Anaerobic benzene degradation

Although many studies have indicated that benzene persists under anaerobic conditions in petroleum-contaminated environments, it has recently been documented that benzene can be anaerobically oxidized with most commonlyconsidered electron acceptors for anaerobic respiration. These include: Fe(III),sulfate, nitrate, and possibly humic substances. Benzene can also be convertedto methane and carbon dioxide under methanogenic conditions. There is evidencethat benzene can be degraded under in situ conditions in petroleum-contaminatedaquifers in which either Fe(III) reduction or methane production is the predominant terminal electron-accepting process. Furthermore, evidence from laboratory studies suggests that benzene may be anaerobically degraded in petroleum-contaminated marine sediments under sulfate-reducing conditions. Laboratory studies have suggested that within the Fe(III) reduction zone of petroleum-contaminated aquifers, benzene degradation can be stimulated with the addition of synthetic chelators which make Fe(III) more available for microbial reduction. The addition of humic substances and other compounds that contain quinone moieties can also stimulate anaerobic benzene degradation in laboratory incubations of Fe(III)-reducing aquifer sediments by providing an electron shuttle between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides. Anaerobic benzene degradation in aquifer sediments can be stimulated with the addition of sulfate, but in some instances an inoculum of benzene-oxidizing,sulfate-reducing microorganisms must also be added. In a field trial, sulfate addition to the methanogenic zone of a petroleum-contaminated aquifer stimulated the growth and activity of sulfate-reducing microorganisms and enhanced benzene removal. Molecular phylogenetic studies have provided indications of what microorganisms might be involved in anaerobic benzene degradation in aquifers. The major factor limiting further understanding of anaerobic benzene degradation is the lack of a pure culture of an organism capable of anaerobic benzene degradation.     

Microbial communities associated with anaerobic benzene degradation in a petroleum-contaminated aquifer.

Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number-PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site's capacity for anaerobic benzene degradation.     

Microbial Communities Associated with Anaerobic Benzene Degradation in a Petroleum-Contaminated Aquifer

Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number–PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site’s capacity for anaerobic benzene degradation.     

Anaerobic Benzene Oxidation by Geobacter Species

The abundance of Geobacter species in contaminated aquifers in which benzene is anaerobically degraded has led to the suggestion that some Geobacter species might be capable of anaerobic benzene degradation, but this has never been documented. A strain of Geobacter, designated strain Ben, was isolated from sediments from the Fe(III)-reducing zone of a petroleum-contaminated aquifer in which there was significant capacity for anaerobic benzene oxidation. Strain Ben grew in a medium with benzene as the sole electron donor and Fe(III) oxide as the sole electron acceptor. Furthermore, additional evaluation of Geobacter metallireducens demonstrated that it could also grow in benzene-Fe(III) medium. In both strain Ben and G. metallireducens the stoichiometry of benzene metabolism and Fe(III) reduction was consistent with the oxidation of benzene to carbon dioxide with Fe(III) serving as the sole electron acceptor. With benzene as the electron donor, and Fe(III) oxide (strain Ben) or Fe(III) citrate (G. metallireducens) as the electron acceptor, the cell yields of strain Ben and G. metallireducens were 3.2 × 10⁹ and 8.4 × 10⁹ cells/mmol of Fe(III) reduced, respectively. Strain Ben also oxidized benzene with anthraquinone-2,6-disulfonate (AQDS) as the sole electron acceptor with cell yields of 5.9 × 10⁹ cells/mmol of AQDS reduced. Strain Ben serves as model organism for the study of anaerobic benzene metabolism in petroleum-contaminated aquifers, and G. metallireducens is the first anaerobic benzene-degrading organism that can be genetically manipulated.     

Anaerobic Benzene Degradation in Petroleum-Contaminated Aquifer Sediments after Inoculation with a Benzene-Oxidizing Enrichment

Sediments from the sulfate-reduction zone of a petroleum-contaminated aquifer, in which benzene persisted, were inoculated with a benzene-oxidizing, sulfate-reducing enrichment from aquatic sediments. Benzene was degraded, with apparent growth of the benzene-degrading population over time. These results suggest that the lack of benzene degradation in the sulfate-reduction zones of some aquifers may result from the failure of the appropriate benzene-degrading sulfate reducers to colonize the aquifers rather than from environmental conditions that are adverse for anaerobic benzene degradation.     

