Bacterial Cultures Preferentially Removing Singly Flanked Chlorine

Nov 2, 2010 - from TCBs, some cultures formed 1,2-DCB from 1,2,3-TCB and/ or1,3-DCBfrom1,2,4-TCB.Stableculturespreferentiallycatalyzing the removal of...
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Environ. Sci. Technol. 2010, 44, 8936–8942

Bacterial Cultures Preferentially Removing Singly Flanked Chlorine Substituents from Chlorobenzenes ¨ L S C H E R , † J A N L I S E C , ‡,§ TINA HO M O H A M E D B A A N I , ‡,⊥ T R A N H O A D U A N , † A N D L O R E N Z A D R I A N * ,†,‡ Department Isotopenbiogeochemie, Helmholtzzentrum fu ¨r Umweltforschung-UFZ, Permoserstrasse 15, D-04318 Leipzig, Germany and Fachgebiet Angewandte Biochemie, Institut fu ¨r Biotechnologie, Technische Universita¨t Berlin, Seestr. 13, D-13353 Berlin, Germany

Received June 10, 2010. Revised manuscript received October 13, 2010. Accepted October 14, 2010.

The wide though not ubiquitous distribution of chlorobenzenedechlorinating bacteria in anaerobic sludge from German sewage plants is demonstrated. The model substrates 1,2,3and 1,2,4-trichlorobenzene (TCB) were dechlorinated to dichlorobenzenes (DCBs) and monochlorobenzene (MCB) via distinct pathways. For easy visualization and differentiation of the pathways, a novel plotting method was developed. While many of the cultures showed a dechlorination pattern similar to that previously found for Dehalococcoides species, removing doubly flanked rather than singly flanked chlorine substituents from TCBs, some cultures formed 1,2-DCB from 1,2,3-TCB and/ or 1,3-DCB from 1,2,4-TCB. Stable cultures preferentially catalyzing the removal of singly flanked chlorines were obtained by repeated subcultivation in sediment-free synthetic medium. This dechlorination pattern is potentially of great benefit for remediation as the accumulation of persistent intermediates such as 1,3,5-TCB from highly chlorinated compounds can be avoided. In addition, the cultures dechlorinated 1,3,5-TCB, pentachlorobenzene (PeCB), and hexachlorobenzene (HCB). Nested PCR demonstrated the presence of low numbers of Dehalococcoides species. However, the observed insensitivity of the dechlorinating bacteria in our cultures to oxygen and sensitivity to vancomycin is not in accordance with the reported properties of Dehalococcoides species, suggesting that other bacteria than Dehalococcoides catalyzed the removal of singly flanked chlorines from TCB.

Introduction Chlorobenzenes constitute important intermediates in a variety of chemical syntheses. Due to direct use as biocides and solvents, or accidental release, chlorobenzene contaminations are ubiquitously distributed in nature. While lowchlorinated benzenes can be mineralized by aerobic bacteria, highly chlorinated benzenes are extremely persistent to aerobic attack (1, 2). Mineralization of HCB by an aerobic * Corresponding author phone: +49 341 235 1435; fax: +49 341 235 1443; e-mail: [email protected]. † Helmholzzentrum fu ¨ r Umweltforschung. ‡ Technische Universita¨t Berlin. § Present address: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany. ⊥ Present address: Technische Universita¨t Darmstadt, Germany. 8936 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 23, 2010

