Environ. Sci. Technol. 2007, 41, 2318-2323
Growth of Dehalococcoides Strains with Chlorophenols as Electron Acceptors L O R E N Z A D R I A N , * ,† S I G R I D K . H A N S E N , † JENNIFER M. FUNG,‡ HELMUT GO ¨ RISCH,† AND STEPHEN H. ZINDER‡ FG Technische Biochemie, Technische Universita¨t Berlin, Berlin, Germany, and Department of Microbiology, Cornell University, Ithaca, New York 14853
Dehalococcoides strains reductively dechlorinate a wide variety of halogenated compounds including chlorinated benzenes, biphenyls, naphthalenes, dioxins, and ethenes. Recent genome sequencing of the two Dehalococcoides strains CBDB1 and 195 revealed the presence of 32 and 18 reductive dehalogenase homologous genes, respectively, and therefore suggested an even higher dechlorinating potential than previously anticipated. Here, we demonstrate reductive dehalogenation of chlorophenol congeners by Dehalococcoides strains CBDB1 and 195. Strain CBDB1 completely converted 2,3-dichlorophenol, all six trichlorophenols, all three tetrachlorophenols, and pentachlorophenol to lower chlorinated phenols. Observed dechlorination rates in batch cultures with cell numbers of 107 mL-1 amounted up to 35 µM day-1. Chlorophenols were preferentially dechlorinated in the ortho position, but also doubly flanked and singly flanked meta- or para-chlorine substituents were removed. We used a newly designed computerassisted direct cell counting protocol and quantitative PCR to demonstrate that strain CBDB1 uses chlorophenols as electron acceptors for respiratory growth. The growth yield of strain CBDB1 with 2,3-dichlorophenol was 7.6 × 1013 cells per mol of Cl- released, and the growth rate was 0.41 day-1. For strain 195, fast ortho dechlorination of 2,3dichlorophenol, 2,3,4-trichlorophenol, and 2,3,6-trichlorophenol was detected, with only the ortho chlorine removed. Because chlorinated phenolic compounds are widely distributed as natural components in anaerobic environments, our results reveal one mode in which the Dehalococcoides species could have survived through earth history.
Introduction Dehalococcoides strains are highly specialized strictly anaerobic bacteria that are known to grow only by respiration using hydrogen as an electron donor and halogenated organic compounds as electron acceptors. Neither fermentation nor respiration with non-halogenated compounds has been observed with any of the described isolates. Recent analyses of the full genomes of strains 195 and CBDB1 support these results (1, 2), showing little evidence for other modes of metabolism. Many toxic aromatic compounds including * Corresponding author. Phone: +49 30 45080266. Fax: +49 30 31427581. E-mail:
[email protected]. † Technische Universita ¨ t Berlin. ‡ Cornell University. 2318
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highly chlorinated dioxins, furans, biphenyls, naphthalenes, benzenes, and brominated diphenyl ethers have thus far been shown to be reductively dechlorinated by Dehalococcoides strains and relatives (3-6), as have chloroaliphatics including chlorinated ethenes, 1,2-dichloroethane, and 1,2-dichloropropane (7-9), making this genus a remarkable group of bacteria. PCR-based studies have shown a wide but not ubiquitous distribution of Dehalococcoides strains in anaerobic environments (10) and demonstrated their pivotal role in the bioremediation of contaminated soils and aquifers (11). A trichloroethene reductive dehalogenase was characterized in Dehalococcoides ethenogenes strain 195, and the encoding gene was identified (tceA, 12). In Dehalococcoides strains VC (13) and BAV1 (14), vinyl chloride reductive dehalogenases were identified by reverse genetics and transcriptional analysis, respectively. Apart from the tceA/ tceB gene pair, 17 reductive dehalogenase homologous (rdh) gene pairs were detected in strain 195 by genome sequencing (1). Fourteen rdh gene pairs have been detected in each of the Dehalococcoides strains CBDB1 and FL2 using degenerate PCR primers (15). For strain CBDB1, this number was recently corrected by full genome sequencing to a total of 32 complete rdh pairs (2). However, apart from the trichloroethene reductive dehalogenase (tceAB) in strain 195, no rdh gene has been linked to a function in strains 195 or CBDB1. The conservation of specific rdh genes between different Dehalococcoides strains (15), the co-evolution of the two rdh subunits (2), and the tight linkage of rdh pairs with regulatory genes suggest that the different rdh genes convey different enzymatic properties to the strains, many of which are not known yet. Chlorophenols, including the biocide pentachlorophenol, are utilized as electron acceptors by several strains of Desulfitobacterium (16-18). Here, we describe physiological experiments that show reductive dechlorination of many different chlorinated phenols by Dehalococcoides strains CBDB1 and 195. In addition, we demonstrate the growth of strain CBDB1 with chlorinated phenols as electron acceptors in synthetic medium.
