzene, 1,2,3-trichlorobenzene, 1,2-dichlorobenzene, or monochlorobenzene was not detected. Control cultures not containing inocula or containing heat-sterilized inocula did not form dechlorination products of hexachlorobenzene or pentachlorobenzene. After 16 days of incubation, pentachlorobenzene concentrations in the aqueous phase were below the detection limit of about 0.5 M and no crystals were seen in the medium, indicating that the crystals were completely consumed. Also, there was no further increase in the concentration of the dechlorination products between days 12 and 16. Therefore, further pentachlorobenzene crystals were added to the cultures at day 16 leading to an increase of the product concentration at day 20 (Fig. 1A). Cultures containing hexachlorobenzene crystals had only low initial aqueous concentrations of hexachlorobenzene (below 3 M). In contrast to cultures with pentachlorobenzene, cultures with hexachlorobenzene contained low concentrations of the substrate even at the end of the incubation period. The steady increase in the concentration of dechlorination products in cultures with hexachlorobenzene crystals (Fig. 1B) indicated a steady dissolution of the hexachlorobenzene crystals throughout the incubation period. Growth of strain CBDB1 based on hexachlorobenzene or pentachlorobenzene dechlorination Growth of strain CBDB1 with hydrogen as the electron donor and acetate as carbon source required the presence of hexachlorobenzene or pentachlorobenzene as electron acceptor. Without chlorinated benzenes, there was no increase in cell number or protein concentration. Both protein concentrations and cell numbers indicated faster growth of strain CBDB1 with pentachlorobenzene than with hexachlorobenzene as electron acceptor (Fig. 2). After 12 days, no crystals of pentachlorobenzene were detectable visually and the pentachlorobenzene concentration in the liquid phase was below the detection limit (< 0.5 M). Also, no further growth of strain CBDB1 was observed. Cultures with hexachlorobenzene grew slowly, as indicated by both growth parameters. However, growth was constant over the entire incubation time, yielding 4.2107 cells/ml and 0.98 g/ml protein at day 36. From the data shown in Fig. 1, molar growth yields (Y) were calculated for days 28, 32 and 36. With pentachloro-

Fig. 1 Product formation in cultures with Dehalococcoides sp. strain CBDB1 amended with crystals of pentachlorobenzene (a) or hexachlorobenzene (b). 1,3,5-Trichlorobenzene, 1,4-dichlorobenzene, 1,3-dichlorobenzene, 1,2,4-trichlorobenzene. No dechlorination was found in uninoculated controls or with autoclaved inocula. Arrow Further addition of pentachlorobenzene crystals

414 Table 1 Specific activities (nkat/mg) towards different chlorobenzene congeners with cultures grown on 1,2,3-trichlorobenzene (TCB), pentachlorobenzene (PeCB), or hexachlorobenzene (HCB). Culture grown on: 1,2,3-TCB PeCB HCB
ankat=nmol

Specific activity towards (nkat/mg)a 1,2,3-TCB 1,2,3,4-TeCBb PeCB 6.7 8.1 8.9 64 310 200 19 22 183 HCB <0.1 0.7 3.36

s-1; values are relative to protein; tests were incubated for 2 h (TCB, TeCB, PeCB) or 4 h (HCB) at 25 C. Numbers are means of duplicate determinations. b TeCB Tetrachlorobenzene.

Fig. 2 Growth of Dehalococcoides sp. strain CBDB1 on H2/pentachlorobenzene or H2/hexachlorobenzene. Cell numbers of strain CBDB1 consuming pentachlorobenzene ( ), and hexachlorobenzene ( ). Total protein yield of strain CBDB1 consuming pentachlorobenzene ( ), and hexachlorobenzene ( ). In inoculated controls containing no chlorobenzenes, no cells could be found and protein concentrations were constantly below the detection limit of about 0.1 g/ml

