Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture 225

1058-8337/02/$.50© 2002 by CRC Press LLCBioremediation Journal 6(3):225–236 (2002)

Effect of Nitrate and Sulfate on Dechlorination bya Mixed Hydrogen-Fed Culture

D. K. Nelson, R. M. Hozalski, L. W. Clapp, M. J. Semmens, and P. J.NovakThe Department of Civil Engineering, University of Minnesota, 122 Civil Engineering Building, 500Pillsbury Drive S.E., Minneapolis, MN 55455–0220

Abstract: A novel hollow-fiber membrane remediation technology developed in our laboratory for hydrogendelivery to the subsurface was shown to support the dechlorination of perchloroethene (PCE) to cis-dichloroethene.In previous research, the presence of nitrate or sulfate has been observed to inhibit biological reductive dechlo-rination. In this study hollow-fiber membranes were used to supply hydrogen to a mixed culture to investigatewhether adequate hydrogen could be added to support dechlorination in the presence of alternative electronacceptors. By continuously supplying hydrogen through the membrane, the hydrogen concentrations within thereactor were maintained well above the hydrogen thresholds reported to sustain reductive dechlorination. It washypothesized that by preventing nitrate and sulfate reducers from decreasing hydrogen concentrations to below thedehalorespirer threshold, the inhibition of PCE dechlorination by nitrate and sulfate might be avoided anddechlorination could be stimulated more effectively. Enough membrane-fed hydrogen was supplied to completelydegrade the alternative electron acceptors present and initiate dechlorination. Nevertheless, nitrate and sulfateinhibited dechlorinating activity even when hydrogen was not limiting. This suggests that competition forhydrogen was not responsible for the observed inhibition. Subsequent microcosm experiments demonstrated thatthe denitrification intermediate nitrous oxide was inhibitory at 13 µM.

Key Words: dechlorination, hollow-fiber membranes, hydrogen, nitrate, sulfate, electron donor.

Introduction

The contamination of aquifers with chlorinated solventssuch as perchloroethene (PCE) has emerged as a majorproblem over the last few decades. One technique forremediating these contaminated aquifers is in situ anaero-bic biological reductive dehalogenation. Indigenous mi-croorganisms are capable of mediating the reduction ofchlorinated solvents such as PCE via either a gratuitousprocess or via a process termed dehalorespiration, wheremicroorganisms are able to conserve the energy pro-duced during PCE reduction (Holliger, 1993; Neumannet al., 1994). The reduction of chlorinated solvents isoften limited in situ by a lack of electron donor. Basedon this concept, several technologies have been used tosupply excess electron donor to contaminated sites tostimulate biological reductive dehalogenation. Theseinclude the addition of fermentable substrates (Carr andHughes, 1998; Fennell et al., 1997; Fennell and Gossett,1998) and direct sparging of hydrogen gas (H2) (Hugheset al., 1997).

One novel technology that has been developedrecently is the use of gas-permeable hollow-fiber mem-brane modules to continuously supply H2 directly togroundwater (Fang et al., 2002; Muenzner et al., ac-cepted). Membrane modules can be designed to pro-vide a large surface area for passive gas transfer whilepresenting minimal resistance to hydraulic flow. Thisinnovative gas transfer technology overcomes the prob-lems of conventional gas transfer devices: inefficiency,nonuniform delivery, and gas loss, by dissolving H2

directly into the water without bubble formation (Côtéet al., 1988; Côté et al., 1989; Ahmed and Semmens,1992a). These membranes are capable of achieving100% gas transfer efficiency (Ahmed and Semmens,1992a; Ahmed and Semmens, 1992b). In addition, theresulting dissolved H2 concentration in the water canbe controlled precisely by adjusting membrane surfacearea and gas pressure (Semmens and Gantzer, 1993).Thus, gas-permeable membranes appear to be a suit-able method for providing H2 as an electron donor forin situ biological dehalogenation.

