Environ. Sci. Technol. 2006, 40, 5704-5709
Stimulatory and Inhibitory Effects of Organohalides on the Dehalogenating Activities of PCB-Dechlorinating Bacterium o-17 H A R O L D D . M A Y , * ,† L E A H A . C U T T E R , † GREGORY S. MILLER,† CHARLES E. MILLIKEN,† JOY E. M. WATTS,§ AND KEVIN R. SOWERS‡ Department of Microbiology and Immunology, Marine Biomedicine and Environmental Science Center, Medical University of South Carolina, Charleston, South Carolina 29425, and Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, and Department of Biology Sciences, Towson University, Towson, Maryland 25212
Bacterium o-17, a microorganism capable of the ortho dechlorination of 2,3,5,6-polychlorinated biphenyl (PCB), is a member of a sediment-free, nonmethanogenic mixed culture. The culture was examined for the ability to dechlorinate 26 PCB congeners, 12 chlorobenzenes (CBZs), and 6 chlorinated ethenes (CEs). Eight of the PCBs and 4 of the CBZs were dechlorinated including single-flanked ortho PCB chlorines, but double-flanked chlorines of PCBs and CBZs were preferentially dechlorinated. The dechlorination of three of the PCBs (2,3,4,5,6-, 2,3,4,6-, and 2,3,5,6-PCB), three of the CBZs (hexa-, penta-, and 1,2,3-CBZ), and PCE could be sustained for three or more sequential transfers of the bacterial community. Two PCBs (2,3,4- and 2,3,5-PCB), two CBZs (1,2,3,5- and 1,2,4,5-CBZ), and trichloroethene were dechlorinated only when a more extensively chlorinated parent compound was present. Aroclor 1260 and 2,4,6PCB, not dechlorinated by the culture, inhibited the dechlorination of 2,3,5,6-PCB. Within the culture only bacterium o-17 was linked to dechlorination by PCR-DGGE analysis, confirming that this dehalogenating species was the catalyst for the dechlorination of the compounds tested. The microorganism is capable of dechlorinating several different congeners of PCBs, CBZs, and CEs, and it remains a rare example of an ortho-PCB dechlorinator. However, its limited ability to dechlorinate more extensively chlorinated congeners and Aroclor plus the inhibitory effects of some PCB congeners upon the bacterium is consistent with the observed infrequency of this reaction in the environment. An assessment of bioremediation potential of this microorganism in situ will require a greater understanding of the synergistic, cometabolic and competitive interactions of PCB dechlorinating microbial communities.
* Corresponding author phone: (843)792-7140; fax: (843)792-2464; e-mail:
[email protected]. Mailing address: Medical University of South Carolina, Department of Microbiology and Immunology, 173 Ashley Ave., 225 BSB, P.O. Box 250504, Charleston, SC 29425-2230. † Medical University of South Carolina. ‡ University of Maryland Biotechnology Institute. § Towson University. 5704
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Introduction Polychlorinated biphenyls (PCBs) are hydrophobic compounds of low volatility and chemical reactivity that adsorb to particles and settle into anoxic soils and sediments where they enter the food chain eventually bioaccumulating in the fatty tissue of animals (for a review of PCBs see ref 1). Due to the persistence and potential toxic threat of PCBs to humans and wildlife, effective procedures for their remediation have been long sought. PCBs, identified by their Aroclor trade names, are listed 11 times among the 275 compounds or mixtures of compounds included in the 2005 CERCLA Priority List of Hazardous Compounds (http:// www.atsdr.cdc.gov/cercla/05list.html). Aroclors 1254 and 1260 are posted as the 13th and 14th most hazardous on the list (ranking is based on a combination of the frequency of occurrence, toxicity, and potential for human exposure at a National Priority Site, i.e., Superfund site). An effective microbial approach to the problem, either through active treatment or natural attenuation, would be attractive since it would be less expensive than approaches such as dredging and capping and could be performed in situ. PCB transforming bacteria, which include