Rapid Anaerobic Benzene Oxidation with a Variety of Chelated Fe(III) Forms

Fe(III) chelated to such compounds as EDTA, N-methyliminodiacetic acid, ethanol diglycine, humic acids, and phosphates stimulated benzene oxidation coupled to Fe(III) reduction in anaerobic sediments from a petroleum-contaminated aquifer as effectively as or more effectively than nitrilotriacetic acid did in a previously demonstrated stimulation experiment. These results indicate that many forms of chelated Fe(III) might be applicable to aquifer remediation.     

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

Laboratory batch experiments were performed with contaminated aquifer sediments and four soluble aromatic components of jet fuel to assess their biodegradation under anaerobic conditions. The biodegradation of four aromatic compounds, toluene, o-xylene, 1,2,4-trimethylbenzene (TMB), and naphthalene, separately or together, was investigated under strictly anaerobic conditions in the dark for a period of 160 days. Of the aromatic compounds, toluene and o-xylene were degraded both as a single substrate and in a mixture with the other aromatic compounds, while TMB was not biodegraded as a single substrate, but was biodegraded in the presence of the other aromatic hydrocarbons. Substrate interaction is thus significant in the biodegradation of TMB. Biodegradation of naphthalene was not observed, either as a single substrate or in a mixture of other aromatic hydrocarbons. Although redox conditions were dominated by iron reduction, a clear relation between degradation and sulfate reduction was observed. Methanogenesis took place during the later stages of incubation. However, the large background of Fe(II) masked the increase of Fe(II) concentration due to iron reduction. Thus, although microbial reduction of Fe(III) is an important process, the evidence is not conclusive. Our results have shown that a better understanding of the degradation of complex mixtures of hydrocarbons under anaerobic conditions is important in the application of natural attenuation as a remedial method for soil and groundwater contamination.     

Microbial Metabolism of Benzene and the Oxidation of Ferrous Iron under Anaerobic Conditions: Implications for Bioremediation

Benzene and toluene were biodegraded when chelated Fe(III) served as the terminal electron acceptor in aquifer sediments contaminated by a petroleum refinery. Benzene biodegradation ceased when Fe(III) was depleted but resumed upon reamendment. Microorganisms from the same sediments degraded toluene, but not benzene, under nitrate reducing conditions. However, the anaerobic oxidation of Fe(II) to Fe(III) was also observed in toluene-degrading incubations. Fe(II) oxidation was dependent on the presence of nitrate and enhanced when organic electron donors were provided. Microbial nitrate-linked Fe(II) oxidation was also documented in other petroleum-contaminated aquifer sediments, sludge from an oil–water separator, a landfill leachate-impacted aquifer and a garden soil. These observations suggest that some of the reported effects of nitrate on hydrocarbon biodegradation may be indirect through the reoxidation of Fe(II).     

Potential for anaerobic bioremediation of BTEX in petroleum-contaminated aquifers

Laboratory incubations of aquifer material or enrichments derived from aquifer material as well as geochemical data have suggested that, under the appropriate conditions, BTEX components of petroleum (benzene, toluene, ethylbenzene and xylene) can be degraded in the absence of molecular oxygen with either Fe(III), sulfate, or nitrate serving as the electron acceptor. BTEX degradation under methanogenic conditions has also been observed. However, especially for benzene, the BTEX contaminant of greatest concern, anaerobic degradation is often difficult to establish and maintain in laboratory incubations. Although studies to date have suggested that naturally occurring anaerobic BTEX degradation has the potential to remove significant quantities of BTEX from petroleum-contaminated aquifers, and mechanisms for stimulating anaerobic BTEX degradation in laboratory incubations have been developed, further study of the organisms involved in this metabolism and the factors controlling their distribution and activity are required before it will be possible to design rational strategies for accelerating anaerobic BTEX degradation in contaminated aquifers. Received 21 November 1995/ Accepted in revised form 20 February 1996     

Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments.

