Appl Microbiol Biotechnol (2001) 57:572–578DOI 10.1007/s002530100805

Abstract Laboratory batch experiments were perform-ed with contaminated aquifer sediments and four solu-ble aromatic components of jet fuel to assess their bio-degradation under anaerobic conditions. The biodegra-dation of four aromatic compounds, toluene, o-xylene,1,2,4-trimethylbenzene (TMB), and naphthalene, sepa-rately or together, was investigated under strictly anaer-obic conditions in the dark for a period of 160 days. Ofthe aromatic compounds, toluene and o-xylene were de-graded both as a single substrate and in a mixture withthe other aromatic compounds, while TMB was not bio-degraded as a single substrate, but was biodegraded inthe presence of the other aromatic hydrocarbons. Sub-strate interaction is thus significant in the biodegrada-tion of TMB. Biodegradation of naphthalene was notobserved, either as a single substrate or in a mixture ofother aromatic hydrocarbons. Although redox condi-tions were dominated by iron reduction, a clear rela-tion between degradation and sulfate reduction was ob-served. Methanogenesis took place during the later stag-es of incubation. However, the large background ofFe(II) masked the increase of Fe(II) concentration dueto iron reduction. Thus, although microbial reduction ofFe(III) is an important process, the evidence is not con-clusive. Our results have shown that a better under-standing of the degradation of complex mixtures of hy-drocarbons under anaerobic conditions is important inthe application of natural attenuation as a remedialmethod for soil and groundwater contamination.

Introduction

The soluble aromatic compounds of jet fuel (mainlymonoaromatic hydrocarbons and naphthalene) are cur-rently the subject of much interest, and considerable ef-fort is being made to improve our understanding of pro-cesses that control their biodegradation and impact ongroundwater quality. While these compounds are knownto be biodegradable under aerobic conditions (e.g.Chiang et al. 1989; Van Agteren et al. 1998), many con-taminated aquifers contain large areas where anaerobicconditions are dominant (Chapelle et al. 1995). There-fore, our present understanding of aromatic compoundsin aerobic environments is not generally applicable toaquifer bioremediation.

Numerous laboratory and field studies have docu-mented that aromatic compounds can be degraded undernitrate-reducing (Hutchins and Wilson 1991; Barbaro etal. 1992; Flyvbjerg et al. 1993), iron-reducing (Lovley etal. 1989; Anderson and Lovley 1999), sulfate-reducing(Haag et al. 1991; Beller et al. 1992; Edwards et al.1992; Thierrin et al. 1992; Coates et al. 1996), and meth-anogenic conditions (Edwards and Grbic-Galic 1994;Weiner and Lovley 1998). Although biodegradation ofaromatic compounds has been shown to occur under an-aerobic conditions, information on the extent of this ac-tivity, or the conditions that encourage it, is limited(Phelps and Young 1999). In particular, studies using un-amended, anaerobic experimental systems with a mini-mum of disturbance, without addition of nutrients, andusing only the naturally occurring bacteria contained ingroundwater and natural sediments from actual pollutionplumes, are scarce (Bjerg et al. 1999).

Biodegradation of aromatic compounds can varymarkedly from site to site, depending upon the relativeabundance of the terminal electron acceptors availableand the characteristics and pollution history of the sedi-ment (Wiedemeier et al. 1999). Kao and Borden (1997)investigated degradation of aromatic compounds in sev-eral aquifer sediments under denitrifying conditions.They found that some experiments resulted in loss of ar-

Z. Zheng (✉ ) · G. Breedveld · P. AagaardDepartment of Geology, University of Oslo, PB 1047 Blindern,0316 Oslo, Norwaye-mail: zuoping.zheng@geologi.uio.noTel.: +47-22856647, Fax: +47-22854215

G. BreedveldNorwegian Geotechnical Institute, PB 3930 Ullevaal Stadion,0806 Oslo, Norway

O R I G I N A L PA P E R

Z. Zheng · G. Breedveld · P. Aagaard

Biodegradation of soluble aromatic compounds of jet fuel under anaerobic conditions: laboratory batch experiments

Received: 26 April 2001 / Received revision: 20 July 2001 / Accepted: 17 August 2001 / Published online: 19 September 2001© Springer-Verlag 2001

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omatic compounds, whereas others showed no, or onlyslight, loss of such compounds. Therefore, according toKorus (1998), published biodegradation results do notprovide a good comparative basis for process designsince experimental conditions vary greatly. In addition,there is a great deal of variability in the fate of aromaticcompounds in anaerobic sediments, with the rate and ex-tent of loss depending on the inoculum used and the ter-minal electron acceptor available (Phelps and Young1999). Variation within a site was shown to be consider-able at a gasoline-contaminated aquifer (Ball and Rein-hard 1996; Reinhard et al.1997).

