Benzene Degradation Coupled with Chlorate Reduction in a Soil Column Study

Perchlorate and chlorate are electron acceptors that during reduction result in the formation of molecular oxygen. The produced oxygen can be used for activation of anaerobic persistent pollutants, like benzene. In this study chlorate was tested as potential electron acceptor to stimulate benzene degradation in anoxic polluted soil column. A chlorate amended benzene polluted soil column was operated over a period of 500 days. Benzene was immediately degraded in the column after start up, and benzene removal recovered completely after omission of chlorate or a too high influent chlorate concentration (22 mM). Mass balance calculations showed that per mole of benzene five mole of chlorate were reduced. At the end of the experiment higher loading rates were applied to measure the maximal benzene degradation rate in this system; a breakthrough of benzene was not observed. The average benzene degradation rate over this period was 31 μmol l⁻¹ h⁻¹ with a maximal of 78 μmol l⁻¹ h⁻¹. The high degradation rate and the necessity of chlorate indicate that oxygen produced during chlorate reduction indeed is used for the activation of benzene. This is the first column study where benzene biodegradation at a high rate coupled with anaerobic chlorate reduction is observed.     

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.     

Physiological and phylogenetic characterization of a stable benzene-degrading, chlorate-reducing microbial community

A stable anoxic enrichment culture was obtained that degraded benzene with chlorate as an electron acceptor. The benzene degradation rate was 1.65 mM benzene per day, which is similar to reported aerobic benzene degradation rates but 20-1650 times higher than reported for anaerobic benzene degradation. Denaturing gradient gel electrophoresis of part of the 16S rRNA gene, cloning and sequencing showed that the culture had a stable composition after the seventh transfer. Five bacterial clones were further analyzed. Two clones corresponded to bacteria closely related to Alicycliphilus denitrificans K601. The three other clones corresponded to bacteria closely related to Zoogloea resiniphila PIV-3A2w, Mesorhizobium sp. WG and Stenotrophomonas acidaminiphila. DGGE analysis of cultures grown with different electron donors and acceptors indicated that the bacterium related to Alicycliphilus denitrificans K601 is able to degrade benzene coupled to chlorate reduction. The role of the other bacteria could not be conclusively determined. The bacterium related to Mesorhizobium sp. WG can be enriched with benzene and oxygen, but not with acetate and chlorate, while the bacterium related to Stenotrophomonas acidaminophila grows with acetate and chlorate, but not with benzene and oxygen. As oxygen is produced during chlorate reduction, an aerobic pathway of benzene degradation is most likely.     

Benzene Degradation at a Site Amended with Nitrate or Chlorate

Aromatic hydrocarbons are widespread in nature and often contribute to the pollution of soils, sediments, and groundwater. The contamination of soil with mobile aromatic compounds, generally termed BTEX (benzene, toluene, ethylbenzene, xylene) is observed at many industrial sites, especially those associated with the petrochemical industry. In situ bioremediation of sites that are contaminated with BTEX can be applied both aerobically and anaerobically. The use of anaerobic in situ bioremediation is advantageous because supply of oxygen is not needed. Nevertheless, anaerobic in situ bioremediation is less commonly used for BTEX contaminated sites. This paper describes push-pull experiments in order to stimulate the degradation of benzene by the addition of nitrate or chlorate. Deuterated benzene was subjected with nitrate-amended groundwater to the aquifer, and the mineralization was traced by the enrichment of deuterium in the groundwater. Nitrate can be used as electron acceptor, and the addition of nitrate at a site in The Netherlands resulted in partial degradation of benzene. This was demonstrated by comparing various push-pull experiments, benzene concentration measurements, stable isotope analyses of benzene and water, and modeling. Chlorate can be used for the in situ production of oxygen, followed by degradation of benzene with oxygen as electron acceptor. The addition of chlorate at the site resulted in the complete removal of benzene demonstrating a complete degradation within 4 weeks. A pull phase was not needed during this run.     

