Dehalococcoides mccartyi Strain JNA in Pure Culture Extensively

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Dehalococcoides mccartyi Strain JNA in Pure Culture Extensively Dechlorinates Aroclor 1260 According to Polychlorinated Biphenyl (PCB) Dechlorination Process N Sarah L. LaRoe,†,§ Ashwana D. Fricker,‡,∥ and Donna L. Bedard*,‡ †

Department of Civil and Environmental Engineering and ‡Department of Biological Sciences, Rensselaer Polytechnic Institute 110 Eighth Street, Troy, New York 12180, United States S Supporting Information *

ABSTRACT: We isolated Dehalococcoides mccartyi strain JNA from the JN mixed culture which was enriched and maintained using the highly chlorinated commercial PCB mixture Aroclor 1260 for organohalide respiration. For isolation we grew the culture in minimal liquid medium with 2,2′,3,3′,6,6′hexachlorobiphenyl (236−236-CB)(20 μM) as respiratory electron acceptor. We repeatedly carried out serial dilutions to extinction and recovered dechlorination activity from transfers of 10−7 and 10−8 dilutions. Fluorescence microscopy, DGGE and RFLP analysis of PCR amplified16S rRNA genes, and multilocus sequence typing of three housekeeping genes confirmed culture purity. No growth occurred on complex media. JNA dechlorinated most hexaand heptachlorobiphenyls in Aroclor 1260 (50 μg/mL) leading to losses of 51% and 20%, respectively. Dechlorination was predominantly from flanked meta positions of 34-, 234-, 235-, 236-, 245-, 2345-, 2346-, and 2356chlorophenyl rings, as indicated by the underscores. The major products were 24−24-CB, 24−26-CB, 24−25-CB, and 25−26-CB. We identified 85 distinct PCB dechlorination reactions and 56 different PCB dechlorination pathways catalyzed by JNA. Dechlorination pathways were confirmed by mass balance of substrates and products. This dechlorination pattern matches PCB Dechlorination Process N. JNA is the first pure culture demonstrated to carry out this extensive and environmentally relevant PCB dechlorination pattern.

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INTRODUCTION Nearly four decades after their manufacture was banned, polychlorinated biphenyls (PCBs) remain on the list of 18 worldwide persistent organic pollutants (or POPs)1 and are ranked fifth on the U.S. Environmental Protection Agency National Priority List of hazardous substances.2 Aquatic sediment systems worldwide harbor hundreds of thousands of metric tons of commercial PCBs and threaten the health of humans and aquatic ecosystems.3 The discovery that PCBs were being dechlorinated in aquatic sediments 4−6 offered promise for a natural means of remediation. It was soon shown that this dechlorination was due to anaerobic microorganisms residing in the sediment,7,8 but identification of the specific organisms responsible has taken much longer. The first PCB-dechlorinating organisms to be identified were two novel anaerobic bacteria derived from estuarine sediments and designated o-17 and DF-1 (the latter was subsequently isolated and named “Dehalobium chlorocoercia” strain DF-1).9−11 These bacteria, which are members of the Chloroflexi, can dechlorinate and use certain PCB congeners chlorinated on a single ring as respiratory electron acceptors.9,10 Subsequently, it was discovered that another member of the Chloroflexi, Dehalococcoides mccartyi strain 195 (formerly Dehalococcoides ethenogenes12), could dechlorinate several PCB congeners chlorinated on a single ring.13 © 2014 American Chemical Society

Bioremediation of PCBs is especially difficult because each of the commercial PCB mixtures, known in the U.S. as Aroclors, typically contains 60−90 kinds of PCB molecules (congeners) that differ in the number and position of chlorines. Commercial PCB mixtures pose a substantial biodegradation challenge in both the number and bioavailability of the substrates because each mixture is so complex and because PCBs are extremely hydrophobic. Most data on microbial dechlorination of Aroclors come from sediment microcosms (reviewed in ref 14 and 15). Eight distinct patterns of Aroclor dechlorination known as PCB dechlorination processes H, H′, LP, M, N, P, Q, and T have been described.14−17 These dechlorination processes describe which PCB congeners and chlorophenyl rings are targeted, which chlorines are removed, and which products are formed. Various studies with sediment microcosms have implicated members of the order Dehalococcoidales, especially the species Dehalococcoides mccartyi, as the agents of Aroclor dechlorination.12,18 For example, a Dehalococcoides phylotype and two phylotypes similar to o-17 and DF-1 were associated with the Received: Revised: Accepted: Published: 9187

February 19, 2014 June 11, 2014 July 7, 2014 July 7, 2014 dx.doi.org/10.1021/es500872t | Environ. Sci. Technol. 2014, 48, 9187−9196