Materials and Methods Pure cultures of Dehalococcoides strains 195 (8) and CBDB1 (3) were cultivated using media described for the two strains previously. Both strictly anaerobic, bicarbonate buffered mineral media were amended with trace elements, vitamins, resazurin, 5 mM acetate, and hydrogen (nominal concentration 4.5 mM). While the medium used for cultivation of strain CBDB1 was purely synthetic and reduced with Ti(III) citrate, the medium of strain 195 was reduced with 1 mM sulfide and amended with two complex additions to obtain growth of strain 195: the sterile filtered supernatant of a mixed anaerobic digestor culture and the cell extract of a dehalogenating mixed consortium (8). Ti(III) citrate was prepared from synthesis grade Ti(III) chloride (Merck--Schuchardt, Hohenbrunn, Germany), aqueous sodium citrate solution, and solid sodium carbonate as described (19). All other chemicals were of analytical grade. Inactivation of Dehalococcoides cells for negative controls was done by shaking 500 µL of an inoculum and 500 µL of air in a syringe until the cell suspension turned pink due to oxidation of the redox indicator resazurin. After a 5 s exposure to this oxic condition, the cell suspension was reduced by taking up about 5 µL of Ti(III) citrate solution into the syringe. The suspension of inactivated cells was then used to inoculate fresh media. 10.1021/es062076m CCC: $37.00
2007 American Chemical Society Published on Web 02/22/2007
For growth experiments, dichlorophenols and trichlorophenols were periodically added to cultures with syringes from 1 mM sterile filtered aqueous stock solutions. For other experiments, chlorophenols were added as a 50 mM solution in methanol resulting in a final concentration of 20 µM chlorophenols and 10 mM methanol. Pentachlorophenol was added from 10 mM methanolic or acetonic solution to a final concentration of 5 µM and re-fed when it was consumed to avoid toxic concentrations. Chlorophenols were analyzed by HPLC (Beckmann) using a 3 µm bead diameter Alltima C8 column (length 53 mm, i.d. 7 mm, Alltech, Deerfield, IL) at ambient temperature. The solvent for isocratic elution was acetonitrile/water/acetic acid (50:50:0.1 v/v/v) at 1.5 mL min-1, and chlorophenols were detected by their absorbance at 220 nm. Alternatively, chlorophenols were analyzed on a 5 µm bead diameter Multisphere 100 RP18 column (Chromatographie-Service, Langerwehe, Germany) with methanol/ water/acetic acid (65:35:0.1 v/v/v) as solvent at a flow rate of 1 mL min-1. HPLC analysis allowed detection of chlorophenols to concentrations as low as 3-5 µM. Chlorophenols with similar retention times (e.g., 2,4- and 3,4-dichlorophenol) were differentiated by gas chromatography after acetylation (20) with acetic anhydride under basic conditions and extraction with hexane (5 mg of NaHCO3, 1 mL sample, 5 µL of acetic anhydride, 100 µL of n-hexane, shaking at 20 °C, 300 rpm for 2 h). The better sensitivity of the gas chromatographic analysis down to about 1 µM was also used to verify complete conversion of chlorophenol congeners. Analysis by gas chromatography (Shimadzu 14b, Tokyo, Japan) and flame ionization detection used a Permabond-FFAP column (25 m, 0.25 mm i.d., 0.25 µm film thickness, Macherey and Nagel, Du ¨ ren, Germany) with the following temperature program: split-less at 70 °C for 1 min, then with a split of 1:50, 15 °C min-1 to 150 °C, 6 °C min-1 to 230 °C, and 10 min at 230 °C. Injector temperature was 220 °C, and detector temperature was 230 °C. The presence of chlorobenzenes was analyzed on the same column by gas chromatography as previously described (3) with a sensitivity of about 0.5 µM. Cell counting was done both by direct microscopy and by quantitative PCR. For microscopic counting, cells were immobilized on agarose-coated slides. The slides were prepared by dispensing 2 mL of a hot autoclaved 2% (wt/v) agarose solution (AppliChem, Darmstadt, Germany) evenly on