PCB dechlorinating anaerobes and PCB oxidizing aerobes, have been demonstrated to be active in the laboratory and in situ (for a review see ref 2). The anaerobic process is critical for PCB degradation as contaminated sediment will be predominantly anoxic, and aerobic bacteria generally do not attack more extensively chlorinated congeners found in commercial PCB mixtures. Thus, the reductive dechlorination of PCBs by anaerobes is critical for the initial transformation of higher halogenated congeners in any bioremedial process. A detailed understanding of the physiological, ecological, and environmental requirements of dechlorinating bacteria is required to facilitate utilization of dechlorinating bacterial populations for significant removal of PCBs from the environment. Multiple, different dehalogenation reactions are required to completely dechlorinate a mixture of PCBs (reviewed in ref 3). Even the dechlorination of chlorobenzenes (CBZs) and chlorinated ethenes (CEs), of which there are far fewer congeners and isomers, requires more than one dechlorination reaction. Several unique microbial dehalogenation reactions have been documented (reviewed by Smidt and de Vos (4) and throughout ref 5), but the identification of the microbial catalysts (microorganisms and specific dehalogenases) responsible for many of these reactions remains unknown. However, significant discoveries have been made regarding the identification of microorganisms that can dechlorinate PCBs, CBZs, and CEs. For example, Dehaococcoides ethenogenes strain 195 (6-9), and other members of the Chloroflexi (10-12) have been shown to dechlorinate members of all three of these groups of chlorinated compounds. In addition, Dehalococcoides sp. strain CBDB1 has been shown to dechlorinate chlorobenzenes (8), Dehalococcoides sp. strain FL2 dechlorinates TCE to ethene (13), Dehalococcoides sp. BAV1 dechlorinates vinyl chloride or DCEs to ethene (14), and Dehalococcoides sp. strain VS has been shown to dechlorinate vinyl chloride and cis-DCE to ethene (15). Strains CBDB1 and 195 also dechlorinate chlorinated dioxins (9, 16), and strain 195 dechlorinates chlorinated napthalenes (9). Whether strains CBDB1, FL2, BAV1, and VS can dechlorinate PCBs remains to be determined. None of these strains have been tested with all 209 PCBs, 12 CBZs, and 6 CEs, but the literature suggests that mixed populations of dehalogenating microorganisms with synergistic dechlorination pathways are required for maximal 10.1021/es052521y CCC: $33.50
2006 American Chemical Society Published on Web 08/18/2006
PCB dechlorination. For example, Fagervold et al. (17) demonstrated the sequential reductive dechlorination of PCB 132 was catalyzed by the complementary activities by two dehalogenating species. Cutter et al. (10) and Wu et al. (12) identified two distinct PCB dechlorinating bacteria within nonmethanogenic, sediment-free, mixed cultures. The growth of the dechlorinating organisms within the enrichment cultures was linked to PCB reductive dechlorination; this was demonstrated by analysis of the microbial community with polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) of 16S rRNA genes. PCR-DGGE revealed four different microorganisms in the o-17 culture including bacterium o-17, which was shown to be responsible for the ortho dechlorination of 2,3,5,6-PCB to 2,3,5-PCB and 3,5-PCB (10). This dechlorination activity is distinctly different from that demonstrated with other PCB dechlorinating microorganisms such as bacterium DF-1 (12) and Dehalococcoides ethenogenes strain 195 (9), which have only been shown to dechlorinate doubleflanked chlorines, defined as having two adjacent chlorinecarbon bonds in PCBs or chlorobenzenes (CBZs). D. ethenogenes strain 195 has not been tested with 2,3,5,6-PCB. Due to its ability to catalyze the reduction of ortho-chlorines of PCBs, which is rarely observed in the environment (2, 3), the catabolic and cometabolic activities of bacterium o-17 were further examined for a broad range of PCBs, CBZs, and CEs.