Environmental Science & Technology

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but were not inoculated. No dechlorination was observed in abiotic controls. To test for culture purity, duplicate bottles of LB broth were inoculated with 10% vol/vol of an actively dechlorinating culture, incubated for 3 weeks, and examined for growth. In addition, two sets of minimal medium plus H2 and acetate, each in duplicate, were inoculated with 5% vol/vol of an actively dechlorinating culture. One set was further amended with 0.1% yeast extract (to grow any non organohalide-respiring bacteria) and the other with a chlorinated substrate. Cultures were grown for a week and then transferred to minimize carryover of chlorinated compounds and microbial cells. DNA was extracted from both sets of cultures, the 16S rRNA genes were amplified with both universal bacterial primers and Dehalococcoides specific primers, and the products were analyzed by agarose gel electrophoresis and DGGE. Molecular Methods. DNA extraction, cloning, restriction fragment length polymorphism (RFLP) analysis, and DNA sequencing were essentially as previously described.21 See SI for minor changes. Multilocus sequence typing and DGGE analysis were done as described.26 PCB Analysis. High resolution congener-specific PCB analyses were carried out as previously described25 on a Hewlett-Packard 5890 GC fitted with a Ni63 electron capture detector. Single Congener Experiments. Ten hexa- and heptachlorobiphenyl congeners were chosen for testing as individual substrates for dechlorination studies with JNA. These were 234−234-CB, 234−245-CB, 235−235-CB, 236−236-CB, 236− 245-CB, 245−245-CB, 2345−24-CB, 2345−236-CB, 2345− 245-CB, and 2346−24-CB. All of these were obtained from AccuStandard and were >99% pure. Triplicate bottles of fresh medium (20 mL) were spiked with a nominal concentration of 20 μM of substrate and then inoculated with 5% (v/v) of an active culture grown with 236−236-CB as sole electron acceptor. In practice, the concentration of the substrate varied from 17 μM to 99 μM. Measured concentrations for each substrate are given in the results section.

dechlorination of Aroclor 1260 in sediment microcosms derived from Baltimore Harbor (Baltimore, MD).19 More recently, a study of nine microcosms enriched from sites in China and Singapore revealed that the changes in the PCB congener distribution of Aroclor 1260 mediated by Dechlorination Processes H, N, and an uncharacterized dechlorination process were associated with Dehalococcoides mccartyi and, in one case, a Dehalogenimonas.20 Despite these studies, there is very limited information on the exact role that individual organisms play in the dechlorination of complex Aroclor mixtures. This is because the isolation of such organisms has been hindered by the inability to maintain dechlorination activity against the extremely insoluble Aroclors in the absence of sediment. In 2006 our group succeeded in developing a sediment-free culture that could extensively dechlorinate Aroclor 1260, the JN culture.21 Subsequently, we showed that the sole dechlorinators present in the JN culture were members of the Pinellas subgroup of Dehalococcoides mccartyi and that these bacteria linked their growth to the dechlorination of Aroclor 1260.22 More recently, Wang and He developed six sediment-free mixed cultures capable of respiratory dehalogenation of Aroclor 1260.20 The dechlorinators were members of all three phylogenetic subgroups of Dehalococcoides mccartyi (Pinellas, Victoria, and Cornell)12,23 as well as a strain of Dehalogenimonas alkenigignens.20 Clearly members of the species Dehalococcoides mccartyi are important catalysts in the dechlorination of commercial PCBs, likely because they have no means of growth other than organohalide respiration12 and have multiple reductive dehalogenases to assist in this restricted lifestyle.24 In order to gain a clear understanding of the genetics underlying PCB dechlorination and identify markers that can pinpoint these genes at field sites, it is necessary to isolate and study pure strains of Aroclor-dechlorinating bacteria. Until now only one isolate, Dehalococcoides mccartyi strain CBDB1, has been shown to have broad PCB-dechlorinating activity against Aroclor 1260, and this matches Dechlorination Process H.25 Here we describe the isolation of Dehalococcoides mccartyi strain JNA, derived from the JN enrichment culture, and show that 2,2′,3,3′,6,6′-hexachlorobiphenyl (236−236-CB) supports growth by strain JNA. We also characterize the dechlorination of Aroclor 1260 and ten individual hexa- and heptachlorobiphenyls by strain JNA in pure culture. We identify the PCB dechlorination reactions that characterize this process and reveal greater insights into the complex dechlorination of highly chlorinated PCBs.

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RESULTS Identifying the Best PCB Congener for Growth. We previously demonstrated that the commercial PCB mixture Aroclor 1260 supports the growth of the JN enrichment culture by respiratory dehalogenation22 but we did not know which congeners were used as respiratory terminal electron acceptors. We sought to identify individual highly chlorinated PCB congeners that could be used as respiratory electron acceptors to aid us in the isolation of a pure culture. We hypothesized that the best candidates would have several features: they would be among the most prominent congeners in Aroclor 1260, they would be efficiently dechlorinated by the JN culture, and they would lose at least two chlorines during dechlorination. The most abundant congeners in Aroclor 1260 are 2345−245-CB, 245−245-CB, 236−245-CB, and 234−245-CB which comprise, respectively, approximately 10.53, 10.38, 8.89, and 8.78 mol % of the mixture.27 All are efficiently dechlorinated by the JN culture, and each congener loses all meta chlorines during dechlorination.21 We chose to test 245−245-CB and 234−245CB. We also tested 234−234-CB and 236−236-CB because they are composed of chlorophenyl rings that are abundant in Aroclor 1260 and are readily dechlorinated. Each culture was inoculated with 1% vol/vol of a JN culture that was actively dechlorinating Aroclor 1260. Each of the