Naphthalene has long been thought to be persistentunder anaerobic conditions (Cerniglia 1992). The bio-degradation of this compound under nitrate-reducing,sulfate-reducing, and iron-reducing conditions has re-cently been documented (Coates et al. 1996; Bedessemet al. 1997; Rockne and Strand 1998; Anderson and Lovley 1999). 1,2,4-trimethylbenezene (TMB), oftenused as a hydrological tracer in anaerobic aquifers, hasbeen found in several studies (Wiedemeier et al. 1996;Barker and Wilson 1997) to be recalcitrant under anaero-bic conditions. However, the compound has also been re-ported to biodegrade under denitrifying conditions(Hutchins and Wilson 1991). Therefore, Wiedemeier etal. (1995) considered that the degree of recalcitrance ofTMB is site-specific and must be evaluated on a case-by-case basis.

The present study was initiated as a result of recentobservation of a jet fuel plume in the aquifer at a fire-fighting training site in the Gardermoen airport, Norway.Our main objective was to find out if there was a differ-ence in the degradation rates of aromatic compounds ofjet fuel as single compounds compared to mixtures ofseveral compounds under anaerobic conditions. We fo-cused on (1) quantifying biodegradation rates of aromat-ic compounds investigated under anaerobic conditions,(2) investigating coupled redox processes associatedwith the biodegradation of organic compounds, and (3)substrate interaction between the different hydrocarbons.

Materials and methods

Aquifer materials

The sandy material contaminated with jet fuel used in this experi-ment was collected from the fire-fighting training site at Garder-

moen. This material was highly anaerobic, and taken from sedi-ments at a depth of 5 m (ca. 1 m below groundwater level) using apiston sampler. Sediments were immediately transfered to glassjars, groundwater was added to remove the headspace, and the jarswere sealed in the field. Upon returning to the laboratory the sam-ple was stored at 10–15°C until needed. The properties of theaquifer sediments are shown in Table 1. Groundwater was takenfrom the same site, but from an uncontaminated well about 50 mfrom the center of contamination. The average composition of thegroundwater in mM was: 0.7 alkalinity, 0.01 Cl–, 0.32 SO4

2–, 0.01 Na+, 0.01 K+, 0.4 Ca2+, 0.2 Mg2+, 0.002 Fe2+, 0.002 Mn2+.We should point out that concentration of sulfate in the groundwa-ter from the contaminated- and uncontaminated-sites is identical.The pH was around 7, and the temperature of the groundwater var-ied from 8 to 10°C. The groundwater used in the experiments wasflushed with N2 for 30 min to remove dissolved oxygen (Butler etal. 1994).

Chemicals

All chemicals used in this study were purchased at the highest pu-rity available (98–99.5%) and used as received.

Incubation strategies

Before starting the experiments, the aquifer sediment was com-pletely homogenised in a sterile bucket inside a N2 atmosphereglove box. Four solutions of single aromatic compounds, toluene,o-xylene, TMB, and naphthalene (Table 2), were prepared by add-ing pure compounds into the N2-purged groundwater to reach aconcentration of 15–20 mg/l of the compound. Simultaneously,two kinds of mixtures of the four aromatic compounds were pre-pared. In mixture (1) approximately 3.5–4.0 mg/l of each singlecompound was present, and in mixture (2) the concentration wasapproximately 0.3–0.4 mg/l per compound. Contaminated sedi-ment (20 g) was dispensed into autoclaved serum flasks (100 ml)and approximately 80 ml organic solution was added under a N2atmosphere. The serum flasks were sealed with Teflon-faced sili-cone septa and capped with aluminum seals (Wheaton Products).For each compound (the single compound batch) and mixture (themixture batch), 10 flasks were prepared, resulting in a total of 110

Table 1 Characterization of the aquifer material

Parameter Value

Sample depth (m) –5Density (cm3/g) 1.8Porosity 0.46Silt clay (<0.063 mm) (%) 2.7Fine sand (0.063–0.25 mm) (%) 31Coarse sand (0.25–2mm) (%) 66Content of organic matter (%) 0.1–0.15Fe(III) content (mg/g) 5.4–15

