Microbial Degradation of Toluene under Sulfate-ReducingConditions and the Influence of Iron on the Process
HARRY R. BELLER,* DUNJA GRBIC-GALIC, AND MARTIN REINHARD
Environmental Engineering and Science, Department of Civil Engineering,Stanford University, Stanford, California 94305-4020
Received 9 August 1991/Accepted 17 December 1991
Toluene degradation occurred concomitantly with sulfate reduction in anaerobic microcosms inoculated withcontaminated subsurface soil from an aviation fuel storage facility near the Patuxent River (Md.). Similarresults were obtained for enrichment cultures in which toluene was the sole carbon source. Several lines ofevidence suggest that toluene degradation was directly coupled to sulfate reduction in Patuxent Rivermicrocosms and enrichment cultures: (i) the two processes were synchronous and highly correlated, (ii) theobserved stoichiometric ratios of moles of sulfate consumed per mole of toluene consumed were consistent withthe theoretical ratio for the oxidation of toluene to CO2 coupled with the reduction of sulfate to hydrogensulfide, and (iii) toluene degradation ceased when sulfate was depleted, and conversely, sulfate reduction ceasedwhen toluene was depleted. Mineralization of toluene was confirmed in experiments with [ring-U-_4Cltoluene.The addition of millimolar concentrations of amorphous Fe(OH)3 to Patuxent River microcosms andenrichment cultures either greatly facilitated the onset of toluene degradation or accelerated the rate oncedegradation had begun. In iron-amended microcosms and enrichment cultures, ferric iron reduction proceededconcurrently with toluene degradation and sulfate reduction. Stoichiometric data and other observationsindicate that ferric iron reduction was not directly coupled to toluene oxidation but was a secondary,presumably abiotic, reaction between ferric iron and biogenic hydrogen sulfide.
In 1986, the U.S. Environmental Protection Agency(EPA) estimated that 35% of the nation's underground motorfuel storage tanks were leaking (30). Together with surfacespill accidents and landfill leachate intrusion, such leakscontribute significantly to groundwater contamination bygasoline, aviation fuel, and other refined petroleum deriva-tives. Benzene and various alkylbenzenes (such as toluene)are relatively water-soluble constituents of gasoline andaviation fuel that present human health concerns. Thisarticle focuses on toluene, an EPA priority pollutant that isa depressant of the central nervous system (27). Toluene isless toxic than benzene (a confirmed carcinogen) and has an
EPA water quality criterion for the protection of humanhealth of 14.3 mg/liter or 0.155 mM (27).Toluene and other alkylbenzenes are readily degradable in
aerobic surface water and soil systems; however, in thesubsurface environment, contamination by organic com-
pounds often results in the complete consumption of avail-able oxygen by indigenous microorganisms and the develop-ment of anaerobic conditions. In the absence of oxygen,
degradation of toluene can take place only with the use ofalternative electron acceptors, such as nitrate, sulfate, or
ferric iron, or fermentatively in combination with methano-genesis. To date, degradation of toluene by aquifer-, sedi-ment-, or sewage-derived microorganisms has been foundunder denitrifying conditions (4, 6, 10, 11, 35, 36), fermen-tative and methanogenic conditions (8, 31, 33, 34), and ferriciron-reducing conditions (14, 15).
Despite recent research advances in the degradation oftoluene under various anaerobic conditions, we are aware ofvery few reports of toluene degradation under sulfate-reduc-ing conditions. Preliminary evidence of the degradation of
* Corresponding author.
786
alkylbenzenes under sulfate-reducing conditions has beenreported in studies of fuel-contaminated aquifer materialfrom Seal Beach, California (9), and in contaminated sub-surface materials collected in Oklahoma (26). More conclu-sive evidence of sulfidogenic toluene degradation at the SealBeach site is reported in this issue by Edwards et al. (5).Despite the limited study of the process, toluene degradationunder sulfate-reducing conditions could be relevant in sub-surface and waterlogged soil environments for the follow-ing reasons: (i) toluene oxidation coupled with sulfate reduc-tion is an exergonic reaction that could yield energy tobacteria (although other anaerobic electron acceptors, suchas ferric iron and nitrate, would yield considerably moreenergy than sulfate); (ii) sulfate is present at millimolarconcentrations in some subsurface environments (e.g., nearlandfills [12, 21, 28] and adjacent to coastal and estuarineareas); and (iii) various pure cultures of sulfate-reducingbacteria are capable of degrading a number of oxygen-containing aromatic compounds (e.g., 1, 3, 25, 29, 32), someof which are proposed intermediates in anaerobic toluenedegradation (e.g., p-cresol, benzoate, and 2- and 4-hydrox-ybenzoate [8, 11, 15]).