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EXPERIMENTAL SECTION Growth Conditions. Cultures were grown with H2 as the electron donor and acetate as the carbon source in a defined mineral salts medium25 with a few modifications (see Supporting Information (SI)). The medium was buffered to pH 7.0 with bicarbonate and reduced with titanium(III) citrate as previously described.25 Single PCB congeners were sorbed to silica, suspended in medium, and added by syringe to give a final nominal concentration of 10 or 20 μM as indicated. In later experiments 236−236-CB was added without silica as a sterile-filtered acetone stock (12 mM) to give a final nominal concentration of 20 μM. This had no negative impact on growth. Cultures were 10 mL in 25 mL serum bottles, 20 or 30 mL in 60 mL serum bottles, or 60 mL in 125 mL serum bottles. All cultures were carried out in triplicate unless otherwise noted. Abiotic controls were identical to experimental samples 9188

dx.doi.org/10.1021/es500872t | Environ. Sci. Technol. 2014, 48, 9187−9196


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tested congeners (10 μM) was dechlorinated by removal of the meta chlorines to the expected tetrachlorobiphenyl (data not shown). Surprisingly, 245−245-CB and 234−234-CB were dechlorinated very slowly, over a period of 70 days, and 234− 245-CB only slightly faster. In contrast, the 236−236-CB was completely dechlorinated to 26−26-CB within 27 days. Cultures with 236−236-CB were respiked to a concentration of 20 μM which was completely dechlorinated over the next 44 days. We transferred all four sets of cultures to fresh medium with the same congener to determine whether the activity could be transferred and enriched. During this transfer, we passed each inoculum (10% vol/vol) through a 0.20 μm SFCA (surfactant-free cellulose acetate) sterile filter (Corning no. 431219) that had been prerinsed with reduced medium. This step was intended to aid in isolation through size exclusion. The dechlorination results are shown in SI Figure S1. After incubation for more than six months, 234−234-CB was not significantly dechlorinated and 245−245-CB was only moderately dechlorinated. The third congener showed more promise: 234−245-CB was dechlorinated to 24−24-CB after a lag of 60 days, and when respiked it was dechlorinated without lag. However, 236−236-CB was clearly the best substrate as it was dechlorinated to 26−26-CB via 236−26-CB in 27 days and could subsequently be spiked and dechlorinated repeatedly. Additional experiments showed that 2345-CB, 2356-CB, and 236-CB were readily dechlorinated and might also be good electron acceptors (each removing the chlorine(s) indicated by the underline, data not shown). No follow-up experiments were done with these congeners. Isolation and Confirmation of Purity. The JN culture has been continuously transferred in cultures with 236−236-CB as the sole electron acceptor for more than five years. During that time the culture has been repeatedly serially diluted to extinction and dechlorination activity successfully recovered from dilutions to 10−7 and 10−8. Figure 1 shows the stoichiometric conversion of 236−236-CB to 26−26-CB in cultures inoculated with an active culture to a final dilution of 10−7. No methane was detected in cultures and no growth was observed on LB medium or minimal medium plus yeast extract without a halogenated organic substrate. Culture purity was confirmed by microscopy (SI Figure S2), and by DGGE analysis (SI Figure S3) and RFLP analysis (110 clones) of PCR amplified16S rRNA genes which showed a single band of DNA. The 16S rRNA sequence of the isolate places strain JNA within the Pinellas subgroup of Dehalococcoides mccartyi along with strain CBDB1. D. mccartyi strains are known to share highly similar or even identical 16S rRNA genes. However, multilocus sequence typing or MLST has been shown to be capable of distinguishing D. mccartyi strains.26 To test whether there were different strains of D. mccartyi in the JNA culture we analyzed the culture by MLST targeting three housekeeping genes: rpoB, adk, and atpD (SI Figure S4). The inserts in 10 clones were sequenced from each library. A single genotype was retrieved for each housekeeping gene suggesting that culture JNA consists of a single D. mccartyi strain, JNA. The 16S rRNA gene sequence and the sequences for the three housekeeping genes have been deposited in GenBank as accession numbers KJ461493 and KJ944280−KJ944282. Aroclor 1260 Dechlorination Findings. JNA was incubated with 50 μg/mL (135 μM) of Aroclor 1260 (AccuStandard) for 124 days. SI Figure S5 shows the change

Figure 1. Stoichiometric dechlorination of 236−236-CB by strain JNA in duplicate cultures. Cultures were inoculated from a culture grown with 236−236-CB as sole electron acceptor to a final dilution of 10−7. The substrate 236−236-CB (pink triangles) was dechlorinated via 236−26-CB (blue squares) to 26−26-CB (green circles). Solid lines and symbols represent one culture and open symbols and dashed lines the other.