Although polycyclic aromatic hydrocarbons (PAHs) have usually been found to persist under strict anaerobic conditions, in a previous study an unusual site was found in San Diego Bay in which two PAHs, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Further investigations with these sediments revealed that methylnaphthalene, fluorene, and fluoranthene were also anaerobically oxidized to carbon dioxide in these sediments, while pyrene and benzo[a]pyrene were not. Studies with naphthalene indicated that PAH oxidation was sulfate dependent. Incubating the sediments with additional naphthalene for 1 month resulted in a significant increase in the oxidation of [14C]naphthalene. In sediments from a less heavily contaminated site in San diego Bay where PAHs were not readily degraded, naphthalene degradation could be stimulated through inoculation with active PAH-degrading sediments from the most heavily contaminated site. Sediments from the less heavily contaminated site that had been adapted for rapid anaerobic degradation of high concentrations of benzene did not oxidize naphthalene, suggesting that the benzene- and naphthalene-degrading populations were different. When fuels containing complex mixtures of alkanes were added to sediments from the two sites, there was significant degradation in the alkanes. [14C]hexadecane was also anaerobically oxidized to 14CO2 in these sediments. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation. These results demonstrate that a wide variety of hydrocarbon contaminants can be degraded under sulfate-reducing conditions in hydrocarbon-contaminated sediments, and they suggest that it may be possible to use sulfate reduction rather than aerobic respiration as a treatment strategy for hydrocarbon-contaminated dredged sediments.     

Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries.

Rates of polycyclic aromatic hydrocarbon (PAH) degradation and mineralization were influenced by preexposure to alternate PAHs and a monoaromatic hydrocarbon at relatively high (100 ppm) concentrations in organic-rich aerobic marine sediments. Prior exposure to three PAHs and benzene resulted in enhanced [14C]naphthalene mineralization, while [14C]anthracene mineralization was stimulated only by benzene and anthracene preexposure. Preexposure of sediment slurries to phenanthrene stimulated the initial degradation of anthracene. Prior exposure to naphthalene stimulated the initial degradation of phenanthrene but had no effect on either the initial degradation or mineralization of anthracene. For those compounds which stimulated [14C]anthracene or [14C]naphthalene mineralization, longer preexposures (2 weeks) to alternative aromatic hydrocarbons resulted in an even greater stimulation response. Enrichment with individual PAHs followed by subsequent incubation with one or two PAHs showed no alteration in degradation patterns due to the simultaneous presence of PAHs. The evidence suggests that exposure of marine sediments to a particular PAH or benzene results in the enhanced ability of these sediments to subsequently degrade that PAH as well as certain other PAHs. The enhanced degradation of a particular PAH after sediments have been exposed to it may result from the selection and proliferation of specific microbial populations capable of degrading it. The enhanced degradation of other PAHs after exposure to a single PAH suggests that the populations selected have either broad specificity for PAHs, common pathways of PAH degradation, or both.     

Effects of co-occurring aromatic hydrocarbons on degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries

Rates of polycyclic aromatic hydrocarbon (PAH) degradation and mineralization were influenced by preexposure to alternate PAHs and a monoaromatic hydrocarbon at relatively high (100 ppm) concentrations in organic-rich aerobic marine sediments. Prior exposure to three PAHs and benzene resulted in enhanced [14C]naphthalene mineralization, while [14C]anthracene mineralization was stimulated only by benzene and anthracene preexposure. Preexposure of sediment slurries to phenanthrene stimulated the initial degradation of anthracene. Prior exposure to naphthalene stimulated the initial degradation of phenanthrene but had no effect on either the initial degradation or mineralization of anthracene. For those compounds which stimulated [14C]anthracene or [14C]naphthalene mineralization, longer preexposures (2 weeks) to alternative aromatic hydrocarbons resulted in an even greater stimulation response. Enrichment with individual PAHs followed by subsequent incubation with one or two PAHs showed no alteration in degradation patterns due to the simultaneous presence of PAHs. The evidence suggests that exposure of marine sediments to a particular PAH or benzene results in the enhanced ability of these sediments to subsequently degrade that PAH as well as certain other PAHs. The enhanced degradation of a particular PAH after sediments have been exposed to it may result from the selection and proliferation of specific microbial populations capable of degrading it. The enhanced degradation of other PAHs after exposure to a single PAH suggests that the populations selected have either broad specificity for PAHs, common pathways of PAH degradation, or both.     

Isolation of Geobacter species from diverse sedimentary environments.

