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5 Identification and Characterization of Three PCB Reductive Dehalogenases

5 Identification and Characterization of Three PCB Reductive Dehalogenases

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J. He and D.L. Bedard



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DEHALGT0124-GT (ADC73492)

PcbA4-CG4 (AII58856)

DET1559-195 (AAW39215)

PcbA5-CG5 (AII60305)

RD11-JNA (AHZ58530)

MbrA-MB (ADF96893)

CbrA-CBDB1 (YP_307261)

PcbA1-CG1 (AII58466)

1350-GY50 (AHB14121)

DcpA-KS (AGS15112)

PceA-195 (YP_181066)

VcrA-VS (YP_003330719)

TceA-195 (YP_180831)

BvcA-BAV1 (AAT64888)

0.2



Fig. 23.5  Phylogenetic analysis of functionally characterized RDases in D. mccartyi including orthologs of the PCB reductive dehalogenases. DEHALGT0124, Det1559, and RD11 are

orthologs of PcbA4 and PcbA5 in D. mccartyi strains GT, 195, and JNA; they have not yet been

functionally characterized. Likewise, 1350-GY50 is an ortholog of Pcb1 in D. mccartyi strain

GY50 but has not been functionally characterized. The tree was constructed with MEGA 6

(Tamura et al. 2013) using the maximum likelihood method in the Jones-Taylor-Thornton (JTT)

model. Branch lengths indicate the number of substitutions per site



23.5.3 Phylogenetic Lineage of PCB RDases

Two of the PCB RDase enzymes, PcbA4 and PcbA5, which attack para- and meta

chlorines, respectively, are phylogenetically similar, sharing 97 % amino acid

sequence identity. The other, PcbA1, clusters in a distant clade and shares only 38 %

amino acid sequence identity with PcbA4 and PcbA5. No PCB RDase has been identified in strains 195 or JNA yet, but both of these strains have orthologs of PcbA4 and

PcbA5 that are potential candidates (Fig. 23.5) (Seshadri et al. 2005; Fricker et al.

2014). CBDB1 has no orthologs of either PcbA1 or the other two PCB RDases, so it

must use an enzyme of yet another lineage. These findings suggest the existence of at

least three different lineages of the PCB RDases in D. mccartyi. Moreover, the results

suggest considerable diversity of regiospecificity within lineages.



23.5.4 PCB Reductive Dechlorinases also Dechlorinate PCE

Transcriptome and enzyme activity assays prove that in addition to dechlorinating

PCBs, PcbA1, PcbA4, and PcbA5 dechlorinate PCE to TCE and then to cis- and



23  The Microbiology of Anaerobic PCB Dechlorination



557



trans-DCE (Wang et al. 2014). This substrate flexibility may provide an insight

into the ability of D. mccartyi strains to evolve and metabolize anthropogenic

compounds that did not exist prior to their industrial production. PCE and TCE are

produced in volcanic gases (Jordan et al. 2000), hence these substrates have been

present on earth for billions of years, whereas PCBs were first manufactured in

1929.

The discovery of the PCB RDases (Wang et al. 2014) increases the functional

diversity of RDases identified in D. mccartyi which include the previously characterized RDases PceA, MbrA, TceA, VcrA, BvcA, DcpA, and CbrA (Fig. 23.5).



23.5.5 Bifunctional PCB/PCE RDases May Facilitate

Bioaugmentation for PCB Remediation

D. mccartyi strains CG1, CG4, and CG5 utilize PcbA1, PcbA4, and PcbA5,

respectively, for PCE respiration when grown with PCE as the sole electron acceptor, yet at the same time they can respire and dechlorinate PCBs (Wang et al.

2014). When grown with PCE as the electron acceptor, the cell density is 12.5to 22-fold higher than when grown with PCBs. In addition, the cells grow much

faster with PCE than with PCBs (several weeks versus several months) (Wang

et al. 2014). PCB dechlorinators with bifunctional PCB/PCE RDases can be selectively enriched, transferred repeatedly, and grown to high cell densities with PCE

as the sole electron acceptor with no possibility of losing their ability to dechlorinate PCBs, i.e., with no risk of losing their PCB RDase gene(s) (Wang et al. 2014).

