Hydroxylation and Carboxylation—Two Crucial Steps of Anaerobic Benzene Degradation by Dechloromonas Strain RCB

Benzene is a highly toxic industrial compound that is essential to the production of various chemicals, drugs, and fuel oils. Due to its toxicity and carcinogenicity, much recent attention has been focused on benzene biodegradation, especially in the absence of molecular oxygen. However, the mechanism by which anaerobic benzene biodegradation occurs is still unclear. This is because until the recent isolation of Dechloromonas strains JJ and RCB no organism that anaerobically degraded benzene was available with which to elucidate the pathway. Although many microorganisms use an initial fumarate addition reaction for hydrocarbon biodegradation, the large activation energy required argues against this mechanism for benzene. Other possible mechanisms include hydroxylation, carboxylation, biomethylation, or reduction of the benzene ring, but previous studies performed with undefined benzene-degrading cultures were unable to clearly distinguish which, if any, of these alternatives is used. Here we demonstrate that anaerobic nitrate-dependent benzene degradation by Dechloromonas strain RCB involves an initial hydroxylation, subsequent carboxylation, and loss of the hydroxyl group to form benzoate. These studies provide the first pure-culture evidence of the pathway of anaerobic benzene degradation. The outcome of these studies also suggests that all anaerobic benzene-degrading microorganisms, regardless of their terminal electron acceptor, may use this pathway.     

Anaerobic benzene degradation by bacteria

Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen‐dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene‐degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the ‘key players’ of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.     

Anaerobic degradation of benzene, toluene, ethylbenzene, and xylene compounds by Dechloromonas strain RCB

Dechloromonas strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (ortho-, meta-, and para-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO2, strain RCB did not completely mineralize para-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 microM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.     

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.     

Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process

An anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic process.     

Anaerobic Benzene Biodegradation: A Microcosm Survey

Benzene-amended microcosms prepared with saturated soil or sediment from five hydrocarbon-contaminated sites and one pristine site were monitored for a year and a half to determine the rate of benzene biodegradation under a variety of electron-accepting conditions. Sustainable benzene degradation was observed under specific conditions in microcosms from four of the six sites. Significant differences were observed between sites with respect to lag times before the onset of degradation, rates of degradation, sustainability of the activity, and environmental conditions supporting degradation. Benzene degradation was observed under sulfate-reducing, nitrate-reducing, and iron(III)-reducing conditions, but not under methanogenic conditions. The presence of competing substrates such as toluene, xylenes, and ethylbenzene was found to inhibit anaerobic benzene degradation in microcosms where sulfate or possibly nitrate was the electron acceptor for benzene degradation, but not in microcosms from where iron(III) was the electron acceptor. The presence of organic matter, indicated by a high fraction organic carbon (f_oc), also appeared to inhibit the biodegradation of benzene; microcosms constructed with soils with the highest f_oc exhibited the longest lag times before the onset of benzene degradation. The initial extent of hydrocarbon contamination did not appear to correlate with anaerobic benzene-degrading activity.     

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.     

Identification of new enzymes potentially involved in anaerobic naphthalene degradation by the sulfate-reducing enrichment culture N47

The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase.     

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.     

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.     

红树林厌氧环境对多环芳烃类有毒物的降解预测

红树林是连接陆地和海洋的重要生态系统,由于潮汐活动,氧化还原条件表现出明显的昼夜间的交替,这一生态体系中不但有大量的动植物种类,同时还有数量极高的不同种类的细菌,包括好氧和厌氧类型,厌养的硫酸(盐)还原菌已证实在降解多环芳烃有机物方面有其独特的生化优势,但从红树林中分离出的此类纯细菌还很少,在降解方面,已初步确定萘的厌氧降解途径异于好氧细菌,厌氧降解时的一系列代谢中间产物也有明显的专一性,羰基化反应是开始的一个重要步骤,而后的每步生化反应还有待进一步验证。从现有的结果可以看出,红树林中厌养的硫酸还原菌应在降解多环芳烃有机物中起到非常重要的作用。     

Degradation of BTEX by anaerobic bacteria: physiology and application

Pollution of the environment with aromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX) is often observed. The cleanup of these toxic compounds has gained much attention in the last decades. In situ bioremediation of aromatic hydrocarbons contaminated soils and groundwater by naturally occurring microorganisms or microorganisms that are introduced is possible. Anaerobic bioremediation is an attractive technology as these compounds are often present in the anoxic zones of the environment. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene and the bacteria involved in anaerobic benzene degradation. Here, we review the existing knowledge on the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria, in particular the physiology and application, including results on the (per)chlorate stimulated degradation of these compounds, which is an interesting new alternative option for bioremediation.     

