Anaerobic microbial reductive dechlorination of tetrachloroethene to predominately trans-1,2-dichloroethene.
ABSTRACT While most sites and all characterized PCE and TCE dechlorinating anaerobic bacteria produce cis-DCE as the major DCE isomer, significant amounts of trans-DCE are found in the environment. We have obtained microcosms from some sites and enrichment cultures that produce more trans-DCE than cis-DCE. These cultures reductively dechlorinated PCE and TCE to trans-DCE and cis-DCE simultaneously and in a ratio of 3(+/-0.5):1 that was stable through serial transfers with a variety of electron donors and occurred in both methanogenic and nonmethanogenic enrichments. Two sediment-free, nonmethanogenic enrichment cultures produced trans-DCE at rates of up to 2.5 micromol L(-1) day(-1). Dehalococcoides populations were detected in both trans-DCE producing cultures by their 16S rRNA gene sequences, and trans-DCE was produced in the presence of ampicillin. Because trans-DCE can be the major product from PCE and TCE microbial dechlorination, high fractions of trans-DCE at chloroethene-contaminated sites are not necessarily from source contamination.
Anaerobic M icrobial Reductive
B E N J A M I N M . G R I F F I N ,†
J A M E S M . T I E D J E ,†A N D
F R A N KE . L O ¨ F F L E R *, ‡
Center for Microbial Ecology, Department of Microbiology and
Molecular Genetics, and Institute for Environmental
Toxicology, Michigan State University,
East Lansing, Michigan 48824-1325, and School of Civil and
Environmental Engineering and School of Biology,
Georgia Institute of Technology, Atlanta, Georgia 30332-0512
While most sites and all characterized PCE and TCE
dechlorinating anaerobic bacteria produce cis-DCE as the
major DCE isomer, significant amounts of trans-DCE are
found in the environment. We have obtained microcosms
fromsome sites and enrichment cultures that produce
more trans-DCE than cis-DCE. These cultures reductively
dechlorinated PCE and TCE to trans-DCE and cis-DCE
simultaneously and in a ratio of 3((0.5):1 that was stable
throughserial transfers witha variety of electrondonors
and occurred in both methanogenic and nonmethanogenic
enrichments. Two sediment-free, nonmethanogenic
enrichment cultures produced trans-DCE at rates of up to
2.5 µmol L-1day-1. Dehalococcoides populations were
detected in both trans-DCE producing cultures by their 16S
rRNA gene sequences, and trans-DCE was produced in
the presence of ampicillin. Because trans-DCE can be the
majorproduct fromPCE andTCE microbial dechlorination,
high fractions of trans-DCE at chloroethene-contaminated
sites are not necessarily fromsource contamination.
Chlorinated ethenes are significant groundwater contami-
and TCE) and daughter products (DCEs and VC). Of the
current or former U.S. Environmental Protection Agency
National Priority List Sites, the Agency for Toxic Substances
and Disease Registry reports tetrachloroethene (perchloro-
ethylene, PCE) at 54% of the sites; trichloroethene (TCE) at
60%; 1,1-dichloroethene (1,1-DCE) at 36%; trans-1,2-dichlo-
roethene (trans-DCE) at 39%; cis-1,2-dichloroethene (cis-
DCE) at 10%; and vinyl chloride (VC) at 37% (1). Under
dechlorination (hydrogenolysis) resulting in the stepwise
conversion of PCE to TCE, DCE isomers, VC, and ethene.
that use PCE or TCE as respiratory electron acceptors for
growth through a process termed (de)chlororespiration
(2-5). These isolates belong to several genera including
(10-13), Dehalococcoides (4, 14), Clostridium (15), Entero-
convert PCE to TCE and a few Dehalococcoidesisolates that
dechlorinate PCE or TCE to VC and ethene (4, 14, 19, 20), all
otherisolatesdechlorinatePCE topredominatelycis-DCE as
been characterized from several organisms including Sul-
PCE-S (22), Desulfitobacterium sp. strain Y51 (23), Dehalo-
coccoidesethenogenes(24), and Dehalobacter restrictus(25).
The reductively dechlorinating enzyme systems contain
corrinoid cofactors in addition to iron-sulfur clusters, and
in Dehalobacter restrictus, dechlorination is suggested to
occur through a radical mechanism (25). A recent compu-
tational study examining the stability and transformation
rates of radical intermediates explains the favored cis-DCE
production by B12catalyzed reactions (26).
Interestingly, trans-DCE is often found at chloroethene-
contaminated sites (1), and its presence has been explained
by source contamination or generation through abiotic
mechanisms acting on polychlorinated ethenes. No biotic
mechanisms that produce predominantly trans-DCE from
PCE or TCE are known. A comprehensive understanding of
themechanismsthat contributeto trans-DCE occurrenceis
relevant for natural attenuation monitoring, point-source
tracking, and the choice of the most promising remediation
cultures that produce trans-DCE and cis-DCE in a ratio of
3:1 from PCE and TCE.