Table 2 Physical and chemical properties of selected aromatic compounds

Compoundsa Chemical Molecular Solubility in LogKoc LogKow Henry’s constantformula weight water (mg/l) (atmm3/mol)

Toluene C6H5CH3 92.14 515 2.06 2.65 6.7e–3

o-Xylene C6H4(CH3)2 106.17 175 2.11 2.95 5.27e–3

TMB C6H3(CH3)3 120.19 51.9 3.57 3.78 5.7e–3

Naphthalene C5H4C5H4 128.18 30 3.11 3.36 4.6e–4

a Toluene, o-xylene and naphthalene data from Montgomery and Welkom (1990); and 1,2,4- trimethylbenzene (TMB) data from Mont-gomery (1991) at standard temperature and pressure of 1 atmosphere and 25°C

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flasks. In addition, sodium azide to a final concentration of 0.2%by weight was added to the solution of those batches which servedas abiotic controls. This inhibited microbial activity and allowedabiotic loss of the aromatic compounds to be estimated. Sodiumazide at this concentration has been shown to be sufficient to inac-tivate bacteria in soil and water (Lyngkilde et al. 1992). Twentyblank batches were prepared without any aromatic compound butwith addition of sediment and groundwater. These water batchflasks allowed us to determine release of organic matter from thesediment. Blank control batches were prepared containing aromat-ic compounds but no sediment. This allowed evaluation of the sta-bility of the organic solutions. All batches were vigorously shakenfor 30 min and then incubated in the dark, submerged in a largewater tank in a cooling room at 5–6°C, to keep a constant temper-ature and avoid gas diffusion.

Sampling and measurements

Samples were taken at weekly intervals in the first month, and bi-weekly intervals after the second month. The serum bottles wereopened in a N2 atmosphere glove box and immediately extractedwith pentane to quantify the concentrations of the aromatic com-pounds. The concentrations of the four aromatic compounds weremeasured in pentane extracts from 10 ml water samples on a gaschromatograph (GC) equipped with a flame ionization detectorwith a detection limit of 0.1 µg/l for all compounds.The quantifi-cation was performed using an internal standard method using 1-Cl-4-F benzene as an internal standard. Subsamples of the suspension were collected using sterilized syringes, filtered(0.45 µm), and analysed for Fe(II) using the ferrozine method(Stookey 1970). Methane production was periodically monitoredafter 3 months and measured by GC as methane accumulation inthe headspace of the flask, which was filled with 1 ml pure N2 be-fore placing on a shaker for 30 min at room temperature. Assum-ing chemical phase equilibrium, the concentration of dissolvedmethane could be calculated according to Henry’s law using theequation KH= Cw/Ca, where Ca is the concentration of methane ingas phase (M), Cw is the concentration of methane in water phase(M), and KH is Henry’s constant (Table 2). After sampling for or-ganic compounds, Fe(II), and methane, the remaining solution wasused for determination of the concentration of cations includingK+, Ca2+, Mg2+, Fe, and Mn2+ and anions including SO4

2–, Cl– andNO3

–, using atomic adsorption and ion chromatography, respec-tively. Solid samples from each batch were frozen immediately forfurther mineralogical and biomass analysis (not reported here).

Determination of biodegradation rates and reaction stoichiometry

The apparent biodegradation rates of the aromatic compounds in-vestigated were estimated by the first-order model using the equa-tion C=C0exp(-Kt), where K is the apparent first-order bio-degrada-tion rate (d–1), t is time, and C0 is the initial concentration. Thismethod is usually used to calculate biodegradation of hydrocar-bons dissolved in groundwater (Wiedemeier et al. 1999). Briefly,biodegradation rates of aromatic compounds were calculated byperforming a linear regression of logarithmic relative concentra-tion data with incubation time.

The stoichiometry of the oxidation of the four aromatic com-pounds and corresponding changes of electron acceptors was de-termined from the molar ratio of Fe(II) production, sulfate con-sumption, and methane production relative to the molar removalof aromatic compounds at the termination of the batch. The theo-retical stoichiometry was calculated assuming complete oxidationof the aromatic compounds to CO2 without production of microbi-al biomass (Table 3)

Results

Biodegradation of aromatic compounds under anaerobic conditions

The relative concentration of four aromatic compoundsinvestigated as a function of incubation time for threekinds of batches is shown in Fig. 1. The relative concen-tration refers to the measured concentration in each batchdivided by the average concentration of abiotic controlsin the entire period of the experiment. During the entireperiod of the experiment (160 days) the concentration ofaromatic compounds in abiotic controls is relatively sta-ble, i.e., below 15% reduction compared to the initialconcentration of aromatic compounds.

