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2 Bacteria Involved in the Dechlorination of Commercial PCB Mixtures in Mixed Cultures
23 The Microbiology of Anaerobic PCB Dechlorination
D. mccartyi clone CW3 (PCB enrichment) (JQ990320)
D. mccartyi clone CG3 (PCB enrichment) (JQ990324)
D. mcccartyi strain CG1 (JQ990322)
D. mccartyi strain CG4 (JQ990325)
D. mccartyi clone CG2 (PCB enrichment) (JQ990323)
D. mccartyi strain 195 (NR_114415)
D. mccartyi strain CG5 (JQ990326)
D. mccartyi strain SG1 (JQ990327)
D. mccartyi clone CW4 (PCB enrichment) (JQ990321)
D. mccartyi strain CBDB1 (NR_074115)
D. mccartyi strain JNA (KJ461493)
Dehalogenimonas lykanthroporepellens strain BL-DC-9 (NR_074337)
Dehalogenimonas alkenigignens strain IP3-3 (JQ994266)
Dehalogenimonas sp. clone CG3 (PCB enrichment) (JQ990328)
Clone m-1 (PCB enrichment) (DQ113418)
Clone OTU-1 (PCB enrichment) (AY559064)
“Dehalobium chlorocoercia” strain DF-1 (AF393781)
Roseiflexus castenholzii DSM 13941 (NR_074188)
Chloroflexus aurantiacus DSM 637 (AJ308501)
Desulfitobacterium dehalogenans JW/IU-DC1 (L28946)
Dehalobacter sp. 12DCB1A (JQ918082)
Dehalobacter sp. clone CW1 (PCB enrichment) (JQ990318)
Dehalobacter sp. clone AD14-PCE (PCB enrichment) (KC342963)
Dehalobacter restrictus strain PER-K23 (NR_121722)
Dehalobacter sp. clone Z40 (PCB enrichment) (AY754831)
Dehalobacter sp. clone Z29 (PCB enrichment) (AY754830)
Geobacter lovleyi strain SZ (NR_074979)
Fig. 23.2 Phylogenetic analysis of the 16S rRNA genes of PCB dechlorinators and putative
PCB dechlorinators from PCB enrichments. V, C, and P refer to the phylogenetic subgroups
of D. mccartyi, Victoria, Cornell, and Pinellas, respectively. The evolutionary history of the
16S rRNA genes of PCB dechlorinating strains was inferred by using the maximum likelihood
method based on the General Time Reversible model (Nei and Kumar 2000). The 16S rRNA
gene sequences of Dehalogenimonas strains BL-DC-9 and P3-3, Dehalobacter strains PER-K23
and 12DCB1A, and the Roseiflexus and Chloroflexus strains were included for comparison, and
the 16S rRNA sequence of Geobacter lovleyi strain SZ was used to root the tree. All other strains
and clones shown have been associated with PCB dechlorination. All clones were obtained from
PCB enrichment cultures. Phylotypes VL-CHL1, , and o-17 are not shown because their published sequences are too short. However, their positions on the tree are represented by clone m-1
(identical to clones VL-CHL1 and SF1 over 466 and 470 bp, respectively) and clone OTU-1
(only 4 bp differences from o-17 over 714 bp)
the community DNA with the D. mccartyi specific primers DHC1F/DHC1377R
(Hendrickson et al. 2002) did not detect any D. mccartyi 16 S rRNA genes in these
enrichments. Zanaroli et al. concluded that phylotype VL-CHL1 represents the
bacterial agent responsible for the dechlorination of Aroclor 1254 in these enrichments. This is the first time that the dechlorination of an Aroclor has been exclusively attributed to a member of the Chloroflexi other than D. mccartyi.
J. He and D.L. Bedard
The tree with the highest log likelihood (−6038.3051) is shown. The percentage of trees in which the associated taxa clustered together (out of 100 replicates)
is shown next to the branches. Initial tree(s) for the heuristic search were obtained
automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of
pairwise distances estimated using the maximum composite likelihood (MCL)
approach, and then selecting the topology with superior log likelihood value. The
tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 27 nearly full-length nucleotide sequences.
All positions containing gaps and missing data were eliminated. There were a total
of 1036 positions in the final dataset. Evolutionary analyses were conducted in
MEGA6 (Tamura et al. 2013).