In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram-negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria. Unlike the type strain, Geobacter metallireducens, three of the five isolates could use H2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process.     

Requirement for a Microbial Consortium To Completely Oxidize Glucose in Fe(III)-Reducing Sediments

In various sediments in which Fe(III) reduction was the terminal electron-accepting process, [¹⁴C]glucose was fermented to ¹⁴C-fatty acids in a manner similar to that observed in methanogenic sediments. These results are consistent with the hypothesis that, in Fe(III)-reducing sediments, fermentable substrates are oxidized to carbon dioxide by the combined activity of fermentative bacteria and fatty acid-oxidizing, Fe(III)-reducing bacteria.     

Rapid Benzene Degradation in Methanogenic Sediments from a Petroleum-Contaminated Aquifer

In methanogenic sediments from a petroleum-contaminated aquifer, [¹⁴C]benzene was converted to ¹⁴CH₄ and ¹⁴CO₂ without an apparent lag. Phenol, acetate, and propionate were intermediates in benzene mineralization. These results suggest that alternative electron acceptors need not be available for there to be significant natural attenuation of benzene in some petroleum-contaminated aquifers.     

Competitive Mechanisms for Inhibition of Sulfate Reduction and Methane Production in the Zone of Ferric Iron Reduction in Sediments

Mechanisms for inhibition of sulfate reduction and methane production in the zone of Fe(III) reduction in sediments were investigated. Addition of amorphic iron(III) oxyhydroxide to sediments in which sulfate reduction was the predominant terminal electron-accepting process inhibited sulfate reduction 86 to 100%. The decrease in electron flow to sulfate reduction was accompanied by a corresponding increase in electron flow to Fe(III) reduction. In a similar manner, Fe(III) additions also inhibited methane production in sulfate-depleted sediments. The inhibition of sulfate reduction and methane production was the result of substrate limitation, because the sediments retained the potential for sulfate reduction and methane production in the presence of excess hydrogen and acetate. Sediments in which Fe(III) reduction was the predominant terminal electron-accepting process had much lower concentrations of hydrogen and acetate than sediments in which sulfate reduction or methane production was the predominant terminal process. The low concentrations of hydrogen and acetate in the Fe(III)-reducing sediments were the result of metabolism by Fe(III)-reducing organisms of hydrogen and acetate at concentrations lower than sulfate reducers or methanogens could metabolize them. The results indicate that when Fe(III) is in a form that Fe(III)-reducing organisms can readily reduce, Fe(III)-reducing organisms can inhibit sulfate reduction and methane production by outcompeting sulfate reducers and methanogens for electron donors.     

Enrichment of Geobacter Species in Response to Stimulation of Fe(III) Reduction in Sandy Aquifer Sediments

Abstract Engineered stimulation of Fe(III) has been proposed as a strategy to enhance the immobilization of radioactive and toxic metals in metal-contaminated subsurface environments. Therefore, laboratory and field studies were conducted to determine which microbial populations would respond to stimulation of Fe(III) reduction in the sediments of sandy aquifers. In laboratory studies, the addition of either various organic electron donors or electron shuttle compounds stimulated Fe(III) reduction and resulted in Geobacter sequences becoming important constituents of the Bacterial 16S rDNA sequences that could be detected with PCR amplification and denaturing gradient gel electrophoresis (DGGE). Quantification of Geobacteraceae sequences with a PCR most-probable-number technique indicated that the extent to which numbers of Geobacter increased was related to the degree of stimulation of Fe(III) reduction. Geothrix species were also enriched in some instances, but were orders of magnitude less numerous than Geobacter species. Shewanella species were not detected, even when organic compounds known to be electron donors for Shewanella species were used to stimulate Fe(III) reduction in the sediments. Geobacter species were also enriched in two field experiments in which Fe(III) reduction was stimulated with the addition of benzoate or aromatic hydrocarbons. The apparent growth of Geobacter species concurrent with increased Fe(III) reduction suggests that Geobacter species were responsible for much of the Fe(III) reduction in all of the stimulation approaches evaluated in three geographically distinct aquifers. Therefore, strategies for subsurface remediation that involve enhancing the activity of indigenous Fe(III)-reducing populations in aquifers should consider the physiological properties of Geobacter species in their treatment design. Received: 19 October 1999; Accepted: 28 December 1999; Online Publication: 25 April 2000     

