Bioremediation Journal, 10:153–168, 2006Copyright ©c 2006 Taylor and Francis Group, LLCISSN: 1040-8371DOI: 10.1080/10889860601021399

Characterization of a Microbial ConsortiumCapable of Rapid and Simultaneous

Dechlorination of 1,1,2,2-Tetrachloroethaneand Chlorinated Ethane and Ethene

IntermediatesElizabeth J. P. Jonesand Mary A. VoytekU.S. Geological Survey, Reston,Virginia, USA

Michelle M. LorahU.S. Geological Survey,Baltimore, Maryland, USA

Julie D. KirshteinU.S. Geological Survey, Reston,Virginia, USA

ABSTRACT Mixed cultures capable of dechlorinating chlorinated ethanesand ethenes were enriched from contaminated wetland sediment at AberdeenProving Ground (APG) Maryland. The “West Branch Consortium” (WBC-2)was capable of degrading 1,1,2,2-tetrachloroethane (TeCA), trichloroethene(TCE), cis and trans 1,2-dichloroethene (DCE), 1,1,2-trichloroethane (TCA),1,2-dichloroethane, and vinyl chloride to nonchlorinated end products etheneand ethane. WBC-2 dechlorinated TeCA, TCA, and cisDCE rapidly and simul-taneously. A Clostridium sp. phylogenetically closely related to an unculturedmember of a TCE-degrading consortium was numerically dominant in theWBC-2 clone library after 11 months of enrichment in culture. Clostridiales,including Acetobacteria, comprised 65% of the bacterial clones in WBC-2,with Bacteroides (14%), and epsilon Proteobacteria (14%) also numerically im-portant. Methanogens identified in the consortium were members of the classMethanomicrobia, which includes acetoclastic methanogens. Dehalococcoidesdid not become dominant in the culture, although it was present at about 1% inthe microbial population. The WBC-2 consortium provides opportunities forthe in situ bioremediation of sites contaminated with mixtures of chlorinatedethenes and ethanes.

KEYWORDS anaerobic dechlorination, bioaugmentation of chlorinated ethanes, biore-mediation, microbial consortium, mixed chlorinated ethenes and ethanes, 1,1,2,2-tetrachloroethane

INTRODUCTIONBioaugmentation, site inoculation with a microbial mixed culture, is a proven

approach for stimulating complete dechlorination of sites contaminated withchlorinated ethenes (e.g., Major et al., 2002; Lendvay et al., 2003). However,cultures have not previously been available for the large-scale treatment ofchlorinated ethane contamination. Of additional concern, chlorinated ethanes

Address correspondence to ElizabethJ. P. Jones, U.S. Geological Survey, 430National Center, Reston, VA 20192,USA. E-mail: ejjones@usgs.gov

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can inhibit the degradation of chlorinated ethenes(Duhamel et al., 2002; Aulenta et al., 2005), and cul-tures are needed for remediation of sites with mixturesof these contaminants. Contamination of groundwa-ter with chlorinated ethenes and ethanes is a seriousproblem due to widespread and historic commercial,industrial, and military use, relative resistance to degra-dation, and associated health hazards (Haggblom andBlossert, 2003). Under anaerobic conditions, chlori-nated ethenes and ethanes can be partially reduced toless chlorinated compounds or completely degraded tononchlorinated end products depending on the phys-iological capability of the indigenous microbial com-munity (Ellis et al., 2000). Based on estimates of nonat-mospheric release in the U.S. during the period 1987 to1991 (http://www.epa.gov/safewater/index.html; Chenet al., 1996), chlorinated ethanes comprise about 75%of the more than one million pounds of chlori-nated ethenes and ethanes annually released to theenvironment. Yet relative to what has been learnedregarding chlorinated-ethene degradation, the studyof microorganisms that catalyze chlorinated ethanedegradation is in its infancy. Isolates capable of reducing1,2-dichloroethane (DCA) and 1,1,1-trichloroethanehave been identified (Maymo-Gatell et al., 1999; DeWildeman et al., 2003; Sun et al., 2002), and one iso-late has been shown to reduce 1,1,2,2-tetrachloroethane(TeCA) to cis 1,2-dichloroethene (cisDCE) (Suyamaet al., 2001). Recent research by Grostern and Ed-wards (2006) on a mixed culture demonstratedgrowth of Dehalobacter sp. concomitant with thereduction of 1,1,2-trichloroethane (TCA) to vinylchloride (VC).

TeCA was developed as a solvent prior to WorldWar I, and large quantities are still used, primarily bythe chemical industry. In addition, historic disposalpractices and release of DNAPLs (dense non–aqueousphase liquids) have led to continuing groundwater con-tamination from subsurface sources. Natural attenu-ation of TeCA has been documented at AberdeenProving Ground (APG) Maryland, where contaminatedgroundwater discharges through anoxic wetland sedi-ments at West Branch Canal Creek (Lorah and Olsen,1999a, 1999b). This sediment provided source ma-terial for developing a dechlorinating culture. TheTeCA degradation pathway (Figure 1) is primarily bi-otic, and includes both hydrogenolysis to less chlori-nated ethanes and dichloroelimination to less chlori-nated ethenes. Abiotic production of trichloroethene

FIGURE 1 Possible pathways of anaerobic 1,1,2,2-tetrachlo-

roethane (TeCA) dechlorination. Compounds for which EPA re-

ports an increased risk of cancer are indicated (*). Pathways are

microbially catalyzed, with the exception of TeCA dehydrohalo-

genation to TCE. Modified from Lorah et al. (1999a).

(TCE) from dehydrochlorination of TeCA generallyaccounts for less than 2% of TeCA removal at APG(Lorah and Olsen, 1999a), and chloroethane (CA),ethene and ethane have not been observed in the sed-iment. Several intermediates of TeCA dechlorinationare possible carcinogens and are listed as contami-nants of concern by the U.S. Environmental Protec-tion Agency. VC is a known human carcinogen that of-ten accumulates at sites where dechlorination is slow orincomplete.

The purpose of the work described here was to de-velop a culture of microorganisms for bioaugmentationtreatment of chlorinated-ethane contaminated ground-water at sites where dechlorination is incomplete orrates are too slow for effective remediation. In this pa-per, we describe a microbial consortium, West BranchConsortium (WBC-2), derived from organic-rich sedi-ments collected in the wetland of West Branch CanalCreek (MD) at APG. We demonstrate the capabilityof WBC-2 to dechlorinate chlorinated ethanes andethenes in culture, both individually and concurrently,and we present a preliminary analysis of its microbialcomposition.