glass slides (21) and allowing them to dry overnight in a clean bench. A volume of 18 µL of cell suspension containing 0.5 µg mL-1 4′,6-diaminido-2-phenyl-indol-dihydrochloride (DAPI) was placed on an agarose-coated slide and immediately covered with a 20 mm × 20 mm cover glass. The liquid volume of 18 µL was chosen because it filled the entire space between the cover glass and the agarose-coated glass slide without flowing out of the area under the cover glass. As a result, the added bacteria were homogeneously distributed on the 20 mm × 20 mm area. Swelling of the agarose forced all cells into a single plane of focus level just below the cover glass and also ensured that the disk-shaped Dehalococcoides cells were all lying flat, which is the only position in which the cells can be seen by light microscopy. Microscopical counting was done using epifluorescence with an Axioskop 2 (Zeiss, Jena, Germany) at 400× magnification. For each sample, 10 micrographs were taken with a Nikon Coolpix 990 digital camera at a fixed focus and aperture. For calibration, a micrometer scale was photographed with the same focus. Processing of the pictures, cell counting, and the calculation of the picture sizes were done with ImageJ software and mostly automated with macros. The successive processing steps were (i) conversion to eight-bit gray scale (pixel values between 0 and 255), (ii) linear adjustment of the brightness so that the mode brightness of the background pixels was at a value of 120, (iii) conversion to a two-bit black and white
FIGURE 1. Strain CBDB1 cultured with 2,3-DCP (O) as the electron acceptor that was dechlorinated to 3-MCP (b). Increasing concentrations of 2,3-DCP were added at the times indicated by arrows. Successive doses were dechlorinated faster. Shown are means of duplicate cultures inoculated with a very small inoculum (0.5% v/v of 8 × 106 cells mL-1 leading to an initial cell number of 4 × 104 mL-1). Variations between the two cultures were less than 10% at each time point. No MCP was produced in abiotic controls or in cultures that were inoculated with inactivated cells. picture using a fixed threshold somewhat brighter than the mode brightness of the background (>135), and (iv) enumeration of the cells with the ImageJ Cell Counter plug-in. The results obtained with this direct counting method were highly reliable as tested with several independent samples of the same culture or with defined dilution steps of a single culture. Standard deviations of the determination were about 10%. Counting of defined dilutions of one culture resulted in correlation factors between dilution and counted cell number of over 0.99. The detection limit was at about 1 × 106 cells mL-1, which corresponded to about two cells per microscopic field. This detection limit could be improved by an initial centrifugation step. Quantitative PCR was done using an ABI 7000 real time PCR machine (Applied Biosystems, Foster City, CA) using the iQ Sybr-Green Supermix (Biorad, Hercules, CA) and primers that targeted sequences within the 16S rRNA gene of Dehalococcoides strains (GGA GCG TGT GGT TTA ATT CGA TGC and GCC CAA GAT ATA AAG GCC ATG CTG). The template was prepared from 200 µL of culture liquid that was centrifuged (15 min, 6000g), and the pellet was resuspended in 500 µL of 10 mM Tris buffer pH 8.0 and again centrifuged (15 min, 6000g). The supernatant was removed so that 50 µL remained in the tube. Cell lysis was achieved using six cycles of freeze-thaw (-80 °C for 1 min, +60 °C for 2 min). DNA was precipitated by adding sodium acetate (pH 5.2) to 0.3 M and 2 volumes of ethanol. The mixture was incubated at -20 °C for 1 h and centrifuged (15 min, 14000g), and the pellet was resuspended in 50 µL of deionized H2O. Of this template solution, 1 µL was used for amplification. Absolute quantification of PCR data was done by including cultures in which cell numbers were determined by direct cell counting.