Concentrations of toluene and o-xylene were substan-tially decreased relative to the abiotic controls during thecourse of incubation as single compounds, indicatingthat biodegradation of these compounds took place(Fig. 1). After 120 days of incubation, concentrations oftoluene and o-xylene reached a stable level, suggestingthat electron acceptors available in the batch, such as

Table 3 Stoichiometries of selected processes involved in major aromatic compound mineralization of jet fuel and relevant geochemicalreactions

Compounds Microbial aromatic compounds Molar ratio of electron acceptormineralization and geochemical reactions to organic compounds

Toluene C7H8 + 36 Fe3+ + 21 H2O → 7 HCO3–+36 Fe2+ + 43 H+ 36:1

C7H8 + 4.5 SO42– + 3 H2O + 2 H+→ 7 HCO3

– + 4.5 H2S 4.5:1C7H8 + 7.5 H2O → 2.5 HCO3

– + 4.5CH4 + 2.5 H+ 4.5:1a

o-Xylene C8H10 + 42 Fe 3+ + 24 H2O →8 HCO3–+42 Fe2+ + 40 H+ 42:1

C8H10 + 5.25 SO42– + 3H2O + 9.5H+ → 8 HCO3

–+5.25 H 2 S 5.25:1C8H10 + 8.25 H2O→ 2.75 HCO3

– + 5.25 CH4 +2.75 H+ 5.25:1a

1,2,4trimethyl- benzene C9H12 +48 Fe3+ + 27 H2O → 9 HCO3– + 48 Fe2+ + 57 H+ 48:1

C9H12 + 6 SO42– + 12 H+ +3 H2O → 9 HCO3

–+6 H2S 6:1C9H12 + 9 H2O → 3 HCO3

– + 6 CH4 + 3 H+ 6:1a

Naphthalene C10H8 + 48 Fe3+ + 30 H2O→ 10 HCO3– + 48 Fe2+ + 58 H+ 48:1

C10H8 + 6 SO42– + 2 H+ + 6 H2O → 10 HCO3

– + 6 H2S 6:1C10H8 + 12 H2O → 4 HCO3

– +6 CH4 + 4 H+ 6:1a

a Molar ratio of methane to organic compounds (after Wiedemeier et al. 1999)

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sulfate, could be depleted. As we discuss later, sulfateconcentration decreased to zero when the concentrationof toluene and o-xylene had decreased by approximately80%. In contrast, concentrations of TMB and naphtha-lene were stable and identical to the abiotic controlsthroughout the single substrate experiments, implyingthat no biodegradation took place.

The aromatic compounds in the mixtures, however, ex-hibited different behavior (Fig. 1). Toluene and o-xyleneshowed a complete degradation in the mixtures. Both thelag phase and final concentrations of toluene and o-xylenewere lower than for the single substrate incubations. Aclear difference in the degradation of TMB was observedin the aromatic hydrocarbon mixture compared to the sin-gle compound batches. While TMB was recalcitrant as asingle compound it was degraded in the mixture. Howev-er, naphthalene was recalcitrant both as a single com-pound and in the mixture.

Biodegradation rates of the aromatic compounds

The biodegradation rates of the aromatic compoundswere estimated based on first-order kinetics as discussedearlier, and are given in Table 4. Since no intermediatecompounds were detected in the experiments, loss of aromatic compounds was assumed to be due to completemineralization into CO2 and water or biomass.

Sulfate concentration

The concentration profiles of sulfate as a function of in-cubation time are shown in Fig. 2. The concentrations of

sulfate are characterized by specific concentration pro-files for the individual batches, suggesting that sulfatereduction played an important role in the oxidation of thearomatic compounds under investigation. However, atthe very initial stage, from the start to 30 days, concen-trations of sulfate tended to gently increase for all batch-es. The same phenomenon was also found in some fuelhydrocarbon-contaminated sites as recently reported byWiedemier et al.(1999). The reason is not clear. A dis-tinct decrease in the sulfate concentration was observedafter 30 days for the single compound incubation of tolu-ene, o-xylene, and mixture (1), clearly indicating thatsulfate was used as the terminal electron acceptor.