E. J. P. Jones et al. 154

MATERIALS AND METHODSDevelopment of the Microbial

ConsortiumSediment was collected from two sites within the wet-

land at West Branch Canal Creek (WB23 and WB30),and prepared anaerobically using the same methodsas for microcosms in previous studies (Lorah andOlsen, 1999a; Lorah et al., 2003b; Lorah and Voytek,2004). APG sediments were collected in March 2003,sieved, slurried with groundwater (1:1.5), and incubated(19◦C) with TeCA (7 μM) in 1-L serum bottles with-out headspace for 1 month. Most of the Fe(III) andsulfate (alternative electron acceptors) were depletedduring this incubation period and methane was be-ing produced. Aliquots (100 ml) of sediment slurrieswere then transferred to 120-ml serum bottles with aN2/CO2 (95:5) headspace and amended with a daugh-ter compound, cisDCE or TCA, for 1 to 2 months. Inall sediment slurry enrichments, the electron donorswere derived only from organic matter in the sedi-ment. Sediment slurries (100 ml each of TCA-enrichedWB23 and cisDCE-enriched WB30) were then trans-ferred into anaerobic culture medium (1800 ml, seecomposition below) with sulfide (50 μM) added asa reductant, and amended with target concentrationsof TeCA (30 μM) or a mixture of TeCA (25 μM),TCA (50 μM), and cisDCE (50 μM). The electrondonor for cultivation was selected in tests on the TeCAamended culture (see below) 3 weeks after inocula-tion from sediment slurry. Cultures were diluted overa 2-year period and contain about 0.1% sediment byvolume.

Evaluation of Electron DonorsAll electron donor tests were performed in duplicate

on sub samples removed from TeCA-depleted stock cul-ture. Because concentrations of intermediates often arelow or undetectable during TeCA degradation by WBC-2, TCA and cisDCE were used as test compounds toensure that the electron donor selected would supportboth chlorinated ethane and chlorinated ethene path-ways. In addition, the ability of each electron donor tosupport the dechlorination of VC, a key compound forcomplete dechlorination of both chlorinated-ethenesand -ethanes, was evaluated. Aliquots of WBC-2 cul-ture (10 ml) were transferred anaerobically to 28-mlpressure tubes (Bellco Glass, Vineland, NJ) filled with

N2/CO2 (80:20). WBC-2 was evaluated for dechlori-nation of test compounds in the following electrondonor treatments: propionate (10 mM); succinate (3mM); lactate (3 mM); pyruvate (3 mM); benzoate (3mM); formate (10 mM); acetate (10 mM); H2 (20 kPaoverpressure, added three times during the incubation)with or without acetate (1 mM) added as a carbonsource; whey (Sigma Chemical; from bovine milk, spraydried powder containing minimum 11% protein andapproximately 65% lactose) (5 g/L); no electron donoradded. The electron donors supplied electron equiva-lents (assuming complete oxidation) equal to about 100times that required for the reduction of the chlorinatedcompounds, cisDCE or TCA (Supelco, Bellefonte, PA),which were added from aqueous emulsions (for a finalconcentration of approximately 1 and 0.75 mM, respec-tively) and monitored for VC and DCA production.The ability to dechlorinate VC was tested by addingVC (4.2 μM) from a gaseous standard (Matheson,Twinsburg, OH) in a separate treatment. All treatmentswere incubated at 19◦C and monitored by sampling theheadspace for analysis with a gas chromatograph (GC)with a flame ionization detector (FID), as describedbelow.

Culture Medium and MaintenanceThe anaerobic medium included (g/L deionized wa-

ter): NaHCO3 (2.5), NH4Cl (0.5), NaPO4 (0.5), KCl(0.1), 10 ml vitamin solution (Balch et al., 1979), and10 ml trace mineral solution (below), with a gas phaseof N2 and CO2 (80:20). The trace mineral solution con-tained (g/L): Nitrilotriacetic acid (1.5), MgSO4·7H2O(3.0), MnSO4·H2O (0.5), NaCl (1.0), FeSO4·7H2O(0.1), CaCl2·2H2O (0.1), CoCl2·6H2O (0.1), ZnCl2(0.13), CuSO4·5H2O (0.01), AlK(SO4)2·12H2O (0.01),H3BO3 (0.01), Na2MoO4 (0.025), NiCl2·6H2O (0.024),Na2WO4·2H2O (0.025). Early in development, somebatches were starved for periods as long as severalmonths, but recovered activity when feeding was re-sumed. Once established, cultures were maintained ei-ther with lactate (1 mM) and TeCA (50 μM) addedfrom aqueous stocks once or twice weekly, or lactate (1.5mM) and TeCA (25 μM), TCA (50 μM), and cisDCE(50 μM). The ratio of electron equivalents for donors toacceptors was 30 and 17, respectively, for stocks main-tained with TeCA and the chlorinated mixture. Thechlorinated stocks were prepared by adding purifiedstandards (Supelco, Bellefonte, PA) to sterile anaerobic

155 Microbial Consortium for Biodegrading Chlorinated Ethanes

deionized water. Chlorinated stock bottles were vigor-ously shaken prior to each amendment in order to emul-sify any undissolved compound. The cisDCE containedapproximately 1% transDCE.

WBC-2 was maintained in 2-L batches of mediumfrom which culture volumes were removed for study.After 1 year, all cultures were restored to full 2 L vol-ume by adding fresh medium. Under contract by theU.S. Army, samples of WBC-2 were given to SiREMLaboratories (Guelph, Ontario, Canada) for propaga-tion of the culture to the large volumes required forfield bioremediation tests conducted by the U.S. Geo-logical Survey, using lactate and the chlorinated mixturedescribed above (GeoSyntec Consultants Inc., 2004).WBC-2 microbial composition (clone analysis, see be-low) was assessed in a culture sample obtained fromSiREM Laboratories that had been scaled up by trans-ferring two times into fresh medium.