Results Growth with 2,3-Di- or 2,3,4-Trichlorophenol. Dehalococcoides strain CBDB1 and strain 195 could be routinely transferred in medium with hydrogen as an electron donor and 2,3-dichlorophenol (DCP) or 2,3,4-trichlorophenol (TCP) as an electron acceptor using small inocula of 0.5-1% in each of the successive transfers without losing dechlorinating activity. Successful transfers were not possible if chlorophenols were omitted. When cultures were repeatedly fed, additional doses of 2,3-DCP or 2,3,4-TCP were dechlorinated faster than the initial amendment (Figure 1). Detailed growth studies were performed with strain CBDB1, which in contrast to strain 195 grew in a purely synthetic medium where no fermentative growth was possible. Cell numbers were monitored primarily by a newly designed direct microscopic VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Growth of strain CBDB1 with 2,3-DCP as an electron acceptor monitored by microscopic cell counting. 2,3-DCP was re-fed to a concentration of 20-50 µM whenever the concentration was below 5 µM. (4) Mean cell number of triplicates and (b) 3-MCP produced. Bars indicate standard deviations. Cell numbers in cultures without 2,3-DCP were constantly below the detection limit.
FIGURE 3. Correlation between cell numbers of strain CBDB1 observed and 3-MCP produced in cultures with 2,3-DCP as electron acceptor. Shown are means of triplicate cultures ( standard deviations. cell counting method and as a complementary method by quantitative PCR. Direct microscopic counting revealed that growth strictly depended on dechlorination of chlorophenols (Figure 2). If no chlorinated phenols were added, the cell number did not reach the detection limit of 1 × 106 cells mL-1. When sequential additions of 20-50 µM 2,3-DCP were made to a culture whenever 2,3-DCP was depleted below 5 µM, growth was detected until a maximum number of 1.7 × 107 cells mL-1 was reached and about 250 µM 3-monochlorophenol (MCP) was produced (Figure 2). During this growth phase, the cell number was proportional to the concentration of 3-MCP produced and amounted to a molar cell yield of 7.6 × 1013 cells per mol of Cl- released (Figure 3). The observed growth rate was about 0.41 day-1, and the specific dechlorination rate was 2.0 × 10-9 µmol day-1 cell-1. The cultures reached the maximum cell number and the maximum dechlorination rate after 13 days of incubation and a production of about 250 µM 3-MCP. In the following week of incubation, the cell number dropped to less than 107 cells mL-1, and the dechlorination rate slowly decreased (Figure 2). However, significant amounts of 2,3-DCP were dechlorinated, indicating that growth and dechlorination were uncoupled during this time. Several independent experiments have yet failed to reveal if this uncoupling of growth and dechlorination was due to depletion of media components or due to toxicity effects of accumulated products. Growth of the strain CBDB1 could also be shown by direct cell counting in cultures incubated with 2,3,4-TCP as an electron acceptor. In this case, growth always occurred parallel to the production of 2,4- and 3,4-dichlorophenol and ceased when 2,3,4-TCP was depleted. However, production of monochlorophenols continued slowly, indicating cometabolic conversion of dichlorophenols. Growth of strain CBDB1 using 2,3-DCP or 2,3,4-TCP as an electron acceptor was confirmed with quantitative PCR 2320
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FIGURE 4. Summary of chlorophenol dechlorination reactions catalyzed by Dehalococcoides strain CBDB1. Bold compounds: compounds that were completely converted if added as a sole electron acceptor; bold arrows: main pathway; thin arrows: side pathway; dashed arrows: slow and incomplete reactions; and dotted arrows: reactions that occurred only if the respective dichlorophenol was formed from a higher chlorinated phenol in the same culture. The asterisks mark reactions where the pathways could not be distinguished. See text for details. as a second method for cell enumeration. These experiments were done twice with duplicate cultures. Initial concentrations of 50 µM 2,3-DCP were completely dechlorinated within 18 days to 3-MCP. During this time, the cell number increased from 9 × 104 to 3 × 106 cells mL-1. After further addition of 50 µM 2,3-DCP and another 9 days of incubation, 2,3-DCP was again completely dechlorinated, and