Discussion
Dehalococcoides sp. strain CBDB1 is the only pure bacterial strain described so far to carry out anaerobic respiration with pentachlorobenzene and hexachlorobenzene. The solubility in water for the chlorobenzene congeners (in M at 22C) used in this study differ widely, as summarized by Holliger et al. (1992): hexachlorobenzene 0.4; pentachlorobenzene 1.0; 1,2,3,4-tetrachlorobenzene 16; 1,2,3-trichlorobenzene 66; 1,4-dichlorobenzene 333; 1,3-dichlorobenzene 469. While trichlorobenzenes and dichlorobenzenes are toxic for chlorobenzene-dechlorinating bacteria below their saturation level in aqueous solution (Holliger et al. 1992; Adrian et al. 1998), solutions containing crystals of hexachlorobenzene and pentachlorobenzene were nontoxic to strain CBDB1 and were dechlorinated efficiently. In contrast to hexachlorobenzene and pentachlorobenzene, 1,2,3,4-tetrachlorobenzene appeared to be toxic for strain CBDB1 if added in crystal form since it completely inhibited growth and dechlorination of other chlorobenzenes. The molecular basis for the inhibitory effect of 1,2,3,4-tetrachlorobenzene is not known. However, the reductive dehalogenase activity was not affected as 1,2,3,4-tetrachlorobenzene was quickly dechlorinated by whole cells of strain CBDB1 when titanium (III)-reduced methyl viologen was added as artificial electron donor (Table 1 and Hlscher et al. 2003). Recently, a mixed culture containing the polychlorinated biphenyl-dechlorinating bacterium DF-1 was described to dechlorinate exclusively doubly flanked chlorine substituents from pentachlorobenzene and hexachlorobenzene added as a solution in acetone. The culture dechlorinated about 36 M pentachlorobenzene to 1,2,3,5-tetrachlorobenzene and 1,3,5-trichlorobenzene within 14 days of incubation. After 56 days, about 85% of the added pentachlorobenzene (about 150 M) was dechlorinated to the only final product, 1,3,5-trichlorobenzene (Wu et al. 2002). Strain CBDB1 formed about 75 M dechlorination products from pentachlorobenzene crystals within 12 days (Fig. 1A). The sole product formed from 1,2,3,5-tetrachlorobenzene is 1,3,5-trichlorobenzene for both strain CBDB1 (Adrian et al. 2000) and the mixed culture containing strain DF-1 (Wu et al. 2002). Whereas the sole dechlorination product formed by the mixed culture containing

benzene as electron acceptor the average molar growth yield was Y=2.90.15 g protein/mol Cl, while for hexachlorobenzene a value of Y=2.10.24 g protein/mol Cl was calculated. 1,2,3,4-Tetrachlorobenzene did not support growth of Dehalococcoides sp. strain CBDB1 even when the compound was supplied in crystal form. When crystals of 1,2,3,4-tetrachlorobenzene were added to cultures amended with hexachlorobenzene, pentachlorobenzene, or trichlorobenzene as electron acceptors, no further increase in cell number or protein concentration occurred and dechlorination was completely inhibited. Measurements of dehalogenase activities Cultures grown on hexachlorobenzene, pentachlorobenzene or 1,2,3-trichlorobenzene were used to measure specific dehalogenase activities towards different chlorobenzene congeners. 1,2,3,4-Tetrachlorobenzene and 1,2,3-trichlorobenzene were tested because they were previously shown to be good electron acceptors for the reductive dehalogenases of strain CBDB1 (Hlscher et al. 2003). Cells grown on hexachlorobenzene or pentachlorobenzene showed the highest specific dechlorination activities towards 1,2,3,4-tetrachlorobenzene followed by specific activity towards pentachlorobenzene and 1,2,3-trichlorobenzene, whereas activity towards hexachlorobenzene was low (Table 1). The highest specific activity towards hexachlorobenzene was found in cultures grown on hexachlorobenzene. In contrast, cultures grown on 1,2,3-trichlorobenzene did not have detectable hexachlorobenzene dechlorinating activity.