Materials and Methods Chemicals. All PCBs (99% purity) were purchased from AccuStandard. All chlorobenzenes and chlorinated ethenes were purchased from Sigma-Aldrich. The purity of cis-DCE was 97% and of trans-DCE was 98%. Purity of 1,3-, 1,2,3,4-, 1,2,4,5-, and penta-CBZ was 98%. The purity of all other CBZs and CEs was 99% or greater. Culture Procedures. A defined estuarine medium (E-Cl) was prepared anaerobically as described by Berkaw et al. (18), except the concentration of Na2S‚H2O was decreased to 10 µM. Volumes of 10 mL E-Cl were dispensed into 30 mL anaerobe tubes (Bellco) or 50 mL E-Cl in 165 mL serum bottles, which were sealed under N2-CO2 (80/20) with 20 mm Teflon-coated butyl stoppers (West Co., Lionville, PA) secured with aluminum crimp seals (Wheaton). The medium was sterilized by autoclaving at 121 °C for 30 min. The o-17 culture was enriched in sediment-free medium as described previously (10, 12, 19). PCR-DGGE, sequencing of DNA fragments (392 bp) from the DGGE, cloning and sequencing of longer 16S rRNA gene fragments (approximately 1500 bp) using DNA primers specific to Archaea and Bacteria domain bacteria, plus analysis for the production of methane, showed that the culture was nonmethanogenic and revealed 4 different species of bacteria including bacterium o-17 (10). Sodium acetate (20 mM) was added to the medium as the electron donor for all experiments. Following a 10% (v/v) inoculation of the medium with a dechlorinating culture, 2,3,5,6-CB in 10 µL of acetone was added to a final concentration of 173 µM PCB in 10 mL of E-Cl. Unless stated otherwise, the culture was maintained with 2,3,5,6-PCB. Control cultures of o-17 without a chlorinated compound were prepared by adding acetone (10 µL) only to E-Cl (10 mL). All cultures were incubated in stationary at 30 °C in the dark, and all experiments were done with at least duplicate cultures. Examination of PCB, CBZ, and CE Dechlorination. Each culture was grown in 10 mL of E-Cl medium and was supplemented with one of the following congeners (supplied in 10 µL of acetone) at a final concentration of 180 µM: 2-, 3-, 4-, 2,3-, 2,4-, 2,5-, 2,6-, 2,2′-, 3,4-, 3,5-, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6-, 3,4,5-, 2,2′,6-, 2,2′,6,6′-, 2,3,4,5-, 2,3,4,6-, 2,3,5,6-, 2,3,4,5,6-. 2,2′,3,5,6,6′-, 2,3,3′,5,5′,6-, 2,2′,3,4,5,6,6′-, and 2,2′,3,3′,5,5′,6,6′-PCB and monochlorobenzene, 1,2-, 1,3-, 1,4-, 1,2,3-, 1,2,4-, 1,3,5-, 1,2,3,4-, 1,2,3,5-, 1,2,4,5-, penta-CBZ.
Hexa-CBZ was added at a final concentration of 35 µM. PCBs and CBZs were extracted from the culture medium (1 mL) into ethyl acetate (5 mL). A gas chromatograph equipped with an electron capture detector was used to analyze the PCBs (10, 18) and CBZs (20). PCBs and CBZs were identified by matching their GC retention times with those of external standards (99% purity, AccuStandard) and were quantified with a piecewise-fit calibration curve generated at 9-16 calibration levels (10, 18). Aroclor 1260 amendment and analysis was performed with a customized external standard PCB mixture according to the methods described by Wu et al. (21). A piecewise-fit calibration curve generated from standards at 4-8 calibration levels was used to quantify each congener within the customized Aroclor mixture. Congener distribution for PCBs and CBZs were calculated and reported in moles percent. For examination of CE dechlorination, the cultures were grown in 50 mL of E-Cl medium amended with filter (0.2 µm polytetrafluoroethylene filter) sterilized CEs (200 µM final concentration) as described previously (11). Baseline separation was achieved with all of the CEs and ethene by GC-FID as previously published in ref 11. External standards of each of the CEs were prepared in E-Cl medium. The standards were allowed equilibrate overnight, and gas samples were