in PCB homologue distribution over time. The hexachlorobiphenyls decreased rapidly in the first month coinciding with the appearance of tetrachlorobiphenyls. Heptachlorobiphenyls were dechlorinated more slowly. After four months, 14%, 51%, and 20%, respectively, of the penta-, hexa-, and heptachlorobiphenyls had been converted to tetrachlorobiphenyls which increased from 0.90 ± 0.17 mol % to 31.98 ± 3.34 mol % of the total PCBs. The average number of chlorines per biphenyl in the Aroclor 1260 dropped from 6.27 ± 0.01 to 5.59 ± 0.10. This was entirely due to meta dechlorination; the average number of meta chlorines per biphenyl dropped from 2.50 ± 0.01 to 1.77 ± 0.08, a 30% decrease. There was no detectable loss of ortho or para chlorines. Figure 2 shows the change in PCB congener distribution following incubation of Aroclor 1260 with strain JNA. Despite being carried for more than five years with 236−236-CB as the sole electron acceptor, strain JNA has retained the broad congener specificity for dechlorinating Aroclor 1260 previously displayed by its parent enrichment culture.21 Large decreases were evident in most penta-, hexa-, and heptachlorobiphenyls and many new tetra- and pentachlorobiphenyls were formed. The major products, 24−24-CB, 24−26-CB, 24−25-CB, and 25−26-CB are the same as previously seen in the JN culture.21,22 The congeners that were substrates and products were quantified and are presented in Table 1 along with the dechlorination pathways deduced from the data presented in this paper. 9189



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indicates that there were insufficient losses of parent congeners to account for these transformations. We were unable to find any errors in the identification, calibration, or integration of these congeners. The most likely explanation is that the particular batch of Aroclor 1260 that was used in our calibration standard actually had more of the 2346-chlorophenyl ringbearing parent congeners than the batch of Aroclor 1260 that was analyzed to produce the weight percent tables for all congeners.27 Because we subsequently incorporated the values from those tables into our calibration standard, the values assigned to these minor 2346-bearing components may have been too low, resulting in poor mass balance. Single Congener Studies. We carried out experiments with selected hexa- and heptachlorobiphenyls to further elucidate PCB dechlorination pathways. Of the 10 congeners tested, only two substrates, 234−245-CB and 2345−245-CB, were efficiently converted to tetrachlorobiphenyls. More than half of the 234−245-CB was converted to 24−24-CB in 40 days by way of both 245−24-CB and 234−24-CB (SI Figure S6). The higher concentration of 245−24-CB suggests that this might be the primary intermediate. A barely detectable amount of 23−24-CB was also produced, suggesting a minute amount of para dechlorination of the 234-chlorophenyl ring. Likewise, 39% of the 2345−245-CB was converted to 24− 24-CB and 24−25-CB in 24 days (Figure 3). Two hexachlorobiphenyl intermediates, 245−245-CB and 2345− 24-CB, were detected in a ratio of about 5 to 1 (Table 2), demonstrating that either the tetra- or the trichlorophenyl ring can be attacked first. Small amounts of 245−24-CB and 235− 24-CB were also present. The final products, 24−24-CB and 24−25-CB, were formed in a ratio of 18 to 1. This means that both hexachlorobiphenyl intermediates were primarily metadechlorinated via 245−24-CB and only about 5% of the total products resulted from removal of the doubly flanked para chlorine on the 2345-ring. Three chlorines were removed for every mole of heptachlorobiphenyl, so despite the lower conversion percentage of this substrate, the total number of moles of chlorine removed (116.8) was similar to that for 234− 245-CB (110.9). The other congeners were not as efficiently dechlorinated (Table 2). For example, at 24 days 1.2 mol % of the 2345−236CB had been converted to 236−245-CB, and only 1.8 mol % had been transformed to the final products, 24−26-CB and 25−26-CB. Other intermediates, 2345−26-CB, 236−245-CB, 235−26-CB, 236−24-CB, and 236−25-CB + 245−26-CB, were present in small but measurable amounts, thus multiple pathways were operative (SI Figure S7). Most of the dechlorination was via loss of meta chlorines; comparison of the sum of products with 245- and 24- chlorophenyl rings to the sum of products with 235- and 25-chlorophenyl rings indicates that only 17% of the dechlorination resulted from removal of the doubly flanked para chlorine of the 2345chlorophenyl group. No 234−26-CB or 23−26-CB were observed. Table 2 gives the products of the dechlorination of 236−245CB (visualized in SI Figure S8), 245−245-CB, and 234−234CB. Further details are presented in the Supporting Information. In separate experiments that were not quantified, 2345−26-CB was reasonably well dechlorinated to 245−26-CB and then 24−26-CB. No para dechlorination to 235−26-CB was detected. A small amount of 2346−24-CB was dechlorinated to 246−24-CB and trace amounts of 236−24CB. And, finally, a modest amount of 235−235-CB was

Figure 2. Change in PCB congener distribution (Process N dechlorination) of Aroclor 1260 (50 μg/mL, equivalent to 135 μM) incubated with D. mccartyi strain JNA in pure culture. Data are the means for triplicate cultures. Panels A and B show the congener distributions at days 0 and 124. Panel C shows the difference. Abiotic controls were identical to day 0.