VL-CHL1 removed ~75 % of the penta-, hexa-, and heptachlorobiphenyls in
Aroclor 1254, converting these to tri- and tetrachlorobiphenyls. The dechlorination removed about 20 % of the chlorine substituents in 30 weeks and was characterized by the removal of flanked meta chlorines from 23- and 234-chlorophenyl
groups and the removal of flanked para chlorines from 34- and 245-chlorophenyl groups (underscores here and throughout the chapter indicate the chlorines
removed). The most prominent products were 2,2′,4,5′-chlorobiphenyl (24-25-CB)
and 25-25-CB, and to a lesser extent, 25-3-CB. These characteristics match PCB
Dechlorination Process H′ which was previously reported in the Acushnet Estuary
of New Bedford Harbor, Massachusetts, USA (Brown and Wagner 1990).
23.2.2 D. mccartyi Mixed Cultures
The low bioavailability of PCBs results in a correspondingly low abundance of
PCB dechlorinating Dehalococcoides in mixed communities obtained from contaminated sites. In addition to hampering PCB bioremediation efforts, the low
abundance of PCB respiring bacteria in available samples poses a challenge in
subsequent bacterial enrichment, isolation, and characterization.
Wang and He (2013a) screened the commercial PCB dechlorination activities in sediment and soil samples originating from four Asian countries (China,
Indonesia, Malaysia, and Singapore), ultimately establishing nine PCB dechlorinating microcosms. To further elucidate PCB dechlorination processes, isolation of
PCB dechlorinators is necessary, which requires the development of sediment-free
cultures first. The nine microcosms were set up with soils, sediments or sludge,
but without addition of any sediment substitutes. In each microcosm, only a single
16S rRNA Dehalococcoides genotype was identified. The D. mccartyi bacteria in
these cultures are affiliated to all three phylogenetic subgroups: Cornell (cultures
CG-2 and CG-4), Victoria (cultures CW-3, CG-1, and CG-3), and Pinellas (cultures CW-2, CW-4, CG-5 and SG-1) (Wang and He 2013b).
Unlike previous Aroclor 1260 dechlorinating microcosms, serial transfers
of these nine microcosms were carried out in defined medium amended with
23 The Microbiology of Anaerobic PCB Dechlorination
Aroclor 1260 and lactate, but without any substitutes for soil, sediment or sludge,
and yielded six sediment-free PCB dechlorinating cultures: CW-4, CG-1, CG-3,
CG-4, CG-5 and SG-1 (Wang and He 2013b). (Throughout this chapter we will
use the term sediment-free to refer to cultures containing no soil, sludge, or sediment). The D. mccartyi organisms in each of these enrichment cultures coupled
their growth with dechlorination of Aroclor 1260. The cell yield of D. mccartyi
supported by PCB respiration reached ~3.3 × 1014 cells per mole of chlorine
removed in these sediment-free cultures, which is comparable to the cell yield of
D. mccartyi supported by respiration of chlorinated ethenes which ranges from
~7.8 × 1013 to 5.6 × 1014 cells per mole of chlorine removed (Löffler et al. 2013).
Previously reported mixed cultures exhibiting PCB dechlorination activity were
all ultimately shown to contain PCB dechlorinating Chloroflexi, either D. mccartyi
or the phylogenetically related, but distinct bacteria (e.g., o-17, DF-1, m1, SF1)
(Wu et al. 2002a; Fagervold et al. 2007; Bedard et al. 2007). PCR amplification
with o-17/DF-1 specific primers did not detect the presence of o-17/DF-1-type or
m1/SF1-type Chloroflexi in any of the six sediment-free enrichment established by
Wang and He (2013b).
In cultures CW-4, CG-1, CG-3, CG-4, CG-5, and SG-1, several distinct PCB
dechlorination patterns were observed, as determined by PCB congener profile
changes from the dechlorination of Aroclor 1260 and by the dechlorination products of two individual PCB congeners, 2345-245-CB and 234-245-CB. Process H
is the dominant PCB dechlorination pattern observed in cultures CW-4 and SG-1.
Dechlorination Process H removes flanked para and doubly flanked meta chlorines (Table 23.1). In culture SG-1 the dechlorination of the 245-chlorophenyl
group was diminished compared to culture CW-4, resulting in more accumulation
of 245-25-CB and less accumulation of 25-25-CB.