226 Nelson et al.

Because dehalorespirers use H2 as an electrondonor (DiStefano et al., 1992), in an anaerobic systemthey compete with H2-utilizing methanogens,homoacetogens, SO4

2– reducers, iron reducers, manga-nese reducers, and NO3

– reducers, all of which can alsouse H2 (Yang and McCarty, 1998). When designingremediation systems involving the application of elec-tron donors using any of the available technologies,adequate electron equivalents must be supplied to sup-port dechlorination while meeting the demands of otherelectron acceptors present, such as NO3

– and SO42–.

Therefore, if supplying H2 directly, more H2 is oftenneeded than is suggested by the stoichiometric require-ment for dehalogenation alone.

In addition to exerting an H2 demand, NO3– and

SO42– have been observed to inhibit dechlorination

such that their complete removal is required prior tothe onset of reductive dechlorination. The H2 thresh-olds for NO3

– and SO42– reduction are lower than the

H2 threshold for dehalorespirers (Table 1). Therefore,some researchers have speculated that NO3

– and SO42–

reducers can outcompete dehalorespirers for reduc-ing equivalents under H2-limited conditions. Thisresults in apparent inhibition of dechlorination untilthese alternative electron acceptors are degraded(Lovley et al., 1994; Chapelle et al., 1996). On theother hand, Townsend and Suflita (1997) have shownthat for one dehalorespirer, Desulfomonile tiedjei,SO4

2– inhibits dehalogenase activity, serves as a pref-erential electron acceptor, and represses dehalogenasesynthesis. Furthermore, products of NO3

– reduction(e.g., N2O) have been shown to inhibit the

methanogens Methanosarcina barkeri strainMS, Methanobacterium bryantii strain Bab1,Methanobacterium thermoautotrophicum, andMethanobacterium formicicum (Balderston, 1976;Kluber, 1998). When supplied with H2 as an electrondonor, M. barkeri strain MS retained about 10% ac-tivity at N2O concentrations of 950 µM; CH4 produc-tion by M. bryantii was completely inhibited at N2Oconcentrations >95 µM (Kluber, 1998). Methano-genesis was completely inhibited in systems contain-ing Methanobacterium thermoautotrophicum andMethanobacterium formicicum supplied with H2 andas little as 0.004 atm (8 µM) N2O (Balderston, 1976).It is possible that these products also inhibit dechlo-rination. Thus, it remains unclear whether NO3

– andSO4

2– inhibit PCE transformation by driving H2 con-centrations below the dehalorespirer threshold or viaother mechanisms. These might include preferentialNO3

– or SO42– utilization and direct or indirect NO3

–

or SO42– inhibition of dehalogenases.

Delivering H2 via hollow-fiber membranes hasbeen shown to support biological reductive dechlori-nation (Muenzner et al., accepted). To further evaluatethe potential of this new technology to stimulate in situPCE biodegradation, the ability of membranes to de-liver adequate H2 to support dechlorination in the pres-ence of alternative electron acceptors was investigated.Furthermore, the hypothesis that dechlorination is in-hibited in the presence of NO3

– or SO42– due to the

suppression of H2 below the dechlorinator thresholdwas tested by using membranes to maintain constant,nonlimiting dissolved H2 concentrations.

Table 1. H2 Concentration Thresholds for Various Anaerobic Microbial Processes

Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture 227

Materials and Methods

CulturesA dechlorinating culture was started by adding 100 gof trichloroethene (TCE)-contaminated Cape Canaveralaquifer material to a reduced medium (Table 2). Thereduced medium was prepared by adding all of theconstituents except Na2S·9H2O, boiling, and then cool-ing the medium under N2. The Na2S·9H2O was thenadded, the pH adjusted to 7.2 with 40% H3PO4, and themedium was filter sterilized through 0.2-µmpolyethersulfone filters in an anaerobic glovebag.

The dechlorinating culture was maintained underan H2 headspace in a 2-L fill-and-draw reactor. H2 wasadded to the reactor three times per week and PCE wasadded to a target concentration of 30 µM. When deg-radation of PCE to cis-dichloroethene (cis-DCE) wascomplete, PCE was respiked to the target concentra-tion. Ten percent of the medium was replaced weekly.