Gas AnalysisCulture headspace was sampled using a gas tight sy-

ringe and injected into one or both of the followingGC-FID systems. For rapid analysis of TeCA, TCA,DCA, TCE, cisDCE, transDCE, and VC, we used aHewlett-Packard model 5890 series II with isothermalseparation at 100◦C on a VOCOL (Supelco, Bellefonte,PA) capillary column (30 m × 0.53 mm). For sepa-ration of methane, ethene, and ethane, and for anal-ysis of VC, transDCE, and cisDCE when interferingpeaks were present, we used a Shimadzu model GC-17A with separation on a Rt Q-Plot (Restek, Belle-fonte, PA) column (30 m × 0.32 mm) using a temper-ature program of 100◦C for 5 min, ramping to 200◦Cat 20◦C/min. Aqueous standards of chlorinated com-pounds were prepared from highly purified neat cali-bration standards (Supelco, Bellefonte, PA). Standardsof chlorinated compounds were prepared by adding10 μL of neat solution to 100 ml water, and prepar-ing aqueous dilutions for headspace analysis in bottlessealed with Teflon coated stoppers (West, Lionville, PA).Dimensionless Henry’s law constants (DHLCs), wereused to calculate expected headspace concentrationsfrom known liquid concentrations. Methane, ethene,and ethane standards were purchased as gas standards(Scott Specialty Gas). Concentrations of chlorinatedcompounds and non-chlorinated end products in sam-ples of WBC-2 culture headspace were converted todissolved values using DHLCs and total concentra-

tions (per volume medium) were calculated. DHLCshave been measured empirically by many researchers,and vary widely. The chosen DHLCs fall in the mid-range of published values. Nonetheless, the DHLCs arethe greatest source of possible error in the concentra-tions reported here, and may exceed 10%. Errors be-tween repeat injections are about 2%. The dimension-less Henry’s law constants (DHLC) applied were 0.019for TeCA, 0.556 for TCA, 0.1821 for DCA, 0.3056 forTCE, 0.1255 for cisDCE, 0.3056 for transDCE, 0.9087for VC (Gossett, 1987), 7.96 for ethene, 19.88 for ethane,and 28.5 for methane (Lampron et al., 1998). All detec-tion limits were less than 0.01 μM.

Cloning, RFLP Screeningand Sequencing, TRFLP

DNA was extracted using the Bio-101 Fast DNASpin Kit for Soil (MP Biomedicals, Irvine, CA) fol-lowing manufacturers instructions, except that prod-uct recovery was maximized at each step. Bacterial andmethanogen DNA were amplified using the polymerasechain reaction (PCR) in a Perkin Elmer Geneamp 2400thermal cycler with 16S rDNA (46f and 519r) primers(Brunk et al., 1996; Lane, 1991) and methyl coenzyme-M reductase (mcrAf and mcrAr) primers (Luton et al.,2002), respectively. 16S rDNA PCR conditions (30 cy-cles) were denaturing at 94◦C (30 s), annealing at 56◦C(30 s), and extension at 72◦C (1 min). For mcr, the condi-tions of Luton et al. (2002) were used. Microbial mem-bers of the consortium were characterized by cloningand sequencing the bacterial 16S rDNA and mcrA am-plicons. Amplicons were purified using the Wizard PCRpurification kit (Promega, Madison, WI) and cloned us-ing the TA cloning kit or the Topo TA cloning kit for se-quencing according to manufacturer’s instructions (In-vitrogen, San Diego, CA). Colonies were picked and16S rRNA and mcrA gene clone fragments (133 and 48,respectively) were recovered using vector primers andmcrA primers, respectively, using PCR. For the bacte-rial 16S rDNA characterization, the PCR products werereamplified using 46f and 519r primers. All PCR am-plicons were digested with restriction enzymes (6 μl ofPCR product with 2.5 U each of MspI and HinPI) ac-cording to manufacturer’s instructions (Promega, Madi-son, WI). Restriction fragments were analyzed by sizeseparation on a 3.5% Metaphor (Cambrex, Rockland,ME) agarose gel, restriction fragment length polymor-phism (RFLP) patterns were distinguished, and the

E. J. P. Jones et al. 156

frequency with which each pattern occurred was de-termined. It should be noted that the frequency ofclones in the library may not correspond directly torelative phylotype numbers in the culture due to unde-fined differences in the number of 16S rDNA copiesper cell. In addition, PCR and nucleic extraction biasesmay contribute to apparent differences in the abun-dance of RFLP patterns. Representative clones for eachpattern were selected for sequencing. Amplicons to besequenced were purified with the Wizard PCR purifi-cation system, and cycle sequencing was performedon both strands using Big Dye v3.1 (Applied Biosys-tems, Foster City, CA) and run on a ABI310 geneticanalyzer. Sequences were edited and assembled us-ing Autoassembler (Applied Biosystems, Foster City,CA). Closest phylogenetic relatives were determinedby BLASTn search of the National Center of Bioin-formatics (NCBI) database (http://www.ncbi.nlm.nih.gov/).

Terminal restriction fragment length polymorphism(TRFLP)-PCR was performed as described above, butusing 46f primer with FAM label attached. A restric-tion digest of 6 μl of PCR product was performed us-ing 5U MnlI (New England Biolabs, Beverly, MA). Di-gested samples were precipitated with 0.1 volume of 3M sodium acetate and 2 volumes of cold 100% ethanoland resuspended in 10 μl sterile water. A 2.5-μl aliquotof the digested sample was added to 12 μl of deion-ized formamide and 0.5 μl ROX500 standard (AppliedBiosystems). Samples were denatured at 95◦C for 5 min.DNA fragments were separated using an ABI310 se-quencer (Applied Biosystem). Terminal restriction frag-ments were detected using 310Genescan analytical soft-ware, version 2.1.1, resulting in a TRFLP profile for eachsample.