This property makes PCB dechlorinators with such bifunctional PCB/PCE RDases

ideal candidates for use in bioremediation of PCBs because they can be easily

grown in large amounts for bioaugmentation using PCE as the electron acceptor

and the PCB RDases will be expressed.



23.6 Other Genera Implicated in PCB Dechlorination

23.6.1 Dehalobacter spp. Involved in PCB Dechlorination

The first evidence that Dehalobacter spp. were involved in PCB dechlorination was reported by Yan et al. (2006a) who implicated three putative PCB

dechlorinating populations in enrichment established from Hudson River sediment and amended with 2345-CB. These investigators studied the impact of

various amounts of aqueous CO2 (supplied as sodium bicarbonate at final concentrations of 0, 100, 500, or 1000 mg/L) on PCB dechlorination and on the

microbial populations. The primary route of dechlorination was 2345-CB → 235CB → 25-CB → 2-CB, but small amounts of 245-CB, 23-CB, and 24-CB were



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also observed (Yan et al. 2006a), all of which are consistent with results typically

reported for enrichments with Hudson River sediment. A single Pinellas-type D.

mccartyi population was present in all enrichment cultures and was not affected

by the sodium bicarbonate concentration. In contrast, two distinct Dehalobacter

populations were much more prominent in the enrichment cultures amended with

100 mg/L sodium bicarbonate. These same cultures also exhibited the most rapid

and extensive dechlorination, i.e., more production of 2-CB (Yan et al. 2006a).

Because increased removal of the unflanked meta chlorine from 25-CB to produce 2-CB (a characteristic unique to Dechlorination Process M) was observed in

the cultures that had the most prominent Dehalobacter populations, the authors

proposed that this particular dechlorination reaction might be catalyzed by the

Dehalobacter populations. The 1445 bp 16S rRNA gene fragments of these

Dehalobacter phylotypes were 99.1 and 99.6 % identical to the 16S rRNA gene of

Dehalobacter restrictus strain PER-K23.

In support of the idea that Dehalobacter may play an active role in dechlorination of PCBs with unflanked chlorines, Nelson et al. (2014) have recently

described three Dehalobacter strains that can dehalogenate dichlorobenzenes with

flanked and unflanked chlorines.

Further evidence implicating Dehalobacter in PCB dechlorination was reported

by Yoshida et al. (2009b) who demonstrated reductive dechlorination of PCBs

and 1,2,3-trichlorodibenzo-p-dioxin in a sediment-free environmental enrichment

culture containing two Dehalobacter phylotypes; a novel dechlorination pathway: 2,3,4-trichlorobiphenyl to 3,4-dichlorobiphenyl and then to 4-chlorobiphenyl was attributed to the Dehalobacter. In addition, Dehalobacter was the only

dechlorinator detected in an Aroclor 1260 dechlorinating microcosm CW-1, which

was developed by Wang et al. from silt and clay near the Yang Tze River in China

(Wang and He 2013b). The pattern of dechlorination in the CW-1 microcosm was

typical of Process H. The 16S rRNA gene sequence of Dehalobacter sp. clone

CW1 (JQ990318) is 99.3 % identical (over 1524 bp) to that of Dehalobacter sp.

strain 12DCB1A which dechlorinates 1,2-dichlorobenzene (Nelson et al. 2014),

and Dehalobacter sp. clone FTH2 which was implicated in the dechlorination of

4,5,6,7-tetrachlorophthalide (Yoshida et al. 2009a).

Finally, a Dehalobacter sp. was found in a subculture of PCB dechlorinating

enrichment culture AD14 (see Sect. 23.2.3). The 1423 bp 16S rRNA gene fragment of Dehalobacter sp. clone AD14-PCE shares 99.2 % sequence identity with

that of Dehalobacter restrictus sp. strain PER-K23 (DSM 9455). Dehalobacter sp.