This article presents evidence that microflora enrichedfrom fuel-contaminated soil can degrade toluene under sul-fate-reducing conditions. In addition, the stimulation oftoluene degradation by amorphous ferric oxyhydroxide[amorphous Fe(OH)3] will be discussed. The microbiallymediated and abiotic interactions among toluene, sulfate,and ferric iron described in this article indicate thatgeochemical site conditions (e.g., oxidation-reduction poten-tial, the presence of sulfate, the presence of iron-containingminerals) are worthy of consideration when assessing thepotential for in situ biological restoration of aquifers contam-inated with refined petroleum products, such as gasoline andaviation fuel.
Construction of microcosms and enrichment cultures. Mi-crocosms and enrichment cultures were prepared understrictly anaerobic conditions in an anaerobic glove box (CoyLaboratory Products, Inc., Ann Arbor, Mich.). The micro-cosms and enrichment cultures were contained in amberglass, 250-ml, screw-cap bottles that were sealed withMininert polytetrafluoroethylene (PTFE) valves (Alltech As-sociates, Inc, Deerfield, Ill.); the Mininert valves provided a
tight seal for the bottles while allowing sampling of theheadspace and culture medium via syringe. The combinedvolume of medium and 30 g of wet solids (in microcosms) or
medium and culture inoculum (in enrichments) was 200 ml.The remaining volume of the bottles was headspace. Fivearomatic hydrocarbons (benzene, toluene, ethylbenzene,and o- and p-xylenes) were initially spiked into bottles as
pure liquids at concentrations in the range of 50 to 100 puMper compound. Xylenes were excluded from later studies.Sterile controls, which contained sediment or culture inoc-ulum, were removed from the glove box after being sealedand were autoclaved at 121°C. Replicate microcosms andcontrols were used routinely. After degradation had begun,regular respiking with toluene (typically 100 to 250 puM per
week) and sulfate was performed as necessary. Incubationwas carried out at 35°C in an anaerobic glove box.
(i) Growth medium. The composition of the growth mediumused for microcosms and enrichment cultures was based on
medium described by Lovley and Phillips (16). This mediumincluded the following compounds at the concentrations(millimolar) specified in parentheses: NaHCO3 (30), NH4Cl(28), NaH2PO4- H20 (4.4), NaCl (1.7), KCl (1.3), CaCI2.2H20 (0.68), MgCl2. 6H20 (0.49), MgSO4. 7H20 (0.41),MnCl2 4H20 (0.025), and Na2MoO4. 2H20 (0.004). Ahigher initial sulfate concentration (2 to 3 mM) was used inlater experiments. The medium (excluding NaHCO3) was
sterilized in an autoclave at 121°C for 20 min and then purgedwith an oxygen-free mixture of 79% N2-21% CO2 for 45 min.The medium was amended with NaHCO3 immediately afterbeing purged and was then flushed in the antechamber of an
anaerobic glove box. The final pH of the medium was
approximately 6.9.(ii) Amorphous Fe(OH)3. The initial goal of the research
described in this article was to enrich ferric iron-reducingmicrobial communities that could degrade monoaromatichydrocarbons. In order to enrich iron-reducing bacteria,soils were screened for hydrocarbon-degrading activity byusing a basal mineral medium that either was amended withferric iron or was not amended with significant concentra-tions of any potential electron acceptor. The phase of ironadded to microcosms and enrichment cultures was Fe(OH)3.Amorphous Fe(OH)3 was originally chosen rather than morecrystalline iron phases because the rate of biological ferriciron reduction tends to increase significantly with decreasingdegree of crystallinity (13). This iron phase was prepared byneutralizing a 0.1 M ferric chloride solution with sodiumhydroxide. The precipitate was aged for 4 h after neutrali-zation; during this period, the pH was adjusted periodicallywith sodium hydroxide to neutralize acidification resultingfrom ferric iron hydrolysis. The precipitate was then rinsedwith Milli-Q water in a 2-liter Buchner funnel to reduce theresidual chloride concentration to a level that would corre-
spond to less than 1 mM in a 200-ml microcosm or enrich-ment culture. The amorphous Fe(OH)3 was prepared withsterilized glassware and reagents prepared in sterilizedMilli-Q water, but the iron phase itself could not be auto-
claved because the elevated heat and pressure would facili-tate crystallization, which was not desired.