Detection and Quantification ofSpecific Members by Quantitative

PCRPrimers were used to detect organisms with abun-

dances too low to be detected in the 16S clone li-brary, including two known dechlorinators, Dehalococ-coides spp., andDesulfuromonas spp., and methanogens.DNA copy number in an extract of WBC-2 DNAwas determined by quantitative PCR (qPCR) usingthe quantitect SYBR green real-time PCR kit (Qiagen,Chatsworth, CA.) and the Opticon real-time PCR sys-tem (MJ Research, now BioRad, Hercules, CA). The

16S rDNA based primers used to target Dehalococcoideswere dhc730f, 5′-GCG GTT TTC TAG GTT GTC-3′

and dhc1350r, 5′-CAC CTT GCT GAT ATG CGG-3′

(Bunge et al., 2001). The Desulfuromonas primers (de-signed for specificity to Desulfuromonas sp. strain BB1and D. chloroethenica 16S rDNA) and conditions are pre-viously described (Loffler et al., 2000). Methanogenswere quantified using mcrA primers (Luton et al., 2002).A standard curve was determined using Ct values of se-rial dilutions of plasmid containing the dhc or mcrA am-plified fragment, or the Desulfuromonas sp. strain BB1amplicon of known concentration (and thus copy num-ber), and the samples were plotted against that curveto determine abundance. Calculations of cell numberswere based on one 16S rDNA copy per cell for De-halococcoides (www.tigr.org),and 1 mcrA copy per cell formethanogens (Nunoura et al., 2006). For the purposesof calculating cell numbers, nucleic acid extractionswere assumed to be perfect, because no measurementof extraction efficiency is available. For microscopiccounts, culture samples were suspended in 0.01% Tri-ton X-100 and stained with 5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) in 1 × phosphate-buffered saline(PBS), filtered onto a black Nuclepore filter (0.2 μm),and viewed using epi-fluorescence (Porter and Feig,1980).

RESULTSElectron Donors Supporting WBC-2

DechlorinationThe electron donor for WBC-2 cultivation was se-

lected by comparing dechlorination in electron donortreatments (summarized in Table 1) with dechlorinationin a control with no added electron donor. Controlswith no added electron donor exhibited decreases inadded TCA and cisDCE characteristic of adsorption,with an initial decrease of 24% and 10%, respectively,followed by no further decrease. Less than 1% of theadded TCA or cisDCE in the controls was reduced toVC and no DCA was produced. H2 did not stimulatereduction of TCA or cisDCE above that observed inthe controls, whether or not acetate was added as acarbon source. The most complete dechlorination wasobtained in treatments with lactate and pyruvate. Thepathway of TCA reduction (production of VC versusDCA from TCA) varied among electron donor treat-ments. Cultures with lactate produced more DCA rel-ative to VC than did cultures with pyruvate. No DCA

157 Microbial Consortium for Biodegrading Chlorinated Ethanes

TABLE 1 WBC-2 Reduction of cDCE, TCA, and VC

Degradation relativeto no electron donor

Electron donor cDCE TCA VC

Succinate + +/− −Lactate ++ + + + + + +Pyruvate + ++ + + +Benzoate − + NDPropionate + − −Formate − − +Acetate − − −H2 − − + + +H2, with 1 mM acetate − − + + +Whey − − ND

+++, complete dechlorination (TCA [0.75 mM], DCE [1 mM] after 27days, or VC [4.2 μM] after 3 days).

++, dechlorination at least 50% complete.+, greater than no electron donor control.+/−, one duplicate greater than control.−, dechlorination not greater than no electron donor control.ND, treatment not done.

was produced in treatments with propionate, acetate,benzoate, or whey.

Dechlorination in CultureWBC-2 was cultivated in batches amended with ei-

ther lactate and TeCA or lactate and a mixture of TeCA,TCA, and cisDCE for 18 months. In the TeCA amendedculture, WBC-2 in a 2-L batch culture completelydechlorinated TeCA (measured at 240 μM) within 2days (Figure 2A). The pathway of TeCA degradationcould not be discerned by monitoring this culture be-cause very little intermediate accumulation occurred.Less than 0.5 μM (0.2% of the added TeCA) accu-mulated as VC, and this VC was degraded by day 2.The end products of dechlorination were ethene andethane. The stoichiometry of TeCA degraded by WBC-2 to nonchlorinated end products (average of valuesfrom three stock cultures) was 2 ± 1 moles TeCAto 1 mole [ethene + ethane]. In a 2-L batch culturemaintained with the chlorinated mixture, TeCA, TCA,and cisDCE, WBC-2 rapidly, simultaneously, and com-pletely reduced all three chlorinated compounds to thenonchlorinated end-products ethene and ethane (Fig-ure 2B). Small amounts of transDCE and VC were ob-served as transient intermediates.

After dechlorination was completed, the fate ofethene was monitored in the cultures in order to de-

termine if ethene reduction could account for the pro-duction of ethane. Ethene was degraded (6 μM day−1)with the production of ethane (Figure 3). After ethenewas depleted, the ethane concentration also decreased,at a rate of 0.5 μM day−1. In another set of treat-ments (data not shown), ethene (336 ± 13 μM) wasadded to the culture to achieve a higher starting etheneconcentration. This ethene was completely degradedafter 18 days with the production of ethane (188 ±40 μM). The lack of stoichiometric accumulation ofethane suggests that ethane and ethene degradation canco-occur.

Pathways of intermediate degradation were deter-mined by incubating subsamples of WBC-2 with TCAor cisDCE. WBC-2 cultures that had been maintainedwith (1) TeCA and (2) the chlorinated mixture (TeCA,TCA, cisDCE) were incubated to deplete chlorinatedcompounds and then compared with respect to theircapabilities to degrade TCA (Figure 4A) and cisDCE(Figure 4B). The two cultures were very similar intheir abilities to degrade the two TeCA intermediates.Degradation products of intermediate dechlorinationare shown for TeCA-maintained WBC-2 as a repre-sentative culture (Figure 5). As in treatments amendedwith TeCA, little intermediate accumulation was ob-served in TCA-amended treatments (Figure 5A). Therate of TCA degradation was 36 μM day−1. The peakVC and DCA concentrations measured were 1.7% and0.3% of the TCA added, respectively, and both inter-mediates were rapidly degraded. Chloroethane was notdetected. cisDCE reduction was accompanied by theproduction of VC (peak accumulation, 24% of addedcisDCE), ethene and ethane (Figure 5B). The rate ofcisDCE degradation in cultures amended with cisDCEwas 54 μM day−1.