Assessment of anaerobic benzene degradation potential using 16S rRNA gene-targeted real-time PCR

Benzene is a common groundwater pollutant that is often recalcitrant under the anaerobic conditions that prevail at hydrocarbon-contaminated aquifers. Thus, determining the potential for anaerobic benzene degradation is important to assess the feasibility of intrinsic bioremediation. In this work we developed a 16S rRNA biomarker to estimate the concentration of putative benzene degraders in a methanogenic consortium that has been enriched on benzene for several years. Primers were designed based on phylogenetic information from this consortium. The primers and probe were obtained by sequencing the dominant denaturing gradient gel electrophoresis band of this consortium, which corresponded to Desulfobacterium sp. clone OR-M2. No hybridization was observed with DNA samples from negative controls (i.e. toluene-degrading and dehalorespiring methanogenic consortia that do not degrade benzene). Samples from an anaerobic aquifer column that was bioaugmented with this benzene-degrading consortium showed a strong correlation between benzene degradation activity and the concentration of the target organism. Although our data do not prove that Desulfobacterium sp. is a benzene degrader, its enrichment as a result of benzene consumption and its correlation to anaerobic benzene degradation activity suggest that it either initiates benzene degradation or is a critical (commensal) partner. Therefore, the utility of this primers and probe set to assess anaerobic benzene degradation potential was demonstrated. This is the first report of the use of real-time quantitative PCR for forensic analysis of anaerobic benzene degradation. Whether this biomarker will be adequately selective and broadly applicable to assess benzene degradation potential under strongly anaerobic (sulfate reducing and methanogenic) conditions will require further research.     

Enhanced anaerobic degradation of benzene by enrichment of mixed microbial culture and optimization of the culture medium

A heterogeneous mixed culture, originally collected from two different sources, namely cow-drug and sludge from the mineral medium containing 1% glucose and then adapted on benzene as the carbon and energy source. Under anaerobic conditions benzene was degraded via benzoic acid as a major intermediate in the benzene degradation pathway. The degradation rate of benzene was improved stepwise by the number of enrichments and optimization of the culture medium. The effects of microaerobic conditions and/or physicochemical treatment with H₂O₂ prior to anaerobic degradation were studied with respect to variations in benzene degradation rate, growth of biomass and gas produced is less than the theoretical value expected and the percentage of methane in the product gas was very small (3%–3.5%). The reason for this is not well understood but it is presumed that the major group of benzene-degrading bacteria present in the culture medium are sulphate reducers and the mixed consortium is unable to degrade certain complex aromatic intermediates in the benzene degradation pathway under the experimental conditions. For an actual explanation of the situation arising in this study, further investigations must be carrie out. However, the mixed culture is capable of oxidizing benzene more rapidly to intermediate compounds and also partly into gas under the culture conditions, compared to the published data for the anaerobic degradation of benzene.     

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.     

Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotype

The anaerobic biodegradation of benzene, a common constituent of petroleum and one of the least reactive aromatic hydrocarbons, is insufficiently understood with respect to the involved microorganisms and their metabolism. To study these aspects, sulfate-reducing bacteria were enriched with benzene as sole organic substrate using marine sediment as inoculum. Repeated subcultivation yielded a sediment-free enrichment culture constituted of mostly oval-shaped cells and showing benzene-dependent sulfate reduction and growth under strictly anoxic conditions. Amplification and sequencing of 16S rRNA genes from progressively diluted culture samples revealed an abundant phylotype; this was closely related to a clade of Deltaproteobacteria that includes sulfate-reducing bacteria able to degrade naphthalene or other aromatic hydrocarbons. Cell hybridization with two specifically designed 16S rRNA-targeted fluorescent oligonucleotide probes showed that the retrieved phylotype accounted for more than 85% of the cells detectable via DAPI staining (general cell staining) in the enrichment culture. The result suggests that the detected dominant phylotype is the 'candidate species' responsible for the anaerobic degradation of benzene. Quantitative growth experiments revealed complete oxidation of benzene with stoichiometric coupling to the reduction of sulfate to sulfide. Suspensions of benzene-grown cells did not show metabolic activity towards phenol or toluene. This observation suggests that benzene degradation by the enriched sulfate-reducing bacteria does not proceed via anaerobic hydroxylation (mediated through dehydrogenation) to free phenol or methylation to toluene, respectively, which are formerly proposed alternative mechanisms for benzene activation.     

Carbon and hydrogen isotopic fractionation during anaerobic biodegradation of benzene

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (epsilon ) for carbon (range of -1.9 to -3.6 per thousand ) and hydrogen (range of -29 to -79 per thousand ) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.     

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

Aims:  To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer.
Methods and Results:  We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2·57 mg l⁻¹ showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12·1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas , Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga .
Conclusions:  Results show that pH 12·1 is inimical to benzene biodegradation, and that oxygen concentrations below 0·03 mg l⁻¹ can support aerobic benzene-degrading communities.
Significance and Impact of the Study:  These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.     

Carbon and Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors () for carbon (range of −1.9 to −3.6‰) and hydrogen (range of −29 to −79‰) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.