bacterium has been reported for the first time only recently (3). The preferred transformation route for highly chlorinated benzenes under anaerobic conditions is the reductive dechlorination leading to the formation of less chlorinated benzenes (1, 2, 4). Anaerobic reductive dechlorination of highly chlorinated benzenes has been detected in a variety of mixed cultures originating from different sources such as river or lake sediments, sewage sludge, or soil. However, a systematic study on the environmental distribution of chlorobenzene-dechlorinating bacteria has not been performed so far. Few bacterial pure cultures are known to reductively dechlorinate chlorobenzenes. All these anaerobic bacteria belong to the phylum Chloroflexi and are able to use chlorobenzenes as terminal electron acceptors for growth. Dehalococcoides sp. strain CBDB1 metabolically dechlorinates 1,2,3-TCB, 1,2,4-TCB, all tetrachlorobenzene (TeCB) isomers, PeCB, and HCB (5, 6). Reductive dechlorination of chlorinated benzenes was also demonstrated for Dehalococcoides ethenogenes strain 195 (7), Dehalococcoides sp. strain DCMB5 (8), and the Dehalococcoides-related bacterium DF-1 (9, 10), all having a smaller spectrum of chlorobenzene substrates than strain CBDB1. With respect to the number of nearest neighbor chlorines, chlorine substituents in multiple chlorinated benzenes can be referred to as isolated, singly flanked, or doubly flanked (4). Like mixed cultures described previously (11-15), Dehalococcoides species and bacterium DF-1 catalyze the removal of doubly flanked chlorine substituents rather than of singly flanked chlorines. This dechlorination pattern follows the thermodynamically more favorable reaction as the removal of doubly flanked chlorine substituents is accompanied by a higher change of the free energy (∆G°′) than the removal of singly flanked chlorine substituents from the same congener (11, 16) (Figure 1). It was proposed that such thermodynamic considerations can be used to predict dechlorination patterns (11, 16). Others have used density functional theory to predict dechlorination pathways of polychlorinated dibenzodioxins (17) or semiempirical molecular orbital calculations (18). However, not all Dehalococcoides species catalyze according to such predicted dechlorination patterns and the determining factor might therefore be the kinetics of the expressed dehalogenating enzymes. For example, HCB dechlorination by strain 195 mainly follows the pattern HCB f PeCB f 1,2,3,5-TeCB f 1,3,5-TCB that was also described for bacterium DF-1. In contrast, HCB dechlorination in strain CBDB1 proceeds via the two isomers 1,2,4,5-TeCB and 1,2,3,5-TeCB and leads to the formation of DCBs and 1,3,5-TCB. Also, dechlorination of 1,2,4-TCB supplied as sole electron acceptor was found for strain CBDB1 but not for other Dehalococcoides strains (7, 8, 19). For a few mixed cultures, preferential dechlorination of singly flanked chlorine substituents from multiple chlorinated benzenes was reported. A methanogenic microbial consortium dechlorinated 1,2,4-TCB, 1,2,3,4-TeCB, 1,2,3,5-TeCB, and PeCB via various pathways, among which the pattern yielding MCB via 1,2,4-TCB was predominant (20). In another study (21), dechlorination of HCB and PeCB to MCB was demonstrated in methanogenic soil slurry microcosms. The pathway found in this study proceeded via 1,2,3,4-TeCB, TCBs (1,2,3-TCB and 1,2,4-TCB), and DCBs (1,2-DCB and 1,4-DCB) to MCB. Such a dechlorination pattern is potentially of great benefit for remediation as HCB and PeCB are not dechlorinated to the aerobically and anaerobically highly persistent congener 1,3,5-TCB. Instead, DCB and/or MCB are formed, which can be mineralized subsequently by aerobic bacteria. 10.1021/es101971m  2010 American Chemical Society

Published on Web 11/02/2010

FIGURE 1. Reductive dechlorination reactions of TCBs to DCBs and MCB and their standard free energies (kJ/mol) with molecular hydrogen as the electron donor (16). However, these mixed cultures needed the addition of undefined soil components to maintain dechlorinating activity, which is an obstacle for further physiological characterization and the isolation of bacteria catalyzing these dechlorination reactions. In the present study the distribution of chlorobenzenedechlorinating bacteria in sludge samples from various German sewage plants was assessed. We here describe the development and characterization of stable mixed cultures that grew in purely synthetic medium and generated 1,2DCB and MCB from TCBs (Figure 1). We also present a plotting method for easy visualization and identification of different dechlorination patterns.