Fig. 1 Changes of aromatic compounds investigated as a functionof incubation time in the individual batches

Fig. 2 Changes of sulfate concentration as a function of time inthe individual batches. Black circles Live, open circles control,crosses blank (water batch), Np naphthalene, TMB 1,2,4-trimethyl-benzene

Table 4 The calculated biodegradation rates for aromatic com-pounds based on the first-order model (day–1)

Experimental Biodegradation rates of aromatic compoundsCompounds

Toluene TMB o-Xylene Naphthalene

Single compound 0.009 NBa 0.009 NBMixture 1 0.051 0.009 0.046 NBMixture2 0.078 0.02 0.026 NB

a No biodegradation

For the aromatic compounds with lower solubility inwater, such as TMB and naphthalene, no biodegradationwas observed in the single substrate batches. Our obser-vations are in agreement with those of Bjerg et al.(1999),who showed that naphthalene and TMB were recalcitrantunder anaerobic conditions over an experimental periodof 537 days. Although biodegradation of naphthalene hasbeen reported in the literature (e.g., Coates et al. 1996;Anderson and Lovley 1999), biodegradation of naphtha-lene was not observed within the time frame of thisstudy, suggesting that the biodegradation of naphthaleneis site-specific or extremely slow. Utilization of naphtha-lene or TMB as single substrates under anaerobic condi-tions might require special enzymes or be energeticallyunfavorable for the microorganisms (Chapelle 2000).

Aromatic compounds are often present in the environ-ment as components of complex mixtures. Therefore,understanding of substrate interactions is important inpredicting the behavior of contaminants in the environ-ment. Several investigations have been conducted tostudy substrate interactions during biodegradation. Ex-periments by Alvarez and Vogel (1991) on the interac-tion of toluene and benzene during biodegradation dem-onstrated that the enhancement of benzene biodegrada-tion is due to toluene-enhanced microbial activity via en-zyme induction. Arcangeli and Arvin (1992) found thataerobic degradation of ethylbenzene and xylene was en-hanced when they were co-metabolized with toluene.Millette et al. (1998) indicated that the least persistentcompounds most likely affect transformation of othermore recalcitrant compounds. Quite recently, Jensen etal. (2001) presented a review on substrate interactionamong aromatic hydrocarbons with emphasis on tolueneacting as the primary substrate in combination with different other aromatics; this study focused mainly onaerobic degradation in a mixture.

The degradation rates of toluene and o-xylene in ourexperiments were faster in mixtures than as single sub-strates as indicated by the slopes of the curves (Fig. 1).TMB was especially persistent as a single substrate, butwas biodegraded in the mixture, indicating that its bio-degradation of was associated with the degradation ofother aromatic compounds. This implies a clear effect ofsubstrate interaction under anaerobic conditions. Severalmechanisms can explain substrate interaction on the ba-sis of microbiological processes, such as co-metabolism,competitive inhibition, and kinetic competition (Jensenet al. 2001). The increase in the rates of toluene and o-xylene biodegradation in the mixture studied might beexplained by the following mechanism: the presence oftoluene and o-xylene aids the proliferation of the bacte-ria, leading to a greater number of microbes, which maystimulate the degradation rate of both compounds. Thissuggests that bacteria utilize the same enzyme to degradeboth compounds. Degradation of TMB is probably of aco-metabolic nature, where toluene and/or o-xylene areprimary substrates generating the energy required forTMB degradation. Proper understanding of this interac-tion will require more detailed studies.

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Methane production

Methane (CH4) is usually produced by fermentative bac-teria in strongly reductive environments (Barker andWilson 1997). The methane concentration in this studywas measured only during later stages of the experiment.The results are shown in Fig. 3. Generally, all biological-ly active batches, with exception of the single compoundincubation of naphthalene, revealed marked methaneproduction, while the inactivated control batch did notshow any production of methane (lower than 0.003 mM;data not shown). This implies that methanogenesis wasoccurring in the biologically active batches.