When WBC-2 cultures were diluted (1:9) into freshmedium, dechlorination was initially slow enough to al-low the observation of intermediates (Figure 6A, B). Di-luted cultures amended with TeCA alone (84 μM) accu-mulated measurable TCA (0.01 μM) at one time point,and trans DCE (6 μM) and VC (0.25 μM) were also bothproduced and degraded. Diluted cultures amended withTeCA, TCA, and cisDCE had a transient accumulationof VC (6% of the added chlorinated compounds) andtransDCE (as much as 4% of added TeCA), and also theabiotic product, TCE (3% of the TeCA parent added).Within 2 to 4 weeks, dechlorination rates increasedto a level comparable to that observed in undilutedcultures, with parent compounds degraded in as little

E. J. P. Jones et al. 158

FIGURE 2 Dechlorination in WBC-2 stock cultures amended with lactate and (A) TeCA only, (B) TeCA, TCA and cisDCE. Control (C)

contained no WBC-2 culture.

as one day and little transient accumulation of interme-diates (Figure 6 A, B).

Microbial Composition of theDechlorinating Consortium

The bacterial community in the source sediments(APG sediments WB23 and WB30, Figure 7A and B)had shifted after a year under culture conditions withlactate as the sole electron donor and TeCA or a mixtureof TeCA, TCA, and cisDCE as the electron acceptors.Both WBC-2 cultures (cultured with TeCA [Figure 7

C] or TeCA, TCA, and cisDCE [Figure 7D]) exhib-ited TRFLP profiles that were different from that ofthe source sediments and overall represented a differ-ent community than was present in the starting ma-terials. This change reflects the selection pressures ex-erted on the community and individual members bythe chlorinated compounds, such that the remainingpeaks represent members tolerant of the chlorinatedcompounds and favored by the culturing conditionsand perhaps directly or indirectly involved in the degra-dation process. The numerically dominant phyloge-netic types in WBC-2 were identified by cloning and

159 Microbial Consortium for Biodegrading Chlorinated Ethanes

FIGURE 3 Degradation of ethene and production of ethane in

WBC-2 culture after depletion of chlorinated compounds. Errors

shown between duplicate treatments.

sequencing 16S rDNA and mcrA genes from a culturegrown with a mixture of TeCA, TCA, and cisDCE. Thefrequency of phylotype occurrence in 16S rDNA andmcrA clone libraries was determined (Figure 8A andB), and phylogenetic placement was determined using

FIGURE 4 TCA (A) and cisDCE (B) degradation by WBC-2 stock cultures grown with (1) TeCA only (solid lines) and (2) a mixture of

TeCA, TCA, and cisDCE (dashed lines), and (none) no culture added. Errors shown between duplicate treatments.

a BLAST search for related sequences. Although mostof the WBC-2 clones were not related to dechlorinat-ing bacteria that have been studied in isolation, manywere related to bacterial clones that have been observedat other dechlorinating sites (Table 2). The 16S rDNAlibrary was dominated by Clostridiales (65%), includ-ing three phylotypes. The phylotype representing thegreatest number of clones was a Clostridium sp., mostclosely related (99%) to an uncultured member of a TCEdechlorinating community (MacBeth et al., 2004). Thesecond most prevalent phylotype was an Acetobacteriumsp. most closely related (97%) to uncultured clonesfrom a 1,2-dichloropropane-dechlorinating enrichment(Ritalahti and Loffler, 2004), and 97% and 96% relatedto the homoacetogens Acetobacterium malicum and A.

wieringae, respectively. Less prevalent was a third phy-lotype, 95% related to an uncultured clone from aTCE-dechlorinating community and 93% related to De-halobacter restrictus, in the evaluated region between 46fand 519r. There was more variability among sequencesof the Bacteroidetes (CFB group), which accounted for14% bacterial clones. Many of these were related to un-cultured clones from dechlorinating populations (seeexamples, Table 2). One clone (delta Proteobacteria)

E. J. P. Jones et al. 160

FIGURE 5 Production and degradation of chlorinated intermediates of (A) TCA (ethene and ethane values are shown from one replicate

only), and (B) cisDCE in WBC-2 culture. Errors shown between duplicate treatments.

was most closely related (98%) to a Geobacter sp. from achlorinated ethene enrichment culture, and 98% relatedto G. lovleyi, a recently described PCE-dechlorinatingisolate (Sung et al., 2006). The gamma Proteobacteriawere 99% related to the top 60 BLAST hits, includingPseudomonas stutzeri and Ps. chloridismutans, that is ableto dechlorinate trichloroacetic acid (Wolterink et al.,2002). For the other phylotypes observed among thebacterial clones (Arcobacter sp. and Desulfobulbus sp.,14% and 2% of the clones, respectively) and all of themcrA clones, the BLAST database did not reveal relat-edness to organisms from dechlorinating populations.

No Dehalococcoides clones were identified. However,Dehalococcoides numbers determined independently us-ing qPCR and compared with the total number of cellsby microscopic count indicated that about 1% of thetotal consortium population was comprised of Dehalo-coccoides spp. Microscopic examination confirmed thatcocci were rare, and the consortium population wascomposed almost entirely of rod-shaped cells. Abso-lute numbers of Dehalococcoides (evaluated by qPCR) inWBC-2 culture samples (generally 105to 106cells perml) were higher than numbers measured in APG sedi-ment (103to 104 cells per ml), although the efficiencyof nucleic acid extraction and the total microbial num-bers are both likely lower for sediment than for culture.

Desulfuromonas spp. were not detected by qPCR usingthe primers specific for that dechlorinating type.

WBC-2 mcrA clone library (Figure 8B) was com-prised of members of the class Methanomicrobia, andincluded both acetate- and H2-utilizing methanogens(accession numbers DQ907209 to DQ907221). Mem-bers of the Methanosarcinaceae family are capable ofutilizing acetate in the production of methane (Sowers,1995). These include Methanosarcina spp., which mayutilize acetate, H2, methanol, or methyl amines, andMethanosaeta spp., which are obligate acetate utilizers.The other Methanomicrobia are related to methanogensthat utilize H2 and formate as electron donors. Thepresence of Methanosaeta spp. in the WBC-2 cultureindicates that acetate is being produced. Although thecultures were methanogenic, methanogens compriseda very small part of the total microbial populationof WBC-2. Total methanogens quantified usingqPCR comprised 0.2% of the total WBC-2 microbialpopulation.