assayed in triplicate by GC-FID. Aqueous values were then determined using Gossett’s published Henry’s Law constants for each CE (22). PCR-DGGE. Samples (1.5 mL) from the o-17 cultures were withdrawn under anaerobic conditions by the Hungate technique using the gassing canula (23). Genomic DNA was extracted using InstaGene matrix (Bio-Rad) according to the manufacturer’s instructions. The culture remained nonmethanogenic and had been shown previously to not contain Archaea rRNA genes (10), thus Bacteria-specific PCR primers only were used throughout this study. Amplification of bacterial 16S rRNA genes by PCR (392-bp) and analysis by DGGE was carried out as described by Cutter et al. (10) in gels containing a 40-70% linear denaturing gradient formed with urea and deionized formamide. A 16S rRNA gene fragment generated from an E. coli clone containing the 16S rRNA gene of unidentified eubacterium RFLP 17 (bacterium o-17, AF058005) was used as a standard to determine relative migration distances. Identity of the DNA fragments was confirmed by sequence analysis. Sequence Analysis. To excise DNA fragments the gels were stained with ethidium bromide and visualized using an UV transilluminator. Excised DNA fragments were amplified with the PCR parameters described by Cutter et al. (10). Purity of the gene fragments was assured by repeated (3-4×) amplification and gel analysis of each excised DNA fragment until only one band with the same migration distance as the original band was detected by DGGE. Following DGGE gel purification, DNA fragments at unique migration distances were sequenced according to the methods of Ferris et al. (24) using an ABI 373 Automated Sequencer (Applied Biosystems, Foster City, CA). Sequences of DNA fragments were submitted to the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (25) and Ribosomal Database Project (26) to determine the similarity with other 16S rRNA genes.
Results and Discussion PCB Dechlorination. The o-17 culture was originally enriched in the presence of 2,3,5,6-PCB and ortho dechlorinates that congener to 3,5-PCB (12), but the results presented here demonstrate that it is also capable of meta dechlorination of some PCBs (Table 1). It preferentially dechlorinates meta before ortho chlorines if the chlorines are double-flanked, e.g., during the dechlorination of 2,3,4-, 2,3,4,5-, 2,3,4,6-, and 2,3,4,5,6-PCBs. Ortho dechlorination was observed only when the ortho chlorines were flanked with a meta chlorine and VOL. 40, NO. 18, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. PCB Dechlorination by the o-17 Culturea mole% mean (range) PCB congener
dechlorination products
2,3,42,3,52,4,52,3,4,5-
2,43,52,4-/2,52,4,52,4-/2,52,4,62,3,53,52,4,62,3,3′,5,5′ 3,3′,5,5′
2,3,4,62,3,5,62,3,4,5,62,3,3′,5,5′,6
35 days
70 days
33.9 (30.2-37.6) 2.9 (1.9-3.9) 33.2 (17.4-49.0) 1.2 (1.1-1.3) 6.3 (6.1-6.5) 16.7 (15.3-18.1) 4.7 (3.7-5.7) 27.6 (10.3-44.9) 14.4 (9.9-18.9) 0.2 (0.2-0.2) 0.3 (0.0-0.6)
32.9 (25.5-40.3) 12.2 (10.3-14.1) 74.1 (74.0-74.2) 0.8 (0.5-1.1) 19.7 (15.8-23.6) 77.5 (77.0-78.0) 4.7 (3.0-6.4) 66.6 (54.5-78.7) 40.9 (31.4-50.4) 0.5 (0.3-0.7) 2.5 (1.2-3.8)
sustained
NDb NDb
NDb
PCBs not dechlorinated 2-, 3-, 4-, 2,3-, 2,4-, 2,5-, 2,6-, 2,2′-, 3,4-, 3,5-, 2,3,6-, 2,4,6-, 3,4,5-, 2,2′,6-, 2,2′,6,6′-, 2,2′,3,5,6,6′-, 2,2′,3,4,5,6,6′-, and 2,2′,3,3′,5,5′,6,6′-PCB a Mole% results are averages from duplicate cultures (triplicates for 2,3,5,6- and 2,3,5-PCB) supplied with each PCB congener individually. Any total mole% less than 100 is due to the residual starting compound. Sustainable refers to whether the dechlorinating activity could be sustained following three sequential transfers of the culture with the starting compound. No dechlorination was detected in sterile controls with 2,3,5,6-PCB after 70 days. b ND ) not determined.
