ORIGINAL PAPER

J. Gerritse á G. Kloetstra á A. Borger á G. DalstraA. Alphenaar á J. C. Gottschal

Complete degradation of tetrachloroethene in coupled anoxicand oxic chemostats

Received: 17 April 1997 / Received revision: 6 June 1997 /Accepted: 7 June 1997

Abstract Anaerobic tetrachloroethene(C2Cl4)-dechlori-nating bacteria were enriched in slurries from chloro-ethene-contaminated soil. With methanol as electrondonor, C2Cl4 and trichloroethene (C2HCl3) were re-ductively dechlorinated to cis-1,2-dichloroethene (cis-C2H2Cl2), whereas, with L-lactate or formate, completedechlorination of C2Cl4 via C2HCl3, cis-C2H2Cl2 andchloroethene (C2H3Cl) to ethene was obtained. In oxicsoil slurries with methane as a substrate, complete co-metabolic degradation of cis-C2H2Cl2 was obtained,whereas C2HCl3 was partially degraded. With toluene orphenol both of the above were readily co-metabolized.Complete degradation of C2Cl4 was obtained in se-quentially coupled anoxic and oxic chemostats, whichwere inoculated with the slurry enrichments. Apparentsteady states were obtained at various dilution rates(0.02±0.4 h)1) and in¯uent C2Cl4-concentrations (100±1000 lM). In anoxic chemostats with a mixture of form-ate and glucose as the carbon and electron source, C2Cl4was transformed at high rates (above 140 lmol l)1 h)1,corresponding to 145 nmol Cl) min)1 mg protein)1)into cis-C2H2Cl2 and C2H3Cl. Reductive dechlorinationwas not a�ected by addition of 5 mM sulphate, butstrongly inhibited after addition of 5 mM nitrate. Ourresults (high speci®c dechlorination rates and loss ofdechlorination capacity in the absence of C2Cl4) suggestthat C2Cl4-dechlorination in the anoxic chemostat wascatalysed by specialized dechlorinating bacteria. Thepartially dechlorinated intermediates, cis-C2H2Cl2 andC2H3Cl, were further degraded by aerobic phenol-me-tabolizing bacteria. The maximum capacity for chlo-

roethene (the sum of tri-, di- and monochloro derivativesremoved) degradation in the oxic chemostat was95 lmol l)1 h)1 (20 nmol min)1 mg protein)1), and thatof the combined anoxic ! oxic reactor system was43.4 lmol l)1 h)1. This is signi®cantly higher than re-ported thus far.

Introduction

Chlorinated ethenes are used in large amounts bylaundries (dry-cleaning) and industry (e.g. metal clean-ing, chemical synthesis). As a consequence, tetrachlo-roethene (C2Cl4) and trichloroethene (C2HCl3) especiallyare common soil and groundwater pollutants (Green-berg et al. 1982; U.S. Environmental Protection Agency1995; VROM Leidraad bodembescherming 1990).Bioreactors are potentially attractive tools for theremoval of chloroethenes from groundwater and soilvapour extracted during control and clean-up of con-taminated locations. For practical application it isessential that all chloroethenes, i.e. C2Cl4, C2HCl3, di-chloroethenes (C2H2Cl2) and chloroethene (vinyl chlo-ride, C2H3Cl), are mineralized at high rates to lowresidual concentrations.

Aerobic bacteria capable of degrading C2Cl4 are notknown; however, under anoxic conditions it can be re-ductively dechlorinated. Indeed, several reports indicatethat C2Cl4 can be completely dechlorinated to harmlessproducts like ethene, ethane, CO2, and chloride (Ball-apragada et al. 1995; De Bruin et al. 1992; DiStefanoet al. 1991; Vogel and McCarty 1985; Wild el al. 1995;Wu et al. 1995). However, a major problem with ap-plication of anoxic dechlorination of C2Cl4 at high ratesis that partially dechlorinated intermediates, especiallycis-1,2-dichloroethene (cis-C2H2Cl2) and C2H3Cl3, tendto accumulate (Carter and Jewell 1993; Gerritse et al.1995; Guiot et al. 1995; Tandoi et at. 1994). Since theseproducts may be even more toxic than C2Cl4 itself(Henschler 1977; Infante and Tsongas 1982), an addi-tional oxic step for their degradation is required. In

Appl Microbiol Biotechnol (1997) 48: 553±562 Ó Springer-Verlag 1997

J. Gerritse (&) á G. Kloetstra á A. Borger á G. DalstraJ. C. GottschalDepartment of Microbiology, University of Groningen,Kerklaan 30, 9751 NN Haren, The NetherlandsTel.: +31 50 3632169Fax: +31 50 3632154e-mail: j.gerritse@biol.rug.nl.