DISCUSSIONWBC-2 TeCA Dechlorination: Pathway

and RatesIn order to develop an effective mixed culture for

bioremediation of TeCA contaminated sites, it is critical

161 Microbial Consortium for Biodegrading Chlorinated Ethanes

FIGURE 6 Dechlorination of added compounds and accumula-

tion of intermediates in cultures transferred (1:9) to fresh medium

and amended (at arrows) with lactate and either (A) TeCA only or

(B) TeCA, TCA, and cisDCE. Data shown are for one representative

of duplicate treatments.

to support the microorganisms catalyzing all branchesof the TeCA degradation pathway. Most of the inter-mediates (with the exception of chloroethane) shownin Figure 1 have been observed to accumulate in APGgroundwater during TeCA degradation (Lorah et al.,2003b). Therefore the remediation of TeCA contam-inated groundwater could require remediation of thechlorinated intermediates as well as mixtures of theparent with chlorinated ethene and ethane interme-diates or cocontaminants. WBC-2 was enriched fromAPG sediments in which intermediates accumulatedand were slowly degraded at rates of 0.1 to 0.6 μMday−1 for TeCA, TCA, and cisDCE (Lorah et al., 2003a,

2003b; Jones et al., 2004). After microbial enrichmentand 1 year in culture, the rate of WBC-2 dechlorinationincreased (rates measured for TeCA, TCA, and cisDCEremoval: 100, 36, and 54 μM day−1 respectively) suchthat almost no intermediates were detected in WBC-2 cultures amended with TeCA or a mixture of TeCA,TCA, and cisDCE. TeCA (as much as 240 μM) wasconverted to ethene and ethane within 2 days, a rate ofdechlorination greater than that observed for anothermixed culture reported to reduce TeCA (6 μM day−1;Aulenta et al., 2005). Ten-times dilutions of WBC-2 cul-ture in fresh medium resulted in the transient accumu-lation of small amounts of TCA and transDCE fromTeCA degradation, but even in the diluted culture, 87%was converted “directly” to ethene and ethane withoutobservation of intermediates. The rate of dechlorina-tion in the diluted cultures increased to the same rateas the parent culture in about 2 weeks, indicating mi-crobial growth. Although intermediates often did notaccumulate, the ability of WBC-2 to degrade all knownintermediates in the TeCA pathway was demonstratedin treatments with individual compounds (e.g., TCAand cisDCE) and in mixtures (e.g., TeCA, TCA, cisDCE,and cis/trans DCE, data not shown).

We used a simple method for monitoring dechlorina-tion, based on partitioning in the headspace. However,the stoichiometry of TeCA to its degradation products isdifficult to determine using this method. Both addingand measuring the initial concentration of TeCA in-volved potentially large errors. We could not rely ondilution of the stock solution to determine the amountof TeCA added, because TeCA in the stock solutionwas not completely dissolved and possibly not com-pletely homogenized by shaking. TeCA measurementswere subject to two known sources of error. First, whencalculating total concentrations from headspace values,errors are magnified for compounds with a very lowDHLC. The DHLC for TeCA (0.019) was an order ofmagnitude lower than any other compound measuredand therefore subject to the greatest error. Conversely,compounds such as methane, ethene and ethane arelargely partitioned to the headspace and subject to theleast error. Secondly, because there is some sediment aswell as cell mass in the cultures, distribution of TeCA be-tween the liquid and gas phases may be complicated byadsorption, resulting in a discontinuity between TeCAuptake and the appearance of end product. All of theseerrors may have contributed to differences between tar-get TeCA additions and measured concentrations (e.g.,

E. J. P. Jones et al. 162

FIGURE 7 Comparison of TRFLP profiles: APG sediments WB23 and WB30, and WBC-2 after 1 year in culture with TeCA or a mixture

of chlorinated compounds.

target addition of 60 μM versus measured concentra-tion, 240 μM, that is reported in Figure 2), as well asthe error in stoichiometry between different bottles (i.e.,2 ± 1). In spite of the large errors, it is evident thatethene and ethane are major products of TeCA degrada-tion. The reduction of ethene to ethane demonstratedby WBC-2 during and after the depletion of chlori-nated compounds and in ethene-amended culture of-fers an explanation for the low recovery of nonchlori-nated end products (2:1 substrate: product). The lackof ethene and ethane accumulation is a common fieldobservation during dechlorination at APG and othercontaminated field sites. This fact has confounded theinterpretation of field data with respect to the degra-dation mechanism and fate of less chlorinated com-pounds. One explanation for this, supported by the datapresented here, is the degradation of nonchlorinatedend products preventing their accumulation, such thatethene and ethane do not accumulate because their rateof removal exceeds their rate of production under mostfield conditions. Characterizing the consortium, WBC-2, has provided an opportunity to learn more about theTeCA degradation pathway and helped explain the lackof accumulation of end products at field sites, as a resultof changes in the rates between degradation steps and

in the microbial community associated with culturingand enrichment.

The reduction of ethene could account for theethane observed during TeCA reduction. However, an-other possible source of ethane is directly throughthe hydrogenolysis of chlorinated ethanes (left halfof Figure 1). 1,1,1-Trichloroethane was degraded tochloroethane and finally to ethane by an isolate relatedto Dehalobacter restrictus (Sun et al., 2002). However, atpresent there is no direct evidence for this pathway inWBC-2. Chloroethane, the immediate precursor for hy-drogenolytic ethane, was never observed, indicating ei-ther that it was not produced, or that rapid degradationprevented its accumulation. Further research will beneeded to definitively determine the source(s) of ethanein the culture during chlorinated ethane reduction.

Many individual reductive dechlorination reactionshave been documented to occur in pure or mixedcultures of microorganisms. The unique contributionof WBC-2 for contaminant treatment is the abilityof the mixed culture to handle a variety of com-pounds (both chlorinated ethenes and ethanes) with-out noticeable inhibition. The dechlorination rates ob-served in WBC-2 for chlorinated ethenes are similarto those observed in cultures used for bioremediation

163 Microbial Consortium for Biodegrading Chlorinated Ethanes

FIGURE 8 Frequency of phylogenetic types in WBC-2 16S

rDNA clone library (A), with Clostridiales groups shaded dark

and Proteobacteria stippled gray, and mcr clone library (B), with

acetate-utilizing methanogens shaded.

treatment of chlorinated ethene contamination (e.g.,KB-1; Duhamel et al., 2002). The advantage offeredby WBC-2 is its ability to degrade chlorinated ethenesand ethanes simultaneously. Although, not shown here,WBC-2 was also able to reduce tetrachloroethene (PCE)in the presence of TeCA (Jones et al., unpublisheddata). WBC-2 may or may not contain dechlorinatingorganisms similar to those studied in pure or mixedcultures with chlorinated ethenes.