Discussion

Anaerobic biodegradation of aromatic compounds hasbeen extensively investigated in recent years. Biodegra-dation under nitrate-, Fe(III)- and sulfate-reducing, aswell as methanogenesis conditions has been shown tooccur. Variations in the biodegradation rates of aromaticcompounds within the same aquifer sediment have beenobserved, suggesting that anaerobic biodegradation ofaromatic compounds is a complex process that may beaffected by several factors, e.g., characteristics and pol-lution history of the sediment, terminal electron accep-tors available, and physico-chemical properties of thearomatic compounds involved as well as substrate inter-actions (Phelps and Young 1999; Wiedemeier et al.1999). Clearly, when the same aquifer sediment is used,the physico-chemical properties of the aromatic com-pounds will largely determine their bioavailability,which further affects the rate and extent of their biodeg-radation. This could be a possible reason why preferredbiodegradation of toluene and o-xylene was observed inour experiments, due to their higher solubility in watercompared to the other compounds studied. Edwards etal.(1992) also found toluene and m-xylene to be pre-ferred as substrates when a mixture of aromatic com-pounds was fed to a sulfate-reducing enrichment cul-ture.

Fig. 3 The production of methane as a function of incubation timein the individual batches

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Changes in redox conditions during the biodegrada-tion of aromatic compounds are often evaluated by mea-suring dissolved chemical species that are characteristicof particular microbial processes; these include the con-centration of dissolved electron acceptors, mainly O2,NO3

–, and SO42–, or the reduced products of electron

acceptor utilization, such as HS–, Fe2+, and CH4(Cozzarelli et al. 2000). As shown previously, Fe(III)and sulfate as well as CO2 are potential electron accep-tors in the batch experiments. Therefore, determinationof redox conditions for batch experiments is based onconsumption of available electron acceptors, and re-duced products of electron acceptors. Because of thesmall amount of Fe(III) utilized and the large back-ground concentration of Fe(II) in the anaerobic sediment,it is difficult to distinguish between the concentrations ofFe(II) due to microbial degradation of aromatic hydro-carbons and the background Fe(II), as the concentrationof Fe(II) was almost identical for abiotic controls andlive batches (data not shown). Although the stoichiomet-ric relationship between the amount of electron acceptorlost and the amount of aromatic compounds degradedduring the course of the experiment supports the hypoth-esis that microbial reduction of Fe(III) iron is a terminalelectron acceptor in our study, the evidence is not con-clusive. The comparision between the cumulative con-sumption of sulfate and the removal of aromatic com-pounds as shown in Fig. 4 also indicates that sulfate wasnot the only electron acceptor in our experiments.

The concentration of sulfate was approximate 32 mg/lin the initial solution, and depleted after about 30 daysfor the batches with toluene and o-xylene. The completeconsumption of sulfate for toluene and o-xylene corre-lates with approximately 80% consumption of these aro-matics (Figs. 1 and 2). This suggests that the availabilityof the electron acceptor played a significant role in thedegradation of these aromatic compounds.

In mixture (1) of aromatic compounds, the concentra-tion of single organic compounds was much lower thanin the single compound incubation, although the totalamount of hydrocarbons available was approximately thesame (15–20 mg/l). In this incubation, sulfate was utilized as electron acceptor. For mixture (2) however,with the lowest concentration of organic compounds(0.3–0.4 mg/l per compound, total 1.2–1.5 mg/l), the

bioavailable Fe(III) seems to be sufficient to oxidize or-ganic compounds. Therefore, sulfate was not used as aelectron acceptor in this incubation. Also in the singlecompound incubations of naphthalene and TMB, theconcentration of sulfate shows little decrease, which is inagreement with the absence of biodegradation. A gradualdecrease of sulfate in the TMB single compound incuba-tion is observed over time. However, the decrease of sul-fate is identical to the abiotic control, implying that sul-fate was not used as the terminal electron acceptor foroxidizing TMB, but was probably oxidizing other organ-ic material present in the sediment.

Interestingly, the available data do not indicate thatsulfate reduction inhibits methanogenesis, because meth-ane was detected in the batches when sulfate was used asthe terminal electron acceptor; thus both redox processesoccur simultaneously in our experiments. Barker andWilson (1997) pointed out that anaerobic fermentation(organic matter is both electron acceptor and donor)probably occurs under all anaerobic conditions, whichmay explain the observed methane levels in our experi-ments. In addition, methane concentration did not show asystematic change with incubation time. The variation inthe methane concentration could be explained by hetero-geneity in the aquifer sediment or accidental leak ofmethane from the batch flasks, as well as uncertainity inthe analytical methods.

In conclusion, our study has shown that a better un-derstanding of the degradation of complex mixtures un-der anaerobic conditions is necessary to apply natural at-tenuation as a remedial method for soil and groundwatercontamination.