Materials and Methods Cultivation. Inocula were obtained from anaerobic stages of several municipal sewage plants from Southern and Eastern Germany. Sampling, transport, and cultivation was done under strictly anaerobic conditions. A synthetic, bicarbonatebuffered mineral medium containing vitamins, 15 µM 1,2,3TCB, and 15 µM 1,2,4-TCB was used as described before (6). The final relative amounts measured in the medium after medium preparation were 58% 1,2,3-TCB and 42% 1,2,4TCB ( 2%, presumably due to different water solubility and adsorption characteristics of the two chlorobenzene isomers to the glass surface and Teflon tubes used. A mixture of the two TCB congeners was used to directly compare dechlorination of doubly flanked, singly flanked, and isolated chlorine substituents. 1,3,5-TCB was added as a solution in acetone, yielding a final concentration of 30 µM 1,3,5-TCB in the cultures, and PeCB and HCB were added as crystals (5). Cultures with a liquid volume of 30 mL were prepared as described previously (6, 22). The medium was reduced with Ti(III) citrate, yielding a final concentration of 0.8 mM Ti(III) and 1.6 mM citrate. Hydrogen was added to a nominal concentration of 7.5 mM with a sterile gastight syringe. When required, 2-bromoethanesulfonic acid, molybdate, or vancomycin were added at final concentrations of 4 mM, 2 mM, and 0.5 mg/L, respectively. Organic acids were used at a concentration of 10 mM (pyruvate, butyrate, and formate) or 5 mM (acetate). Inoculation was done with nitrogenflushed syringes or, in the case of the initial inoculation with

thick sludge, with a pipet with a cut off tip. Subcultivation was done by transferring 2% (v/v) of a parent culture to a bottle containing sterile, reduced, fresh medium and resulted in the dilution of contaminating compounds from the initial inoculum and dilution of bacteria that could not grow under the given cultivation conditions. Cultures were subcultured when 50-100% of the added TCBs were dechlorinated. This was the case after about 2-4 weeks of incubation. Cultures were incubated in the dark at 29 °C without shaking. To expose cells to a positive redox potential, an inoculum was taken up with a syringe and sterile air was bubbled through the liquid until the redox indicator resazurin turned pink. Five minutes after the color change the inoculum was injected into a culture flask containing reduced medium. Analytical Procedures. Chlorobenzene concentrations were determined by GC/FID after hexane extraction (22). For graphic representation of the results, the relative concentrations of the different chlorobenzene congeners measured at various sampling times in a given culture were plotted over the mean degree of chlorination, defined as the total molar amount of all chlorine substituents measured in a culture per mole of chlorinated benzenes (Figure 2). At the time of inoculation, all cultures set up with TCBs had a mean degree of dechlorination of 3, as all chlorobenzenes had three chlorine substituents. With incubation time, chlorine substituents were successively removed, yielding a mean degree of chlorination of 2.5 when 50% TCBs were dechlorinated to DCBs, 2 when only DCBs were present, or 1 when all TCBs were dechlorinated to MCB. Data from all cultures showing the same dechlorination pattern were compiled in one graph. Methane was analyzed by gas chromatography and thermal conductivity detection (22). Molecular Methods. Dehalococcoides-specific nested PCR was done as described (23) using primers DHC1172R and DHC728F for the second amplification (24) and a primer annealing temperature of 56 °C. Unique bands of the expected size were cloned by TA cloning (Invitrogen, De Schelp, Netherlands) according to the instructions of the manufacturer and sequenced (M. Meixner-Sequenzierservice, Berlin, Germany). Chemicals. Titanium(III) citrate was prepared from synthesis-grade titanium(III) chloride (Merck-Schuchard, VOL. 44, NO. 23, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Chlorobenzene dechlorination patterns detected in this study. Shown are compilations of chlorobenzene analyses from all cultures with the respective pattern of the indicated culture line. (A) Pure CBDB1 cultures compiled as a reference for cultures preferentially dechlorinating doubly flanked chlorine substituents; (B) MA cultures showing MA/Z-type dechlorination; (C) MB cultures showing MB-1-type dechlorination; (D) MB cultures showing MB-2-type dechlorination. Hohenbrunn, Germany) as described (25). All other chemicals used were analytical grade. Gases were obtained in 99.999% (N2, H2) or 99.8% (CO2) quality from MesserGriesheim (Berlin, Germany); traces of oxygen were removed with a reducing column (Ochs, Go¨ttingen, Germany).