Evidence for a Distinct DechlorinatingPopulation

The TeCA dechlorination pathway includes reactionsthat are a part of the more thoroughly studied PCE-dechlorination pathway (see right half of Figure 1),and thus might be expected to support the growthof similar organisms. Indeed, the predominance ofClostridia and CFB in the WBC-2 population (Table 2)is also characteristic of TCE-dechlorinating commu-nities (MacBeth et al., 2004, Richardson et al., 2002),

and organisms similar to the ethene-dechlorinating De-halococcoides and Dehalobacter are observed in WBC-2.However, the dechlorinating abilities of WBC-2, en-riched in the presence of chlorinated ethanes, are dif-ferent than cultures enriched with chlorinated ethenes,both with respect to the response to added electrondonors and in the relative importance of Dehalococ-coides within the microbial population. Based on lim-ited studies (this paper and Aulenta et al., 2005), theelectron donor needs for chlorinated ethane–enrichedcultures appear to be different from those of culturesenriched for chlorinated ethene reduction. Most bac-terial isolates capable of reductive dechlorination ofchlorinated ethenes, including Dehalococcoides and De-halobacter, use H2 as the preferred electron donor. Sunet al. (2002) showed that a Dehalobacter isolate requiredH2 plus acetate to reduce 1,1,1-trichloroethane. Fur-thermore, He et al. (2002) suggested that a volatilefatty acid (VFA) such as propionate can provide a slowrelease of H2 to support hydrogenotrophic dechlori-nators at the expense of methanogenesis. The chlori-nated ethane–enriched culture, WBC-2, was not stim-ulated to reduce cisDCE or TCA with H2, H2 plusacetate, or propionate added as the electron donor.The failure of H2 to stimulate dechlorination suggeststhat the organisms involved are not the same as thechlorinated ethene–reducing organisms that have beenstudied in isolation. Aulenta et al. (2005) found thatbutyrate, a VFA that supported a PCE-dechlorinatingculture, did not do so in the presence of TeCA. TeCAapparently inhibited organisms that could release H2

from butyrate. Thus, another mechanism by whichchlorinated ethanes may inhibit chlorinated ethene–enriched populations is through the inhibition of keyconsortium members that may be indirectly involved indechlorination.

Dehalococcoides spp., which include the only isolatedbacteria identified to completely biodegrade chlori-nated ethenes, have been targeted as an indicator ofdechlorinating capability (Hendrickson et al., 2002). Anumber of field studies support the idea that com-plete degradation of chlorinated alkenes is dependentupon the presence of specific microorganisms from thegenus Dehalococcoides (e.g., Hendrickson et al., 2002;Lowe et al., 2002). Several laboratory mixed culturesenriched for chlorinated ethene reduction are reportedto have populations of Dehalococcoides spp. represent-ing more than 30% of the total bacteria (Gu et al.,2004; Richardson et al., 2002; Duhamel et al., 2004),

E. J. P. Jones et al. 164

TABLE 2 Frequency of WBC-2 Clones and Their Closest BLAST Matches to a Dechlorinating Isolate or to Clone Sequences from a

Dechlorinating Environment

Phylotype Phylogenetic BLAST hits1

frequency placement Clone id (% similiar) Source

48/133 Clostridium acc #DQ907197 ∗AY667266 (99%) TCE-dechlorinating community

35/133 Acetobacteria acc #DQ907202 ∗AY185312 (97%)AY185315 (97%)AY185311 (96%)

1,2-Dichloropropane-dechlorinatingenrichment

2/133 Dehalobacter acc #DQ907207 ∗AF422637 (95%) TCE-reducing communityAY754830 (93%) PCB-dechlorinating cultureDQ663785 (93%) 111-trichloroethane degrading

mixed culture

19/133 Bacteroides acc #DQ907199 ∗DQ080146 (95%) 2,3,4,5-Tetrachlorobiphenyl culturecontaining Dehalococcoides

AY553955 (95%) PCB contaminanted harborsediment

AY780553 (95%) Chlorinated ethene-dechlorinatingenrichment

AJ488070 (90%) Chlorobenzene degradingconsortium

acc #DQ907201 ∗AY217446 (97%) TCE-dechlorinating communityAY217435 (97%) TCE-dechlorinating community

1/133 Geobacter acc #DQ907206 ∗AY780563 (98%) Chlorinated ethene enrichmentculture

AF223382 (98%) Isolate that dechlorinatestrichloroacetic acid

AF447133 (98%) Population that dechlorinatesAF447134 (98%) saturated PCEAY914177 (98%) Isolate that dechlorinates PCEAY667270 (97%) TCE contaminated aquifer

7/133 Pseudomonas acc #DQ907203 AY017341 (99%) Chlorate-reducing isolate

∗Top BLAST hit.

and it is widely believed that Dehalococcoides is key tothe dechlorination in these cultures. WBC-2 dechlori-nated chlorinated ethenes at rates similar to these otherlaboratory mixed cultures (e.g., 54 μM day−1cisDCEversus approximately 30 μM day−1 for KB-1 [Duhamelet al., 2002]). However, Dehalococcoides spp. compriseonly a minor part (about 1%) of the cell populationin WBC-2. This, coupled with the presence of consor-tium members that appear to be closely related to clonesof unknown function in other dechlorinating popula-tions, suggests that organisms other than Dehalococcoidesspp.may play a greater role in TeCA dechlorination.

These observations suggest that exposure to chlori-nated ethanes results in the selection of a different pop-ulation of organisms for chlorinated ethene reduction.Cultures that were enriched using chlorinated ethenes(i.e., in the absence of chlorinated ethanes) were inhib-ited in the presence of chlorinated ethanes (Duhamelet al., 2002; Aulenta et al., 2005). WBC-2 was able

to degrade chlorinated ethanes and ethenes simulta-neously with little VC accumulation. This capabilitymakes the microbial consortiumWBC-2 a potentiallyvaluable tool for bioremediation of sites contaminatedwith mixtures of chlorinated ethenes and ethanes. In ad-dition, the simultaneous reduction of all componentsof the TeCA degradation pathway can reduce the totaltreatment time and help prevent transport of hazardouscompounds out of the treatment zone.

