Anaerobic Benzene Biodegradation Research Articles

Sulfate Land Application Enhances Biodegradation in a Petroleum Hydrocarbon Smear Zone

AbstractDelivery of sulfate to petroleum hydrocarbons (PHCs) source zones and groundwater plumes is desirable to enhance biodegradation rates when treatment has become limited due to depletion of sulfate. Sulfate land application involves spreading sulfate salts on ground surface and allowing their dissolution and infiltration of sulfate into subsurface. The objectives of this pilot‐scale investigation were to capture the vertical transport of sulfate beneath an application area, confirm that sulfate reduction was occurring, and explore how the added sulfate affected biodegradation of benzene and toluene. Approximately 4000 kg of gypsum was spread over a 30 m × 30 m study area above a smear zone located approximately 2 m below‐ground surface. Precipitation was augmented by two irrigation events. Groundwater samples, collected over 1058 days from multilevel wells and a conventional long‐screened monitoring well, were analyzed for benzene, toluene, ethylbenzene, and xylenes (BTEX), sulfate, bromide, dissolved inorganic carbon (DIC) and methane. Compound‐specific isotope analyses (CSIA) for benzene and toluene, and isotope analyses of13C‐DIC and34S‐SO42−were performed. Following application, an increase in sulfate concentration was noted in the smear zone.34S‐SO42−enrichment and13C‐DIC depletion indicated that sulfate reduction and mineralization of PHCs were enhanced. CSIA results provided unequivocal evidence of anaerobic biodegradation of benzene and toluene. After 1058 days when sulfate was depleted, methane concentrations were about three times greater than baseline conditions suggesting syntrophic benefit of the delivered sulfate. Observations from this investigation support the viability of sulfate land application to enhance biodegradation rates in shallow PHC smear zones.

Anaerobic Benzene Biodegradation Linked to the Growth of Highly Specific Bacterial Clades.

Reliance on bioremediation to remove benzene from anoxic environments has proven risky for decades but for unknown reasons. Research has revealed a strong link between anaerobic benzene biodegradation and the enrichment of highly specific microbes, including Thermincola in the family Peptococcaceae and the deltaproteobacterial Candidate Sva0485 clade. Using aquifer materials from Canadian Forces Base Borden, we compared five bioremediation approaches in batch microcosms. Under conditions simulating natural attenuation or sulfate biostimulation, benzene was not degraded after 1-2 years of incubation and no enrichment of known benzene-degrading microbes occurred. In contrast, nitrate-amended microcosms reported benzene biodegradation coincident with significant growth of Thermincola spp., along with a functional gene presumed to catalyze anaerobic benzene carboxylation (abcA). Inoculation with 2.5% of a methanogenic benzene-degrading consortium containing Sva0485 (Deltaproteobacteria ORM2) resulted in benzene biodegradation in the presence of sulfate or under methanogenic conditions. The presence of other hydrocarbon co-contaminants decreased the rates of benzene degradation by a factor of 2 to 4. Tracking the abundance of the abcA gene and 16S rRNA genes specific for benzene-degrading Thermincola and Sva0485 is recommended to monitor benzene bioremediation in anoxic groundwater systems to further uncover growth-rate-limiting conditions for these two intriguing phylotypes.

Indigenous microbial communities in Albertan sediments are capable of anaerobic benzene biodegradation under methanogenic, sulfate-reducing, nitrate-reducing, and iron-reducing redox conditions.

Alberta is a major center for oil and gas production, and correspondingly harbors hundreds of unresolved contamination sites by environmental hazards such as benzene (C6 H6 ). Due to its cost-effectiveness, bioremediation has become a promising strategy for C6 H6 removal. Contamination sites typically take on an anaerobic context, which complicates the energetics of contamination sites and is a subject that is scarcely broached in studies of Albertan sediments. This study examines the innate potential for indigenous microbial communities in Albertan sediments to remove C6 H6 in a multitude of reduced conditions. Community profiles of these sediments were analyzed by 16S rRNA gene amplicon sequencing, and removal rates and reaction stoichiometries were observed by gas chromatography and ion chromatography. Organisms belonging to known primary degrader taxa were identified, including Geobacter (iron-reducing), and Peptococcaceae (nitrate-reducing). Furthermore, benzene removal patterns of the cultures were similar to those observed in previously reported microcosms, with lag times between 70 and 168days and removal rates between 3.27 and 12.70µM/day. Such information could support a more comprehensive survey of Albertan sediment consortia, which may eventually be utilized in informing future remediation efforts in the province. PRACTITIONER POINTS: ●Clay and sand sediments originating from Northern Alberta could remove benzene under methanogenic, sulfate-reducing, iron-reducing, and nitrate-reducing conditions. ●Degradation profiles were broadly comparable to those of reported cultures from other geographical locales. ●Key degrader taxa observed included Geobacter (Fe3+ -reducing) and Peptococcaceae ( -reducing). ●Knowledge gained can be the start of a more extensive survey of Albertan sediments. Eventually, this collection of information can be used to generate robust C6 H6 -degrading cultures that can be implemented for bioaugmentation and be implemented in informing remediation strategies in soil and water matrices for priority contamination cases such as leaking underground storage tanks and orphan wells.