double-flanked meta chlorines were unavailable (e.g., a 2,3,5or 2,3,5,6-PCB). An exception to this is the inability of o-17 to dechlorinate 2,3-PCB, which was consistent with the inability to dechlorinate any dichlorobiphenyls tested. Although flanking of the chlorines favored dechlorination, additional chlorines on the adjacent ring has a detrimental effect, especially when the congener possesses 4 ortho chlorines. The culture did ortho dechlorinate a 2,3,5,6substitution if the adjacent-ring chlorines were at the 3 and 5 positions, but the dechlorination rate of 2,3,3′,5,5′,6-PCB was poor relative to that of congeners with no additional substitutions on the opposing ring. The trichlorobiphenyls 2,3,4-CB and 2,3,5-CB were dechlorinated (double-flanked meta and single-flanked ortho dechlorination, respectively), but this activity was not sustained following sequential transfer of the culture. Surprisingly, dechlorination of 2,3,5PCB was limited when it was the only congener added to the medium. This was unexpected since 2,3,5,6-PCB is dechlorinated to 3,5-PCB with 2,3,5-PCB as a transient intermediate (Table 1) (10). The o-17 culture targeted first the doubleflanked meta chlorine of 2,3,4,5-PCB and subsequently dechlorinated the intermediate product 2,4,5-PCB to either 2,4- or 2,5-PCB, which could not be separated chromatographically, as the final products. When added individually to the culture 2,4,5-PCB was also dechlorinated to 2,4- or 2,5-PCB. However, far more dichlorobiphenyl was generated when 2,4,5-PCB rather than 2,3,4,5-PCB was the starting compound, indicating that the dechlorination of the doubleflanked meta chlorine was a rate-limiting step. Regardless of whether the product of 2,4,5-PCB dechlorination is 2,4- or 2,5-PCB, the dichlorobiphenyl product indicates a dechlorination activity by the o-17 culture not observed with any of the other congeners, i.e., the dechlorination of either a single-flanked para or meta chlorine. Overall, the positioning of the chlorines on the biphenyl rings appeared to influence dechlorination more than the number of chlorines, which indicates that solubility (availability, generally higher with fewer chlorines) and oxidation state (increases with more chlorines) are of less importance. Inhibition of PCB Dechlorination by PCBs. Individual congener studies do not address how microorganisms contribute to the dechlorination of a complex mixture of PCBs in the environment. Therefore, the o-17 culture was incubated with Aroclor 1260, previously shown to be meta and ortho dechlorinated when added to live sediments from Baltimore Harbor (21), the original source of bacterium o-17 5706
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(10). The culture was incubated with 800 ppm Aroclor 1260 and sterile Baltimore Harbor sediment or was maintained sediment-free and incubated with or without the addition of 2,3,5,6-PCB. Congener 2,3,5,6-PCB was added as a “priming” congener since some single PCB and polybrominated biphenyl congeners have been demonstrated to stimulate the dechlorination of Aroclors (21, 27-29), and because this congener will support the growth and activity of the dechlorinating bacterium o-17 (10). However, in the presence of Aroclor 1260 the culture did not dechlorinate the Aroclor or 2,3,5,6-PCB after 6 months, suggesting that a congener(s) within the Aroclor could inhibit dechlorination of 2,3,5,6PCB as well as other PCB dechlorination. To test whether an individual congener, even one produced by the o-17 culture during PCB dechlorination, would inhibit the dechlorination of 2,3,5,6-PCB, the culture was incubated for 5 months with 173 µM each of 2,3,5,6-PCB and 2,4,6-PCB. This meta dechlorination product from 2,3,4,5,6-PCB completely inhibited the dechlorination of 2,3,5,6-PCB, indicating that the dechlorinating bacterium could block its own ability to attack other PCBs as a result of generating an inhibitory congener. Such action by the dehalogenating bacterium, and the restriction of the ortho dechlorination to congeners with single-flanked ortho chlorines, is consistent with numerous