An NO3– and SO4

2– reducing (NSR) culture was startedby adding 150 g TCE-contaminated Cape Canaveral aqui-fer material to an NO3

– and SO42– containing (NS) medium

(with 640 µM NO3– and 850 µM SO4

2–; Table 2). Toremove oxygen, the medium was cycled through an anaero-bic glovebag degassing chamber three times, vacuumed to20 in Hg and then flushed with N2 or 93%/7% N2/H2. Themedium was then filter-sterilized through 0.2-µmpolyethersulfone filters in the anaerobic glovebag.

The NSR culture was maintained in a second 2-Lfill-and-draw reactor in a manner similar to that for thedechlorinating culture. The only differences were thatthe PCE target concentration was 12 µM and 20% ofthe medium was replaced weekly.

Batch Experiments Employing Hollow-Fiber Membrane BioreactorsThe experiments were performed in semibatch reac-tors fabricated from widemouth 460-mL glass jars

Table 2. Comparison of Medium Compositions

228 Nelson et al.

fitted with Teflon®-lined screw-on lids. FiveSwagelok™ stainless steel bulkhead fittings wereplaced through the lids to provide ports for inlet andoutlet gas flow, liquid sampling, and the installation ofan oxidation reduction potential (ORP) probe (Figure1). Stainless steel washers and Viton™ o-rings pro-vided a gas-tight seal at each bulkhead fitting.Mininert™ valves were used to sample both the liquidand the outlet gas (off-gas). The hollow-fiber mem-branes were threaded through 1-mm guide holes drilledin Teflon® rods suspended from the lid. Approximately3.5 m of microporous polyethylene hollow-fiber mem-brane (340-µm ID, 390-µm OD; Mitsubishi-Rayon,NY) were used to supply H2 or N2 to the reactors. Thereactors were stirred and behaved as completely mixedreactors.

The experiments were performed using four par-allel reactors inoculated with equal masses (as volatilesuspended solids of washed NSR culture and dechlo-rinating culture. The cultures were washed to removenutrients and dechlorination byproducts prior to inocu-

lation by centrifuging an aliquot of the culture at 350× g for 7 min and resuspending the pellet in freshmedium three times. The initial biomass in each of thereactors was approximately 65 mg/L as volatile sus-pended solids, 4 × 109 cells/mL, as determined by theacridine orange direct count method. Three of the re-actors were supplied with H2 via hollow-fiber mem-branes (1%, 50%, or 100%), and the fourth reactorreceived 100% N2 (control). The reactors were oper-ated in a headspace-free manner and contained 460 mLof NS medium (Table 2). Because the hollow-fibermembranes allowed gas transfer both into and fromthe water, volatile compounds (such as methane (CH4))were slowly stripped and removed with the off-gas.Therefore, the off-gas from each reactor was analyzedfor volatile compounds. Liquid samples were analyzeddaily to determine aqueous PCE, TCE, and cis-DCEconcentrations. When PCE concentrations declinedbelow 0.6 µM, neat PCE was added to each reactor toa target concentration of 6 µM. NO3

–, NO2–, and SO4

2–

analyses were performed three times per week. H2 and

FIGURE 1. Semi-batch membrane reactor. Reactors were fitted with stainless steel bulkheadsfor sampling and an ORP probe for in-place measurement of oxidation-reduction potential. Offgas went to a gas mixing vessel and then to an in-line H2 detector.

Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture 229

CH4 analyses were performed weekly. The pH of thereactors was measured three times per week and ad-justed with 2 M H3PO4 when necessary to maintain apH of 7.0. NaHCO3 was added to each reactor once perweek to maintain a CO2 concentration of approxi-mately 3 mM to compensate for CO2 removed viastripping and microbial utilization.