Stimulating the anaerobic degradation of aromatic hydrocarbons in contaminated sediments by providing an electrode as the electron acceptor

The possibility that electrodes might serve as an electron acceptor to simulate the degradation of aromatic hydrocarbons in anaerobic contaminated sediments was investigated. Initial studies with Geobacter metallireducens demonstrated that although toluene was rapidly adsorbed onto the graphite electrodes it was rapidly oxidized to carbon dioxide with the electrode serving as the sole electron acceptor. Providing graphite electrodes as an electron acceptor in hydrocarbon‐contaminated sediments significantly stimulated the removal of added toluene and benzene. Rates of toluene and benzene removal accelerated with continued additions of toluene and benzene. [¹⁴C]‐Toluene and [¹⁴C]‐benzene were quantitatively recovered as [¹⁴C]‐CO₂, demonstrating that even though the graphite adsorbed toluene and benzene they were degraded. Introducing an electrode as an electron acceptor also accelerated the loss of added naphthalene and [¹⁴C]‐naphthalene was converted to [¹⁴C]‐CO₂. The results suggest that graphite electrodes can serve as an electron acceptor for the degradation of aromatic hydrocarbon contaminants in sediments, co‐localizing the contaminants, the degradative organisms and the electron acceptor. Once in position, they provide a permanent, low‐maintenance source of electron acceptor. Thus, graphite electrodes may offer an attractive alternative for enhancing contaminant degradation in anoxic environments.     

Anaerobic Oxidation of Toluene, Phenol, and p-Cresol by the Dissimilatory Iron-Reducing Organism, GS-15

The dissimilatory Fe(III) reducer, GS-15, is the first microorganism known to couple the oxidation of aromatic compounds to the reduction of Fe(III) and the first example of a pure culture of any kind known to anaerobically oxidize an aromatic hydrocarbon, toluene. In this study, the metabolism of toluene, phenol, and p-cresol by GS-15 was investigated in more detail. GS-15 grew in an anaerobic medium with toluene as the sole electron donor and Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. [ring-¹⁴C]toluene was oxidized to ¹⁴CO₂, and the stoichiometry of ¹⁴CO₂ production and Fe(III) reduction indicated that GS-15 completely oxidized toluene to carbon dioxide with Fe(III) as the electron acceptor. Magnetite was the primary iron end product during toluene oxidation. Phenol and p-cresol were also completely oxidized to carbon dioxide with Fe(III) as the sole electron acceptor, and GS-15 could obtain energy to support growth by oxidizing either of these compounds as the sole electron donor. p-Hydroxybenzoate was a transitory extracellular intermediate of phenol and p-cresol metabolism but not of toluene metabolism. GS-15 oxidized potential aromatic intermediates in the oxidation of toluene (benzylalcohol and benzaldehyde) and p-cresol (p-hydroxybenzylalcohol and p-hydroxybenzaldehyde). The metabolism described here provides a model for how aromatic hydrocarbons and phenols may be oxidized with the reduction of Fe(III) in contaminated aquifers and petroleum-containing sediments.     

Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns

Toluene and m-xylene were rapidly mineralized in an anaerobic laboratory aquifer column operated under continuous-flow conditions with nitrate as an electron acceptor. The oxidation of toluene and m-xylene was coupled with the reduction of nitrate, and mineralization was confirmed by trapping 14CO2 evolved from 14C-ring-labeled substrates. Substrate degradation also took place when nitrous oxide replaced nitrate as an electron acceptor, but decomposition was inhibited in the presence of molecular oxygen or after the substitution of nitrate by nitrite. The m-xylene-adapted microorganisms in the aquifer column degraded toluene, benzaldehyde, benzoate, m-toluylaldehyde, m-toluate, m-cresol, p-cresol, and p-hydroxybenzoate but were unable to metabolize benzene, naphthalene, methylcyclohexane, and 1,3-dimethylcyclohexane. Isotope-dilution experiments suggested benzoate as an intermediate formed during anaerobic toluene metabolism. The finding that the highly water-soluble nitrous oxide served as electron acceptor for the anaerobic mineralization of some aromatic hydrocarbons may offer attractive options for the in situ restoration of polluted aquifers.