Acknowledgement This project was funded by the NorwegianResearch Council, through the Strategic University Programme,“Contaminant spreading and behavior in soil and groundwater” atthe University of Oslo.

References

Alvarez PJJ, Vogel TM (1991) Substrate interactions of benzene,toluene, and para-xylene during microbial degradation by purecultures and mixed culture aquifer slurries. Appl Environ Microbiol 57:2981–2985

Anderson RT, Lovley DR (1999) Naphthalene and benzene degra-dation under Fe(III)-reducing conditions in petroleum-contam-inated aquifers. Bioremediation J 3:121–135

Arcangeli JP, Arvin E (1992) Modeling of toluene biodegradationand biofilm growth in a fixed film reactor. Water Sci Technol26:617–626

Ball HA, Reinhard M (1996) Monoaromatic hydrocarbon transfor-mation under anaerobic conditions at Sea Beach, California:laboratory studies. Environ Toxicol Chem 15:114–122

Barbaro JR, Barker JF, Lemon LA, Mayfield CI (1992) Biotrans-formation of BTEX under anaerobic, denitrifying conditions:field and laboratory observations. J Contam Hydrol 11:245–272

Barker JF, Wilson JT (1997) Natural biological attenuation of aro-matic hydrocarbons under anaerobic conditions. In: Ward CH,Cherry JA, Scalf MR (eds). Subsurface restoration. Ann ArborPress, Ann Arbor, Mich., pp 289–300

Bedessem ME, Swoboda-Colber NG, Colberg PJS (1997) Naph-thalene mineralization coupled to sulfate reduction in aquifer-derived enrichments. FEMS Microbiol Lett 152: 213–218

Fig. 4 Schematic of the relationship between sulfate and aromaticcompounds tested as a single compound incubation

Korus R A (1998) Scale-up of processes for bioremediation. InHurst CJ, Knudsen GR, McInerney MJ, Stetzenbach LD, Walter MV (eds) Manual of environmental microbiology.ASM Press, Washington, D.C., pp 856–863

Jensen BK, Broholm K, Jørgensen C (2001) Toluene as the mainprimary substrate in aromatic hydrocarbon substrate interac-tions under different redox systems. Proceedings of the FirstEuropean Bioremediation Conference, Greece, pp 553–556

Lovley DR, Baedecker MJ, Lonergan DL, Cozzarelli IM, PhillipsEJP, Siegel DI (1989) Oxidation of aromatic contaminantscoupled to microbial iron reduction. Nature 339:297–299

Lyngkilde J, Christensen TH, Gillham RW, Larsen T, Kjeldsen P,Skov B, Fover S, Hannesin SO (1992) Degradation of specificorganic compounds in leachate-polluted groundwater. In:Christensen TH, Cossu R, Stegman R (eds), Landfilling ofwaste: leachate. Elsevier, Amsterdam, pp 485–495

Millette D, Butler BJ, Frind EO, Comeau Y, Samon R (1998) Sub-strate interaction during aerobic biodegradation of creosote-related compounds in columns of sandy aquifer material. J Contam Hydrol 29:165–183

Montgomery JH (1991) Groundwater chemicals field guide. Lewis, Boca Raton, Fla.

Montgomery JH, Welkom LM (1990) Groundwater chemicalsdesk reference. Lewis, Boca Raton, Fla.

Phelps CD, Young LY (1999) Anaerobic biodegradation of BTEXand gasoline in various aquatic sediments. Biodegradation10:15–25

Reinhard M, Shang S, Kitanidis PK, Orwin E, Hopkins GD, Leb-ron CA (1997) In situ BTEX biotransformation under en-hanced nitrate- and sulfate-reducing conditions. Environ SciTechnol 31:28–36

Rockne KJ, Strand SE (1998) Biodegradation of bicyclic andpolycyclic aromatic hydrocarbons in anaerobic enrichments.Environ Sci Technol 32:3962–3967

Stookey LL (1970) Ferrozine-a new spectrophotometric reagentfor iron. Anal Chem 42:779–781

Thierrin J, Davis GB, Barber C, Patterson BM, Pribac F, PowerTR, Lambert M (1992) Natural degradation rates of BTEXcoupounds and naphthalene in a sulfate-reducing ground waterenvironment. Hydrol Sci J 38:309–332

Van Agteren MH, Keuning S, Janssen DB (1998) Handbook onbiodegradation and biological treatment of hazardous organiccompounds. Kluwer, Dordrecht.