Microcosm ExperimentsA batch microcosm experiment was performed toinvestigate whether acetate could stimulate dechlo-rination in the presence of NO3

– and SO42–. Four

140-mL serum bottles were filled with 50 mL of theNS medium, to which approximately 200 mg/L ofacetate was added. The headspace of two of themicrocosms was flushed with H2 and two wereflushed with N2. PCE was added to a concentrationof 29 µM. The bottles were inoculated with thedehalorespirer stock culture that had been centri-fuged and rinsed three times (approximately 109

cells/mL) and were incubated upside down on aplatform shaker (120 rpm) at room temperature. NO3

–,SO4

2–, acetate, PCE, TCE, and cis-DCE were mea-sured every 2 to 3 days.

An additional batch microcosm experiment wasconducted to investigate whether N2O could directlyinhibit dechlorination. Six 140-mL serum bottles werefilled with 50 mL of the reduced medium. Theheadspace of each bottle was flushed with H2 and29 µM PCE was added. The bottles were inoculatedwith the washed dechlorinating stock culture (3 × 109

cells/mL), sealed with Teflon®-lined stoppers and alu-minum crimp caps and incubated upside down on aplatform shaker (120 rpm) at room temperature. PCE,TCE, and cis-DCE were measured three times perweek. When the cultures began to dechlorinate PCE,2.0% N2O was added as a gas to the microcosmheadspace of two bottles and 0.5% N2O was added to2 others, corresponding to equilibrium aqueous-phaseconcentrations of 400 µM and 100 µM, respectively.

Analytical MethodsPCE, TCE, and cis-DCE. PCE, TCE, and cis-DCEwere analyzed using a gas chromatograph (GC)(Hewlett Packard 6890 series) equipped with a pho-toionization detector. Headspace samples (0.25 mL)were injected onto an HP-5 capillary column (0.53mm × 30 m). The carrier gas flow (ultra pure he-lium) was 11.0 mL/min. Method detection limits forPCE, TCE, and cis-DCE were 0.0043, 0.0044, and0.0036 µM, respectively (Standard Method 1030C;APHA, 1998). External GC calibration standardsfor PCE, TCE, and cis-DCE were prepared gravi-metrically.

Aqueous Phase CH4 and H2. Aqueous phase CH4 andH2 concentrations were measured with a GC equippedwith a pulse discharge helium ionization detector(Valco). Liquid samples of 1 mL were added to he-lium-purged 21-mL sealed vials for headspace analy-sis. Headspace samples were taken (1 mL at 2 psig)with a Tekmar 7000 headspace autosampler. Theanalytes were separated on HP-Plot Q (0.53 mm × 30 m)and HP-Plot Molecular Sieve 5A (0.53 mm × 30 m)PLOT columns connected by a column-switching dia-phragm valve (Valco). The carrier gas (helium) flowwas 6.0 mL/min. The method detection limits for dis-solved CH4 and H2 in the aqueous phase were 0.090and 0.13 µM, respectively. Aqueous CH4 and H2 cali-bration standards for headspace analysis were pre-pared using a gas-sampling tube (Fisher Scientific).

Gas-Phase H2. H2 concentrations in the reactor off-gaswere measured every 30 min using an in-line Qubit H2

detector (Qubit Inc., Kingston, Ontario) interfaced with acomputer via LABVIEW software (National Instruments).Because the H2 detector required oxygen to function, amixing vessel was designed to blend the anoxic reactoroff-gas with air. The reactor off-gas flow and flow froman air tank were measured with variable-area flowmeters(Gilmont) and mixed in a gas-tight vessel (a wide mouth250-mL glass jar) before entering the Qubit H2 detector.Gas-phase H2 concentrations were calculated over timefrom a mass balance using the measured gas flow rates.The gas-phase detection limit for the Qubit H2 detectorwas approximately 0.0004 atm.

Anion Analysis. NO3–, NO2

–, SO42–, and acetate con-

centrations were measured using a Dionex DX-100 ionchromatograph (IC) equipped with IonPac AG4A andAS4A 4-mm columns and an anion micromembrane™eluent suppressor. Samples of 1 mL volume were fil-tered (0.45 µm) and then manually injected into a150-µL sample loop. Eluent (1.8 mM Na2CO3 and1.7 mM NaHCO3) was pumped at 2 mL/min throughthe column. The method detection limits for NO3

–,NO2

–, SO42–, and acetate were 0.145, 6.46, 0.281, and

6.64 µM, respectively.