The dechlorination observed in culture CG-4 shared some elements of Process
H, but was either a different dechlorination process or a combination of a less
robust Process H and Process T. Either of the doubly flanked chlorines of the
2345-chlorophenyl group could be removed to yield both 235- and 245-chlorophenyl groups. The doubly flanked meta chlorine of 234- groups and the flanked para
chlorine of 245-groups were also removed, but these latter two activities were
much less prominent than in Process H. The dechlorination primarily converted
heptachlorobiphenyls to pentachlorobiphenyls. The CG-5 culture showed the most
extensive dechlorination of Aroclor 1260 via Dechlorination Process N.
Both cultures CG-1 and CG-3 exhibited novel PCB dechlorination patterns
attacking primarily doubly flanked chlorines on heptachlorobiphenyls bearing
2345- and 234-chlorophenyl groups (Wang and He 2013b). Culture CG-3 primarily removed the meta chlorine of 234-chlorophenyl groups, and either the meta
or para chlorine of 2345-chlorophenyl groups (both are doubly flanked). Culture
CG-1 primarily attacked meta chlorines of 234- and 2345-chlorophenyl groups
(where the underlined chlorines are removed).
J. He and D.L. Bedard
23.2.3 Mixed Culture AD14 (D. mccartyi and
Members of D. mccartyi have also been found to dechlorinate PCBs in mixed
cultures containing other obligate dechlorinating bacteria. A possible synergistic relationship between D. mccartyi and Dehalobacter was observed in culture
AD14, a sediment-free Aroclor 1260 dechlorinating culture amended with lactate
(Wang and He 2013a). This culture was established with sludge from an anaerobic
digester in a wastewater treatment plant in which concentrations of PCBs, polybrominated diphenyl ethers, chlorophenols, chlorinated ethenes, and chlorinated
ethanes were below the detection limit (<0.1 μM). The PCB dechlorination pattern of culture AD14 most closely resembles Process H (Table 23.1) (Wang and
High throughput pair-end Illumina sequencing of 16S rRNA genes was performed in order to obtain a snapshot of the microbial community structure of culture AD14. D. mccartyi and Dehalobacter sp. were present in low abundance, 2.1
and 2.2 %, respectively, of the total sequences (Wang and He 2013a). The growth
of both organisms was correlated with chlorine removal from PCBs, as determined by quantitative polymerase chain reaction (qPCR) analysis of 16S rRNA
genes during dechlorination of Aroclor 1260. The qPCR data also showed that the
Dehalobacter sp. had a longer lag phase than the D. mccartyi genotype, suggesting a possible requirement for intermediate PCB dechlorination products generated by D. mccartyi. The Illumina sequencing data (34,724 pair-end reads) showed
the absence of other known reductive dechlorinating bacteria such as o-17/DF-1
type or m1/SF1 type Chloroflexi, Desulfitobacterium, Geobacter, Sulfurospirillum,
The low proportions of potential PCB dechlorinators in culture AD14 suggested that further enrichment of the dechlorinating bacteria was necessary prior
to any attempt to characterize the RDase genes and gene products responsible for
dechlorination of Aroclor 1260 in this culture. The PCB dechlorinating bacteria
were enriched via addition of more bioavailable organohalides such as tetrachloroethene (PCE), 1,2-dichloroethane, and 2,4,6-trichlorophenol. The low relative
abundance of both the Dehalobacter and D. mccartyi (each ~2 %) in the original
culture AD14 increased to more than 50 % when grown with PCE. Along with the
relative increase in abundance of certain populations, this highly enriched PCE-fed
subculture AD14-PCE retained PCB dechlorination activity. This provides confirmation that D. mccartyi and Dehalobacter were responsible for the Aroclor 1260
dechlorination in the original microcosm, consistent with the original microcosm
Illumina sequencing result (Wang and He 2013a).
A significant finding is that PCB dechlorination was not inhibited by the presence of other organohalides that are found as co-contaminants with Aroclor 1260:
octabromodiphenyl ether mixture, PCE, 1,2-dichloroethane, and 2,4,6-trichlorophenol. This may be important for the development of effective in situ bioremediation technologies.
23 The Microbiology of Anaerobic PCB Dechlorination
D. mccartyi strains AD14-1 and AD14-2 were isolated from the sediment-free
enrichment culture AD14-PCE. However, neither of these isolates was capable of
dechlorinating PCB congeners in Aroclor 1260. This loss of metabolic ability may
be attributed to loss of the PCB dechlorinators, loss of functional reductive dehalogenase genes for PCB dechlorination during the isolation process, or to PCB
dechlorination requiring the cooperation of both Dehalobacter and D. mccartyi.