A. AlphenaarTAUW milieu bv, P.O. Box 133, 7400 AC Deventer,The Netherlands

contrast to C2Cl4, the partially dechlorinated ethenescan be co-metabolized by aerobic bacteria, which pro-duce oxygenases during growth on methane, isoprene,ammonia, toluene or phenol, for example (Dol®ng et al.1993; Ensley 1991)

Several studies have indicated that sequential anoxic/oxic systems allow e�cient and complete biodegradationof chlorinated aliphatic and aromatic compounds (Be-unink and Rehm 1988, 1990; Field et al. 1995; Fathepureand Vogel 1991; Fogel et al. 1995; Gerritse and Got-tschal 1992; Gerritse et al. 1995; Guiot et al. 1995;Zitomer and Speece 1993). Recently we obtained com-plete degradation of C2Cl4 in two steps in a coupledanoxic/oxic ®xed-bed column system (Gerritse et al.1995). In the ®rst anoxic column C2Cl4 was reductivelydechlorinated into mainly cis-C2H2Cl2, which was fur-ther mineralized co-metabolically under oxic conditionsby methanotrophic bacteria in the second column. It wasshown that co-metabolism of chloroethene by the meth-anotrophs represented the rate-limiting step in the de-gradation. Inactivation of methanotrophs, which hasbeen correlated with (lethal) toxicity of chloroethenetransformation products formed during co-metabolism,clearly precludes e�ective use of these microbes forchloroethene removal (Alvarez-Cohen and McCarty1991; Fennell et al. 1993; Henry and Grbic�-Galic�1991;Newman and Wackett 1991; Oldenhuis et al. 1991).Bacteria metabolizing certain aromatic compounds, likephenol or toluene, appear to be considerably more re-sistent to intermediates from chloroethane metabolismthan are methanotrophs (Folsom and Chapman 1991;Landa et al. 1994). Therefore, a system that combinesaerobes that metabolise such aromatics with anaerobicC2Cl4-dechlorinating bacteria should, in principle, en-able complete mineralisation of C2Cl4 at high rates.

The aims of this investigation were (1) to set up atwo-step anoxic ! oxic bioreactor system, capable ofreliable puri®cation of high loads of chloroethene-con-taminated groundwater, (2) to obtain further kinetic andphysiological data from such cultures, and (3) to studythe e�ect of alternative electron acceptors on dechlori-nation.

Sequentially coupled anoxic and oxic chemostatswere used, which allowed detailed study of the micro-biology of dechlorinating populations. In the ®rst che-mostat C2Cl4 was partially dechlorinated by anaerobicbacteria and in the second step the dechlorinationproducts were co-metabolized by aerobic phenol-grownbacteria.

Materials and methods

Sample collection

Samples were collected in July 1994 from bore-holes at atetrachloroethene �C2Cl4�-contaminated site below a laundrybuilding in Breda, The Netherlands. At this location C2Cl4 had beenused for more than 20 years for dry-cleaning procedures, and haspenetrated into the sandy soil to a depth of about 10 m. The major

C2Cl4-dechlorination products detected in the groundwater at thislocation were C2HCl3, cis-C2H2Cl2 and C2H3Cl. Soil samples weretaken from a depths between 0 and 6 m below the soil surface at thecentre of the C2Cl4 plume (groundwater oxygen content: 0.0±1.5 mg/l, total chloroethene concentration: 250±400 lM) and atabout 50 m down-¯ow with the groundwater (groundwater oxygencontent: 0.5 mg/l, total chloroethene concentration: 1±10 lM). Thegroundwater at this location had a temperature of 17±20 °C and apH of 6.4±6.7, and the water table was located 2±3 m below thesurface. Soil samples (about 500 g) and groundwater (about 100 ml)were transferred into sterile screw-cap bottles. The bottles were®lled completely, reduced with 5 ml 2% w/v Na2S solution, storedon ice and transported to the laboratory.

Media and growth conditions

Mineral medium, with a low chloride concentration, used for bio-reactor experiments was prepared under a N2 gas phase and con-tained (per litre): 1.15 g (NH4)H2PO4, 0.1 g MgSO4 á 7H2O, 0.05 gCa(NO3)2 á 4H2O, 1 mg EDTA, 2 mg FeSO4 á 7H2O, 0.1 mgZnSO4 á 7H2O, 0.03 mg MnCl2 á 4H2O, 0.3 mg H3BO3, 0.2 mgCoCl2 á 6H2O, 0.01 mg CuCl2 á 2H2O, 0.02 mg NiCl2 á 6H2O,0.03 mg NaMoO4 á 2H2O, 0.03 mg Na2SeO3 á 5H2O, and 0.03 mgNa2WO4 á 2H2O. For batch incubations, resazurin (0.1 mg/l),yeast extract (0.1 g/l) and, after autoclaving, 10 ml/l 10% w/vKOH solution and 1 ml/l ®lter-sterilized vitamins solution(Heijthuijsen and Hansen 1986) were added.