AD14-PCE couples growth with dechlorination of Aroclor 1260 (Wang and He,

2013b). Interestingly, Dehalobacter sp. clone AD14-PCE had a longer lag phase

than the PCB dechlorinating D. mccartyi in the same culture, suggesting the possible synergistic involvement of the Dehalobacter in removal of chlorines from the

less-chlorinated PCB congeners produced by the D. mccartyi in the same culture.

Dehalobacter spp. have recently been found to harbor multiple RDase genes,

with up to 25 RDase genes present on a single genome (Kruse et al. 2013). The

Dehalobacter spp. are currently the only non-Chloroflexi bacteria known to carry

out PCB dechlorination activities.



23  The Microbiology of Anaerobic PCB Dechlorination



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23.6.2 Dehalogenimonas alkenigignens

The Dehalogenimonas genus of the Chloroflexi phylum, which is closely related

to Dehalococcoides, also appears capable of dechlorinating and respiring PCBs.

For example, Illumina sequencing analysis and qPCR suggested the involvement of members of the Dehalogenimonas genus as well as D. mccartyi in the

reductive dechlorination of Aroclor 1260 in enrichment culture CG-3 (see

Sect. 23.2.2) (Wang and He 2013b). Dechlorination in this sediment-free enrichment most closely approximates a mixture of Dechlorination Processes H and

T, mainly attacking the doubly flanked meta and para chlorines (Wang and He

2013a). Subsequent two-step denaturing gradient gel electrophoresis (Wang and

He 2012) and sequencing showed that the full-length 16S rRNA gene sequence of

the Dehalogenimonas in culture CG-3 (Accession no. JQ990328) differs from that

of Dehalogenimonas alkenigignens strain IP3-3(T), by a single nucleotide over

1493 bp. qPCR analysis showed that growth of Dehalogenimonas alkenigignens

strain CG-3 was coupled to the dechlorination of Aroclor 1260 in this sedimentfree culture, and that D. alkenigignens strain CG-3 was the dominant dechlorinator

in the culture, representing 2.16 % of the total microbial population versus 0.37 %

for D. mccartyi (Wang and He 2013a).



23.7 Conclusions and Outlook

The last seven years have been exciting and enlightening for those of us interested

in PCB dechlorination. There are now six pure strains of D. mccartyi, representing all three phylogenetic subgroups, and a pure strain of “Dehalobium chlorocoercia”, all of which have been demonstrated to dechlorinate the commercial

PCB mixture Aroclor 1260, and at least four of which can use Aroclor 1260 for

respiration. Complete genomes have been published for five of the D. mccartyi

strains. In addition, there is strong evidence that both Dehalogenimonas spp. and

Dehalobacter spp. can dechlorinate and respire PCBs.

It is now clear from many examples that D. mccartyi organisms capable of

dechlorinating Aroclor 1260 are widespread at freshwater sites including locations

in China, Germany, Singapore, and the USA. In contrast, non-Dehalococcoides

Chloroflexi of the o-17/DF-1 and m1/SF1 groups have not been found in any

freshwater sites to date even though they have been targeted with specific primers. On the other hand, a member of the Chloroflexi with a 446 bp 16S rRNA gene

fragment sequence identical to that of clone m1 is apparently responsible for the

dechlorination of Aroclor 1254 in a marine site (Zanaroli et al. 2012). Now that

we know that the Dehalobacter and Dehalogenimonas genera also play a role in

Aroclor dechlorination, it will be interesting to see the precise PCB dechlorination

processes that they catalyze.



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Pure strains of D. mccartyi that dechlorinate Aroclor 1260 by Processes H, N,

Z, and variations of these are now available. However, no organisms capable of

removing unflanked chlorines as in PCB Dechlorination Processes M, LP, and Q,

have yet been identified. It will be important to identify and isolate such organisms

because they hold the key to further dechlorinating the less chlorinated PCBs generated by Processes H, H′, N, P, T, and Z.