reports that noncoplanar ortho congeners tend to accumulate in the environment (28). It was anticipated that at least some of the dechlorinating activities observed with the live sediment containing Aroclor 1260 would be observed with the o-17 culture. Wu et al. (21) demonstrated with live microcosms of Baltimore Harbor sediment that dechlorination of Aroclor 1260 would begin within 1 month. After 6 months, the live microcosms had a 55% reduction of all meta chlorines and 12% reduction of all ortho chlorines of the Aroclor congeners when 2,3,5,6-PCB was added as a “priming” congener to the live microcosms, and 99% of the 2,3,5,6-PCB added was dechlorinated (21). Since the concentration of Aroclor used in the present study and that by Wu et al. (21) was identical (800 ppm), it is likely that the o-17 culture requires a consortium of PCB dechlorinating bacteria in order to contribute to the dechlorination of the Aroclor, or the this culture did not participate in the dechlorination of Aroclor 1260 in the study by Wu et al. These other dechlorinating species would dechlorinate PCBs that cannot be dechlorinated by bacterium o-17, but more importantly they may dechlorinate congeners that inhibit the PCB dechlorination carried out by bacterium o-17.
TABLE 2. Chlorobenzene Dechlorination by the o-17 Culturea chlorobenzene
dechlorination products
1,2,31,2,4,5pentachlorobenzene
1,31,2,41,2,3,5-1,3,5-
hexachlorobenzene
pentachlorobenzene 1,3,5-
mole% mean (range)
sustained
100 55.6 (22.6-88.5) 2.0 (1.8-2.1) 15.8 (12.5-19.1) 53.0 (49.5-56.5) 14.0 (4.5-23.4)
yes no yes yes
chlorobenzenes not dechlorinated monochlorobenzene, 1,2-, 1,3-, 1,4-, 1,2,4-, 1,3,5-, 1,2,3,4-, and 1,2,3,5-CBZb aMole%
results are the range from duplicate cultures supplied with each chlorobenzene individually and incubated for 28 days. A total mole% less than 100 is due to the residual starting compound. Sustainable refers to whether the dechlorinating activity could be sustained following three sequential transfers of the culture with the starting compound. b Although it was not dechlorinated when added individually to the medium, 1,2,3,5-CBZ was observed as an intermediate during the dechlorination of pentachlorobenzene to 1,3,5-CBZ.
The inhibition of the dechlorination of 2,3,5,6-PCB by 2,4,6-PCB suggests that single-flanked ortho dechlorination and double-flanked meta dechlorination are carried out by different dehalogenases. This hypothesis is supported by (1) the significant qualitative difference between these dechlorinating activities, (2) the fact that the o-17 culture favors double-flanked meta dechlorination over single-flanked ortho dechlorination, and (3) 2,4,6-PCB does not inhibit the meta dechlorination of 2,3,4,5,6- or 2,3,4,6-PCB, just as 3,5-PCB does not inhibit the ortho dechlorination of 2,3,5,6-PCB. The o-17 culture has been tested with concentrations of 3,5-PCB or 2,4,6-PCB greater than that of the starting congener following repetitive feedings and dechlorination of 2,3,5,6PCB or 2,3,4,5,6-PCB. Under these conditions the culture continued to dechlorinate the parent compound at the same rate and to the same extent as when the product PCB was not added to the culture (data not shown). Dechlorination of Chlorinated Ethenes and Chlorobenzenes. The only chlorinated ethene to be dechlorinated when added individually to the o-17 culture was tetrachloroethene. The dechlorination products after 50 days of incubation were cis-dichloroethene (92.8-93.2% of the products) and transdichloroethene (6.8-7.2%) with no remaining tetrachloroethene and no other products detected. Trichloroethene was not dechlorinated when added as the sole chlorinated substrate. This activity with tetrachloroethene was sustained in cultures that were sequentially transferred more than three times. The primary production of cis-dichloroethene during the dechlorination of tetrachloroethene is common among dechlorinating anaerobic bacteria, but it stands in contrast to the dechlorination of tetrachloroethene by bacterium DF1, a PCB-dechlorinating bacterium which produces more trans-dichloroethene than