Enrichment of chloroethene-metabolizing bacteria

For pre-enrichment of anaerobic and aerobic bacteria capable of(co-)metabolizing chlorinated ethenes, a slurry was prepared bymixing soil under a N2 ¯ow with basal medium (50% v/v) that hadbeen prepared in groundwater collected at the sampling site insteadof demineralized water. From this slurry, 25-ml fractions weretransferred into 137-ml screw-cap bottles that were sealed with aViton-rubber septum, which was placed below a butyl-rubberseptum. After addition of C2Cl4, C2HCl3 and cis-C2H2Cl2 (nomi-nal concentration 10±40 lM each from a stock solution in meth-anol, resulting in a methanol concentration of 1 mM) and carbonsubstrate, the soil slurries were incubated at 20 °C, 100 rpm undereither anoxic (gas phase N2) or oxic (gas phase air) conditions.Carbon substrates, prepared in separately autoclaved 1 M stocksolutions, which were used for anoxic slurries, were methanol(25 mM), L-lactate (10 mM) or formate (25 mM). Weekly, theanoxic slurries were supplied with an additional 10% of the initiallyadded carbon substrate. Methane (nominal concentration 25 mM),phenol (5 mM) or toluene (nominal concentration 5 mM) wassupplied to oxic slurries for the enrichment of aerobic chloroetheneco-metabolizing bacteria. No signi®cant disappearance of C2Cl4,C2HCl3 or cis-C2H2Cl2 was observed in anoxic or oxic controlincubations that were not amended with carbon substrates.

Bioreactor set-up

Two coupled chemostats were used for complete degradation ofC2Cl4 (Fig. 1). The e�uent of the anoxic chemostat was pumpedinto a second oxic chemostat. C2Cl4 was supplied to the ®rst che-mostat by pumping medium via two mixing vessels, one of whichcontained a few millilitres of C2Cl4 in order to saturate the mediumpassing through this vessel. Di�erent C2Cl4 in¯uent concentrationswere obtained by applying di�erent ratios of medium ¯ow ratesthrough the two vessels. Medium in the anoxic chemostat was re-duced and maintained at pH 7.0±7.2 by automatic titration with a

1M KOH solution, containing 5 g l)1 Na2S á 9H2O. The head-

space of the anoxic chemostat was gassed with N2 (50±150 ml h)1)

and stirred at 250 rpm. The oxic chemostat was gassed with air(¯owrate 1500±4000 ml h)1), stirred at 1000 rpm and kept atpH 7.0±7.2 by titration with 1 M NaOH. Both chemostats were

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made of glass, stainless steel and Viton rubber tubing and septa.Viton tubing that had been inserted inside silicone tubing was usedfor peristaltic medium pumps. The chemostats had a workingvolume of about 1 l (anoxic chemostat) and 1.5 l (oxic chemostat)and were operated at 20 °C. A mixture of formate (10 mM) andglucose (1 mM) was routinely used as the carbon and electrondonor for the anaerobic dechlorinating bacteria, whereas 5 mMphenol was added to the reservoir medium as a substrate for theaerobic bacteria. For analysis of steady-state mass balances theoperating conditions were maintained constant for at least ®vevolume changes. The degradation rates of the chemostats weredetermined by measuring the disappearance of chloroethenes andchloride formation in steady states obtained with increasing me-dium ¯ow and in¯uent chloroethene concentrations. Concentra-tions of chloroethenes were quanti®ed both in the liquid and thegas phase of the reactors. Since the volume of the oxic chemostatwas larger than that of the anoxic chemostat, the dilution rate ofthe latter chemostat was 1.5-fold lower at the same medium ¯owrate.

Analytical procedures

Sulphide was analysed colorimetrically, as described by TruÈ per andSchlegel (1964). Chloride was measured with an ion-selective elec-trode (Orion, Boston, USA) after oxidation of the samples with0.3% H2O2 for 30 min. The electrode was calibrated with samplescontaining known concentrations of NaCl (Gerritse et al. 1995).

Protein was measured according to Lowry et al. (1951) with bovineserum albumin as a standard. Formate was measured with a col-orimetric assay according to Lang and Lang (1972). Phenol wasmeasured with a Jasco HPLC (Tokyo, Japan) equipped with aUV975 UV/VIS detector and an Alltech Lichrospher 110RP8 col-umn (Deer®eld, Ill., USA) (Pieper et al. 1988). Volatile organicacids were measured on a Varian capillary gas chromatograph(type 3600), using an Alltech 30 m econocap FFAP wide-borecolumn (Alltech, Breda, Netherlands). The temperature of thesplitless injector and the ¯ame ionization detector was 250 °C.Separation of acetate, propionate, isobutyrate, butyrate, isovale-rate, valerate, and caproate was obtained with a linear temperaturegradient (10 °C min)1) from 110 °C to 140 °C. Before analysis,samples were centrifuged (10 min, 9000 g) and supernatants wereacidi®ed by addition of 0.8% (®nal concentration) H3PO4.Isovalerate (10 mM) was used as an internal standard. H2 and CH4

were determined by gas chromatography on a Shimadzu 104 gaschromatograph, equipped with a thermal conductivity detector(temperature 300 °C, polarization current 50 lA) and a 6-mMolsieve 3A column kept at 50 °C (Alltech, Breda, Netherlands).Chlorinated ethenes were determined in triplicate through head-space analyses by capillary gas chromatography (Gerritse et al.1995). For mass balances of chemostat steady states, chloroetheneswere measured both in liquid and gas-phase samples. Volatilecompounds present both in the liquid and the gas phase areexpressed as nominal concentrations in the liquid.