Anaerobic degradation of monoaromatic hydrocarbons

Over the last two decades significant advances have been made in our understanding of the anaerobic biodegradability of monoaromatic hydrocarbons. It is now known that compounds such as benzene, toluene, ethylbenzene, and all three xylene isomers can be biodegraded in the absence of oxygen by a broad diversity of organisms. These compounds have been shown to serve as carbon and energy sources for bacteria growing phototrophically, or respiratorily with nitrate, manganese, ferric iron, sulfate, or carbon dioxide as the sole electron acceptor. In addition, it has also been recently shown that complete degradation of monoaromatic hydrocarbons can also be coupled to the respiration of oxyanions of chlorine such as perchlorate or chlorate, or to the reduction of the quinone moieties of humic substances. Many pure cultures of hydrocarbon-degrading anaerobes now exist and some novel biochemical and genetic pathways have been identified. In general, a fumarate addition reaction is used as the initial activation step of the catabolic process of the corresponding monoaromatic hydrocarbon compounds. However, other reactions may alternatively be involved depending on the electron acceptor utilized or the compound being degraded. In the case of toluene, fumarate addition to the methyl group mediated by benzylsuccinate synthase appears to be the universal mechanism of activation and is now known to be utilized by anoxygenic phototrophs, nitrate-reducing, Fe(III)-reducing, sulfate-reducing, and methanogenic cultures. Many of these biochemical pathways produce unique extracellular intermediates that can be utilized as biomarkers for the monitoring of hydrocarbon degradation in anaerobic natural environments.     

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 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 μM 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.     

Fermentative toluene degradation in anaerobic defined syntrophic cocultures

A syntrophic coculture of a new sulfate-reducing isolate, strain TRM1, with Wolinella succinogenes degraded toluene with either fumarate or NO3- as the terminal electron acceptor. Neither strain TRM1 nor W. succinogenes could metabolise toluene under these conditions in pure culture. Syntrophic degradation was 2-3 times slower than toluene utilisation by strain TRM1 in pure culture with sulfate as electron acceptor. The culture did not produce benzoate or fatty acids like acetate or propionate in detectable amounts. An increase in biomass of the syntrophic toluene-degrading culture was shown in a growth curve with nitrate as the terminal electron acceptor. Both partner organisms were detected microscopically at the end of the growth experiment. Syntrophic degradation of toluene with W. succinogenes and fumarate as the terminal electron acceptor was also demonstrated with the iron reducer Geobacter metallireducens. The results provide the first example of a fermentative oxidation of an aromatic hydrocarbon in a defined coculture.     

Anaerobic Growth of Microorganisms with Chlorate as an Electron Acceptor

The ability of microorganisms to use chlorate (ClO₃^-) as an electron acceptor for respiration under anaerobic conditions was studied in batch and continuous tests. Complex microbial communities were cultivated anaerobically in defined media containing chlorate, all essential minerals, and acetate as the sole energy and carbon source. It was shown that chlorate was reduced to chloride, while acetate was oxidized to carbon dioxide and water and used as the carbon source for synthesis of new biomass. A biomass yield of 1.9 to 3.8 g of volatile suspended solids per equivalent of available electrons was obtained, showing that anaerobic growth with chlorate as an electron acceptor gives a high energy yield. This indicates that microbial reduction of chlorate to chloride in anaerobic systems is coupled with electron transport phosphorylation.     

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.     

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.     

Degradation of BTX by dissimilatory iron-reducing cultures

The ability of indigenous bacteria to anaerobically degrade monoaromatic hydrocarbons has received attention as a potential strategy to remediate polluted aquifers. Despite the fact that iron-reducing conditions are often dominating in contaminated sediment, most of the studies have focussed on degradation of this class of pollutants with other terminal acceptors. In this work, we enriched bacteria from an iron-reducing aquifer in which a plume of pollution has developed over several decades and we show that benzene, toluene, meta- and para-xylene (BTX) could be degraded by the enriched cultures containing intrinsic iron-reducing microorganisms. To our knowledge, this is the first time that para-xylene degradation by dissimilatory iron-reducing bacteria has been reported in sediment free enrichment cultures. BTX degradation rates in enrichment cultures progressively increased in time and were found in good agreement with theoretical values calculated assuming complete BTX oxidation with Fe(II) as final electron acceptor. In addition, using labelled ((13)C(1)) benzene and toluene we could unambiguously identify intermediates of their respective degradation pathways. We provide evidence for benzene degradation via phenol formation under iron-reducing conditions, whereas toluene and meta-xylene were transformed into the corresponding benzylsuccinates.     

Behaviour of toluene, benzene and naphthalene under anaerobic conditions in sediment columns

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 μM) at 20°C. Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [¹⁴C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO₂.     