Oxidation Reduction Potential. The ORP within eachreactor was measured using dedicated redox probes(Cole Parmer) interfaced with a pH/ORP controller.The probes were calibrated using a published protocol(ASTM Standard D1498-93; ASTM, 1989).

Biomass. Biomass was quantified by both volatilesuspended solids (Standard Method 2540 E; APHA,1998) and the acridine orange direct count method(Hobbie et al., 1977).

230 Nelson et al.

Results and DiscussionDechlorination Inhibition Associatedwith NO3

– and SO42–

Semibatch bioreactor experiments employing gas-per-meable membranes to supply H2 were conducted un-der both H2-limiting and non-H2-limiting conditions.This was done to determine whether competition forH2 resulted in the sequential reduction of NO3

– andSO4

2– prior to the onset of dechlorination. NO3–, NO2

–,and SO4

2– profiles are shown for the 100%, 50%, and1% H2-fed reactors in Figure 2. No NO3

– or SO42– loss

was observed in the 100% N2-fed reactor (results notshown). CH4 and TCE formation over time in themembrane off-gas is shown in Figure 3 for the 100%and 50% H2-fed reactors. No CH4 or TCE formationoccurred in either the 1% H2-fed reactor or the 100%N2-fed reactor (results not shown).

Sequential reduction of NO3– and SO4

2– was ob-served in the semibatch membrane reactors. CH4 wasproduced after Day 10 in both the 100% and 50% H2-fed reactors, coinciding with SO4

2– reduction (Figures2 and 3). When SO4

2– was completely removed fromthe 100% H2-fed reactor after Day 22, CH4 productionincreased dramatically and TCE formation began. Inthe 50% H2-fed reactor, CH4 and TCE production oc-curred prior to complete SO4

2– removal. These resultssuggested that methanogenesis and dechlorination werecompletely inhibited by NO3

– and somewhat inhibitedby SO4

2–. In addition, the ORP values in reactors fed100% H2 (Figure 4) clearly showed that reducing con-ditions were required before TCE formation occurred.Similar patterns of rapidly decreasing ORP prior toNO3

– reduction and between NO3– and SO4

2– reduc-tion, with stable ORP during NO3

– and SO42– reduc-

tion, were also observed in the 50% and 1% H2-fedreactors. This was corroborated in replicate experi-ments (data not shown).

Absence of H2 Limitation in 50% and100% H2-Fed ReactorsH2 was never depleted, and therefore never limiting, inthe medium of the 100% H2-fed reactor (Figure 5a).Excluding the first-day measurement, the average aque-ous-phase H2 concentration was 121.2 ± 27.0 µM (stan-dard deviation) when compared with a theoretical equi-librium H2 concentration (i.e., without biological H2

consumption) of 811 µM. H2 was detected in the reac-tor off-gas throughout the experiment at between 0.48atm and 0.99 atm (Figure 5b). Therefore, H2 availabil-ity did not limit PCE degradation or CH4 production inthe 100% H2-fed reactor.

H2 was also never depleted, and therefore neverlimiting, in the medium of the 50% H2-fed reactor(Figure 5a). The average aqueous H2 concentrationwas 43.9 ± 2.4 µM during NO3

– reduction and 4.3 ± 6.4 µMduring SO4

2– reduction and methanogenesis, whencompared with a theoretical equilibrium H2 concentra-tion (i.e., without biological H2 consumption) of 405 µM.The aqueous-phase H2 concentration did approach thedetection limit of 0.13 µM, dropping as low as 0.15 µMon Day 28. The H2 threshold for the dechlorinatingculture used in this experiment was not measured.Nevertheless, the dissolved H2 concentration in the50% H2-fed reactor was always two to three orders ofmagnitude greater than the reported dechlorination H2

threshold of 0.13 to 2 nM (Smatlak and Gossett, 1996;Yang and McCarty, 1998; Löffler et al., 1999). H2 inthe off-gas from the 50% H2-fed reactor was below thedetection limit during most of SO4