(iii) Soil inoculum. Patuxent River soil was collected at theNaval Air Station, Patuxent River, Md., in September 1987.The site was extensively contaminated with aviation fuel(e.g., JP-5). The sample was collected from a Quaternarystratum of an outcrop through which hydrocarbon-contami-nated groundwater was seeping. Exposed soil was removedbefore sampling. The soil was received in water-saturatedform and stored at 4°C until its use in microcosms roughly 2years after collection. The homogenized soil slurry used forinoculation contained approximately 7 ,umol of sulfate per g.
Analytical methods. (i) Aromatic hydrocarbon analysis.Aromatic substrates were measured by a static headspacetechnique with an HP model 5890A gas chromatograph(Hewlett-Packard Company, Palo Alto, Calif.) with an HNUmodel PI 52-02A photoionization detector (10.2 eV lamp;HNU Systems, Inc., Newton, Mass.) and a DB-624 fusedsilica capillary column (30 m length by 0.53 mm insidediameter, 3.0-,um film thickness; J & W Scientific, Folsom,Calif.). Analyses were isothermal (65°C) with splitless injec-tion (the split was turned on after 0.5 min). Sampling andanalysis of headspace from microcosms, enrichment cul-tures, and standards were performed identically: 300 ,ul ofheadspace was sampled through the Mininert valve of mi-crocosms, enrichment cultures, or standards with 500-pugas-tight syringe that included a PTFE plunger tip and aside-port needle.The aqueous concentration of aromatic compounds in
microcosms and enrichment cultures was determined bycomparing peak areas in samples with those of externalstandards. Standards for headspace analyses were preparedby spiking a methanolic stock solution of the aromaticcompounds into a Mininert-sealed bottle that contained 200ml of water. The transfer of the methanolic stock solutionwas made with a gas-tight 500-ptl syringe. The amount ofstock solution (and correspondingly, the mass of each com-pound) added to the standard bottle was determined gravi-metrically by weighing the syringe immediately before andafter spiking. The aqueous concentrations of aromatic com-pounds in standards were calculated by using Henry's Lawconstants (25°C) obtained from the literature (18), whichwere empirically corrected for the incubation temperature of35°C, and a mass balance expression.A potential source of systematic error in the headspace
analyses was the presence of methanol in the standards.However, it was assumed that the presence of methanol atthe applicable methanol concentrations would not signifi-cantly affect the gas-liquid partitioning of the compounds inthe standards; this assumption was based on detailed studiesof Henry's Law constants of other volatile organic com-pounds that entailed spiking with methanolic stock solutions(7). Another potential source of systematic error was thesorption of toluene to solids. Controlled sorption experi-ments were not performed to quantify this effect. However,the sorption of toluene would be of concern only for micro-cosms; in enrichment cultures, the solid/liquid (mass/mass)ratio was on the order of 0.03 or less and could be expectedto result in a very small mass of sorbed toluene relative tothe mass of dissolved toluene. Mass balances indicated thatthe effect of all observed losses of toluene (including sorp-tion to solids) on aqueous concentrations in enrichmentcultures would be within the range of analytical error (i.e.,within 10%). Thus, quantitative data (e.g., stoichiometricratios) in this article focus on enrichment cultures ratherthan microcosms.
(ii) Fe(II) analysis. HCl-soluble Fe(II) was measured asdescribed by Lovley and Phillips (17). Briefly, this methodinvolved the dissolution of 0.1 to 0.2 g of culture medium in0.5 M HCI, the use of a spectrophotometric reagent forferrous iron (ferrozine), and measurement of theA.62 with anHP model 8451A diode array spectrophotometer (Hewlett-Packard Company). An advantage of first dissolving theculture medium in HCI was that Fe(II) in poorly crystallineprecipitates (such as amorphous ferrous sulfide) could bedetermined in addition to soluble Fe(II), which was impor-tant because precipitates were the predominant Fe(II)phases in the systems studied. Standards were prepared bydissolving a known amount of ammonium iron(II) sulfatehexahydrate (stored in the dark in an anaerobic glove box) in0.5 M HCI.