Results Screening for Chlorobenzene Dechlorinating Activity. Inocula from sewage sludge of a variety of locations in Germany were analyzed for their ability to catalyze the removal of singly flanked or isolated chlorine substituents from multiple chlorinated benzenes under a variety of cultivation conditions. All cultures contained hydrogen, bicarbonate, and citrate as potential sources of electrons and/or carbon. As additional cosubstrates, pyruvate, butyrate, or a mixture of butyrate, formate, and acetate were tested (Table 1). To all cultures 1,2,3- and 1,2,4-TCB were added as electron acceptors. Many cultures transformed either 1,2,3-TCB or 1,2,4TCB or both to DCBs. In some cultures, 1,2-DCB was detected, originating either from dechlorination of singly flanked residues of 1,2,3-TCB or from dechlorination of the isolated chlorine of 1,2,4-TCB. From the progress of the formation of DCBs and the disappearance of TCBs it was evident which reactions occurred. Evidence for the dechlorination of singly flanked chlorines was found with the cultures from Bad Wo¨rishofen and from one of the two cultures set up from two different samples from Marienfelde (denominated Marienfelde 2). With pyruvate as a cosubstrate, the Bad Wo¨rishofen culture completely dechlorinated the 1,2,3-TCB/ 1,2,4-TCB mixture to 1,2-DCB and 1,3-DCB. The high relative 8938

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amount of 66% 1,2-DCB indicated that 1,2-DCB originated both from dechlorination of the isolated chlorine of 1,2,4TCB and from dechlorination of a singly flanked residue of 1,2,3-TCB. In contrast, with butyrate as the sole cosubstrate, the Bad Wo¨rishofen culture did not use 1,2,4-TCB but exclusively dechlorinated 1,2,3-TCB to 1,3-DCB. In the culture from Marienfelde 2, little dechlorination occurred with pyruvate as cosubstrate. With butyrate, however, this culture dechlorinated 1,2,3-TCB to 1,2-DCB, as evidenced by the high relative amount of 54% 1,2-DCB formed, demonstrating preferential dechlorination of singly flanked chlorine substituents. In addition, the culture from Marienfelde 2 dechlorinated 1,2,4-TCB to 1,3-DCB. When using the cosubstrate mixture, significant amounts of 1,2-DCB were not produced by any of the cultures. However, the culture from Kempten showed a preference for the removal of singly flanked chlorines, dechlorinating mainly 1,2,4-TCB to 1,4DCB (Table 1). MCB was not detected in any culture during 8 weeks of incubation. No dechlorination occurred in autoclaved controls. Several cultures were subcultured in order to obtain cultures in purely synthetic medium without abiotic residues from the initial inoculum. In the subcultures the patterns were reproduced, showing that the pattern was determined by the type of inoculum used. However, after three transfers with 2% inocula each, all cultures were lost (data not shown). In a second approach in which we focused on the development of sediment-free cultures preferentially dechlorinating singly flanked chlorine substituents by repeated subcultivation in synthetic medium, we incubated a further set of sludge samples with hydrogen as electron donor (7.5

TABLE 1. Relative Amounts of TCBs and DCBs in Cultures 8 Weeks after Inoculation with 2% (v/v) Sewage Sludgea chlorobenzenes (%)b substrate

origin of the inoculum

1,2,3-TCB

1,2,4-TCB

1,2-DCB

1,3-DCB

1,4-DCB

pyruvate, 10 mM

Bad Wo¨rishofen Ingolstadt 1c Ingolstadt 2 Landsberg Marienfelde 1 Marienfelde 2 Schrobenhausen Schrobenhausend Bad Wo¨rishofen Ingolstadt 1 Ingolstadt 2 Landsberg Marienfelde 2 Schrobenhausen Schrobenhausend Ansbach