415

Fig. 3 Proposed pathway of hexachlorobenzene and pentachlorobenzene reductive dechlorination by Dehalococcoides sp. strain CBDB1. The values indicate the relative amounts of product formation and are related to the total amount of products detected. Other reactions are not catalyzed by strain CBDB1, as determined using the different chlorobenzene congeners separately. Bold arrows Major dechlorination pathway

strain DF-1 from pentachlorobenzene is 1,2,3,5-tetrachlorobenzene, strain CBDB1 in addition forms 1,2,4,5-tetrachlorobenzene from pentachlorobenzene. 1,2,4,5-Tetrachlorobenzene is exclusively converted to 1,2,4-trichlorobenzene by strain CBDB1 and finally leads to the formation of 1,3- and 1,4-dichlorobenzene as reported earlier (Adrian et al. 2000). In this second pathway, singly flanked chlorine substituents are also removed (Fig. 3). Strain CBDB1 could be indefinitely subcultured in a medium containing acetate, hydrogen, and pentachlorobenzene or hexachlorobenzene as electron acceptors, indicating that pentachlorobenzene and hexachlorobenzene are used in a respiratory process for energy conservation. Increases of cell numbers and cell protein (Fig. 2) concurrent with dechlorination of pentachlorobenzene (Fig. 1A) or hexachlorobenzene (Fig. 1B) as well as the cessation of growth after depletion of pentachlorobenzene further sup-

port the conclusion that growth is coupled to pentachlorobenzene or hexachlorobenzene dechlorination. The growth yields with hexachlorobenzene and pentachlorobenzene of 23 g protein per mol chloride released are similar to those described previously for other bacteria growing by dehalorespiration. Reported growth yields range from 0.2 g/mol Cl for a vinyl-chloride-respiring Dehalococcoides species (He et al. 2003) to about 5 g/mol Cl for two other chloroethene-respiring Dehalococcoides species (Maym-Gatell et al. 1997, Cupples et al. 2003). Variations may be due to differences in the efficiency of energy conservation, but may also partly result from biases of protein assays. The dechlorination reactions observed with strain CBDB1 could be catalyzed by a single enzyme with broad substrate specificity. However, taking also into account the results of Tiedje et al. (1987), Holliger et al. (1992), Chang et al. (1998), and Wu et al. (2002), who found that in their mixed cultures hexachlorobenzene and/or pentachlorobenzene is dechlorinated exclusively to 1,3,5-trichlorobenzene, it could also be that the two different identified pathways for pentachlorobenzene dechlorination in strain CBDB1 are catalyzed by different enzymes. One enzyme would exclusively dechlorinate doubly flanked substituents catalyzing the dechlorination of pentachlorobenzene via 1,2,3,5-tetrachlorobenzene to 1,3,5-trichlorobenzene. Other enzymes present in strain CBDB1, but not in the DF-1 containing culture, could dechlorinate pentachlorobenzene to 1,4- and 1,3-dichlorobenzene via 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene. Our results indicate that the specific activity of strain CBDB1 towards hexachlorobenzene is highest in cultures grown with hexachlorobenzene and that the specific activity towards 1,2,3,4-tetrachlorobenzene is three to five times higher in cultures grown with pentachlorobenzene or hexachlorobenzene than with 1,2,3-trichlorobenzene. This suggests that different dehalogenase activities are inducible by different chlorobenzene congeners. Wu et al. (2002) found a longer adaptation time for the dechlorination of hexachlorobenzene than for pentachlorobenzene possibly also indicating the need to induce the responsible dehalogenases. In contrast, 1,2,3-trichlorobenzene dehalogenase activity is present in cells of Dehalococcoides sp. strain CBDB1 regardless of whether growth occurred with 1,2,3-trichlorobenzene, pentachlorobenzene, or hexachlorobenzene, although 1,2,3-trichlorobenzene is not an intermediate in hexachlorobenzene and pentachlorobenzene dechlorination.
Acknowledgements We thank the DAAD for a doctoral fellowship to G.J., and the DFG for financial support to L.A. We are grateful to G. Wagner for technical assistance and T. Hlscher for helpful discussions and suggestions for quantification of protein.