(iii) Sulfate analysis. Sulfate in filtered culture medium wasdetermined by ion chromatography (Dionex Series 4000iwith a Nelson Analytical Chromatography Software system)equipped with an HPIC-AS4A column (Dionex Corporation,Sunnyvale, Calif.), an anion micro membrane suppressor,and a conductivity detector. Analyses were isocratic, with a0.75 mM sodium bicarbonate-2.2 mM sodium carbonateeluant flowing at a rate of 2 ml/min. Ions were identified andquantified by comparing retention times and peak areas,respectively, with those of external standards.
(iv) 14C analysis. 14C-labeled toluene was fed to selectedenrichment cultures to investigate the extent of toluenemineralization. For spiking into samples, [ring-U-14C]tolu-ene (>98% purity; 10.2 mCi/mmol; Sigma Chemical Co., St.Louis, Mo.) was diluted into unlabeled toluene (>99.9%purity; Aldrich Chemical Co., Inc., Milwaukee, Wis.) to afinal specific activity of 67.5 ,uCi/mmol. 14C activity inculture liquid and headspace samples was analyzed with aTri-Carb model 4530 liquid scintillation system (PackardInstruments Co., Inc., Downers Grove, Ill.). All sampleswere automatically blank-corrected and were corrected forsample-specific quenching by using an external standardmethod (with a 226Ra gamma source) and a quenching curvedeveloped from a series of quenched standards.Sample processing methods were similar to those de-
scribed by Grbi6-Galic and Vogel (8). Three 1-ml samples ofculture liquid were collected and subjected to differenttreatments. One 1-ml sample of culture liquid was collectedwith a 1-ml gas-tight syringe and was injected into a vialcontaining 10 ml of Universol scintillation fluid (ICN Bio-medicals, Inc., Irvine, Calif.) and 0.6 ml of 1 N NaOH; thissample represented the sum of '4C activity from all possiblespecies in the culture liquid (i.e., toluene, volatile interme-diates, C02, nonvolatile intermediates, and biomass). An-other 1-ml sample was injected into 0.6 ml of 1 N NaOH andwas purged with N2 for 15 min, after which 10 ml ofscintillation fluid was added. This sample represented thesum of 14C activity of CO2, nonvolatile intermediates, andbiomass. A third 1-ml sample was injected into 0.6 ml of 1 NHCl, purged with N2 for 15 min, and mixed with 10 ml ofscintillation fluid. This fraction represented the 14C activityof nonvolatile intermediates and biomass. Three 1-ml sam-ples of headspace were treated analogously, except that 11ml of scintillation fluid was used rather than 10 ml. 14CO2was calculated as the difference between the 14C activities ofthe purged NaOH-treated and purged HCl-treated samples.Volatile 14C (other than 14C02) was calculated as the differ-ence between the unpurged NaOH-treated and purgedNaOH-treated samples.
Experimental design. The two microcosm experimentsperformed with Patuxent River material (experiments Ml
and M2) were originally attempts to screen for activity ofiron-reducing bacteria. Briefly, the experimental design con-sisted of setting up parallel series of microcosms, one withand one without added amorphous Fe(OH)3. The initialconcentration of iron differed in the two experiments: thefirst experiment (Ml) involved ca. 12 mM Fe(III), and thesecond experiment (M2) involved ca. 20 mM Fe(III). Enrich-ments of certain iron-amended microcosms were prepared inan anaerobic glove box by shaking the microcosm, removing20% (by volume) of the combined liquid and solids, addingthis inoculum to a new bottle, and diluting to 200 ml withfresh medium. Series of enrichment cultures were preparedafter 88 and 235 days of incubation of selected microcosmsfrom experiment M2; these enrichment series will be re-ferred to as experiments El and E2, respectively. Secondaryenrichments (experiment E3) were prepared after 119 daysof incubation of an enrichment culture from experiment E2.Iron was added over time to certain enrichment cultures atconcentrations of ca. 2 to 4 mM per addition. Xylenes wereexcluded from experiments E2 and E3.