cis-dichloroethene (11). Hexa-CBZ and penta-CBZ were dechlorinated to 1,3,5CBZ by the o-17 culture, and these activities were sustained after three sequential transfers (Table 2). A short-lived intermediate product (1,2,3,5-CBZ) was detected at low concentration when penta-CBZ was added to the culture and not detected in the cultures supplied hexa-CBZ. The only other CBZ to be rapidly and sustainably dechlorinated was 1,2,3-CBZ, and as with the dechlorination of penta- and hexa-CBZ, the dechlorination was restricted to that of a double-flanked chlorine. The extent of dechlorination of tetrachlorobenzenes when added individually to the o-17 culture was limited. Congener 1,2,3,4-CBZ, which contains two double-flanked chlorines, was not dechlorinated at all; neither was congener 1,2,3,5-CBZ dechlorinated, which is the transient intermediate formed during penta-CBZ dechlorination. Congener 1,2,4,5-CBZ was the only tetrachlorobenzene to be dechlorinated by the culture when added individually to the medium. The product of this reaction was 1,2,4-CBZ, indicating that in this case a single-flanked chlorine
FIGURE 1. PCR-DGGE of the o-17 culture supplemented with PCBs, chlorobenzenes, tetrachloroethene (PCE), and no chlorinated compound (No Cl Cmpd). Details of DGGE methodology are presented in the Materials and Methods section. The o-17 clone is amplified 16S rDNA from an E. coli clone containing the 16S rDNA from bacterium o-17 (10).
on the benzene ring was targeted. However, dechlorination of 1,2,4,5-CBZ could not be sustained following sequential transfer of the culture. Similar to the results with PCBs, the overall pattern of CBZ dechlorination by the o-17 culture appeared to be more dependent on chlorine position than the solubility of the congeners. The exceptions observed with some of the tetrachlorobiphenyls may be the result of their being inhibitory, e.g., 1,2,3,4-CBZ has been shown to be inhibitory of the growth and dechlorinating activity of Dehalococcoides sp. strain CBDB1 (8). Another possible explanation for the results observed with some of the CBZs and chlorinated ethenes is cometabolic dechlorination dependent upon the presence of other chlorinated substrates. PCR-DGGE Analysis of the o-17 Culture. Similar to results previously reported (10), the o-17 culture grown by dechlorination of 2,3,5,6-PCB was shown by PCR-DGGE to contain 4 microorganisms (represented by DNA fragments A-D), although two of these (fragments B and C) were infrequently detected (Figure 1). Sequencing the DNA from these fragments confirmed that they were most similar to the following: fragment A was 100% similar to bacterium o-17 (unidentified eubacterium RFLP 17, AF058005), fragment B was 98% similar to Pseudomonas grimontii (AF268029), fragment C was 96% similar to Aminobacterium columinense (AF069287), and fragment D was 97% similar to Desulfovibrio aminophilus (AF067964). Fragments A and D dominated the DGGE profile, and fragment A (from bacterium o-17) was only observed when a PCB (or pentachlorobenzene or tetrachloroethene) was added to the medium and dechlorinated. No other putative DNA fragments from any of the gels in Figure 1 had 16S rRNA gene sequence, indicating that they were PCR artifacts (i.e., nonspecific products). VOL. 40, NO. 18, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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A detection limit for the 16S rRNA gene fragment by DGGE for bacterium o-17 was determined by PCR amplification of serially diluted DNA. Assuming one copy of 16S rRNA gene per cell the detection limit was calculated to be 6.3e3 cells/ mL of culture. Fagervold et al. (17) showed by MPN-PCR that the number of PCB dechlorinating microorganisms range between 1e4 and 1e6 cells per mL in active cultures, which is within the range of the PCR-DGGE assay. Substituting 2,3,4,5,6-PCB for 2,3,5,6-PCB resulted in a shift from ortho to meta dechlorination as described above. However, the DGGE profile remained unchanged indicating that the same microorganism responsible for ortho dechlorination (bacterium o-17) was carrying out