23.2.4 D. mccartyi Strain in Mixed Culture Dechlorinates
Aroclor 1260 Exclusively by Removal of Doubly
D. mccartyi strain 195 grows to much higher cell densities when grown in mixed
culture with butyrate as the electron donor and carbon source and with fermented
yeast extract as a supplement; therefore those conditions were used in the following experiments. Zhen et al. (2014) tested the ability of strain 195, the only known
dechlorinator in the culture, to dechlorinate 1 µg/ml of Aroclor 1260, Aroclor
1254, or Aroclor 1242 in the presence, or absence, of periodic supplements of
25 µM 1,2,3,4-tetrachlorobenzene. This chlorobenzene is dechlorinated to 1,2,3and 1,2,4-trichlorobenzene and appears to support growth of strain 195 by organohalide respiration (Fennell et al. 2004).
In 250 days, strain 195 dechlorinated 13 of the 24 major PCB congeners in
Aroclor 1260. These congeners constitute 44 % of the total PCBs in Aroclor 1260.
In the absence of 1,2,3,4-tetrachlorobenzene these congeners were decreased by
42 % in 250 days, but when 1,2,3,4-tetrachlorobenzene was added on days 0, 65,
108, and 156, the same congeners were decreased by 84 %. The congeners dechlorinated were primarily hepta-, octa-, and nonachlorobiphenyls which showed
decreases of 21.5, 6.5, and 0.6 mol%, respectively; corresponding increases
occurred in penta- and hexachlorobiphenyls (Zhen et al. 2014).
The congeners that were dechlorinated were composed mainly of 234-, 2345-,
2346-, and 23456-chlorophenyl rings (the targeted chlorines are underlined).
The primary products were 235-245-CB, 245-24-CB, 235-236-CB/2356-25-CB,
and 235-24-CB which increased by 8.2, 6.1, 5.6, and 4.9 mol%, respectively
(Zhen et al. 2014). Three additional congeners, 235-235-CB, 245-246-CB, and
235-25-CB increased by 2.4 to 3.0 mol%. The authors showed stoichiometric mass balances for dechlorination substrates and products. On the basis of
these, they concluded that the 23456-chlorophenyl group, which has three doubly flanked chlorines, and the 2345-chlorophenyl group which has two doubly
flanked chlorines, are both primarily attacked at the para chlorine to yield 2356and 235-chlorophenyl groups, respectively. The latter conclusion was confirmed
by an experiment using 2345-4-CB as a substrate. Both 235-4-CB and 245-4CB were products, but they were produced in a ratio of 49:1 (Zhen et al. 2014).
This well characterized dechlorination pattern is novel and we assign it the name
Dechlorination Process Z (Table 23.1).
J. He and D.L. Bedard
Dechlorination experiments of strain 195 with the less-chlorinated Aroclor
1254, which has an average of about 5.1 chlorines per biphenyl, showed dechlorination of hexa- and heptachlorobiphenyls with doubly flanked chlorines to
tetra- and pentachlorobiphenyls (Zhen et al. 2014). However, the impact of
the dechlorination was far less than that for Aroclor 1260 because the Aroclor
1254 has a much smaller proportion of congeners with doubly flanked chlorines
than Aroclor 1260 (Fig. 23.1). Dechlorination experiments with Aroclor 1242,
which has an average of about 3.5 chlorines per biphenyl, showed very little
Several attempts to determine if strain 195 can use PCB congeners in Aroclor
1260 for respiration failed, as did an attempt using 2345-4-CB as an electron
acceptor. The authors concluded that strain 195 most likely does not use any of the
PCBs in Aroclor 1260 for organohalide respiration (Zhen et al. 2014).
23.3 Pure Culture of “Dehalobium chlorocoercia” Strain
DF-1 Exclusively Removes Doubly Flanked Chlorines
Strain DF-1, informally named “Dehalobium chlorocoercia”, was the first PCB
respiring bacterium to be isolated (May et al. 2008b). It was isolated from sediments of Charleston Harbor (South Carolina, USA). Strain DF-1 is a member of
the Chloroflexi related to the Dehalococcoides, and like them appears to be an
obligate organohalide respirer, but it is significantly smaller, with a mean size of
137 ± 51 nm, and it can only be grown as a co-culture with cells of, or cell extract
from, a Desulfovibrio sp. (May et al. 2008b). Similar to D. mccartyi strain 195,
its PCB dechlorinating specificity, as determined by incubation with single congeners substituted on only one ring, is limited to removal of doubly flanked meta
chlorines from 234- and 2346-chlorophenyl rings and doubly flanked para chlorines from 345- and 2345-chlorophenyl rings (where the underlined chlorines are
removed) (Wu et al. 2002b).