the cell number was determined by quantitative PCR to be 6.3 × 106 cells mL-1. From these data, the molar growth yield was 6.3 × 1013 cells per mol of Cl- released. An increase of the cell number was also determined with strain 195 incubated on 2,3-DCP or 2,3,4-TCP. With 2,3-DCP as an electron acceptor, cultures of strain 195 developed a cell number of 1 × 107 cells mL-1 after 40 days of incubation when 120 µM 3-MCP was produced, corresponding to a molar yield of 8.3 × 1013 cells per mol of Cl-. Cultures that did not contain chlorophenols or cultures that contained 2,4-DCP did not show an increase in cell number during the time of incubation as determined by the same quantitative PCR method. Dechlorination of Other Chlorophenol Congeners. To test the dechlorination spectrum of strains CBDB1 and 195, cultures were amended with single chlorophenol congeners as an electron acceptor together with abiotic and inactivated controls. Strain CBDB1 dechlorinated all highly chlorinated phenols including pentachlorophenol, all three tetrachlorophenol (TeCP) congeners, and all six TCP congeners (Figure 4). In addition, 2,3-DCP and at low rates 2,6-DCP and 2,4-
DCP were transformed by strain CBDB1. The products of all observed reactions were lower chlorinated phenols. Production of phenol was not observed. Between the different congeners, fastest dechlorination was observed with 2,3-DCP, 2,3,4-TCP, 2,3,5-TCP, 2,3,6-TCP, 3,4,5-TCP, 2,3,4,6-TeCP, and pentachlorophenol. In these cultures, a concentration of 20 µM parent compound was completely dechlorinated within 1 week of incubation. 2,3,4,5-TeCP and 2,3,5,6-TeCP were completely converted within 2 weeks of incubation. Slow dechlorination with products detectable after 3 weeks of incubation was observed with 2,4,5-TCP and 2,4,6-TCP. The congeners 2,4-DCP and 2,6-DCP were dechlorinated only partially (about 50%) after an extended incubation time of several months. The three monochlorophenols, and the three dichlorophenols 3,4-DCP, 3,5-DCP, and 2,5-DCP, were not dechlorinated by strain CBDB1 when added as the sole electron acceptor. However, slow dechlorination of 3,4-DCP and 2,5-DCP was seen when they were generated in the culture by dechlorination of higher chlorinated phenols. No abiotic dechlorination occurred under the used cultivation conditions, and also, controls with autoclaved inocula or with inocula that were inactivated by exposure to oxygen did not transform any of the chlorophenols. All these experiments were done with triplicate cultures and three independent negative controls for each chlorophenol congener. With strain CBDB1, dechlorination of chlorophenols was fastest in the ortho position if the neighboring meta position was also chlorinated. As an example, 2,3-DCP was rapidly and stoichometrically dechlorinated to 3-MCP. Other ortho dechlorinations included that of 2,3,5,6-TeCP via 2,3,5-TCP to 3,5-DCP, 2,3,5-TCP to 3,5-DCP, and 2,3,6-TCB to a mixture of 2,5-DCP and 3-MCP. Also, doubly flanked chlorine substituents (chlorine substituents that had chlorine substituents on both sides) were rapidly dechlorinated by the CBDB1 strain. As an example of this type of dechlorination, 3,4,5-TCB was stoichiometrically dechlorinated to 3,5-DCP. If both possibilities existed as in 2,3,4-TCP, 2,3,4,5-TeCP, 2,3,4,6-TeCP, and pentachlorophenol, ortho dechlorination occurred, but doubly flanked chlorine substituents were also removed. From 2,3,4-TCP, both 3,4-DCP and 2,4-DCP were produced with minor amounts of 3-MCP and 4-MCP also formed. Pentachlorophenol dechlorination resulted in the production of a mixture of 3,5-DCP, 3,4-DCP, 2,4-DCP, 3-MCP, and 4-MCP, indicating that several dechlorination pathways were catalyzed. Similarly, the dechlorination of 2,3,4,5-TeCP yielded a mixture of 3,5-DCP and 2,4,5-TCP, while 2,3,4,6-TeCP was dechlorinated to 3,4-DCP, 2,4-DCP, and 2,5-DCP. Reduction of the chlorophenols to the corresponding chlorobenzenes was never observed, even if the hydroxyl group was flanked by two chlorine substituents (e.g., in 2,6-DCP). Slow dechlorination of singly flanked chlorine substituents in the ortho position was observed (2,4,5-TCP to 3,4-DCP, 2,4,6-TCP to 2,4-DCP, 2,4-DCP to 4-MCP, and 2,6-DCP to 2-MCP). Dehalococcoides strain 195 dechlorinated a smaller spectrum of chlorophenols, all in the ortho position and only if a chlorine substituent was present in the flanking meta position. Dechlorination was detected with 2,3-DCP, 2,3,4TCP, and 2,3,6-TCP but not with other di- or trichlorophenols or pentachlorophenol. Like strain CBDB1, strain 195 did not dechlorinate monochlorophenols. Tetrachlorophenols were not tested with strain 195. 