Four major hexa- and heptachlorinated biphenyls were dechlorinated via various intermediates to produce the major product, 24−24-CB. This process involved as many as 10 distinct dechlorination reactions and seven different pathways (Table 1). Similarly, five additional hexa- and heptachlorinated biphenyls were dechlorinated to produce 24−26-CB involving 14 distinct dechlorination reactions and 10 separate pathways. Most of these dechlorination reactions were verified with single congeners (see section on single congeners) and they illustrate the complexity of the PCB dechlorination catalyzed by strain JNA. Altogether, this table includes 77 distinct PCB dechlorination reactions and 51 different dechlorination pathways. The stoichiometric mass balance between substrates and products for the congeners showing the greatest losses (Table 1) is a clear indication that the quantitation and congener assignments are correct. On the other hand, the mass balances for the dechlorination reactions leading to 26−4-CB and 26− 26-CB reveal that the amount of these products was less than expected. These congeners are the most volatile products of Aroclor 1260 and therefore the most subject to loss during handling. The dechlorination of congeners bearing 2346- rings to congeners bearing 246-rings had poor mass balance. The formation of 246−24-CB, 246−25-CB, and 246−26-CB clearly demonstrates that the dechlorination of 2346-chlorophenyl rings to 246-chlorophenyl rings did occur, but the quantitation 9190


132

174

187

177

81

97

93

98

9191

90, 101

87 146

141 180

170

56

64 80

84 105

109

174

140

75

97

170

109

95 151 135

153 180

82 105

48 70 71

138

IUPAC no.

87

Pk no.

parent congener

1.17 0.08 0.06

234−2345 (5%)e

0.20 0.47

2345−25 245−2345 (5%)e

234−25 245−235

2.13

0.15

2345−236 (17%)e

245−25 and 24−235

2.65 1.53 0.91

0.40

0.86

25−236 25−2356 235−236

234−2356

245−2356

0.74

2345−236 (83%)e

4.97

245−236e 2.46

1.17

2345−234 (95%)e

234−236

4.39 1.47

4.84

observed decrease mol%

245−245 2345−245 (95%)e

234−245

a

pathway

245−25 → 24−25 24−235 → 24−25 234−25 → 24−25 245−235 → 24−235 → 24−25 245−235 → 245−25 → 24−25 2345−25 → 245−25 → 24−25 25−2345 → 245−235 → 24−235 → 24−25 245−2345 → 245−235 → 245−25 → 24−25 234−2345 → 234−235 → 24−235 → 24−25 234−2345 → 24−2345 → 24−235 → 24−25

25−236 → 25−26 25−2356 → 25−236 → 25−26 235−236 → 235−26 → 25−26 235−236 → 25−236 → 25−26 2345−236 → 235−236 → 25−236 → 25−26 2345−236 → 235−236 → 235−26 → 25−26 2345−236 → 2345−26 → 235−26 → 25−26

245−236 → 24−236 → 24−26 245−236 → 245−26 → 24−26 234−236 → 24−236 → 24−26 234−236 → 234−26 → 24−26 2345−236 → 245−236 → 24−236 → 24−26 2345−236 → 2345−26 → 245−26 → 24−26 245−2356 → 245−236 → 24−236 → 24−26 245−2356 → 24−2356 → 24−236 → 24−26 234−2356 → 234−236 → 24−236 → 24−26 234−2356 → 24−2356 → 24−236 → 24−26

234−245 → 234−24 → 24−24 234−245 → 24−245 → 24−24 245−245 → 245−24 → 24−24 2345−245 → 245−245 → 245−24 → 24−24 2345−245 → 2345−24 → 245−24 → 24−24 2345−234 → 245−234 → 245−24 → 24−24 2345−234 → 2345−24 → 245−24 → 24−24

b

Table 1. Mass Balance of A1260 Dechlorination and Dechlorination Pathways

4.11

5.24

9.61

11.87

expected total increase mol%c

30

22 45

73

47

49

23

58

31

Pk no.

49

53 94

147

102

91

51

99

47

IUPAC no.