DF-1 was grown with 2345-CB and added to nonsterile sediment contaminated with 4.62 µg/g of weathered Aroclor 1260 in order to determine if bioaugmentation with strain DF-1 would dechlorinate weathered PCBs in the presence
of indigenous bacteria. The addition of DF-1 resulted in significant losses of
hepta- and octachlorobiphenyls with doubly flanked chlorines (May et al. 2008a).
Specifically, 2345-245-CB (PCB 180), 2345-234-CB (PCB 170), and 2346-234CB (PCB 171) plus 2345-34-CB (PCB 156), where underscores indicate the chlorines targeted, decreased by 4.90, 2.55, and 2.12 mol%, respectively. However,
there were no corresponding increases in the expected products: 235-245-CB
(PCB 146), 235-24-CB (PCB 90), 234-246-CB (PCB 140), 2346-24-CB (PCB
139), 246-24-CB (PCB 100), and 235-34-CB (PCB 109) (May et al. 2008a).
Instead, there were large increases in 235-4-CB (PCB 63), and in the peak containing 235-25-CB (PCB 92). The authors proposed that dechlorination products
23 The Microbiology of Anaerobic PCB Dechlorination
from DF-1 may have stimulated indigenous bacteria to carry out additional
dechlorination (May et al. 2008a). Indeed, further dechlorination of the putative
DF-1 products 235-34-CB and 235-245-CB, two congeners that DF-1 should not
be able to dechlorinate, would form 235-4-CB and 235-25-CB, respectively.
The chlorophenyl ring specificity of a PCB dechlorinator is not always exactly
the same for highly chlorinated PCBs as it is for congeners substituted on only one
ring, because the chlorine configuration on the opposite ring can affect the position
of dechlorination (Adrian et al. 2009; LaRoe et al. 2014) (see Sect. 23.4.1). Strain
DF-1 is a unique and interesting PCB dechlorinator. It would be of interest to the
field to know its precise specificity for Aroclor 1260 as has been determined for
the six PCB dechlorinating D. mccartyi strains.
23.4 Pure Cultures of D. mccartyi Exhibit Diverse
Complex Patterns of Dechlorination of Commercial
23.4.1 D. mccartyi Strain CBDB1
Strains of D. mccartyi that harbor different suites of RDase genes frequently have
identical or nearly identical 16S rRNA genes (Löffler et al. 2013). Therefore, none
of the previously described studies could determine whether the complex PCB
dechlorination patterns observed in mixed cultures resulted from the action of
a single strain or several strains of D. mccartyi. Definitive proof that a single D.
mccartyi strain in pure culture can exhibit a complex pattern of dechlorination was
first demonstrated with strain CBDB1, a strain originally isolated by growth with
trichlorobenzenes as sole electron acceptor (Adrian et al. 2000, 2009).
Adrian et al. (2009) identified 43 PCB congeners with 3–8 chlorines that were
dechlorinated by CBDB1. Most of these congeners are components of Aroclor
1260, although a few are components of Aroclor 1248 and some are not present
in either Aroclor and were tested as single congeners (Adrian et al. 2009). Seven
chlorophenyl rings were dechlorination substrates: singly flanked and doubly
flanked para chlorines were removed from 34-, 245-, 2345-, and 345-chlorophenyl rings; primarily doubly flanked meta chlorines were removed from 234 and
2346-chlorophenyl rings; and either the doubly flanked meta (23456-) or para
chlorines (23456-) from 23456-chlorophenyl rings (Adrian et al. 2009). The primary dechlorination products from Aroclor 1260 were 235-25-CB, 25-25-CB,
and 24-25-CB. The observed dechlorination corresponds to PCB Dechlorination
Process H which was observed in situ in sediments of the Acushnet Estuary of
New Bedford Harbor (Massachusetts, USA) and the Hudson River (New York,
USA) (Brown and Wagner 1990). This conclusively proved that a single D. mccartyi strain is capable of carrying out a complex PCB dechlorination pattern that
occurs in the environment.