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2 Bacteria Involved in the Dechlorination of Commercial PCB Mixtures in Mixed Cultures

2 Bacteria Involved in the Dechlorination of Commercial PCB Mixtures in Mixed Cultures

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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)

m-1/SF1 group

o-17/DF-1 group

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

Dehalobacter sp.)

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

He 2013a).

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,

or Anaeromyxobacter.

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

Flanked Chlorines

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

PCB Mixtures

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.

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2 Bacteria Involved in the Dechlorination of Commercial PCB Mixtures in Mixed Cultures

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