Results

Batch enrichment of anaerobic andaerobic chloroethene-metabolizing bacteria

Reductive dechlorination of C2Cl4 and C2HCl3 to cis-C2H2Cl2 occurred within 20 days in anoxic slurries ofchloroethene-contaminated soil, amended with mineralnutrients and either methanol, L-lactate or formate. Inanoxic slurries without nutrients and/or an electrondonor, chloroethene dechlorination was not observed.In slurries with methanol as electron donor, no signi®-cant dechlorination of cis-Cl2H2Cl2 occurred (Fig. 2A).After about 30 days, methanogenesis started, and morethan 80% of the methanol was converted into methane.Interestingly, with L-lactate (Fig. 2B) or formate(Fig. 2C) cis-C2H2Cl2 was completely dechlorinated viaC2H3Cl into ethene. Signi®cant methane productiononly occurred after C2H3Cl had been dechlorinated,suggesting that the dechlorinating bacteria in these en-richments had a better a�nity for electron donors sup-plied, or produced (e.g. H2 from L-lactate fermentation)than the methanogens.

In oxic slurries incubated in the absence of an addedcarbon substrate, no degradation of either C2Cl4,C2HCl3 or cis-C2H2Cl2 was detected. However, after alag of more than 2 months, complete degradation ofC2H3Cl occurred in oxic soil, and a liquid enrichmentculture of aerobic bacteria growing on vinyl chloride asthe sole substrate could be obtained. With methane as asubstrate for enrichment of aerobic methanotrophicbacteria, consumption of 25 mM methane coincidedwith the disappearance of 12 lM cis-C2H2Cl2 anddegradation of only 6 lM of the 9 lM C2HCl3 addedinitially (Fig. 2D). Toluene (5 mM ) was consumed

Fig. 1 Schematic drawing of the coupled anoxic! oxic chemostatsystem

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within 10 days in oxic slurries (Fig. 2E), which paral-lelled rapid cis-C2H2Cl2 and C2HCl3 degradation. Sim-ilar results were obtained in oxic enrichments with

phenol as the primary growth substrate (results notshown). Degradation of C2Cl4 was not observed in anyof the oxic slurry incubations.

Fig. 2A±E Reductive dechlorination of C2Cl4 and C2HCl3 to cis-C2H2Cl2 in (A) an anoxic slurry enrichment with methanol aselectron donor, or complete dechlorination with (B) formate or(C) lactate as substrates. Co-metabolism of C2HCl3 and cis-C2H2Cl2in oxic slurries incubated with (D) methane or (E) toluene as theprimary growth substrate. n C2Cl4, e C2HCl3, s cis-C2H2Cl2, ,C2H3Cl, + ethene, h methane, j toluene

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Reductive dechlorination of tetrachloroethenein an anoxic chemostat

The C2Cl4-dechlorinating slurries (10 ml enrichments onmethanol, L-lactate, and formate respectively) were usedas an inoculum for an anoxic chemostat. With formate(10 mM) plus glucose (1 mM, to provide a source ofeasily assimilable carbon substrate) as electron donors inthe reservoir medium, reductive dechlorination of C2Cl4(about 100 lM) started within 2 weeks. When all theC2Cl4 was dechlorinated the medium was started, andthe C2Cl4 supply increased stepwise (in¯uent C2Cl4concentration from approximately 100 lM to approxi-mately 1000 lM) resulting, after at least ®ve volumechanges, in steady states (i.e. no apparent changes ine�uent chloroethene concentrations) at dilution ratesranging from 0.02 h)1 to 0.4 h)1. In all steady states themajor dechlorination products were cis-C2H2Cl2 andC2H3Cl (Fig. 3), whereas production of 1,1-dichloro-ethene, trans-1,2-dichloroethene and ethene was lessthan 1 mol% of the C2Cl4 supplied. Although methaneproduction was observed during start-up of the chemo-stat, the amount of methane formed dropped rapidly(less than 1 mol% of the supplied organic carbon sub-strate) after several days of continuous cultivation.Acetate (2±7 mM) was the major volatile organic acidproduced at all dilution rates. Small amounts of propi-onate (0±0.9 mM) and butyrate (0±0.3 mM) were alsoformed. Accumulation of H2 (detection limit about1 lmol l)1) was not observed. The residual C2Cl4 con-centrations in the gas and liquid phases increasedslightly with increasing dilution rate, i.e. increasingC2Cl4 load (Fig. 3). However, the e�ciency of C2Cl4removal dropped dramatically at loads above150 lmol l)1 h)1 (Fig. 4A). At dilution rates above0.4 h)1, wash-out of the dechlorinating population oc-curred. The maximum dechlorination rate obtained with