RESULTS AND DISCUSSION
Microcosms containing Patuxent River material and en-richment cultures of these microcosms yielded analogousresults. The following trends were apparent in both micro-cosms and enrichment cultures: (i) toluene degradation,sulfate reduction, and ferric iron reduction (in systemsamended with ferric iron) proceeded concomitantly at ratesthat were highly correlated with each other; (ii) the presenceof amorphous Fe(OH)3 enhanced toluene degradation; (iii)degradation of other aromatic hydrocarbons added to thesystems (benzene and ethylbenzene) was not observed; (iv)toluene degradation, sulfate reduction, and ferric iron reduc-tion were not observed in sterile controls. In this section,data presented for microcosms will focus on the stimulationof toluene degradation by iron, and data for enrichmentcultures will be used to explore quantitative relationshipsbetween toluene degradation, sulfate reduction, and ferriciron reduction. The quantitative discussion will focus onenrichment cultures rather than microcosms for two rea-sons: to minimize the potentially confounding effects of anyelectron donors or acceptors present in the native soil, andto minimize the potentially confounding sorptive effects ofsolids in the system.
Microcosms: toluene degradation and its apparent stimula-tion by iron. The results of the first microcosm experimentwith Patuxent River material (experiment Ml) are shown inFig. 1 in terms of aqueous toluene concentration versus timeover the first 2 months of incubation. The data in Fig. 1represent averages for three groups of microcosms: (i) twoautoclaved controls (one with added iron and one without),(ii) three microcosms without added amorphous Fe(OH)3,and (iii) four microcosms with added amorphous Fe(OH)3[ca. 12 mM Fe(III) on day 0]. No toluene degradation wasobserved in the controls. Although rates of toluene degrada-tion for microcosms with and without added iron were verysimilar for the first 3 to 4 weeks of incubation, the micro-cosms with added iron had faster toluene degradation ratesstarting on day 30. These differences in rates continuedthroughout the next 30 days of incubation. Note that themicrocosms with added iron received additional toluene onday 44, whereas those without added iron did not. The effectof the presence of iron on toluene degradation rate wasreproducible in this study. As an indication of the variabilityamong replicates, the average amount of toluene that had
Time (days)FIG. 2. Toluene versus time in individual Patuxent River micro-
cosms (experiment M2). Solid arrows represent amendments of ca.10 mM Fe(III). "With iron" represents microcosms with ca. 20 mMFe(OH)3 added on day 0; "without iron" represents microcosmsthat were not amended with Fe(OH)3.
0 10 20 30 40 50 60
Time (days)FIG. 1. Average toluene concentrations versus time in Patuxent
River microcosms (experiment Ml). The number of replicates isindicated parenthetically, and reproducibility is discussed in thetext. "With iron" represents replicates with ca. 12 mM Fe(OH)3added on day 0; "without iron" represents replicates that were notamended with Fe(OH)3. Arrows indicate times of toluene addition.
been degraded by day 37 in microcosms with added iron(0.248 + 0.0125 mM, mean + standard deviation [SD]) wassignificantly greater (P < 0.001) than the average amongreplicates without added iron (0.164 + 0.0156 mM). Suchsignificant differences continued after day 37. Enrichmentcultures also showed stimulation of toluene degradation inthe presence of iron, although the effect was more rapid(within days of iron addition) in enrichment cultures than inexperiment Ml microcosms. Both ferric and ferrous ironwere found to stimulate toluene degradation in enrichmentcultures (data not shown).
In the second microcosm experiment (experiment M2), asin the first, the presence of amorphous Fe(OH)3 had an effecton toluene degradation, but the effect was qualitativelydifferent. Toluene concentration versus time is shown in Fig.2 for the first 47 days of incubation of five microcosms andtwo autoclaved controls (one with added iron and onewithout). Toluene was not degraded in any microcosmsduring the first month of incubation. However, the additionof ca. 10 mM amorphous Fe(OH)3 to the three microcosmsthat initially contained added iron (microcosms C8, C9, andC10) initiated toluene degradation within a few days in eachof those microcosms. Two microcosms that did not receiveamorphous Fe(OH)3 over the period shown (microcosms C5and C6) did not degrade toluene until roughly 40 days laterthan the iron-amended microcosms. Toluene degradationwas not observed in the controls. Toluene degradation andsulfate reduction were strongly correlated in this microcosmexperiment. For the three iron-amended microcosms shownin Fig. 2, regressions of cumulative sulfate reduction versuscumulative toluene degradation yielded r2 values of >0.97(calculated over a 1.5-month period following the initiationof toluene degradation; n = 6).