M aterials andM ethods
Chemicals. The following analytical grade chlorinated com-
pounds were used in this study: PCE, TCE, trans-DCE, cis-
DCE, all of which were obtained from Supelco (Bellefonte,
(Ronkonkoma, NY). Other chemicals used in thestudy were
obtained from Sigma-Aldrich (Milwaukee, WI).
Inoculum Sources,Microcosm Preparation,andGrowth
Conditions. Microcosms that produced a mixture of trans-
DCE and cis-DCE were derived from sediment and soil
materials collected from the Tahquamenon River and the
Hue ´ in Vietnam, and a swampy area in Chitwan National
Park, Nepal, as described (27, 28). In addition, PCE dechlo-
rinating mircososms were established from a soil and
slurry was maintained under nitrate reducing conditions to
reduce readily bioavailable electron donors by 10 repeated
additions of 0.5 mM nitrate. Following complete removal of
nitrate and nitrite, approximately 10 g of slurry was added
to 90 mL of basal salts medium reduced with cysteine and
sulfide (0.2 mM each) (3) in 160 mL serum bottles. All
microcosms were amended with lactate (5 mM) as a source
from each sitewereautoclaved for60min at 121°C on three
consecutive days and served as sterile controls.
Chloroethenes were analyzed in headspace samples
(by gas chromatography) and electron donor consumption
(by high performance liquid chromatography) as described
previously, and substrates were replenished as needed
*Correspondingauthorphone: (404)894-0279;fax: (404)894-8266;
†Michigan State University.
‡Georgia Institute of Technology.
Environ. Sci. Technol. 2004, 38, 4300-4303
(27-30). Transfers were 1% (vol/vol) into fresh basal salts
medium. Initially, PCE was added as electron acceptor but
replaced with TCE following the observation that PCE was
dechlorinatedtoTCE beforeanytrans- andcis-DCE formation
occurred. Culture bottles were incubated in an inverted,
stationary position at 22-25 °C unless otherwise noted.
the dechlorinating populations, cultures were pasteurized
immediately following inoculation. The temperature was
measuredinparallelin100mL ofmediumina160mL serum
Timing began at 77 °C, and the cultures were incubated at
a temperature not exceeding 80 °C for 10 min. The bottles
were then cooled in an ice water bath to room temperature,
before vitamins and chloroethenes were amended.
of electron donor addition on trans-DCE and cis-DCE
production. TCE (20 µmol) and 200 µmol of hydrogen plus
1 mM acetate, or 5 mM of formate, acetate, succinate,
medium. To examinetheeffect oftemperatureon reductive
theeffect of cell wall synthesis inhibitors on dechlorination,
ampicillin was added from a sterile anoxic aqueous stock
solution to a final concentration of 400 mg/L to freshly
inoculated TCE and lactatefed cultures. Theeffect of pH on
dechlorination was determined in basal salts medium
amended with 10 mM TES (N-tris[hydroxymethyl]methyl-
2-aminoethane sulfonic acid) as an additional buffer and
adjusted to a pH of 6.8, 7.5, or 8.2.
Nucleic Acid-Based Characterization of trans-DCE Pro-
ducing Cultures. DNA was extracted from the lactate-
amended Tahquamenon and Perfume River cultures after
they had consumed the initial dose of TCE (20 µmol). The
culture fluids (100 mL) were forced through sterile 0.22 µm
polycarbonate membrane filters. The membranes with the
cake of biomass were cut with sterile razor blades to fit into
the tubes provided in the MoBio UltraClean Soil Kit (Solana
To screen for the presence of known chloroethene-
dechlorinating populations, PCR amplification was per-
formed with specific primers targeting the 16S rRNA genes
ofthefollowing genera: Dehalobacter (31), Desulfuromonas
(32), Desulfitobacterium (2 sets (33)), and Dehalococcoides
(primer set 3 described in ref 34). PCR reactions were 20 or
50µL (totalvolume),andamplification wasperformedusing
the conditions described in each of the previous references.