the anoxic chemostat was 260 lmol chloride formedl)1 h)1, which corresponded to a speci®c dechlorinationrate of 145 nmol Cl) min)1 (mg cell protein))1 (Fig. 4A).Phenol (5 mM), which was added to the reservoir me-dium as a substrate for the aerobic bacteria in the oxicchemostat (see below), was not degraded in the anoxicchemostat.

Electron donors and acceptors

The e�ect of alternative electron acceptors and donorson C2Cl4 dechlorination was studied with the anoxicchemostat culture at a dilution rate of 0.02 h)1. Addi-tion of 5 mM Na2SO4 to the reservoir medium resultedin sulphide production (1±2 mM), indicating the activityof sulphate-reducing bacteria, but did not signi®cantlyalter the pattern of dechlorination products. In contrastwhen, in addition to sulphate, 5 mM KNO3 was alsoadded to the reservoir, both reductive dechlorinationand sulphate reduction were suppressed within hours.

Fig. 3 Relative amounts (mol%) of chloroethenes in the anoxicchemostat in steady state at various C2Cl4 (PCE) loads. VC C2H3Cl,cDCE cis-C2H2Cl2, TCE C2HCl3

Fig. 4A,B Rates of chloride formation and conversion e�ciency ofchloroethene transformation in steady states of (A) the anoxic and (B)the oxic chemostat. A e C2Cl4 (PCE) removed (%) s volumetric rateof chloride formation (lmol l)1 h)1), n speci®c rate of chlorideformation (nmol min)1 mg protein)1). B n cis-C2H2Cl2 removed(%), e C2H3Cl removed (%), s volumetric rate of chloride forma-tion (lmol l)1 h)1), . speci®c rate of chloride formation (nmolmin)1 mg protein)1).

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When CCl4 was mixed with C2Cl4 in the in¯uent medi-um (®nal concentration about 10% v/v), partial inhibi-tion of dechlorination of C2Cl4 occurred transiently (forabout 2 days) in parallel with the formation of CHCl3and CH2Cl2, indicating that the anaerobic communitywas also capable of dechlorinating CCl4.

If formate or glucose was supplied separately as thesole carbon and energy donors, complete C2Cl4 de-chlorination continued. However, when these substateswere replaced by either methanol (20 mM) or butyrate(10 mM), dechlorination stopped within several days.When the anoxic chemostat was fed during several vol-ume changes with a medium without C2Cl4, and C2Cl4was subsequently added to the medium, it took 1±2weeks for reductive dechlorination to resume its originalactivity.

Dilution series of the anoxic chemostat were made inmedia with a mixture of formate (10 mM), acetate(1 mM) and glucose (2 mM) as electron donors, with250 lM either C2Cl4, C2HCl3, cis-C2H2Cl2 or C2H3Cl.Dechlorination of C2Cl4 and C2HCl3 in the 10)1 dilu-tion tubes started within several days. Within 1 month,C2Cl4 and C2HCl3 dechlorination occurred down to the10)7 dilution tubes. Similarly, dechlorination of cis-C2H2Cl2 and C2H3Cl was observed down to the 10)7

dilutions, but only after 120 and 287 days of incubationrespectively. This corresponded to a most probablenumber of at least 1:2� 107 C2Cl4-, C2HCl3-, cis-C2H2Cl2- or C2H3Cl-dechlorinating bacteria (ml che-mostat liquid))1, which accounted for about 6% ofthe microscopically determined cell numbers (i.e.2� 108 mlÿ1).

Co-metabolism of partially dechlorinated ethenesin an oxic phenol-limited chemostat

Two 50-ml batch cultures with aerobic bacteria enrichedon either toluene or phenol were inoculated into an oxicchemostat with phenol (5 mM) as the carbon and energysource. After an initial phase of batch growth, with adoubling time of 3.9 h, the oxic chemostat was fed withthe e�uent of the anoxic C2Cl4 dechlorinating chemo-stat. Figure 5 shows an example of a mass balance ofchloroethene metabolism by the combined anoxic andoxic chemostats (at dilution rates of 0.11 h)1 and0.07 h)1 respectively) with an in¯uent C2Cl4 concentra-tion of 300 lM. The disappearance of cis-C2H2Cl2 andC2H3Cl, parallelled by the formation of chloride,showed that chloroethenes formed through C2Cl4 de-chlorination in the anoxic chemostat were metabolizedin the second oxic chemostat. The extent of cis-C2H2Cl2degradation remained above 75% for loads up to about100 lmol l)1 h)1 (Fig. 4B). cis-C2H2Cl2 was removed toa higher extent than C2H3Cl. Degradation of residualC2Cl4 was not observed in the oxic chemostat. In allsteady states, phenol was degraded to concentrationsbelow the detection limit (about 0.5 lM). Organic acids,produced in the anoxic chemostat (see above) were de-