References
Adrian L, Manz W, Szewzyk U, Grisch H (1998) Physiological characterization of a bacterial consortium reductively dechlorinating 1,2,3- and 1,2,4-trichlorobenzene. Appl Environ Microbiol 64:496503

416 Adrian L, Szewzyk U, Wecke J, Grisch H (2000) Bacterial dehalorespiration with chlorinated benzenes. Nature 408:580 583 Becker K, Kaus S, Krause C, Lepom P, Schulz C, Seiwert M, Seifert B (2002) German Environmental Survey 1998 (GerES III): environmental pollutants in blood of the German population. Int J Hyg Environ Health 205:297308 Beurskens JEM, Connie DGC, van den Heuvel H, Swart M, de Wolf J, Dolfing J (1994) Dechlorination of chlorinated benzenes by an anaerobic microbial consortium that selectively mediates the thermodynamic most favorable reactions. Environ Sci Technol 28:701706 Bunge M, Adrian L, Kraus A, Opel M, Wilhelm GL, Andreesen RJ, Grisch H, Lechner U (2003) Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium. Nature 421: 357360 Cam C (1958) Cases of skin porphyria related to hexachlorobenzene intoxication. Saglik Dergisi 32:215216 Chang BV, Su CJ, Yuan SY (1998). Microbial hexachlorobenzene dechlorination under three reducing conditions. Chemosphere 36:27212730 Cupples AM, Spormann AM, McCarty PL (2003) Growth of a Dehalococcoides-like microorganism on vinyl chloride and cisdichloroethene as electron acceptors as determined by competitive PCR. Appl Environ Microbiol 69:953959 Fathepure BZ, Tiedje JM, Boyd SA (1988) Reductive dechlorination of hexachlorobenzene to tri- and dichlorobenzenes in anaerobic sewage sludge. Appl Environ Microbiol 54:327330 Fisher BE (1999) Most unwanted. Environ Health Perspect 107: A18-A23 Gustafson DL, Long ME, Thomas RS, Benjamin SA, Yang RSH (2002) Comparative hepatocarcinogenicity of hexachlorobenzene, pentachlorobenzene, 1,2,4,5-tetrachlorobenzene, and 1,4-dichlorobenzene: application of a medium-term liver focus bioassay and molecular and cellular indices. Toxicol Sci 53: 245252 He J, Ritalahti KM, Yang KL, Koenigsberg SS, Lffler FE (2003) Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium. Nature 424:6265 Holliger C, Schraa G, Stams AJM, Zehnder AJB (1992) Enrichment and properties of an anaerobic mixed culture reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-dichlorobenzene. Appl Environ Microbiol 58:16361644 Hlscher T, Grisch H, Adrian L (2003) Reductive dehalogenation of chlorobenzene congeners in cell free extracts of Dehalococcoides sp. strain CBDB1. Appl Environ Microbiol 69:2999 3001 IPCS (International Programme for Chemical Safety) (1997) Environmental Health Criteria 195. Hexachlorobenzene. ISBN 9241571950, ISSN 0250863X, World Health Organisation, Geneva, Switzerland Maym-Gatell X, Chien YT, Gossett JM, Zinder SH (1997) Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 276:15681571 Oliver BG, Nicol KD (1982) Chlorobenzenes in sediments, water, and selected fish from lakes Superior, Huron, Erie, and Ontario. Environ Sci Technol 16:532536 Thomas RS, Gustafson DL, Pott WA, Long ME, Benjamin SA, Yang RSH (1998) Evidence for hepatocarcinogenic activity of pentachlorobenzene with intralobular variation in foci incidence. Carcinogenesis 19:18551862 Tiedje JM, Boyd SA, Fathepure BZ (1987) Anaerobic degradation of chlorinated aromatic hydrocarbons. J Ind Microbiol Suppl 27:117127 Wu QZ, Milliken CE, Meier GP, Watts JEM, Sowers KR, May HD (2002) Dechlorination of chlorobenzenes by a culture containing bacterium DF-1, a PCB dechlorinating microorganism. Environ Sci Technol 36:32903294 Yeh DH, Pavlostathis SG (2001) Development of hexachlorobenzene-dechlorinating mixed cultures using polysorbate surfactants as a carbon source. Water Sci Technol 43:4350 Zehnder AJB, Wuhrmann K (1976) Titanium(III)citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194:11651166