Genomic DNA from pureculturesofDehalobacterrestrictus,
Desulfuromonasmichiganensis, Desulfitobacterium hafniense,
and a Dehalococcoides containing mixed culture were used
as positive controls for PCR amplification. Whenever am-
plification with the specific primers occurred, the PCR
productswerecloned in Escherichia coli using theTOPO TA
cloning kit (Invitrogen, Carlsbad, CA). Inserts from at least
three positive clones from each culture were sequenced by
Michigan State University’s Genomic Technology Support
Microcosms that reduced PCE to primarily trans-DCE as
dechlorination end product were obtained with sediment
and soil materials collected from geographically diverse
before complete conversion to trans-DCE and cis-DCE
occurred in a consistent ratio of 3((0.5):1 (Table 1). No
dechlorination and production of DCEs occurred in killed
sediment microcosms, sediment-free cultures, and non-
methanogenic cultures transferred over 25 times. The 3:1
ratioofDCE isomerswasstablethrough all transfers, andno
further reduction of DCEs to VC was observed even upon
Physiological Studies. trans-DCE formation wasstudied
in more detail in cultures from the Tahquamenon and
Perfume Rivers because these cultures were obtained first,
activity after more than 30 and 25 transfers, respectively.
Time courses for the dechlorination of TCE showed the
simultaneousproduction ofbothtrans- andcis-DCE (Figure
1). The formation of 1,1-DCE was never observed. The lag
time before the onset of dechlorination varied from culture
to culture but was always greater than 7 days and could be
up to several weeks. Lactate-fed cultures reduced TCE at
was constant over the period of active dechlorination, and
in the culture.
The effects of a variety of electron donors on the
production oftrans-DCE wereexploredin moredetail in the
Tahquamenon culture. The trans- to cis-DCE ratio (mean
ratio, (SD, n measurements) with different electron donors
wasasfollows: hydrogen plusacetate(3.33,0.05,4),formate
(3.14, 0.21, 4), acetate (3.21, -, 1), succinate (2.93, 0.06, 3),
TABLE1. Sum m ary of M icrocosm s andEnrichm ent Cultures that ProducedM ixtures of trans-DCEandcis-DCE
sediment-free methane productiontrans/cis DCEb
Tahquamenon River, MI
Perfume River, Vietnam
Red Cedar River, MI
Pine River, MI
Chitwan National Park, Nepal
mixed MI soil and sediment inocula
aTransfers were 1% (vol/vol) into fresh minimal medium.bRatio is the average of at least five measurements (SD <0.5), except for the Chitwan
sample, which is from a single time point and thus only has one significant figure in the ratio.c1,2-Dichloropropane.
River enrichment culture. Lactate (5 mM) was added as electron
donor, and25µmol ofTCE was addedtoa 100mLculture. Despite
complete reduction of TCE upon extended incubation, no vinyl
chloride, ethene, ethane, or methane was detected.
pyruvate (2.92, 0.41, 2), lactate (3.16, 0.31, 5), and glycerol
(3.25, 0.28, 3). No dechlorination occurred with propionate
or benzoate as electron donors. The average trans- to cis-
DCE ratio was 3.13, and trans-DCE was the dominant
dechlorination end product under all electron donor condi-
No dechlorination wasobserved in theTahquamenon or
of incubation. Pasteurized inocula from stationary cultures
wereno longer ableto dechlorinatewith all electron donors
tested. All cultures produced trans-DCE around pH 7.5, but
the Tahquamenon River culture did not dechlorinate at pH
8.2, and only one replicate exhibited dechlorination activity
at pH 6.8. The ratios of trans- to cis-DCE isomers produced
from TCE by the Perfume River culture were 2.68 (SD 0.05)
at pH 6.8, 2.57 (SD 0.06) at pH 7.5, and 2.46 (SD 0.11) at pH
8.2. This slight, but significant (p ) 0.027, t-test), decreasing
trend in the trans- to cis-DCE ratio with increasing pH was
linear (R2) 0.9994) in the pH range tested. Dechlorination
of TCE to trans-DCE and cis-DCE occurred in lactate-fed
Tahquamenon and Perfume River cultures treated with
ampicillin indicating that the organisms involved in the
Nucleic Acid-Based Characterization of trans-DCE Pro-
ducing Cultures. Genomic DNA from the Tahquamenon
River and Perfume River enrichment cultures was screened
from primers targeting the Desulfuromonas or Desulfito-
bacterium groups were not detected. In both cultures,
the Dehalococcoides-targeted primers pairs. The sequences
from the 1377 base pair PCR products from both cultures
were 99% identical to Dehalocococcoides sp. strain FL2 and
Dehalococcoidessp.strain CBDB1, which both belong to the
Pinellas subgroup of the Dehalococcoides cluster (33). Ad-
ditionally, Dehalobacter targeted primers produced a band
of the expected size from the Perfume River culture DNA.