2,3-DCP was completely converted to 3-MCP, 2,3,4-TCP to 3,4-DCP, and 2,3,6-TCP to an equimolar mixture of 2,5-DCP and 3-MCP. Toxicity. Toxicity tests were performed by adding increasing initial concentrations of 2,3,4-TCP to cultures containing a small inoculum of Dehalococcoides strain CBDB1 (0.5% v/v). With an initial 2,3,4-TCP concentration of 75 µM or above, no product formation was detected in the cultures within 13 weeks of incubation, while cultures that were set
up with 10, 25, or 50 µM significant concentrations above 3 µM dichlorophenol were produced within 7, 14, and 21 days, respectively (data not shown). After several doses of 20-50 µM 2,3,4-TCP, higher additions of up to 80 µM could be added without inhibition of dechlorination in the cultures. Cultures of both investigated Dehalococcoides strains could repeatedly be fed with 2,3-DCP or 2,3,4-TCP up to accumulated product concentrations of about 250-300 µM 3-MCP or 3,4-DCP, respectively, without apparent inhibition.
Discussion This paper describes a new class of compounds that is used for respiratory dehalogenation by Dehalococcoides spp., the chlorophenols. With the two congeners 2,3-DCP and 2,3,4TCP, we provide experimental proof for such respiratory growth for strain CBDB1 and strong evidence for strain 195. Other congeners that were rapidly dechlorinated seem to serve similarly as respiratory electron acceptors. In contrast, 2,4-DCP and 2,6-DCP were only slowly and incompletely dechlorinated, and also, 2,5-DCP and 3,4-DCP that were only dechlorinated when they were formed in the culture from higher chlorinated phenols appear to be transformed cometabolically by strain CBDB1. Chlorophenols are the first known compounds with functional groups other than chlorine substituents that were identified to serve as electron acceptors for Dehalococcoides strains. This is particularly important as chlorophenols have been demonstrated to be abundant in many different natural environments. As one of many examples, halogenated phenolic compounds are known to be products of aerobic lignin degradation (22). Therefore, chlorinated phenols could have contributed together with other naturally occurring halogenated aliphatic or aromatic compounds to the evolution, survival, and spread of bacteria of the Dehalococcoides cluster before man-made chlorinated compounds were available. The possibility that the dechlorination potential was developed only recently in response to man-made contamination was ruled out by genomic studies, showing that the topologies of 16S rDNA and reductive dehalogenase gene trees were corresponding to each other (15). Ortho dechlorination of chlorophenols was previously described for several Desulfitobacterium spp. (reviewed by refs 17 and 23), Anaeromyxobacter species (24), and Desulfovibrio dechloroacetivorans (25). In contrast, Desulfomonile tiedjei dechlorinated highly chlorinated phenols at the meta position (26). Also, a number of Desulfitobacterium species dechlorinated chlorophenols at the meta (27) or meta and para-positions (16). These bacteria dehalogenate a wide spectrum of chlorinated phenols but not chlorobenzenes, PCBs, or PCDD/PCDFs. Desulfitobacterium dehalogenans was able to reductively dechlorinate chlorophenols and hydroxylated PCBs but not unmodified PCBs (28). This narrow spectrum of chlorinated compounds used as electron acceptors is reflected by a lower number of rdh genes encoded in the genomes of Desulfitobacterium species as compared to Dehalococcoides strains (29). In contrast, several studies show that some Dehalococcoides related strains are able to dechlorinate PCBs (5, 30-32). Without identifying the dechlorinating bacteria in their mixed culture, Cho et al. (33) have found a supporting effect of different halogenated aromatic compounds including chlorinated phenols on the reductive dechlorination of PCBs, a process described as priming. Our finding that Dehalococcoides strains also grow by respiratory dehalogenation of chlorophenols now suggests that this priming effect is due to the growth of Dehalococcoides strains on the halogenated aromatic priming compound. With two