24−25

25−26 235−26

2356−24

245−26

24−236

24−26

245−24

24−24

product congener

4.52

4.68 0.17

0.76

0.10

0.98

8.95

1.39

10.60

observed increase mol%

110.0

92.6

112.3

99.0

mass balanced %

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9192

183

94

158

171

99

88

144 175

72 91

parent congener

2346−34

2346−236

2346−245

2346−234

2346−25 2346−235

236−236 2356−236

2356−34

236−34

a

0.39

0.00

0.17

0.26

0.21 0.09

1.52 0.83

1.75

1.76

observed decrease mol% pathway

0

0.39

2346−236 → 246−236 → 246−26 2346−236 → 2346−26 → 246−26 2346−34 → 246−34

246−24 246−24 246−24 246−24 0.43

→ → → →

2346−234 2346−234 2346−245 2346−245

246−234 2346−24 246−245 2346−24

0.30

2346−25 → 246−25 2346−235 → 246−235 → 246−25 2346−235 → 2346−25 → 246−25 → → → →

2.33

3.51

expected total increase mol%c

236−236 → 236−26 → 26−26 2356−236 → 236−236 → 236−26 → 26−26 2356−236 → 2356−26 → 236−26 → 26−26

236−34 → 26−34 → 26−4 236−34 → 236−4 → 26−4 2356−34 → 236−34 → 26−34 → 26−4 2356−34 → 2356−4 → 236−4 → 26−4

b

59

36 59

43

42

17 40

39

16

Pk no.

119

104 150

100

103

54 96

71

32

IUPAC no.

246−34

246−26 236−246

246−24

246−25

26−26 236−26

26−34

26−4

product congener

0.23

0.39 0.23

1.59

0.61

1.61 0.10

0.37

1.18

observed increase mol%

b

66.7

None

370.0

203.3

73.4

44.2

mass balanced %

The order in which the chlorophenyl groups in congeners are presented was chosen for consistency within each group and is not necessarily the order that correct nomenclature would call for. Reactions are marked in bold italics the first time they appear in this table. There are 77 distinct dechlorination reactions and 51 different pathways shown. cExpected increase = the sum of the observed decreases. d Defined as the sum of expected increases in products divided by the sum of decreases in the parent congeners. eParentheses indicate fraction of the congener dechlorinated by loss of the meta or para chlorine on the 2345-chlorophenyl group based on single congener studies.

a

136 179

66 84

176

163

87

86

110

IUPAC no.

67

Pk no.

Table 1. continued

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acceptor for organohalide respiration in strain JNA was unexpected because 236−236-CB is a relatively minor component of Aroclor 1260 (2.0 mol %). Furthermore, it has four ortho chlorines and exists as two distinct stereoisomers which could conceivably require two distinct dehalogenases.28 Nevertheless, since all of the 236−236-CB was stoichiometrically converted to 26−26-CB (Figure 1), it is clear that both isomers were dechlorinated equally well. The single congener data suggest two other potential respiratory terminal electron acceptors in Aroclor 1260: 2345−245-CB, and 234−245-CB. Further studies would be necessary to determine if either of these can support growth of strain JNA. Insights into PCB Dechlorination by Strain JNA. Both rings were attacked for all PCB congeners whenever both rings had susceptible meta chlorines. The dechlorination was highly specific for the removal of flanked meta chlorines but there was no detectable dechlorination of 2345-chlorophenyl rings to 234-. There was little para dechlorination of the 2345-ring for single congeners even though the para chlorine is flanked by two chlorines which often facilitates dechlorination. There was no measurable loss of para chlorines from Aroclor 1260. No ortho dechlorination was ever observed. It is not clear why 234−245-CB was dechlorinated more readily than 245−245-CB or 234−234-CB. However, this finding is consistent with our previous determinations that the ring substitution pattern is not the only factor controlling whether or not a chlorophenyl ring will be dechlorinated or how well it will be dechlorinated;16,25 the chlorination pattern on the opposite ring also plays a role. There were several discrepancies in how well certain congeners were dechlorinated as components of the commercial PCB mixture Aroclor 1260 versus as individual substrates. As components of Aroclor 1260, 234−245-CB, 236−245-CB, and 245−245-CB were all dechlorinated with comparable efficiency, whereas the latter two congeners were not efficiently dechlorinated when presented as individual substrates. In addition, 2345−236-CB was more efficiently dechlorinated than 2345−245-CB in the Aroclor mix even though it was a much poorer substrate when presented by itself. These observations confirm previous findings that individual congeners may be dechlorinated with different dynamics when presented in mixtures vs as single congeners.16 This suggests that many congeners in Aroclor 1260 may be dechlorinated by cometabolism which should be taken into account when trying to predict PCB dechlorination in the environment. Comparison of Aroclor 1260 Dechlorination in the Parent and Pure Culture. The dechlorination specificity remains the same as previously described for the mixed culture JN with the exception that there was at most 5−17% removal of the doubly flanked para chlorine of the 2345-chlorophenyl ring in the single congener experiments, far less than the 60% reported for the JN culture.21 In fact, for Aroclor 1260 incubated with JNA there was no measurable loss of para chlorines whereas previous experiments with the parent JN enrichment showed a 9.4% loss of para chlorines.21 In addition, JNA exhibited no measurable dechlorination of octachlorobiphenyls and less dechlorination of heptachlorobiphenyls including two major Aroclor components: 2345−245-CB and 2345−234-CB. This reduced dechlorination activity may be at least partially due to the lower cell density of the pure culture. It has been noted that pure cultures of dechlorinators within the order Dehalococcoidales do not grow as robustly as the mixed cultures