0 0 4 28 0 54 37 60 0 6 0 59 0 60 60 0

0 25 42 35 41 32 40 40 46 40 42 41 5 40 40 41

66 19 0 0 0 7 0 0 0 0 0 0 54 0 0 0

34 56 54 37 59 7 23 0 54 54 58 0 41 0 0 59

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Bad To¨lz Buchloe Buchloed Dingolfing Donauwo¨rth Garmisch Gunzenhausen Kaufbeuren Kempten Memmingen Memmingend Miersbach Miersbachd Obenfeiblach Obere Iller Pfaffenhofen Schongau Starnberg

61 61 59 60 0 62 0 60 56 63 60 0 60 60 60 0 60 0

39 39 41 40 37 38 37 40 18 37 40 24 40 40 40 38 40 31

0 0 0 0 0 0 0 0 2 0 0 1 0 0 0 0 0 0

0 0 0 0 56 0 59 0 0 0 0 75 0 0 0 57 0 69

0 0 0 0 7 0 4 0 24 0 0 0 0 0 0 5 0 0

butyrate, 10 mM

butyrate, 10 mM; formate, 10 mM; acetate, 5 mM

a The medium contained Ti(III) citrate (1.6 mM with respect to citrate) and other substrates as specified. b In percent of total chlorobenzenes. c Numbers behind sampling sites differentiate samples that were taken at the same time from different sampling ports of the respective plant. d Autoclaved control. All inocula came from the anaerobic stage of municipal sewage sludge plants. MCB formation was not detected in any culture.

mM nominal concentration), 5 mM acetate as carbon source, and Ti(III) as reducing agent. This set of cultures did not contain formate or butyrate to further limit the range of organisms able to grow in the cultures. As electron acceptors, again a mixture of 1,2,3-TCB and 1,2,4-TCB was added. Ten inocula from 5 sewage plants (Zo¨rbig, Manschow, Waren, Kla¨den, and Wassmannsdorf, all located in Eastern Germany) were set up each in triplicate (data not shown). Inocula from the same source mostly exhibited similar rates and patterns of dechlorination even when they were sampled at different anaerobic stages of the sewage plant. Cultures with inocula from Zo¨rbig, Manschow, or Wassmannsdorf dechlorinated 30-80% of the added TCBs within 4 weeks of incubation; however, as with the cultures described above the preferred dechlorination pattern was the dechlorination of 1,2,3-TCB to 1,3-DCB. Only a few cultures showed formation of 1,2DCB and/or traces of MCB. Such cultures were selected for subcultivation. After four successive transfers with 1% inoculum, always selectively transferring those inocula that showed the highest 1,2-DCB and MCB formation, culture lines from two inocula from Manschow (MA, MB) and one from Zo¨rbig (Z) reproducibly formed 1,2-DCB and MCB. These three culture lines were regularly subcultured so that data of about 20 successive subcultivations accumulated. Dechlorination rates varied among cultures of the same culture line. However, most cultures dechlorinated 50-100%