independent methods, we demonstrated that strain CBDB1 grows by using chlorophenols as electron acceptors in respiratory dehalogenation. The measured yield of 7.6 × 1013 cells per mol of Cl- released obtained with VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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chlorophenols is similar to growth yields found for various Dehalococcoides strains with different electron acceptors including strain CBDB1 growing on hexachlorobenzene (8.2 × 1013 cells per mol of Cl- released) (34), strain BAV1 with vinyl chloride (6.3 × 1013 cells per mol of Cl- released) (7), strain FL2 on trichloroethene (7.8 × 1013 cells per mol of Clreleased) (35), and 2.5 × 1014 for strain GT using vinyl chloride (36). Tetrachloroethene dechlorinating Dehalococcoides strains in mixed cultures showed considerably higher growth yields of 5.2 × 1014 (37) and 5.6 × 1014 cells per mol of Cl- released (38), respectively. Conversion of these molar cell yields to molar protein yields requires a factor describing the protein content of a single cell. In the study mentioned previously (34), we measured both protein concentrations and cell numbers of strain CBDB1 growing in medium with hexachlorobenzene as the electron acceptor. From these data, a conversion factor of 2.3 × 10-14 g of protein cell-1 can be calculated. Using this conversion factor, the observed growth of strain CBDB1 with 2,3-DCP as the electron acceptor gave a protein yield of 1.73 g of protein per mol of Cl- released, which compares well with molar growth yields reported for Desulfitobacterium spp., Dehalobacter, or Sulfurospirillum spp. growing by respiratory dehalogenation (1-4 g of protein per mol of Cl- released). While most studies used quantitative PCR to enumerate Dehalococcoides cell numbers, we quantified via a direct microscopic method. While quantitative PCR can be more easily parallelized and also allows for the quantification of Dehalococcoides cells in mixed cultures or cultures with complex media, the direct cell counting method described here has the important advantage of not requiring any manipulation of the culture liquid such as centrifugation steps or DNA extraction that can strongly influence the results of a PCR determination (36). In addition, the quantification of a few samples can be achieved much faster with the direct cell counting method. Comparing the two Dehalococcoides strains 195 and CBDB1, our results extend previous findings (3-5, 8, 34) that strain CBDB1 can dechlorinate a wider spectrum of chlorinated aromatic compounds than strain 195. In contrast, strain 195 is better adapted to growth with chlorinated ethenes (3, 8). This difference in the substrate spectrum is also reflected in the genomes of the two organisms (1, 2). While strain CBDB1 encodes nearly twice the number of rdh genes than strain 195, it does not encode the tceAB operon that was found in strain 195. It was suggested that the different orthologue clusters of rdh genes encode different enzymatic activities (2, 15). With the present study, we provide potential functions for several of these orthologue clusters in Dehalococcoides strains. Our findings that some dechlorination reactions are shared between strains CBDB1 and 195 such as the dechlorination of 2,3-DCP or 2,3,4-TCP while other reactions are only catalyzed by strain CBDB1 coincides with the identification of both orthologue clusters that encompass rdh genes from both strains and of orthologue clusters with an rdh gene only from strain CBDB1. The physiological substrates of the different rdh gene products, however, remain to be identified. For that, cultivation with chlorophenols is a promising new tool, as the strains can be cultivated on significantly different electron acceptors to cell numbers of more than 107 cells mL-1 and successively be differentially analyzed for protein or mRNA levels.
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(17) (18)
Acknowledgments
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The authors thank B. Krostitz-Schroeer for excellent technical assistance. This work was supported by NSF Grant MCB 0236044 to S.H.Z. and DFG AD178/1 to L.A.
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Received for review August 30, 2006. Revised manuscript received January 17, 2007. Accepted January 19, 2007. ES062076M
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