Figure 3. Dechlorination pathways for 2345−245 heptachlorobiphenyl by strain JNA in pure culture. The value given under each congener represents its mole percent of total PCBs ± standard deviation after incubation for 24 days. The question marks indicate possible reactions that could not be verified. a235−245-CB was a significant contaminant of the substrate which decreased over the course of the incubation and likely contributed to the formation of 24−25-CB. b235−24-CB and 245−25-CB coelute. Congeners are drawn to show dechlorination most clearly, hence in some cases the position of the rings and the congener designation do not conform with normal nomenclature.

dechlorinated via 235−25-CB to 25−25-CB. Trace amounts of 235−23-CB were also detected but no 23−23-CB or 23−25CB was detected. Collectively, the data from Aroclor 1260 and individually incubated congeners indicate that dechlorination occurred almost exclusively from the flanked meta positions of 34-, 234-, 235-, 236-, 245-, 2345-, 2346-, and 2356-chlorophenyl rings as indicated by the underscores. Only trace amounts of para dechlorination were detected except for congeners bearing 2345-chlorophenyl rings which were partially dechlorinated to 235-chlorophenyl rings. The single congener experiments revealed five additional dechlorination pathways and eight dechlorination reactions not shown in Table 1. These were 234−234-CB → 234−24-CB, 235−235-CB → 235−25-CB → 25−25-CB, 2345-CB → 245CB → 24-CB, 2345-CB → 235-CB, and 2356-CB → 236-CB → 26-CB. Excluding the trace dechlorination reactions described for single congeners, we can attribute 85 distinct PCB dechlorination reactions and 56 different dechlorination pathways to strain JNA.

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DISCUSSION PCB Congeners As Growth Supporting Terminal Electron Acceptors. We previously demonstrated that the parent culture of strain JNA could metabolically dechlorinate Aroclor 1260,22 but we did not determine which of the congeners in Aroclor 1260 could support growth. The discovery that 236−236-CB is an excellent terminal electron 9193



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Table 2. Conversion of PCB Substrates to Products substrate

conc μM

daya

mol % ± std dev

total mol % converted to tetra-CB

234−245-CB

17

40

24−24-CB 245−24-CB 234−24-CB

55.44 ± 9.61 4.43 ± 0.49 0.26 ± 0.02

55.44 ± 9.61

2345−245-CB

20

24

24−24-CB 24−25-CB 245−245-CB 2345−24-CB 245−24-CB 235−24-CB +245−25-CB

36.77 2.16 1.58 0.34 1.52 1.96

± ± ± ± ± ±

38.93 ± 9.81

236−245-CB

54

56

24−26-CB 245−26-CB 236−24-CB

245−245-CBb

40

24

24−24-CB

234−234-CB

17

32

24−24-CB

4.50 ± 0.91

4.50 ± 0.91

2345−236-CB

99

24

24−26-CB 25−26-CB 236−245-CB 2345−26-CB otherc

1.53 0.26 1.22 0.39 0.90

± ± ± ± ±

1.78 ± 0.96

product

7.06 0.51 0.03 0.02 0.26 0.53

4.23 ± 1.88 0.60 ± 0.10 0.80 ± 0.14

4.23 ± 1.88

1.1% 23.12%

0.81 0.16 0.28 0.11 0.31

a Data are for the day of incubation showing the highest value for the product. bThis congener was inefficiently converted to 24−24-CB in two replicates(average of 1.1% conversion), but in the third replicate 23.12% of the 245−245 was converted to 24−24-CB. cSum of four pentachlorobiphenyl intermediates.

from which they were derived.11,29 Most likely some of the bacteria in mixed cultures supply nutrients and cofactors such as cobalamins, which enhance growth and dechlorination activity.30,31 In addition, the reduced para dechlorination activity by JNA suggests that an organism or a reductive dehalogenase gene responsible for para dechlorination in the parent culture may have been lost upon purification. Such an activity was very recently described: D. mccartyi strain 195 in mixed culture primarily dechlorinated hepta- and octachlorobiphenyls in Aroclor 1260 by removing doubly flanked chlorines including the para chlorine of 2345-chlorophenyl groups.32 Loss of a similar organism or dehalogenase gene during purification would explain both the loss of para dechlorination activity and the less robust dechlorination of hepta- and octachlorobiphenyls in strain JNA. Process N Dechlorination. Previously, Process N was observed in the environment, primarily in the Housatonic River (Lenox, MA)33 and in sediment microcosms derived from sites in New York, Maryland, Massachusetts, and China.8,19,20,34 Process N dechlorination of Aroclor 1260 was previously attributed to D. mccartyi organisms of the Pinellas subgroup in two sediment-free mixed cultures: the JN culture,22 and the CG-5 culture.20 In at least one study it was proposed that Process N resulted from the combined activity of several organisms.19 Now for the first time we have established that the set of at least 80 PCB dechlorination reactions known as Process N (77 in Table 2 plus those for 234−234-CB, 235− 235-CB, and 235−25-CB revealed by single congener experiments) can be mediated by a single strain of bacteria, D. mccartyi strain JNA, in pure culture.