2– reduction andmethanogenesis (Figure 5b) when aqueous H2 concen-trations were below 5 µM (Days 11 to 44, Figure 5a).NO3

– and SO42– reduction proceeded at a rate similar to

that in the 100% H2-fed reactor (Figure 2), furtherindicating that the 50% H2-fed reactor was not H2

limited.Extremely slow rates of NO3

– reduction were ob-served in the 1% H2-fed reactor (Figure 2) with no H2

detected in the off-gas or in the reactor liquid through-out the entire experiment, suggesting H2-limited con-ditions. Without the biological consumption of H2, theaqueous phase concentration should have been 8.11 µMat equilibrium. No degradation of NO3

– or SO42– was

observed in the control reactor fed 100% N2 (resultsnot shown).

The preceding semibatch reactor results indicatedthat H2 could be supplied via the hollow-fiber mem-branes at rates sufficient to deplete alternative electronacceptors and initiate dechlorination within a shorttime period (approximately 10 days). The rates ofNO3

–, NO2–, and SO4

2– reduction were dependent onthe rate of H2 delivery when H2 was limiting; when H2

was present in excess, however (as in the 100% and50% H2-fed reactors), sequential NO3

– and SO42– re-

duction occurred rapidly and was presumably limitedby intrinsic biodegradadation kinetics. Again, similarresults were observed in replicate experiments with50% H2-fed, 1% H2-fed, and 100% N2-fed reactors.

Dechlorination Inhibition by N2OThe semibatch reactor results also indicated that PCEdechlorination was inhibited in the presence of NO3

–

and SO42–. Furthermore, this inhibition was not attrib-

utable to denitrifiers and sulfidogens driving H2 con-

Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture 231

FIGURE 2. Sequential reduction of NO3- (�), NO2

- (�), and SO42- (�) in (a) 100% H2-fed, (b) 50% H2-

fed, and (c) 1% H2-fed reactors.

232 Nelson et al.

FIGURE 3. CH4 (�) and TCE (�) production over time in (a) 100% H2-fed and (b) 50% H2-fed reactorsas measured in the reactor off-gas.

Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture 233

centrations below the 0.13 to 2 nM threshold fordechlorination. One potential explanation was thatNO3

– and SO42– reduction byproducts inhibited dechlo-

rination. For example, N2O is an intermediate in thebiological reduction of NO3

–, and has been shown toinhibit the methanogens Methanosarcina barkeristrain MS, Methanobacterium bryantii strain Bab1,Methanobacterium thermoautotrophicum, andMethanobacterium formicicum (Balderston, 1976;Kluber, 1998).

To test this hypothesis, a microcosm experi-ment was performed. Aqueous N2O concentrationsof 100 µM completely inhibited dechlorination (Fig-ure 6). Although this N2O concentration is roughly10 times greater than the maximum reported N2Oconcentration in groundwater (Mühlherr andHiscock, 1998, Ronen et al., 1988), much highertransient N2O concentrations may exist in ground-water. For example, Schalk-Otte and co-workers(2000) found that when feeding NO2

– and acetate toa culture of the denitrifier Alcaligenes faecalis, upto 160 µmol/L/h N2O was produced under transientstarvation conditions. Thus, the N2O inhibition mi-crocosm experiment suggested that NO3

– may in-hibit dechlorination in contaminated groundwatervia the production of N2O during denitrification.Although not evaluated experimentally, SO4

2– maysimilarly inhibit dechlorination via production ofHS–/H2S during sulfate reduction.