graded in the oxic chemostat to less than 0.5 mM. At adilution rate of 0.4 h)1, a maximum rate of chlorideformation of 95 lmol l)1 h)1 (20 nmol min)1 mg pro-tein)1) was obtained. Higher dilution rates were nottested, since under these conditions wash-out of theC2Cl4-dechlorinating community from the anoxic che-mostat occurred. When phenol was omitted from themedium, the rate of chloride production in the oxicchemostat dropped to below 1 lmol l)1 h)1, indicatingthat phenol-oxidizing bacteria were indeed responsiblefor co-metabolic chloroethene degradation.

In¯uence of some process conditions

The coupled chemostats were routinely operated at20 °C and pH 7.0. At a C2Cl4 load of 50 lmol l)1 h)1,more than 80% of chloroethenes were also removed attemperatures and pH values in the range 15±30 °C and6.0±8.5 respectively.

The addition of a polyurethane support for the at-tachment of bacteria (100 pores in)2, 15.5 pores cm)2;surface/volume ratio 500 m2/m3) prevented washout ofdechlorinating biomass and enabled chloroethene de-gradation (64% removal) at hydrolic retention times ofthe anoxic and oxic chemostats of 1.8 h.

Discussion

Microorganisms in anoxic soil slurries, obtained from aC2Cl4-contaminated location, readily dechlorinatedC2Cl4 and C2HCl3 when they were supplied with ap-propriate electron donors. This rapid dechlorination was

Fig. 5 Example of a mass-balance of C2Cl4 (PCE) mineralization bythe combined anoxic! oxic chemostats. VC C2H3Cl, cDCE cis-C2H2Cl2, TCE C2HCl3

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not unexpected since dechlorination products, C2HCl3,C2H2Cl2, C2H3Cl and ethene, were detected in thegroundwater at the sampling site, indicating in situ ac-tivity of dechlorinating microbes. The fact that additionof an electron donor and mineral nutrients was requiredto obtain measurable dechlorination suggests that thein situ activity of dechlorinating anaerobes is limited by(one of) these compounds.

It is not clear why slurries supplied with methanoldechlorinated C2Cl4 only to cis-C2H2Cl2 whereas, withformate or L-lactate, complete dechlorination to ethenewas obtained. Weekly additions of additional methanolensured that limiting availability of an electron donorwas not the reason for the lack of complete dechlori-nation. Possibly methanol-utilizing cis-C2H2Cl2-dechlo-rinating bacteria were not present in the samples, orwere outcompeted for this electron donor by meth-anogens. Indeed, the dechlorinating anaerobic bacteriareported thus far use electron donors like L-lactate,formate, H2 and/or ethanol, but not methanol (DeWe-erd et al. 1990; Gerritse et al. 1996a,b; Holliger et al.1993; Scholz-Muramatsu et al. 1995; Utkin et al. 1994).Studies of Fennell et al. (1997) indicated that hydrogenwas the direct electron donor for PCE dechlorinators inan anaerobic mixed culture enriched on methanol. Theyobserved that, during growth on butyric or propionicacids, a low H2 partial pressure was created and de-chlorination was favoured over methanogenesis. Incontrast, ethanol and lactate were oxidized at higher H2

concentrations, which stimulated methanogenesis andresulted in less complete C2Cl4 dechlorination. Compe-tition for H2 was also suggested to dictate the ¯uxof electrons to dechlorinating, sulphate-reducing ormethanogenic bacteria in mixed cultures transforming3-chlorobenzoate or chlorophenols (Dol®ng and Tiedje1991; in Dol®ng and Beurskens 1995). The observedin¯uence of the electron donor on dechlorination is in-teresting, since the use of methanol might provide ameans to direct (in situ) dechlorination to cis-C2H2Cl2,thus reducing accumulation of more toxic and volatileC2H3Cl. Alternatively, more complete dechlorination ofC2Cl4 to ethene may be obtained with electron donorslike formate or lactate, which would avoid the need foran oxic step for further degradation of partially de-chlorinated intermediates.