We can also now better define PCB Dechlorination Process N. The dechlorination was almost exclusively from the flanked meta position of 34-, 234-, 235-, 236-, 245-, 2345-, 2346-, 2356-, and likely 345- and 23456-chlorophenyl rings. The chlorophenyl rings that are substrates for JNA comprise 88.65 mol % of all the chlorophenyl ring substitutions in Aroclor 126027 explaining why strain JNA can target so many congeners in this PCB mixture. Furthermore, most congeners can be dechlorinated by multiple pathways. This improved description of Process N will assist in the development of more accurate model systems to predict the fate of PCBs in the environment. Comparison of PCB Dechlorination Processes H and N as Expressed by D. mccartyi Strains CBDB1 and JNA. Previously we demonstrated that D. mccartyi strain CBDB1 in pure culture could carry out all of the complex PCB dechlorination reactions attributed to Dechlorination Process H.25 Until that time, Process H had been seen only in the environment and in sediment microcosms.6,8,15 Here we have shown that a second complex dechlorination process seen in the environment, Process N, can be mediated by a single bacterial strain. Both dechlorination processes share the ability to remove the doubly flanked meta chlorine from 234- and 2346-chlorophenyl rings. However, Process N removes a meta chlorine from 34-, 245-, and 2345-chlorophenyl rings, while Process H exclusively removes the para chlorine from these groups. Furthermore, Process N removes meta chlorines from 235-, 236-, and 2356-chlorophenyl rings, whereas these groups are not substrates for Process H. Two other dechlorination processes, H′ and P, also favor para dechlorination and are very similar to Process H.15 The result is that Process N is the most effective process for dechlorinating Aroclor 1260 and other 9194



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Notes

highly chlorinated PCB mixtures. Under optimal conditions it will dechlorinate most hexa- and heptachlorinated congeners to tetrachlorobiphenyls. These in turn can be further dechlorinated by Process LP or Process Q which can remove the isolated para chlorine on the 24- and 246-chlorophenyl rings left by Process N.14−17 The potential net effect of Process N combined with either Process LP or Process Q is to convert Aroclor 1260 to di- and trichlorobiphenyls which can be degraded by aerobic bacteria.17,35−37 Biostimulation and bioaugmentation of D. mccartyi catalyzed dechlorination of chlorinated ethenes have met with great success in the field.38 Perhaps we can look forward to similar success for D. mccartyi catalyzed PCB dechlorination in the future. One of the outstanding features of the various, fully characterized strains of D. mccartyi is their high specialization to use halogenated organics as respiratory substrates. This is attributed to the suite of multiple reductive dehalogenase homologous genes that is a hallmark of these organisms.24 For example, CBDB1 has 32 reductive dehalogenase homologous genes;39 however, function has been assigned to only four of them thus far. It will be exciting to determine which reductive dehalogenases are responsible for mediating PCB Dechlorination Processes H and N. In summary, we have isolated a novel strain of D. mccartyi, strain JNA, that carries out organohalide respiration with 236− 236-CB and have shown that it retains the broad substrate range for highly chlorinated PCBs previously reported for the JN mixed culture.21,22 Isolation of strain JNA has allowed an extensive and detailed characterization of PCB Dechlorination Process N, including determination of the dechlorination pathways for many of the PCB congeners in Aroclor 1260. This information will facilitate an understanding of the genetics underlying PCB dechlorination when the JNA genome is sequenced. Accordingly, these findings will ultimately lead to the development of specific probes that can be used to determine whether the genes that specify the reductive dehalogenase(s) responsible for Process N are present at contaminated sites, leading to more accurate predictions for the potential for in situ remediation.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are very grateful to Jianzhong He and Shanquan Wang for fluorescence microscopy, and DGGE and MLST analyses of strain JNA. We thank Steve Zinder for methane analysis of our samples, for the gift of strain 195 DNA for PCR standards, and for many helpful suggestions. We thank Richard Bopp for assistance collecting sediments from Woods Pond in Lenox, MA, and we thank Lorenz Adrian for encouragement and many helpful suggestions. This research was supported by NSF grant 0641743 to DLB and by NSF graduate fellowships DGE0237084 and DGE-0750272 to SLL.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details, detailed description of dechlorination of various congeners, figure comparing dechlorination of four PCB congeners tested as potential terminal electron acceptors for JNA, figure showing change in PCB homologue distribution during incubation of Aroclor 1260 with JNA, figures showing fluorescence microscopy, agarose gel electrophoresis and DGGE analysis of 16S rRNA, MLST analysis of three housekeeping genes, and figures showing the pathways of dechlorination and quantitative product distribution for 234− 245-CB, 236−245-CB, and 2345−236-CB. This material is available free of charge via the Internet at http://pubs.acs.org

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses §

Anchor QEA, 4300 Route 50, Suite 202, Saratoga Springs, NY 12866. ∥ Department of Microbiology, 175 Wing Hall, Wing Rd., Cornell University, Ithaca, NY, 14853. 9195



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Dehalococcoides mccartyi Strain JNA in Pure Culture Extensively

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