of the supplied TCBs (i.e., 15-30 µM TCB) within 3 weeks of incubation. These culture lines were studied in more detail. Dechlorination Patterns. The dechlorination patterns of culture lines MA, MB, and Z were determined by compiling the results from chlorobenzene analyses of all cultures of the respective culture line. The relative concentrations of the different chlorobenzene congeners were plotted over the mean degree of chlorination of all chlorobenzene congeners present in the culture (Figure 2). All cultures initially showed a degree of chlorination of 3 when only TCBs were present and reduced it with incubation time if dechlorination occurred. As a reference for a culture preferentially catalyzing the removal of doubly flanked chlorine substituents, data from about 150 chlorobenzene analyses of pure cultures of strain CBDB1 were compiled (Figure 2A). Strain CBDB1 uses 1,2,3-TCB first, which is stoichiometrically dechlorinated to 1,3-DCB. Only after depletion of 1,2,3-TCB, 1,2,4-TCB is dechlorinated to a mixture of 1,4- and 1,3-DCB. 1,2-DCB and MCB are not produced by strain CBDB1; therefore, no degree of chlorination below 2 is obtained with this strain. Culture line MA showed a stable dechlorination pattern in which singly flanked chlorine substituents were preferentially removed (Figure 2B). The culture line reproducibly dechlorinated 1,2,3-TCB to 1,2-DCB and 1,2,4-TCB to 1,3DCB. Both dechlorination reactions occurred simultaneously. 1,4-DCB was not produced. After most TCBs were dechloVOL. 44, NO. 23, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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rinated to DCB, both 1,2-DCB and 1,3-DCB were simultaneously dechlorinated to MCB. Experiments with only one of the TCB congeners as substrate confirmed that 1,2,4-TCB was transformed predominantly to 1,3-DCB and minor amounts of 1,2-DCB, while 1,2,3-TCB was dechlorinated predominantly to 1,2-DCB. The same dechlorination pattern was also obtained in cultures of culture line Z. This dechlorination pattern will therefore be referred to as the MA/Z-type dechlorination pattern. In contrast, the cultures of the second culture line from Manschow (MB) did not show a homogeneous dechlorination pattern as a whole, but each culture showed one out of three distinct dechlorination patterns even after 10 successive transfers. While some cultures produced DCBs and MCB as described for the MA/Z-type pattern above, other cultures produced mainly 1,3-DCB from both TCBs and only minor amounts of 1,2-DCB in a ratio of about 6:1 (Figure 2C). 1,4DCB was not produced in these cultures. In contrast to the MA/Z-type pattern, the TCBs were not simultaneously dechlorinated. Instead, 1,2,3-TCB was dechlorinated first mainly to 1,3-DCB and subsequently 1,2,4-TCB was transformed. After the TCBs were depleted, 1,3-DCB was dechlorinated to MCB while 1,2-DCB was not further dechlorinated by these cultures. This dechlorination pattern will be named the MB-1 type. A third pattern (MB-2) found in some cultures of culture line MB was similar to pattern MA/Z with the crucial difference that 1,4-DCB instead of 1,3-DCB was formed from 1,2,4-TCB while 1,2,3-TCB was dechlorinated to 1,2-DCB (Figure 2D). After TCB dechlorination, first 1,2-DCB and then also 1,4-DCB was dechlorinated to MCB. Using standard PCR, Dehalococcoides species could not be detected in culture lines MA, MB, or Z. Only by applying a much more sensitive nested PCR approach using universal and Dehalococcoides-specific primers successively, Dehalococcoides 16s rDNA was detected. Specific Inhibitors. The methanogenic activity in all cultures was completely inhibited by addition of 4 mM 2-bromoethanesulfonic acid; however, the dechlorinating activity and dechlorination pattern remained unchanged. In contrast, the effect of vancomycin on the dechlorinating activity and pattern varied among the culture lines. Dechlorination in cultures showing MA/Z- or MB-1-type patterns was strongly inhibited in the presence of vancomycin, while cultures dechlorinating according to the CBDB1-type remained active. When a culture of culture line MB, showing predominantly MA/Z-type dechlorination but also formation of trace amounts of 1,4-DCB, was transferred to vancomycincontaining medium the sole dechlorination products were 1,3- and 1,4-DCB. Aerobic incubation of an inoculum for 5 min, a procedure known to completely inactivate the chlorobenzene-dechlorinating strain CBDB1 (6), did not change the dechlorination pattern or the dechlorinating activity in cultures with dechlorination type MA/Z or MB-1. In contrast, a 5 min aerobic incubation of inocula from MB2-type culture lines prevented 1,4-DCB formation from 1,2,4TCB, while formation of 1,2-DCB and 1,3-DCB from 1,2,3TCB remained unchanged. Dechlorination of 1,3,5-TCB, PeCB, and HCB. Cultures of culture line MA showing the MA/Z-type dechlorination were further investigated to determine degradation of 1,3,5TCB, PeCB, and HCB. 1,3,5-TCB was transformed to 1,3DCB, which was slowly dechlorinated to MCB (Figure 3). The dechlorination patterns observed in the cultures set up with PeCB or HCB were similar to each other: none of the cultures produced 1,3,5-TCB as an intermediate or end product. In contrast, all cultures produced 1,2,3,4- and 1,2,3,5TeCB, 1,2,4-TCB, and 1,2- and 1,4-DCB. 8940

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FIGURE 3. Dechlorination of 1,3,5-TCB by cultures of culture line MA: (closed circles) 1,3,5-TCB, (open circles) 1,3-DCB, (closed triangles) MCB. Shown are means of triplicate cultures ( SD.

