Anaerobic Benzene Degradation Research Articles

Use of stable isotopes to identify benzoate as a metabolite of benzene degradation in a sulphidogenic consortium.

An enriched sulphidogenic consortium capable of mineralizing benzene was used to study the metabolic pathway of anaerobic benzene degradation. Benzoate was detected in active cultures and benzene was confirmed to be the source of this benzoate by the addition of deuterated benzene (D6) and subsequent detection of deuterated benzoate (D5) in active cultures but not in autoclaved controls. Benzoate was utilized by this culture at 1/12 the rate of benzene, while its presence did not inhibit benzene utilization. The benzene utilization rate was reduced, however, in the presence of 2-fluorobenzoate. When the culture was supplemented with [(13)C]-bicarbonate, the carboxyl group on benzoate was not labelled with [(13)C]-carbon, suggesting that this transformation relies on a more complex set of reactions than simple addition of carbonate.

Anaerobic Bioremediation of Benzene under Sulfate-Reducing Conditions in a Petroleum-Contaminated Aquifer

The addition of sulfate to an anaerobic petroleum-contaminated aquifer in which benzene was a major soluble contaminant resulted in removal of benzene from the groundwater. The loss in benzene was associated with a decrease in sulfate along the groundwater flow path, relative to a conservative bromide tracer. Studies with [2-14C]acetate and molybdate demonstrated that sulfate reduction was the predominant terminal electron-accepting process (TEAP) in the sulfate-amended sediments, and studies with [14C]benzene indicated that benzene oxidation was dependent upon sulfate reduction. Abundant ferrous iron in the subsurface likely prevented the generation of free sulfide in the groundwater during the field trial. Comparisons of benzene and sulfate depletion in the treatment zone indicated that benzene degradation could account for 53% of the sulfate depletion. These results suggest that the addition of sulfate stimulated the activity of benzene-degrading, sulfate-reducing microorganisms. This is the first field study demonstrating that it is possible to stimulate anaerobic benzene degradation in a petroleum-contaminated aquifer.

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 commonly considered electron acceptors for anaerobic respiration. These include: Fe(III), sulfate, nitrate, and possibly humic substances. Benzene can also be converted to methane and carbon dioxide under methanogenic conditions. There is evidence that benzene can be degraded under in situ conditions in petroleum-contaminated aquifers 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.

Naphthalene and Benzene Degradation under Fe(III)-Reducing Conditions in Petroleum-Contaminated Aquifers

Naphthalene was oxidized anaerobically to CO2 in sediments collected from a petroleum-contaminated aquifer in Bemidji, Minnesota in which Fe(III) reduction was the terminal electron-accepting process. Naphthalene was not oxidized in sediments from the methanogenic zone at Bemidji or in sediments from the Fe(III)-reducing zone of other petroleum-contaminated aquifers studied. In a profile across the Fe(III)-reducing zone of the Bemidji aquifer, rates of naphthalene oxidation were fastest in sediments with the highest proportion of Fe(III), which was also the zone of the most rapid degradation of benzene, toluene, and acetate. The comparative studies attempted to elucidate factors that might account for the fact that unsubstituted aromatic hydrocarbons such as benzene and naphthalene were degraded under Fe(III)-reducing conditions at Bemidji, but not at the other aquifers examined. These studies indicated that the ability of Fe(III)-reducing microorganisms to degrade benzene and naphthalene at the Bemidji site cannot be attributed to groundwater components that make Fe(III) more available for reduction or other potential factors that were evaluated. However, unlike the other aquifers evaluated, uncontaminated sediments at the Bemidji site could be adapted for anaerobic benzene degradation merely with the addition of benzene. These findings indicate that Bemidji sediments naturally contain Fe(III) reducers capable of degradation of unsubstituted aromatic hydrocarbons.

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.

Enhanced anaerobic benzene degradation with the addition of sulfate

Potential mechanisms for stimulating anaerobic benzene degradation in methanogenic sediments from a petroleum‐contaminated aquifer were evaluated. In short‐term (<2 weeks) incubations, addition of sulfate slightly stimulated benzene degradation and caused a small decrease in the ratio of methane to carbon dioxide production from benzene. However, in longer‐term (>100 days) incubations, sulfate significantly stimulated benzene degradation with a complete shift to carbon dioxide as the end product of benzene degradation. The addition of Fe(III) and humic substances had short‐term and long‐term effects that were similar to the effects of the sulfate amendments. In studies in which anaerobic groundwater was pumped through columns of aquifer sediments, addition of sulfate to the groundwater significantly enhanced the removal of benzene from the groundwater. The stoichiometry of sulfate and benzene removal from the groundwater passing through the sediment columns was consistent with benzene oxidation to carbon dioxide with sulfate serving as the primary electron acceptor. These results demonstrate for the first time that addition of sulfate may be an effective strategy for enhancing anaerobic benzene removal in some petroleum‐contaminated aquifers.

Enhanced Anaerobic Benzene Degradation with the Addition of Sulfate

Enhanced Anaerobic Benzene Degradation with the Addition of Sulfate

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 (foc), also appeared to inhibit the biodegradation of benzene; microcosms constructed with soils with the highest foc 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.

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.

Anaerobic Degradation of Benzene in Diverse Anoxic Environments

Benzene has often been observed to be resistant to microbial degradation under anoxic conditions. A number of recent studies, however, have demonstrated that anaerobic benzene utilization can occur. This study extends the previous reports of anaerobic benzene degradation to sediments that varied with respect to contamination input, predominant redox condition, and salinity. In spite of differences in methodology, microbial degradation of benzene was noted in slurries constructed with sediments from various geographical locations and range from aquifer sands to fine-grained estuarine muds, under methanogenic, sulfate-reducing, and iron-reducing conditions. In aquifer sediments under methanogenic conditions, benzene loss was concomitant with methane production, and microbial utilization of [14C]benzene yielded 14CO2 and 14CH4. In slurries with estuarine and aquifer sediments under sulfate-reducing conditions, the loss of sulfate in amounts consistent with the stoichiometric degradation of benzene or the con...

Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction

Enrichment cultures from marine sediments mineralized benzene while using sulfate as the terminal electron acceptor. Parallel cultures using river marsh sediment displayed no activity. Mineralization was confirmed by release of 14CO 2 from radiolabeled benzene. The dependence on sulfate reduction was demonstrated by stoichiometric balances and the use of specific inhibitors. This work supports recent observations that anaerobic benzene degradation takes place coupled to sulfate reduction.

Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction.

Enrichment cultures from marine sediments mineralized benzene while using sulfate as the terminal electron acceptor. Parallel cultures using river marsh sediment displayed no activity. Mineralization was confirmed by release of 14CO2 from radiolabeled benzene. The dependence on sulfate reduction was demonstrated by stoichiometric balances and the use of specific inhibitors. This work supports recent observations that anaerobic benzene degradation takes place coupled to sulfate reduction.

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 city waste-water treatment plant, was grown in 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 composition. It was noticed that the amount of 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 carried 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.

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

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

Degradation of BTEX and their aerobic metabolites by indigenous microorganisms under nitrate reducing conditions

Batch incubations, seeded with four different aquifer materials, were used to survey the catabolic capacity of indigenous microorganisms under nitrate reducing conditions. Benzene, toluene, ethylbenzene, xylenes (BTEX), and selected potential metabolites of their incomplete aerobic degradation, were tested as substrates for nitrate-based respiration. Toluene and its potential aerobic metabolites, benzoate, protocatechuate, 3-methylcatechol, 4-methylcatechol, succinate, and adipate were degraded in strictly anoxic (O2 < 0.1 mg/l) nitrate reducing incubations. Toluene degradation was directly coupled to nitrate reduction. Ortho-xylene removal was toluene dependent. Meta- and para-xylenes were degraded in nitrate reducing enrichments from only one of the four aquifer samples. Benzene, ethylbenzene, catechol and gentisate were not degraded within up to four months in any of the incubations, even though nitrate reduction occurred. Anaerobic benzene degradation was not observed. Incubations receiving nitrate as an adjunct electron acceptor to oxygen degraded significantly more benzene than incubations amended with only oxygen, although benzene was only degraded until the dissolved oxygen was depleted. Possibly, more oxygen was available to degrade benzene when nitrate was added because denitrifiers utilizing nitrate as terminal electron acceptor oxidized benzoate, which had been added to increase the biochemical oxygen demand of the system. Benzoate oxidation with nitrate apparently spared oxygen for benzene degradation.

Degradation of BTEX and their aerobic metabolites by indigenous microorganisms under nitrate reducing conditions

Batch incubations, seeded with four different aquifer materials, were used to survey the catabolic capacity of indigenous microorganisms under nitrate reducing conditions. Benzene, toluene, ethylbenzene, xylenes (BTEX), and selected potential metabolites of their incomplete aerobic degradation, were tested as substrates for nitrate-based respiration. Toluene and its potential aerobic metabolites, benzoate, protocatechuate, 3-methylcatechol, 4-methylcatechol, succinate, and adipate were degraded in strictly anoxic (O2 &amp;lt; 0.1 mg/l) nitrate reducing incubations. Toluene degradation was directly coupled to nitrate reduction. Ortho-xylene removal was toluene dependent. Meta- and para-xylenes were degraded in nitrate reducing enrichments from only one of the four aquifer samples. Benzene, ethylbenzene, catechol and gentisate were not degraded within up to four months in any of the incubations, even though nitrate reduction occurred. Anaerobic benzene degradation was not observed. Incubations receiving nitrate as an adjunct electron acceptor to oxygen degraded significantly more benzene than incubations amended with only oxygen, although benzene was only degraded until the dissolved oxygen was depleted. Possibly, more oxygen was available to degrade benzene when nitrate was added because denitrifiers utilizing nitrate as terminal electron acceptor oxidized benzoate, which had been added to increase the biochemical oxygen demand of the system. Benzoate oxidation with nitrate apparently spared oxygen for benzene degradation.

The kinetics of the degradation of chloroform and benzene in anaerobic sediment from the River Rhine

In anaerobic methanogenic sediment microcosms14C labelled chloroform was degraded mainly to carbon dioxide. At a concentration of 4 μg.l−1 the mineralization followed first order kinetics with a half life of 12 days at 10°C and 2.6 days at 20°C. At a concentration of 400 μg.l−1 the mineralization rate increased with time and followed logarithmic kinetics with a μmax of 0.02.d−1 at 10°C. The logarithmic kinetics can be explained by growth of the bacteria on the higher concentration of chloroform with a generation time of 35 days. Shaking and oxygenation did not inhibit the mineralization of chloroform, probably because of bacterial consumption of the dissolved oxygen. 14C labelled benzene was mineralized only for a small percentage to14C labelled carbon dioxide while other, not acid extractable, degradation products were formed. Under anaerobic conditions after one day when 5% of the benzene was degraded to carbon dioxide, the mineralization ceased, while the disappearance of benzene proceeded. With air in the headspace of the incubation bottles 25% of the benzene was mineralized to carbon dioxide. The anaerobic degradation of benzene at a concentration of 100 μ.l−1 showed similar kinetics as the degradation at 1 μg.l−1. Hence no adaptation of the microflora in the sediment occurred during the 63 days of the experiment at 100 μg.l−1.