The discovery and characterization of the first three PCB reductive dehalogenases constitute a new milestone in the field. PcbA1, PcbA4, and PcbA5 dechlorinate Aroclor 1260 with distinct regiospecificities and prove that individual

RDases can carry out the complex dechlorination of dozens of PCB congeners

described by PCB dechlorination processes (Wang et al. 2014). PcbA4 and PcbA5

differ by only 14 amino acids out of 482, yet their specificity is entirely different (Wang et al. 2014). Studying and perhaps altering the amino acid sequences

of these RDases should yield new insights into PCB dechlorination and perhaps

increase their substrate range (Bedard 2014).

The discovery that PCB dechlorination and PCE dechlorination are both catalyzed by all three PCB dechlorinases has important implications for PCB remediation because it means that PCE can be used to grow large amounts of PCB

dechlorinators actively expressing PCB RDases for use in bioaugmentation. This

makes PCB remediation much more feasible.



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Chapter 24



“Dehalobium chlorocoercia” DF-1—from

Discovery to Application

Harold D. May and Kevin R. Sowers



Abstract “Dehalobium chlorocoercia” strain DF-1 is an organohalide respiring ultramicrobacterium isolated from a tidal estuary of Charleston Harbor

using a polychlorinated biphenyl (PCB) congener as the sole electron acceptor.

Organohalide respiration occurs by dechlorination of PCB congeners with doubly flanked chlorines, but this strain is also capable of dechlorinating chlorobenzenes with doubly flanked chlorines and tetra- and tri-chloroethene to a mixture

of cis- and trans-1,2-dichloroethene. The range of PCB congeners dechlorinated

from an Aroclor is limited in comparison with other PCB respiring strains; however, “D. chlorocoercia” strain DF-1 is capable of dechlorinating PCBs at environmentally relevant concentrations that are typically below saturation in water. In

sediment-free medium an unidentified water-soluble factor from a Desulfovibrio

sp. is required for growth. “D. chlorocoercia” strain DF-1 is osmotolerant, enabling it to grow and dechlorinate PCBs in sediments ranging from freshwater to

marine. What follows is a description of “D. chlorocoercia” strain DF-1 and some

of its related PCB respiring species from the perspective of environmental detection, dechlorination pathways and kinetics, biostimulation, electrostimulation, and

finally bioaugmentation to enhance PCB degradation in sediments.



H.D. May 

Marine Medicine and Environmental Science Center, Department of Microbiology and

Immunology, Medical University of South Carolina, Charleston, SC, USA

e-mail: mayh@musc.com

K.R. Sowers (*)

Institute of Marine and Environmental Technology, Department of Marine Biotechnology,

University of Maryland Baltimore County, Baltimore, MD, USA

e-mail: sowers@umbc.edu

© Springer-Verlag Berlin Heidelberg 2016

L. Adrian and F.E. Löffler (eds.), Organohalide-Respiring Bacteria,

DOI 10.1007/978-3-662-49875-0_24



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24.1 The Discovery of PCB Dechlorinating

Microorganisms

Polychlorinated biphenyls (PCBs) remain among the more difficult to treat environmental pollutants due to their chemical stability and low aqueous solubility,

particularly as more chlorines are added to the biphenyl ring. The complexity

of commercial PCB mixtures, which contain combinations of 209 possible congeners, further contributes to their recalcitrance in the environment. Identifying

microorganisms that are capable of attacking these chemical structures proved

to be a daunting task and began with the discovery of aerobic bacteria that could

cleave the biphenyl ring of lesser-chlorinated congeners (Ahmed and Focht 1973;

Furukawa 1976). Transformation of higher chlorinated PCB congeners was also

observed in anaerobic sediments as early as 1984 (Brown et al. 1984). Identifying

the microorganisms that dechlorinated PCBs proved to be difficult, but as

described below anaerobic bacteria that could link their growth to PCB respiration

were eventually identified as Dehalococcoides and closely related microorganisms

within the Chloroflexi.