Identifying Microbial RolesAlthough we have identified numerically impor-

tant components of the microbial consortium WBC-2,the specific roles of consortium members have yet tobe determined. Although tentative roles could be as-signed to Dehalococcoides (cisDCE, transDCE, VC, andDCA reduction), Dehalobacter (TCA reduction to VC,DCA reduction to ethene), and Acetobacterium (DCA

165 Microbial Consortium for Biodegrading Chlorinated Ethanes

reduction to ethene) based on studies of related organ-isms (Duhamel et al., 2002; He et al., 2003; Grosternand Edwards, 2006; De Wildeman et al., 2003), WBC-2 exhibited some capabilites, such as TeCA reductionto transDCE, and TCA reduction to DCA, for whichno organisms have been implicated. In addition, indi-rect roles, such as satisfying the “undefined nutritionalneeds” of dechlorinating organisms, may be critical tothe consortium function. Additional research on WBC-2 is planned to identify the roles of individual consor-tium members.

The observation of closely related phylotypes inWBC-2 and other dechlorinating communities, suchas Clostridium sp., Acetobacterium sp., and CFB providessome evidence for the involvement of previously un-recognized bacteria in dechlorination processes. How-ever, enrichment of organisms in a dechlorinating sys-tem provides only circumstantial evidence for direct in-volvement. In WBC-2, lactate fermentation, homoace-togenesis, methanogenesis, sulfur cycling, syntrophy,and chemoautotrophy could support organisms in theculture without deriving energy from dechlorination.Thus, organisms could persist in mixed culture withoutplaying a direct role in dechlorination, and further workwould be needed to confirm whether or not each playsa significant role. For example, Acetobacteria are ableto grow by converting H2+ CO2 to acetate, but theymay also be directly or indirectly involved in dechlori-nation. An Acetobacterium sp. has been isolated that cancometabolically reduce DCA to ethene (De Wildemanet al., 2003). This strain lost its ability to dechlorinateafter about 10 transfers probably due to undefined nu-tritional requirements. Although the 16S rDNA of thedechlorinating Acetobacterium strain was > 99% relatedto A. wieringae, the type strain was unable to dechlori-nate DCA (De Wildeman et al., 2003), illustrating thelimitation of phylogenic identification alone. Aceto-bacteria could also support dechlorination indirectly,for example through the production of corrinoid fac-tors, which play a role in some dechlorinating reactions(Magnuson et al., 1998; Holscher et al., 2004), or in syn-trophic relationships, through the production of acetate(He et al., 2002).

The possible role of methanogens in dechlorinationby WBC-2 is not known. Although the presence andactivity of methanogens are often considered to be in-hibitory to or at least contraindicative of dechlorina-tion activity, some evidence suggests that methanogensmay play an important role in dechlorination. Dechlo-

rination is associated with methanogenic environments(Vogel and McCarty, 1985) but is often thought to bea cometabolic process that is less efficient than de-halorespiration. Bromoethane sulfonic acid, a potentinhibitor of methanogenesis, inhibited VC degrada-tion in APG sediments (Lorah et al., 2003b). Work withpure cultures of Methanosarcina sp. and Methanosarcinamazei has shown that methanogens can dechlorinatePCE to TCE, and dechlorination occurs only dur-ing active methane production (Fathepure and Boyd,1988). Methanogens also produce corrinoid factors andunique enzymes and vitamins that may support dechlo-rination. For example, extracellular factors produced byMethanosarcina have been shown to enhance dechlo-rination of carbon tetrachloride (Novak et al., 1998).Anaerobic methane oxidation (“reverse methanogen-esis” carried out by methanogens) has been coupledto sulfate reduction (Boetius et al., 2000) and denitri-fication (Raghoebarsing et al., 2006). Although not yetshown to be coupled to dechlorination, such a reac-tion is theoretically possible. Production of methaneby WBC-2 could also stimulate co-metabolic dechlori-nation by methane oxidizers in aerobic environments(Chang and Alvarez-Cohen, 1996) during actual fieldapplications. WBC-2 presents an ideal platform for in-vestigating the role of methanogens in dechlorination,because dechlorination and methanogenesis co-occurin WBC-2.

Bioremediation with WBC-2Bioremediation is a promising treatment for sites

contaminated with chlorinated ethanes or mixturesof chlorinated ethanes and ethenes. Until now, how-ever, large-scale cultures capable of dechlorinating com-pounds such as TeCA and TCA have not been readilyavailable. The microbial consortium WBC-2 was en-riched from contaminated sediment at APG and is ca-pable of rapid and complete dechlorination of TeCAunder anaerobic conditions with almost no detectableintermediates. WBC-2 has been tested in small-scalebioaugmentation tests (Lorah et al., 2004), in which anaddition of 5% (v/v) culture to sediment greatly de-creased both the concentration of hazardous interme-diates and the time required for complete dechlorina-tion. In a field test at Aberdeen Proving Ground, ap-plication of a horizontal permeable barrier seeded withWBC-2 resulted in complete remediation of groundwa-ter contaminated with chlorinated ethanes, ethenes and

E. J. P. Jones et al. 166

methanes prior to discharge to the land surface or creek(Lorah et al., 2005; Majcher et al., 2005). WBC-2 rep-resents a useful addition to the bioremediation toolboxwith potential uses for bioremediation of sites contam-inated with chlorinated ethanes and mixed chlorinatedcompounds.

ACKNOWLEDGEMENTSThe development of WBC-2 was supported by the

National Research Program of the U.S. Geological Sur-vey and the U.S. Army Environmental Conservationand Restoration Division at Aberdeen Proving Ground(John Wrobel). The authors acknowledge SiREM Lab-oratory (Guelph, Canada) for providing DNA from thescaled-up WBC-2 culture for use in cloning. They thankFrank Loffler for kindly providing Dehalococcoides plas-mid. Any use of trade, product, or firm names in thisarticle is for descriptive purposes only and does not im-ply endorsement by the U.S. Government.

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