Enrichment cultures: interrelationships among toluene deg-
radation, sulfate reduction, and iron reduction. In PatuxentRiver microcosms and enrichment cultures, toluene degra-dation, sulfate reduction, and ferric iron reduction appearedto be strongly linked. An example of these relationships in aPatuxent River enrichment culture (experiment El) is shownin Fig. 3, in which the cumulative appearance of Fe(II) (anindication of ferric iron reduction) is shown along withcumulative sulfate reduction and cumulative toluene degra-dation. As shown in the figure, not only did these processesoccur simultaneously, but their relative rates were approxi-mately constant. An autoclaved control prepared identicallyto and incubated concurrently with the enrichment cultureshown had C/Co (final/initial concentration) values for tolu-ene, sulfate, and Fe(II) of 0.99, 0.97, and 1.08, respectively,after approximately 3 months of incubation, indicating a lackof discernible toluene degradation, sulfate reduction, andiron reduction in a sterile system. Plots similar to Fig. 3 wereobtained for microcosms. The linkage between toluene deg-
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FIG. 3. Cumulative Fe(I1) appearance and toluene and sulfatedisappearance in an enrichment culture from experiment El. Theregression equation relating toluene and sulfate consumption for thisenrichment (days 0 to 84) was: sulfate consumed (mM) = [3.79 x
(9)C C7H8 + 6.39 Fe(OH)3 (s) + 4.09 S042- + 0.17 NH4+ = 3.2 S0 (s) +0.83 FeS (s) + 5.56 FeCO3 (s) + 0.6 HCO3- + 0.17 C5H702N +9.31 H20 + 7.34 OH-
Biotic toluene oxidation with sulfate (no cell growth)
Biotic toluene oxidation with sulfate (cell growth)
Biotic toluene oxidation with amorphous Fe(OH)3 (no cellgrowth; magnetite formation)
Abiotic oxidation of sulfide to S by amorphous Fe(OH)3; FeS(s)formation
Abiotic oxidation of sulfide to S" by amorphous Fe(OH)3; noFeS(s) formation
Abiotic oxidation of sulfide to S0 by amorphous Fe(OH)3; limitedFeS(s) formation; FeCO3(s) formation
Toluene oxidation with sulfate; formation of S (s) and FeS (s)
Toluene oxidation with sulfate; formation of S0 (s) but not FeS (s)
Toluene oxidation with sulfate; formation of S0 (s), FeS (s), andFeCO3 (s)
a Combination of equations 2 and 4.b Combination of equations 2 and 5.c Combination of equations 2 and 6.
radation, sulfate reduction, and ferric iron reduction raisesthe question of which compound, sulfate or ferric oxyhy-droxide, was serving as the terminal electron acceptor fortoluene degradation.
Several lines of evidence suggest that toluene degradationwas directly linked to sulfate reduction in microcosms andenrichments: (i) the two processes were synchronous, (ii) theobserved stoichiometric ratios of sulfate consumed to tolu-ene consumed were consistent with the theoretical ratio forthe complete oxidation of toluene to bicarbonate coupledwith the reduction of sulfate to hydrogen sulfide, and (iii)toluene degradation ceased when sulfate was depleted, andconversely, sulfate reduction ceased when toluene wasdepleted.The synchronism of toluene degradation and sulfate re-
duction is apparent in Fig. 3 and is supported by the verystrong r2 values for regressions of cumulative toluene deg-radation versus cumulative sulfate reduction over time (typ-ically, r2 > 0.98 for enrichment cultures). The regressionequation for the enrichment shown in Fig. 3 had an r2 valueof >0.999 and a slope of 3.79 (see Fig. 3 legend). Thus, theratio of sulfate consumed to toluene consumed for theenrichment culture shown in Fig. 3 was ca. 3.8. The ratio inenrichment cultures overall averaged 4.07 ± 0.26 (mean ±SD) and ranged from 3.6 to 4.4 (calculated for 11 enrichmentcultures over intervals of at least 20 days of incubation).These values observed in enrichment cultures approximatethe theoretical ratios, ranging from 4.5 (toluene oxidation tobicarbonate with no bacterial cell growth; equation 1 inTable 1) to 4.1 (toluene oxidation to bicarbonate with esti-mated cell growth; equation 2 in Table 1); the estimation
, 3.%0'3.0 ,%s,\ %"''% %% %%
Control Enrichment
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FIG. 4. 14C activity in a control (autoclaved medium only) andsecondary Patuxent River enrichment culture spiked with ca. 37,umol (5.59 x 106 dpm) of [nng U-'4C]toluene. '4C02 constituted83.6% of the 4C activity in the enrichment culture on day 8, whereasnonvolatile 4C (presumably biomass) constituted 11.4%. Theseresults agree well with equation 2 (Table 1), which estimates that88% of the carbon in toluene will be mineralized and the remaining12% will be converted to biomass. The total '4C activity in theenrichment culture on day 8 was 95.6% of that on day 0. The sulfateconsumed/toluene consumed (molar) ratio over this period was 3.8(assuming complete toluene consumption, as suggested by gaschromatography analysis). Toluene was unaltered in the control.