Growing PCB dechlorinating microorganisms remains difficult for several

reasons, including the low solubility of PCBs in aqueous growth medium, the

slow growth rates of organohalide respiring bacteria on PCBs and the requirement for anoxic, low redox growth conditions. This impeded research on anaerobic dechlorination of PCBs for many years, but the advent of molecular tools

to detect microbes without cultivation in pure culture provided the first indication that the anaerobic microorganisms responsible for PCB dechlorination

belonged to the Chloroflexi, which included Dehalococcoides mccartyi (formerly

Dehalococcoides ethenogenes), known at that time to dechlorinate chlorinated

ethenes. This initial discovery was accomplished by sequential transfer of estuarine sediment in minimal medium with short chain fatty acids as electron donors

and a single PCB congener as an electron acceptor (Pulliam Holoman et al.

1998). Cloning and sequencing of 16S rRNA genes were then used to identify

the predominant microorganisms within highly enriched sediment microcosms.

Phylotype o-17 was identified as the microbe responsible for the ortho  dechlorination of 2,3,5,6-tetrachlorobiphenyl to 3,5-dichlorobiphenyl through a combination of restriction and comparative sequence analyses of PCR-amplified 16S rRNA

genes (Cutter et al. 1998, 2001). Despite identification of a PCB dechlorinator

within the enriched microbial community, isolation continued to elude investigators due to the inability to grow the PCB dechlorinator in the absence of sediment.

At the time, reductive dechlorination of PCBs was only observed in enrichment

cultures that contained sediment, which also provided nutrients that supported the

growth of non-PCB dechlorinating microorganisms. Cutter et al. (1998) finally

developed a sediment-free culture of strain o-17 by incrementally diluting out

sediment with sequential transfer in sediment-free minimal medium that contained

acetate and 2,3,5,6-tetrachlorobiphenyl. An initial lag of 100 days in sedimentfree medium was decreased to less than 50 days and dechlorinating activity was

maintained throughout subsequent transfers. Ultimately, a coculture was obtained



24 “Dehalobium chlorocoercia” DF-1—from Discovery to Application



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containing the 2,3,5,6-tetrachlrobiphenyl-respiring strain o-17 and a non-dechlorinating Desulfovibrio sp. Similarly, sequential dilutions in sediment-free minimal medium with formate and 2,3,4,5-tetrachlorobiphenyl were used to identify

and isolate “D. chlorocoercia” strain DF-1 as a dechlorinator of PCBs with doubly flanked chlorines (Wu et al. 2002b; May et al. 2008a, b). A suite of molecular

approaches was subsequently used to identify other PCB dechlorinating bacteria

(Fagervold et al. 2005, 2007) with broader dechlorinating capabilities. The subsequent discovery that pure cultures of D. mccartyi dechlorinated PCBs expanded

the diversity of PCB organohalide respiring bacteria even further (Fennell et al.

2004; Zhen et al. 2014; LaRoe et al. 2014; Wang et al. 2014; Adrian et al. 2009).

Combined, these investigations indicated that a broad range of extensively chlorinated PCB congeners could be anaerobically dechlorinated, including Aroclor mixtures, by organohalide respiring microorganisms within the Chloroflexi. Many of

the lesser-chlorinated congeners that remain following the transformative action of

organohalide respiring anaerobes have the potential to be subsequently degraded

via ring cleavage and mineralization by aerobic PCB degrading bacteria (Bedard

2003).