Weiner JM, Lovley DR (1998) Rapid benzene degradation inmethanogenic sediments from a petroleum-contaminated aqui-fer. Appl Environ Microbiol 64:1937–1939

Wiedemeier TH, Swanson MA, Wilson JT, Kampbell DH, MillerRN, Hansen JE (1995) Patterns of intrinsic bioremediation attwo United States Air Force bases. In: Hinchee RE, Wilson JT,Downey DC (eds) Intrinsic bioremediation. Battelle Press, Co-lumbus, Ohio, pp 31–51

Wiedemeier TH, Swanson MA, Wilson JT, Kampbell DH, MillerRN, Hansen JE (1996) Approximation of biodegradation rateconstants for monoaromatic hydrocarbons (BTEX) in groundwater. Ground Water Monit Rem 16:186–194

Wiedemeier TH, Rifai HS, Newell CJ, Wilson JT (1999) Naturalattenuation of fuels and chlorinated solvents in the subsurface.Wiley, New York

578

Beller HR, Grbic-Galic D, Reinhard M (1992) Microbial degrada-tion of toluene under sulfate-reducing conditions and the influ-ence of iron on the process. Appl Environ Microbiol 58:786–793

Bjerg PL, Rugge K, Cortsen J, Nielsen PH, Christensen TH (1999)Degradation of aromatic and chlorinated aliphatic hydrocar-bons in the anaerobic part of the Grindsted Landfill leachateplume: in situ microcosm and laboratory batch experiments.Ground Water 37:113–121

Butler IB, Schoonen MAA, Rickard DT (1994) Removal of dis-solved oxygen from water: a comparison of four commontechniques. Talanta 41:211–215

Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydro-carbons. Biodegradation 3:351–368

Chapelle FH (2000) Ground-water microbiology and geochemis-try. Wiley, New York.

Chapelle FH, McMahon PB, Dubrovsky NM, Fuji RF, OaksfordET, Vroblesky DA (1995) Deducing the distribution of termi-nal electron-accepting processes in hydrologically diversegroundwater systems. Water Resour Res 31:359–371

Chiang CY, Salanitro JP, Chai EY, Colthart JD, Klein CL (1989)Aerobic biodegradation of benzene, toluene, and xylene insandy aquifer: data analysis and computer modeling. GroundWater 27:823–834

Coates JD, Anderson RT, Woodward JC, Phillips EJP, Lovley DR(1996) Anaerobic hydrocarbon degradation in petroleum/con-taminated harbor sediments under sulfate-reducing and artifi-cially imposed iron-reducing conditions. Environ Sci Technol30:2784–2789

Cozzarelli IM, Suflita JM, Ulrich GA, Harris SH, Scholl MA,Schlottmann JL, Christenson S (2000) Geochemical and mi-crobiological methods for evaluating anaerobic processes in anaquifer contaminated by landfill leachate. Environ Sci Technol34:4025–4033

Edwards EA, Grbic-Galic D (1994) Anaerobic degradation of tol-uene and o-xylene by a methanogenic consortium. Appl Envi-ron Microbiol 60:313–322

Edwards EA, Wills LE, Reinhard M, Grbic-Galic D (1992) Anaer-obic degradation of toluene and xylene by aquifer microorgan-isms under sulfate-reducing conditions. Appl Environ Micro-biol 58:794–800

Flyvbjerg J, Arvin E, Jensen BK, Olsen SK (1993) Microbial deg-radation of phenols and aromatic hydrocarbons in creosote-contaminated groundwater under nitrate-reducing conditions. J Contam Hydrol 12:133–150

Haag FM, Reinhard M, McCarty PL (1991) Degradation of tolu-ene and p-xylene in anaerobic microcosms: evidence for sul-fate as a terminal electron acceptor. Environ Toxicol Chem10:1379–1389

Hutchins SR, Wilson JT (1991) Laboratory and field studies onBTEX biodegradation in a fuel-contaminated aquifer underdenitrifying conditions. In: Hinchee RE, Olfenbuttel RF (eds),In situ bioreclamation. Butterworth-Heinemann, Stoneham,Mass., pp 157–172

Kao CM, Borden RC (1997) Site-specific variability in BTEX bio-degradation under denitrifying conditions. Ground Water 35:305–311