Time (days)FIG. 5. Mutual dependence of toluene degradation and sulfate
reduction for a Patuxent River enrichment culture (experiment E2).Arrows indicate additions of toluene or sulfate. Datum pointsrepresent the averages of duplicate measurements.
of cell growth in equation 2 was derived by using thecalculation methods described by McCarty (19, 20). Theobserved values of the sulfate consumed/toluene consumedratio and the consistency of the ratio over months ofmonitoring provided preliminary evidence that toluene wasoxidized to bicarbonate by sulfate reducers. Experiments inwhich [ring-U-14C]toluene was fed to enrichment culturesconfirmed that oxidation to bicarbonate was occurring inaccordance with equation 2 (shown for a secondary enrich-ment culture in Fig. 4).
Further evidence of the link between toluene degradationand sulfate reduction was the dependence of toluene degra-dation on the presence of sulfate and, conversely, thedependence of sulfate reduction on the presence of toluene(shown for an enrichment culture from experiment E2 in Fig.5). When sulfate was depleted (days 0 to 3 of this experi-ment), toluene was not appreciably degraded. After sulfatewas spiked into the enrichment culture (day 3), concurrenttoluene degradation and sulfate reduction occurred until day7, at which time toluene was depleted. In the absence oftoluene (days 7 to 10), sulfate reduction was not apparent.
If toluene oxidation and sulfate reduction were directlylinked, the concurrent process of ferric iron reduction re-quires explanation (e.g., identification of the electron do-
TABLE 2. Theoretical and observed stoichiometric ratios relatingtoluene degradation, ferric iron reduction, and sulfate reduction
Type and Fe(III) reduced/ Fe(III) reduced/equation no.' toluene consumed sulfate consumed
Theoretical3 367 2.7 0.678 8.2 2.09 6.4 1.6
Observed" 6.1 1.6
See Table 1 for equations.^ Observed ratios were calculated by using the enrichment culture shown in
Fig. 3.
nor). Two possible explanations for ferric iron reduction are(i) direct coupling with toluene oxidation (where ferric ironcould have served as a terminal electron acceptor for tolu-ene) and (ii) coupling with sulfate reduction (where ferriciron could have served as the electron acceptor for theabiotic oxidation of biogenic hydrogen sulfide). Stoichiomet-ric ratios were used to examine the likelihood of these twoexplanations, although data for other variables would berequired to reach definitive conclusions. The ratio of Fe(III)reduced to toluene consumed was used to investigate thepossibility that ferric iron-reducing bacteria were oxidizingtoluene. The observed ratios were considerably lower thanthe theoretical ratio of 36 expected for toluene oxidation tobicarbonate coupled to ferric iron reduction (e.g., equation 3in Table 1, as reported by Lovley and Lonergan [15]). Forexample, the observed ratio for the enrichment shown inFig. 3 was 6.1 rather than 36 (Table 2), which suggests thatnot nearly enough ferric iron had been reduced to accountfor complete toluene oxidation. Thus, the stoichiometrysuggests either that ferric iron reducers were not involved intoluene oxidation or that they carried out incomplete oxida-tion. Indeed, the ratio of sulfate consumed to toluene con-sumed (discussed earlier) further suggests that ferric ironreducers played little, if any, role in toluene degradation.Moreover, in experiments E2 and E3, rapid toluene degra-dation over months of incubation with no initial lag periodwas observed in enrichment cultures that were amendedwith ferrous iron rather than ferric iron (data not shown).