Discussion Detection of dechlorinating activity in most of the sludge samples investigated in our study suggests that chlorobenzene-dechlorinating bacteria are widely, though not ubiquitously, distributed in anaerobic sewage sludge. The specificity of the dechlorination reactions, the high dechlorination rates, the fact that dechlorinating activities could be transferred from one culture to the next, and the finding that autoclaved inocula lost their dechlorinating activity demonstrated that viable microorganisms were required for dechlorination. Our results show that the type of substrate used as the source of carbon and electrons can change the dechlorination pattern (e.g., comparing the results from Bad Wo¨rishofen). The fate of different electron donors in mixed cultures and their influence on dehalogenating bacteria was analyzed in detail and extensively discussed by others (26). In the present study, it was achieved for the first time to establish stable culture lines in synthetic medium with the capacity to dechlorinate singly flanked rather than doubly flanked chlorine substituents from chlorobenzenes. The use of synthetic medium facilitates the characterization and identification of the key players involved in dechlorination. The described dechlorination pattern directs the anaerobic reductive dechlorination of highly chlorinated benzenes in such a way that the accumulation of the persistent congener 1,3,5-TCB is avoided. Therefore, the cultures described here are of much greater practical value for the cleanup of ground waters or process effluents contaminated with chlorinated benzenes than cultures preferentially dechlorinating doubly flanked chlorine substituents. The apparent ability to dechlorinate 1,3,5-TCB suggests that these cultures also have potential for bioremediation at sites contaminated with 1,3,5TCB. In the cultures, MCB constituted the final dechlorination product. Dechlorination of MCB to benzene under anaerobic conditions has been rarely found (27, 28). In contrast, aerobic mineralization of MCB is an extensively studied process and has been demonstrated as a second decontamination step after anaerobic reductive dechlorination (29). To make use of the described dechlorination pattern for bioremediation, cultures dechlorinating preferentially singly flanked substituents could be applied in bioaugmentation processes after further enrichment and mass cultivation. Alternatively, the focus could be on the isolation of pure cultures from the described mixed cultures and characterization of their physiology to allow a rational approach in which the conditions at a contaminated site are controlled