the meta dechlorination of 2,3,4,5,6-PCB to 2,4,6-PCB. A similar result was observed when the culture was dechlorinating 2,3,4,6-PCB to 2,4,6-PCB (data not shown), and again, bacterium o-17 was not detected when the culture was transferred without PCB. Likewise, the fragment A in Figure 1 from cultures grown with 2,3,4,5,6-PCB, PCE, and penta-CBZ was extracted, sequenced, and confirmed as matching (100%) the 16S rRNA gene for bacterium o-17. Only limited dechlorination of congener 2,3,5-PCB was detected when added alone to the o-17 culture (Table 1), and this dechlorinating activity could not be sustained following sequential transfer. Correspondingly, the PCR-DGGE analysis of the o-17 culture grown with 2,3,5-PCB demonstrated that bacterium o-17 was difficult to detect in these cultures (Figure 1). This observation is consistent with the limited, nonsustainable activity of the culture when grown with only 2,3,5-PCB. Interestingly, the 16S rRNA gene for bacterium o-17 was observed when the culture was incubated with 2,3,3′,5,5′,6-PCB but dechlorination was very low. Sequential transfer with this congener would be required to confirm if dechlorination is linked to growth. Evidence for Catabolic and Cometabolic PCB Dechlorination. The complexity of PCB dechlorination increases when cometabolism is considered. Cometabolism is defined here as the transformation of a chlorinated compound during the growth-supporting dechlorination of a second compound; the cometabolized compound is incapable of supporting growth in the absence of the other compound. Sustainable dechlorination with a PCB congener, i.e., dechlorination that can be sequentially transferred, plus the detection of bacterium o-17 by PCR-DGGE only when dechlorination is sustained, suggests that dechlorination of that particular congener may be linked to the energy metabolism and growth of the bacterium. This was demonstrated with several PCBs, CBZs, and PCE added as the sole electron acceptor. However, 2,3,5-PCB, 1,2,3,5-CBZ, and TCE were dechlorinated only when another more extensively chlorinated compound of the same class was present, suggesting that these compounds were cometabolized. Consistent with this hypothesis was the inability to detect bacterium o-17 by DGGE when it was incubated with these compounds. It is also likely that 2,3,4-PCB and 1,2,4,5-CBZ were dechlorinated cometabolically since the dechlorination of these compounds could not be maintained beyond one sequential transfer of a culture initially maintained with 2,3,5,6-PCB. These results suggest that the presence of a growth-linked chlorinated compound may expand the range of PCB dechlorination for cometabolically reduced congeners that do not support growth. This is consistent with the stimulation of PCB dechlorination in live sediment microcosms by the addition of “priming” compounds (28). The o-17 culture possesses a distinct combination of dechlorinating capabilities including the ability to dechlorinate double-flanked meta and single-flanked ortho chlorines of PCBs, double-flanked chlorines of chlorobenzenes, and tetrachloroethene to cis- and trans-dichloroethene. Since, the culture is also sensitive to the presence of inhibitory PCBs, 5708
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such results demonstrate that the bacterial composition of a dehalogenating population has a critical role in influencing both the pathways and the extent of dehalogenation by the individual species within the consortium. An understanding of the synergistic, cometabolic, and competitive interactions of these dechlorinating microbial populations in the environment will yield important information for the assessment of bioremediation potential in situ.
Acknowledgments This work was supported by the Office of Naval Research, U.S. Department of Defense, Grants N00014-06-1-0090 to K.S. and N00014-06-1-0091 to H.M.
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Received for review December 16, 2005. Revised manuscript received July 22, 2006. Accepted July 25, 2006. ES052521Y
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