Molecular oxygen controls nitrate transport of Escherichia coli nitrate-respiring cells

Escherichia coli cells grown anaerobically in the presence of nitrate reduce the nitrate as a terminal electron acceptor in place of molecular oxygen by an induced respiratory-type electron transferring system residing in the inner membrane structure. When oxygen is introduced to a suspension of nitrate-respiring cells, the oxygen is immediately reduced preferentially and the cellular uptake of nitrate ceases abruptly. In contrast, we found that the cells exhibited no oxygen control on uptake of chlorate, a competitive substrate analogue, indicating operation of an oxygen-sensitive transport system specific to nitrate. This was further evidenced by the fact that chlorate inhibition of reduction of nitrate was brought about only when the transport of both chlorate and nitrate was facilitated by the aid of carrier-type chlorate (or nitrate) ionophore. We demonstrated that oxygen inhibition on reduction of nitrate was abolished within the cells treated by octyl glucoside resulting in a removal of permeability barrier specific to nitrate. We conclude that the transient control by molecular oxygen is primarily due to the inhibition of nitrate transport into the cytoplasmic side. Since nitrate induces the nitrate-respiring system, the repression of the nitrate reductase operon by molecular oxygen is consistently interpreted on the basis of the "inducer exclusion mechanism."     

Enhanced anaerobic biodegradation of benzene-toluene-ethylbenzene-xylene-ethanol mixtures in bioaugmented aquifer columns

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential. bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.     

Reduction of (per)chlorate by a novel organism isolated from paper mill waste

As part of a study on the microbiology of chlorate reduction, several new dissimilatory chlorate-reducing bacteria were isolated from a broad diversity of environments. One of these, strain CKB, was selected for a more complete characterization. Strain CKB was enriched and isolated from paper mill waste with acetate as the sole electron donor and chlorate as the sole electron acceptor. Strain CKB is a completely oxidizing, non-fermentative, Gram-negative, facultative anaerobe. Cells of strain CKB are 0.5 x 2 microm and are highly motile, with a single polar flagellum. In addition to acetate, strain CKB can use propionate, butyrate, lactate, succinate, fumarate, malate or yeast extract as electron donors, with chlorate as the sole electron acceptor. Strain CKB can also couple chlorate reduction to the oxidation of ferrous iron, sulphide, or the reduced form of the humic substances analogue 2,6-anthrahydroquinone disulphonate. Fe(II) is oxidized to insoluble amorphous Fe(II) oxide, whereas sulphide is oxidized to elemental sulphur. Growth is not associated with this metabolism, even when small quantities of acetate are added as a potential carbon source. In addition to chlorate, strain CKB can also couple acetate oxidation to the reduction of oxygen or perchlorate. Chlorate is completely reduced to chloride. Strain CKB has an optimum temperature of 35 degrees C, a pH optimum of 7.5 and a salinity optimum of 1% NaCl. Strain CKB can grow in chlorate and perchlorate concentrations of 80 or 20 mM respectively. Under anaerobic conditions, strain CKB can dismutate chlorite into chloride and O2, and is only the second organism shown to be capable of this metabolism. Oxidized minus reduced spectra of whole-cell suspensions of strain CKB showed absorbance maxima at 423, 523 and 552nm, which are indicative of the presence of c-type cytochrome(s). Analysis of the complete sequence of the 16S rDNA indicates that strain CKB is a member of the beta subclass of the Proteobacteria. The phototroph Rhodocyclus tenuis is the closest known relative. When tested, strain CKB could not grow by phototrophy and did not contain bacteriochlorophyll. Phenotypically and phylogenetically, strain CKB differs from all other described bacteria and represents the type strain of a new genus and species.     

Enhanced Anaerobic Biodegradation of Benzene-Toluene-Ethylbenzene-Xylene-Ethanol Mixtures in Bioaugmented Aquifer Columns

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential. bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.     

The effect of oxygen on chemotaxis to naphthalene by Pseudomonas putida G7

Chemotactic bacteria can be attracted to electron donors they consume. In systems where donor is heterogeneously distributed, chemotaxis can lead to enhanced removal of donor relative to that achieved in the absence of chemotaxis. However, simultaneous consumption of an electron acceptor may result in the formation of an acceptor gradient to which the bacteria also respond, thus diminishing the positive effect of chemotaxis. Depletion of an electron acceptor can also reduce the rate of electron donor consumption in addition to its effect on chemotaxis. In this study, we examined the effect of oxygen on chemotaxis to naphthalene and on naphthalene consumption by Pseudomonas putida G7. The organism was able to move up an oxygen gradient when there was a naphthalene gradient in the opposite direction. In the absence of an oxygen gradient, low levels of oxygen attenuated chemotaxis to naphthalene but did not affect random motility. The rate of naphthalene consumption decreased at dissolved oxygen concentrations similar to those at which chemotaxis was attenuated. These results suggest that low dissolved oxygen concentrations can reduce naphthalene removal by P. putida G7 in systems where naphthalene is heterogeneously distributed by simultaneously attenuating chemotactic motion toward naphthalene and decreasing the rate of naphthalene degradation.     

Behaviour of toluene, benzene and naphthalene under anaerobic conditions in sediment columns

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 M) at 20°C.Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [¹⁴C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO₂.     

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.