in a way that favors bacteria dechlorinating singly flanked substituents. While some information is available about microorganisms preferentially dechlorinating doubly flanked chlorine substituents, nothing is known about the microorganisms preferentially dechlorinating singly flanked chlorines from chlorobenzenes. In the present study, the latter organisms were enriched from sludge samples of sewage plants in Manschow (culture lines MA and MB) and Zo¨rbig (culture line Z). The culture lines MA and Z always showed the same MA/Z-type dechlorination pattern, suggesting that one dechlorinating population was dominant. In contrast, in the different cultures of culture line MB, three distinct dechlorination patterns, MA/Z, MB-1, and MB-2 type, were observed, indicating the presence of several different, probably competing, dechlorinating microorganisms. Using nested PCR, low numbers of Dehalococcoides species were detected in the enrichment samples. The physiological role, however, of these Dehalococcoides species in the cultures is unclear. We suppose that the microorganisms preferentially catalyzing the removal of singly flanked chlorine substituents do not belong to the Dehalococcoides group. This assumption is supported by experiments with aerobically treated inocula. Cultures catalyzing MA/Z- or MB-1-type dechlorination were much less sensitive to oxygen exposure than pure cultures of strain CBDB1. Dehalococcoides species, such as the chlorobenzene-dechlorinating strains CBDB1 and 195, are known for their extremely high oxygen sensitivity (6, 30), with the possible exception of Dehalococcoides sp. strain FL2 (31), which appears to be more robust but has so far not been shown to dechlorinate chlorobenzenes (19). A second result supporting our hypothesis is the observation that MA/ Z- and MB-1-type dechlorination patterns were sensitive to vancomycin, an antibiotic that has been shown not to inhibit growth of Dehalococcoides species (6, 30). Vancomycin acts by inhibiting synthesis of a peptidoglycan cell wall, which is absent in known Dehalococcoides species (6, 30, 32-34). Therefore, Dehalococcoides species most probably do not contribute or contribute only to a minor extent to TCB dechlorination in MA/Z- or MB-1-type cultures, dechlorinating 1,2,3-TCB to 1,3-DCB as described for strain CBDB1, while the major share of TCBs is dechlorinated to 1,2- and 1,3-DCB by other microorganisms. Interestingly, in some MA/Z-type cultures with a low background dechlorination of 1,2,4-TCB to 1,4-DCB, much 1,4-DCB was produced after addition of vancomycin. In these vancomycin-containing cultures the Dehalococcoides population might completely be responsible for TCB dechlorination. Therefore, the treatment with vancomycin or oxygen is a useful tool for specific enrichment of CBDB1-type or MA/Z/MB-1-type dechlorinating microorganisms, respectively. In MB-2-type cultures, oxygen exposure had a significant effect on dechlorination, completely inhibiting dechlorination of 1,2,4-TCB to 1,4-DCB, while 1,2- and 1,3-DCB were still produced. Therefore, the MB-2 type represents a mixed pattern, which could result from the activity of Dehalococcoides species and other dechlorinators. Finally, it should be noted that the microbial TCB dechlorination examined here was accomplished by Bacteria and not by methanogenic Archaea since it was not inhibited in the presence of the specific inhibitor 2-bromoethanesulfonic acid. It has been shown that chlorobenzene-dechlorinating Dehalococcoides and Dehalococcoides-related strains are also responsible for dechlorination of polychlorinated dioxins (7, 8, 35) and/or polychlorinated biphenyls (PCBs) (7, 9, 36-38). Especially for PCBs many different dechlorination patterns involving the 209 congeners have been described in environmental and laboratory studies [for an overview, see refs 39, 40]. The dechlorination pathways observed at different field sites contaminated with complex

PCB mixtures have been classified into so-called dechlorination processes. These processes differentiate between several distinct dechlorination patterns, e.g., removing only flanked meta chlorines or removing unflanked para chlorines. However, there is still a need to develop methods for the characterization and visual comparison of apparent PCB dechlorination pathways (40). The graphic representation of dechlorination patterns described in our study could be a relatively simple means to aid in this task. Different chlorobenzene dechlorination patterns were clearly differentiated in our plots. The obtained patterns do not depend on the dechlorination rate, which can vary in cultures of the same culture line due to the size of the inoculum, the number of dechlorinating bacteria in the inoculum, redox conditions in the culture, the dechlorinating species present, and other parameters. Thus, differences in dechlorinating cultures from the same source are normalized, allowing compilation of a lot of data from one source without losing clarity. As described here for chlorobenzenes, relative concentrations of different PCB congeners can be plotted over the mean degree of chlorination of all PCBs present in a culture or sample. Similarly, our graphical representation can be used to characterize field sites where several sampling wells are available without correlating the patterns directly to time. Strain CBDB1, strain 195, and bacterium DF-1 preferentially or exclusively remove doubly flanked chlorine substituents from both PCBs and chlorobenzenes (5-7, 9, 37). Thus, it is important to investigate whether the cultures described in the present study catalyze the preferential dechlorination of singly flanked chlorine substituents also from PCBs or other chloroaromatics such as polychlorinated dioxins or chlorophenols. In this way, for environmental cleanup purposes, these cultures could complement cultures preferentially removing doubly flanked chlorines in their degradation capacity. The physiological information obtained in this work will be used for further characterization and identification of the bacteria preferentially dechlorinating singly flanked chlorine substituents using molecular and cultivation approaches.

Acknowledgments The authors thank Bernd Krostitz-Schroeer for excellent technical assistance. This work was supported by the European Research Council (ERC Microflex project, No. 202903-2) to L.A., the VIED to T.H.D., and the Deutsche Forschungsgemeinschaft (DFG AD178/1).

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