In some investigations it was demonstrated that thecomplete dechlorination of C2Cl4, all the way to etheneor ethane, can be achieved in anoxic soil columns or inlaboratory-scale ®xed-bed or up¯ow reactors (Ball-apragda et al. 1995; De Bruin et al. 1992; Vogel andMcCarty 1985; Wild et al. 1995; Wu et al. 1995). How-ever, complete dechlorination in these reactors was onlyobtained at relatively low rates, typically in the range2.6±9.1 lmol l)1 h)1 (Table 1). In anoxic systems withhigher C2Cl4 loads, dechlorination appears to be an in-complete process usually yielding cis-C2H2Cl2 and/orC2H3Cl as end-products (Carter and Jewell 1993; Fogelet al. 1995; Gerritse et al. 1995; Guiot et al. 1995). Thesepartially dechlorinated products can be co-metabolized

by aerobic bacteria. Methanotrophic co-metabolic con-version of chloroethenes has appeared unstable partic-ularly at high loading rates (Gerritse et al. 1995).However, toluene- or phenol-induced co-metabolism is aconsiderably more stable process than methanotrophy(Folsom and Chapman 1991; Landa et al. 1994). In ourstudy, stable, complete mineralization of C2Cl4 could bemaintained for more than 6 months in sequentiallycoupled anoxic and oxic chemostats under variousprocess conditions. Relatively high concentrations ofC2Cl4 (above 500 lM) were degraded at overall hy-draulic retention times of less than 3 h. With C2Cl4loads below 60 lmol l)1 h)1, the residual concentrationsof C2Cl4, C2HCl3, cis-C2H2Cl2 and phenol in the che-mostat e�uent, after anoxic! oxic treatment, were inthe same range or below the Dutch intervention valuesfor groundwater pollution, i.e. 40 lg l)1 (C2Cl4), 500lg l)1 (C2HCl3), 1300 lg l)1 (cis-C2H2Cl2) and 2000lg l)1 (phenol) respectively (VROM Leidraad bodem-sanering 1990). However, for C2H3Cl, with a very lowintervention value of 0.7 lg l)1, residual concentrationsremained 20±750 times too high. This indicates that itmay be more important to avoid anaerobic formation ofthis intermediate than to improve its aerobic degrada-tion. Aerobic bacteria that can use C2H3Cl as a growthsubstrate may be more competent degraders of thisproduct than C2H3Cl- co-metabolizing aerobes growingon phenol. Although aerobic batch enrichments onC2H3Cl were obtained, an active population of bacteriagrowing on vinyl chloride apparently did not establish inour oxic chemostat. Studies by Hartmans and De Bont(1992) suggest that the maximum speci®c growth rate(lmax) of C2H3Cl-using bacteria like Mycobacteriumaurum �lmax � 0:03±0:06 hÿ1� may be too low to main-tain a C2H3Cl- using population in chemostats operatedat relatively high dilution rates.

The results demonstrate that the performance of thepresent two-step system compares favourably with pre-viously used completely anoxic reactors and also se-quentially coupled anoxic/oxic (methanotrophic) reactorsystems (Table 1).

The following observations indicate that C2Cl4 de-chlorination in the anoxic chemostat was catalysedmainly by specialized dechlorinating bacteria, likeDesul®tobacterium spp., Dehalobacter restrictus or De-halospirillum multivorans that can use C2Cl4 or C2HCl3as electron acceptors for growth (dehalorespiration)(Gerritse et al. 1996a; Scholz-Muramatsu et al. 1995;Holliger et al. 1993), and not by C2Cl4-co-metabolizinganaerobes such as methanogens or acetogens (Egli et al.1988; Fathepure et al. 1987; Terzenbach and Blaut 1994).

1. The speci®c rate of dechlorination in the anoxicchemostat, up to 145 nmol min)1 mg)1 protein, wasin the same range as found for pure cultures of C2Cl4-respiring anaerobes (Gerritse et al 1996; Holliger etal. 1993; Neumann et al. 1994), which is more than10 000-fold higher than values reported for co-meta-bolic dechlorination.

559

Table1Someexamplesoftetrachloroethenedegradationinvarioustypesofbioreactor.t H

Rhydrolicretentiontime;Vmaxmaximumvolumicrateofchloroethenetransform

ation;Qmax

maximumspeci®crateofchloroethenetransform

ation;VSSvolatilesuspended

solids;VFAvolatileorganicacids;NDnotdetermined.Thereactionsshowthemajorproductsdetected.

E�ciency

100�

R(chloroethenesin¯uent )

chloroethenee�uent )/R(chloroethenesin¯uent ).Sequentialmineralization

Rchloroethenemineralizationovertotalanoxic+

oxicreactorvolume

Reactor

Conditions

Tem

p.

t HR

Substrate(s)

Reaction(s)

Vmax

Qmax

E�ciency

Reference

system

(°C)

(h)

!products

(lmoll)1h

)1)[lmolh

)1

(gVSS))1]

(%)

Fluidized-bed

Anoxic

15

1.8

Sucrose!

VFACH

4C2Cl 4!

C2H

3Cl

39.9

1.34

98.8

CarterandJewell1993

Fluidized-bed

Anoxic

20±22

24

Canemolasses

VFA

C2Cl 4C2HCl 3!

C2H

49.1

0.19

>99:9

Wuetal.1995

Fluidized-bed

Anoxic

35

12

Lactate

C2Cl 4!