If ferric iron reduction was not directly coupled to toluenedegradation, it may have resulted from an oxidation-reduc-tion reaction between ferric iron and biogenic hydrogensulfide. Geochemical studies of the abiotic, anoxic or oxy-gen-limited reactions of goethite or amorphous Fe(OH)3 withhydrogen sulfide (2, 22, 24) have demonstrated the relativelyrapid formation of ferrous sulfide and elemental sulfur asmajor products and, in Pyzik and Sommer (22), thiosulfate asan additional product. In accordance with these observa-tions, possible abiotic reactions between ferric iron andhydrogen sulfide that result in elemental sulfur and ferroussulfide formation are shown in Table 1 (equations 4, 5, and6). Equations 4 to 6 have the same sulfide oxidation product(i.e., elemental sulfur) but different proportions of availablesulfide relative to iron. In equation 4, there is sufficientsulfide present to precipitate all reduced iron as ferroussulfide, whereas in equation 5 there is sufficient sulfidepresent only to reduce all available ferric iron, but not toprecipitate any of the reduced iron. Equation 6 represents acase in which there is an intermediate amount of sulfidepresent, and a portion of the reduced iron is precipitated asferrous sulfide. In equation 6, the remainder of the reducediron is precipitated as siderite, in recognition of solubilityequilibria that would pertain to the 30 mM bicarbonate-buffered medium used in this study.
Sulfide was not measured in this study, so it was notpossible to directly examine the stoichiometry of iron reduc-tion in relation to hydrogen sulfide. However, if it is assumedthat all consumed sulfate was reduced to hydrogen sulfide (awell-founded assumption based on the literature on sulfatereduction, the odor of acidified culture samples collectedduring this study, and the appearance of a black precipitatein culture medium that was probably iron sulfide [231), thenthe ratio of Fe(III) reduced to sulfate consumed can be usedto examine the possibility of iron reduction via hydrogensulfide. Equations 7, 8, and 9 in Table 1 represent completeoxidation of toluene with sulfate as the electron acceptor(with cell growth) combined with the oxidation of biogenic
hydrogen sulfide to elemental sulfur with ferric iron as theelectron acceptor. More specifically, equations 7, 8, and 9represent the combination of equation 2 with equations 4, 5,and 6, respectively. Equation 2 was chosen because it isconsistent with the results for the enrichment culture shownin Fig. 3 (i.e., the ratio of sulfate consumed to tolueneconsumed in this enrichment [3.8] was similar to the ratio inequation 2 [4.09]). Equation 2 is also supported by experi-ments with radiolabeled toluene (Fig. 4). Table 2 illustratesthat, for the enrichment culture shown in Fig. 3, equation 9is generally consistent with the observed ratios of Fe(III)reduced/sulfate consumed and Fe(III) reduced/toluene con-sumed. The fact that equation 9 is generally consistent withthe observed stoichiometry in terms of iron, sulfate, andtoluene indicates the plausibility that toluene was oxidized tocarbon dioxide by a sulfate-reducing consortium and that theresulting sulfide was responsible for ferric iron reduction.The abiotic reaction proposed for hydrogen sulfide andamorphous Fe(OH)3 (equation 6) is only one of a number ofpossible reactions that could explain the stoichiometry. Forexample, a similar approach could be used in which thiosul-fate, rather than elemental sulfur, was the sulfide oxidationproduct.The data collected in this study have been used to indicate
the importance of iron in either initiating or acceleratingtoluene degradation and to provide support for a proposedmechanism of iron reduction; however, the data are notsuitable for explaining how iron stimulated toluene degrada-tion. Further study will be required to address this issue.Hypotheses that could explain the nature of iron's effectinclude the following: (i) iron may have reduced sulfidetoxicity to toluene-degrading bacteria (e.g., via the oxidationand precipitation mechanisms illustrated by equation 4 inTable 1), and (ii) iron might have been a limiting nutrient thatwas required for toluene degradation (e.g., amendments ofiron could have compensated for the indigenous ferrous ironthat was removed from solution by precipitation as ferroussulfide or other phases). Although amorphous Fe(OH)3 itselfis extremely insoluble, it is possible that the reduction ofmillimolar amounts of ferric iron (e.g., Fig. 3) resulted inmicromolar concentrations of Fe(II) that remained solubleand available to bacteria.The microbiological and geochemical complexities of
these systems merit further investigation. Ongoing researchis focusing on experiments to clarify the role of iron instimulating toluene degradation, to isolate the toluene-de-grading bacteria in pure culture (or, if syntrophy is required,to isolate the critical members of the community), and toelucidate the metabolic pathway of toluene degradation.
ACKNOWLEDGMENTSWe thank Ron Hoeppel of the Naval Civil Engineering Labora-
tory (Port Hueneme, Calif.) for providing the Patuxent River mate-rials used as the inoculum in this study.
This research was supported by U.S. Environmental ProtectionAgency grant EPA CR-815721.
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