24.2 Isolation and Characterization of “Dehalobium

chlorocoercia” Strain DF-1

“D. chlorocoercia” strain DF-1 was initially enriched in microcosms containing

sediment from the Ashley River, a tributary of Charleston Harbor, SC, and minimal medium with fumarate as electron donor and 2,3,4,5-tetrachlorobiphenyl as

the sole electron acceptor (Wu et al. 2000). Initial attempts to isolate strain DF-1

on solidified medium or in agar shake tubes were unsuccessful. Serial dilutions

in sediment-free minimal medium with formate and 2,3,4,5-tetrachlorobiphenyl as the sole electron donor and acceptor, respectively, yielded a coculture with

a Desulfovibrio sp. (Wu et al. 2002b). Strain DF-1 could only be maintained in

coculture with the Desulfovibrio sp. throughout subsequent serial transfers. “D.

chlorocoercia” strain DF-1 was finally isolated after serial dilution in minimal

medium with titanium (III) nitrilotriacetate (TiNTA) substituted for cysteine as

a chemical reductant, thereby minimizing partially oxidized sulfur product that

could be used by the Desulfovibrio sp. as an electron acceptor (May et al. 2008a,

b). However, growth and activity by strain DF-1 was no longer detected after three

sequential serial dilution series and could only be restored and maintained by

reinoculating cells or adding cell-free extracts from the Desulfovibrio sp. to the

medium. Growth of strain DF-1 could also be maintained with cells or cell-free

extracts from Desulfovibrio vulgaris but nutrients such as yeast extract, peptone,

B-vitamins including cyanocobalamin, which is reported to stimulate growth of

D. mccartyi strains (Löffler et al. 2013), could not be substituted. The factor in

Desulfovibrio spp. required for maintaining growth of “D. chlorocoercia” strain

DF-1 is water-soluble and heat-stable, but the structure is unknown at this time.



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H.D. May and K.R. Sowers



Fig. 24.1  TEM micrographs of negatively stained “D. chlorocoercia” strain DF-1.

Bar = 0.1 µm. Right panel reprinted with permission from (May et al. 2008). Copyright 2008

American Society for Microbiology



“D. chlorocoercia” strain DF-1 is closely related to D. mccartyi within the

organohalide respiring Chloroflexi. Strain DF-1 is an ultramicrobacterium with

cells 75–339 nm in diameter when grown with 2,3,4,5-tetrachlorobiphenyl and

often observed in clusters (Fig. 24.1). The small cell size may reflect the slow

growth rates, but it would also maximize the membrane surface area-to-volume

ratio, which would be an advantage for uptake of hydrophobic compounds such as

a PCBs. Electron micrographs of the organism also reveal a possible matrix structure surrounding the cells, which if hydrophobic, would be consistent with the tendency of the organisms to clump or cluster. “D. chlorocoercia” strain DF-1 was the

first organohalide respiring bacterium shown to link its growth to reductive dechlorination of PCBs in pure culture (May et al. 2008a, b). This strain requires hydrogen or formate as an electron donor and select organohalides as electron acceptors

(May et al. 2008a, b). Growth and organohalide respiration by strain DF-1 is

restricted to the dechlorination of PCB congeners with doubly flanked meta and

para chlorines, but the microorganism is also capable of dechlorinating chlorobenzenes with doubly flanked chlorines (Wu et al. 2002a) and PCE and TCE to a mixture of cis- and trans-1,2-dichloroethene in a ratio of 1.2-1.7 (Miller et al. 2005).

More recently, D. mccartyi strain CBDB1 was also reported to dechlorinate TCE

to a mixture of cis- and trans-1,2-dichloroethene in a ratio of 0.3 (Marco-Urrea

et al. 2011). Prior to these reports high amounts of trans-dichloroethene had only

been observed in sediment enrichments and environmental samples. This observation suggests that organohalide respiring microorganisms similar to strain DF-1

and D. mccartyi strain CBDB1 are a potential source of trans-DCE, which is often

detected in the environment. The optimal temperature range for growth is 30–33 °C

with no growth or dechlorination observed at 10 or 35 °C. The pH optimum is 6.8

with growth observed in the range of 6.5–8.0. “D. chlorocoercia” strain DF-1 is



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