C2CH

3ClC2H

47.9

ND

ND

Ballapragdaetal.1995

Fixed-bed

Anoxic

20

48

Acetate

C2Cl 4!

C2HCl 3C2H

2Cl 2C2H

3Cl

CO2

2.58

ND

99.98

Vogeland

McC

arty1985

Fixed-bed

Anoxic

10±20

2.4

Lactate

C2Cl 4!

C2H

43.7

ND

>95

DeBruin

etal.1992

Fixed-bed

Anoxic

20

35.6

Glucose!

VFACH

4C2HCl 3!

C2H

46.2

ND

>99:9

Wildetal.1995

Fluidized-bed

Anoxic/oxic

35

6:2�1

Sucrose

methanolVFA

C2Cl 4!

C2HCl 3cis-C2H

2Cl 2

15.2

2.36

94

Guiotetal.1995

Sequential

mineralization

C2Cl 4!

Clÿ

15.1

0.78

31

Fixed-bed

Anoxic

20

2±8

Methanol!

CH

4C2Cl 4!

C2H

2Cl 2C2H

3ClC2H

460.3

ND

ND

Fogeletal.1995

!oxic

8CH4H2O2

cis-C2H

2Cl 2C2H

3Cl!

ND

1.0

ND

>60

Sequential

mineralization

16

C2Cl 4!

ND

0.5

Fixed-bed

Anoxic

18±20

6.9

Form

ate!

CH

4C2Cl 4!

cis-C2H

2Cl 2C2H

3Cl

7.4

ND

>99:6

Gerritseetal.1995

!oxic

55.6

CH4

cis-C2H

2Cl 2C2H

3Cl!

Cl)

1.1

ND

ND

Sequential

mineralization

111.2

C2Cl 4!

Clÿ

0.23

99

Fixed-bed

Anoxic

22

37.5

Acetate

C2Cl 4!

C2HCl 3

cis-C2H

2Cl 2

2.8

ND

79.8

Fathepure

andVogel

1991

!oxic

2.24

Glucose

H2O2

C2HCl 3!

CO2ND

ND

ND

68

Sequential

mineralization

39.7

96

Chem

ostat

Anoxic

20

4.2

Form

ateglucose!

VFA

C2Cl 4!

cis-C2H

2Cl 2C2H

3Cl

140.8

4695a

88.3

Thisstudy

!oxic

2.6

Phenol

cis-C2H

2Cl 2C2H

3Cl!

Clÿ

66.9

508a

66.7

Sequential

mineralization

7.9

C2Cl 4!

Clÿ

43.4

474a

60

aAssumingthatcellsconsisted

for60%

(w/w)protein

560

2. After the chemostat had been operated for severalvolume changes without C2Cl4, resumption of re-ductive dechlorination showed a lag of several daysupon re-addition of C2Cl4 to the in¯uent medium.Apparently the dechlorinating population had beenpartly washed out from the chemostat in the absenceof C2Cl4.

3. Cell counts showed that a considerable fraction of theviable anaerobic population (6%) consisted ofdechlorinating bacteria, and a C2Cl4 to cis-C2Cl4-dechlorinating Desul®tobacterium sp. was isolatedfrom the highest-dilution tubes. Some properties ofthis novel anaerobe were presented recently (Gerritseet al. 1996b).

The fact that reductive dechlorination was not in-hibited by sulphate, whereas strong inhibition did occurafter addition of nitrate, may suggest that dehalore-spiring bacteria were outcompeted by denitri®ers, butnot by sulphate reducers. An alternative explanationcould be switch-over from use of C2Cl4 to nitrate by thedechlorinating bacteria. Indeed, some reductively de-chlorinating bacteria can also use nitrate as an alterna-tive electron acceptor (Christiansen and Ahring 1996;Scholz-Muramatsu et al. 1995; Utkin et al. 1994). Fi-nally, inhibition due to the formation of NOÿ2 mightcause an even more direct explanation for interferencewith reductive dechlorination. The presence of alterna-tive electron acceptors like oxygen, nitrate or sulphategenerally appears to suppress reductive dechlorination(Bagley and Gossett 1990; Dol®ng and Beurskens 1995).Nevertheless, reductive dechlorination can occur under(micro-)oxic conditions (Enzien et al. 1994; Gerritse andGottschal 1992; Van den Tweel et al. 1987). Detailedstudies on pure cultures of dechlorinating, and de®nedmixed cultures of dehalorespiring and non-dechlorina-ting anaerobes, grown in the presence of mixtures ofvarious electron acceptors, are much needed to improveour understanding of dehalorespiring bacteria and tocontrol their dechlorination and competitiveness betterunder in situ conditions.

Acknowledgements This work was ®nanced by NOVEM B.V.(project no. 351710/1110) and grants from Nedlin Groep B.V. andTauw milieu B.V.

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