The sequence from the 800 base pair amplicon was 98%
identical to the 16S rRNA gene sequence of Dehalobacter
that are proving amenable to remediation by microbial
reductive dechlorination (35, 36). Problems arise, however,
iftoxic and persistent intermediatessuch as1,1-DCE, trans-
DCE, cis-DCE, or VC accumulate. Chloroethene dechlori-
nation research hasfocused on cis-DCE and VC degradation
because these compounds are detected at many PCE/ TCE
contaminated sites and are frequently produced as dechlo-
produce the cis-DCE isomer as the major intermediate or
were detected at some contaminated sites (1). An example
spill consisted of TCE, but trans-DCE constitutes the major
DCE contaminant detected today (37). Apparent accumula-
tion of one DCE isomer could be due to its preferential
production or to isomer-specific degradation. Dehalococ-
coidespopulations that only metabolically dechlorinate the
that trans-DCE also serves a growth-supporting electron
acceptor in chlororespiration (19, 20).
We evaluated PCE and TCE dechlorination endpoints in
microcosms established with soil, aquifer, and sediment
materials collected from numerous sites (refs 27, 29, 32, 36,
DCE asthemajor dechlorination end product wasobserved
in microcosms from diverse source materials on several
occasions (Table 1). These observations are consistent with
the more infrequent accumulation of trans-DCE at PCE/
Ourfindingsdemonstrated that trans-DCE accumulation in
these cultures is in fact due to a microbial process favoring
trans-isomer over cis-isomer formation and not because of
the preferential consumption of the cis-isomer, as no DCE
TheratioofDCE isomerswasremarkably stable, with the
trans-DCE and cis-DCE ratio remaining 3:1 during serial
transfers and with different electron donors. Only a slight
decrease in the ratio was observed with increasing medium
pH in theculturestudied.Theratioof3:1transtocisisomers
is interesting because there is no precedence in microbial
systemsfor thisproduct distribution, and it isnot explained
by current understanding of dehalogenase catalyzed reduc-
tion. Theformation oftrans-DCE and theconsistency ofthe
ratio produced suggest a yet unknown underlying biochemi-
cal mechanism. Abiotic Zn(0) catalyzed hydrogenolysis of
TCE produces trans-DCE and cis-DCE in a 2.5:1 ratio (38).
There was no significant source of zinc or other zerovalent
metals in the medium used in the biological systems
described in this study, and formation of dechlorination
not seeded with an inoculum.
The production of trans-DCE in a 3:1 ratio with cis-DCE
as described in this study is distinct from the accumulation
of trans-DCE by Dehalococcoides ethenogenes strain 195.
than trans-DCE and subsequently consumed cis-DCE at a
faster rate (39). trans-DCE only accumulated to a low
percentage of the total chloroethene mass. In all cultures
investigatedin thisstudy,trans- andcis-DCE wereproduced
simultaneously and in a constant 3:1 ratio with no further
reduction to VC. Hence, in addition to possible source
contamination with trans-DCE, there are at least two
biological TCE reduction mechanisms that may lead to the
accumulation of trans-DCE in PCE/TCE contaminated
Dehalococcoides populations may be involved in trans-
DCE production in the Tahquamenon and Perfume River
transferred twice in the absence of TCE lost the ability to
dechlorinate, and Dehalococcoides populations were no
longer detectable. This observation is consistent with the
known physiology of Dehalococcoides populations, which
require a chlorinated compound as growth-supporting
lag periods before the onset of dechlorination were long,
ranging from 1 to several weeks, a phenomenon that has
also been observed in Dehalococcoides cultures that grew
with VC as terminal electron acceptor (19, 20). Further, the
involvement of Dehalococcoides populations is supported
by the fact that dechlorination of TCE to trans- and cis-DCE
also occurred in the presence of ampicillin. Resistance to
cell wall biosynthesis inhibitors such as ampicillin is a
characteristic trait oftheknown Dehalococcoidesisolates(4,
14, 20, 40).
Both the Tahquamenon and Perfume River cultures
contained 16S rRNA gene sequences most closely related to
Dehalococcoides populations of the Pinellas group. The
known and characterized Dehalococcoides isolates of the
Pinellas group are closely related by 16S rRNA sequence
identity but exhibit distinct physiologies (e.g., the range of
chloroorganic compounds used as growth-supporting elec-
tron acceptors (4, 20, 41)). None of the known isolates,
however, producetrans-DCE asa major intermediatein the
dechlorination of PCE and TCE, and if Dehalococcoides
populationsareindeed involved, ourfindingswould further
populations to act on chlorinated ethenes.
DCE formation by the mixed cultures at various stages of
enrichment cannot be completely ruled out. Pure cultures
of the dechlorinating populations are needed to definitively
DCE can be the major end product from PCE and TCE
at a site should not automatically be attributed to source
This work was supported by an EPA STAR fellowship to
B.M.G., by a National Science Foundation CAREER award
(0090496) to F.E.L., and by the Strategic Environmental
Research and Development Program (contract DACA72-00-
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Received for review December 21, 2003. Revised manuscript
received May 6, 2004. Accepted May 20, 2004.