Other Potential Causes for ObservedDechlorination InhibitionOne alternative explanation for the observed lag priorto dechlorination in the semibatch membrane reactorexperiments is that the dechlorinating organisms tooklonger to acclimate than denitrifiers or sulfidogenswhen transferred to new growth medium. In a separatestudy performed in our laboratory with H2-suppliedsemibatch membrane reactors and the same dechlori-nating stock culture, 6-µM PCE was dechlorinated to4 to 5 µM cis-DCE after 10 days, with a first-order cis-DCE production rate of 0.65 day–1 when no competingelectron acceptors were present (Muenzner et al., ac-cepted). In the present study, although similar lag pe-riods were observed before dechlorination began, only0 to 0.006 µM TCE was produced after 10 days, witha first-order TCE production rate of 0.10 and 0.05 day–1

for the 100% and 50% H2-fed reactors, respectively(Figure 3). Therefore, although the role of acclimationto the new growth medium was not specifically inves-tigated in the present study, the dechlorinators failed todechlorinate PCE to the extent observed by Muenznerand co-workers (accepted) in very similar experimentsto which no NO3

– and SO42– were added. This suggests

that acclimation to the new growth medium after trans-fer from the stock culture was not the reason for theobserved inhibition.

Another potential explanation for the observed lagprior to dechlorination is that the dechlorinators actu-

FIGURE 4. ORP values over time in 100% H2-fed (�) and 100% N2-fed control (◊) reactors. Bracketsindicate where NO3

- and SO42- reduction occurred in the 100% H2-fed reactor. ORP trends for the 1%

H2-fed and 50% H2-fed reactors were very similar and are not shown for clarity.

234 Nelson et al.

FIGURE 5. H2 concentrations in the reactor (a) liquid and (b) off-gas for (◊) 50% H2-fed, and (�) 100% H2-fedreactor. The theoretical equilibrium liquid H2 concentrations for the 50% H2 and 100% H2 reactors were 405 µMand 811 µM, respectively, assuming no biological H2 consumption. Periods of NO3

- and SO42- reduction in both

reactors are indicated by brackets. The dissolved H2 concentrations and off-gas H2 concentrations for the 1% H2-fed reactor were always below the detection limits and are therefore not shown.

Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture 235

ally utilized acetate, produced by homoacetogens, astheir electron donor (Dollhopf and Löffler, 2000) andsufficient acetate concentrations did not immediatelydevelop in the semibatch reactors. Homoacetogenslikely competed for H2 and other nutrients in the 50%and 100% H2-fed reactors because the dissolved H2

concentrations were well above the threshold forhomoacetogenesis (Table 1). Additional microcosmexperiments, however, showed that the stock culturewas unable to dechlorinate PCE over an 11-day periodwhen NO3

– and SO42– were present and acetate was

supplied (data not shown). Thus, the possibility thatdechlorination was inhibited by the absence of acetatecan be ruled out.

Finally, the observed lag prior to dechlorinationwas possibly due to the higher ORP levels when NO3

–

and SO42– were present (Figure 4). Although the mi-

crocosm experiments indicated that high N2O concen-trations can inhibit PCE dechlorination, dechlorinationinhibition also coincided with high ORP values (Fig-ure 4). This indicates that the possibility that high ORPlevels inhibited dechlorination cannot be ruled out.Nonetheless, because high ORP values in the environ-ment are associated with the presence of NO3

– andSO4

2–, it can be concluded that there was a causalrelationship between the presence of NO3

– and SO42–

and inhibition of PCE dechlorination.

Environmental SignificanceThe results from this study clearly showed that ad-equate H2 can be delivered via hollow-fiber mem-branes to reduce alternative electron acceptors and

initiate dechlorination. NO3–, SO4

2– or their transfor-mation byproducts will inhibit PCE degradation whenpresent. The rate of H2 transfer using membranes canbe readily controlled by adjusting the surface area ofthe membrane, the H2 partial pressure in the feed gas,and the gas flow rate (Ahmed and Semmens, 1992;Semmens and Gantzer, 1993). Thus, it should be pos-sible to provide the reducing equivalents required toremove competing electron acceptors as well as PCE.Therefore, this technology appears to hold promise forremediating electron donor-limited sites that are con-taminated with chlorinated solvents.

AcknowledgmentsThis work was principally funded by the United StatesDepartment of Defense’s Strategic Environmental Re-search and Development Program, with additionalsupport provided by the University of Minnesota.

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