Enhancing Microbial Sulfate Reduction of Hydrocarbons in Groundwater Using Permeable Filled Borings

AbstractAt a service station closed in 1993, groundwater contained benzene that persisted above the cleanup goal of 1 mg/L in zones depleted of background sulfate. The benzene and other petroleum hydrocarbons (PHCs) were present as much as 36 feet (11 m) below the water table and therefore remediation of a thick saturated zone interval was required. Microcosms using site sediments demonstrated that anaerobic benzene biodegradation occurred only if sulfate was added, suggesting sulfate addition as a remediation approach. Twenty‐four boreholes (9.1″ diameter and 56′ deep) were drilled around four monitoring wells, in which benzene concentrations exceeded 1 mg/L. The boreholes were backfilled with a mixture of gravel and 15,000 pounds of gypsum (which releases sulfate as it dissolves) to create “Permeable Filled Borings” (PFBs). Concurrently, nine high pressure injections (HPIs) of gypsum slurry were conducted in other site locations (312 pounds of gypsum total). PFBs were expected to release sulfate for up to 20 years, whereas HPIs were expected to produce a short‐lived plume of sulfate. Concentrations of benzene and sulfate in groundwater were monitored over a 3‐year period in six monitoring wells. In two wells near PFBs, benzene concentrations dropped below the cleanup goal by two to three orders of magnitude; in one well, sulfate concentrations exceeded 500 mg/L for the most recent 18 months. Benzene concentrations in two other PFB monitoring wells declined by a factor of 2 to 4, but remained above 1 mg/L, presumably due to high‐dissolved PHC concentrations and possibly greater residual PHC mass in adjacent sediments, and therefore greater sulfate demand. However, hydrogen and sulfur isotopic enrichment in benzene and sulfate, respectively, confirmed biodegradation of benzene and stimulation of sulfate‐reducing conditions. Thus, it is hypothesized that the PHC mass in adjacent sediments will decline over time, as will dissolved PHC concentrations, and eventually benzene concentrations will decrease below the cleanup goal. Benzene in two HPI monitoring wells was below the cleanup goal for all but one sampling event before HPIs were conducted and remained below the cleanup goal after HPIs; there was no stimulation of sulfate‐reducing conditions. It is concluded that sulfate released from PFBs contributed to declining benzene concentrations.

Diagnostic Tools to Assess Mass Removal Processes During Pulsed Air Sparging of a Petroleum Hydrocarbon Source Zone

AbstractDuring remediation of contaminated aquifers, diagnostic tools can help evaluate whether an intended mass removal process was successfully initiated and acted on specific contaminants of concern. In this study, several diagnostic tools were tested in a controlled‐release in situ air sparging experiment that focused on the treatment of target hydrocarbons (e.g., benzene, toluene, ethylbenzene, and xylenes). The tools included compound‐specific isotope analysis (CSIA), expression of functional genes (mRNA), and metabolites characteristic of aerobic and anaerobic biodegradation. Total and compound‐specific mass balances were established and used, along with traditional monitoring parameters, to validate the results from the various tools. CSIA results indicated biodegradation as the main process contributing to benzene and toluene removal. Removal process‐specific isotope shifts were detected in groundwater as well as in the system effluent gas. CSIA, metabolite, and mRNA biomarkers consistently indicated that both aerobic and anaerobic biodegradation of benzene and toluene occurred, but that their relative importance evolved over time and were related to the treatment system operation. While the indicators do not allow quantification of the mass removed, they are particularly useful to identify if a removal process has been initiated, and to track relative changes in the predominance of in situ contaminant attenuation processes resulting from remediation efforts.

Anaerobic Biodegradation of Ethylic and Methylic Biodiesel and Their Impact on Benzene Degradation

The aim of the present work was to study the anaerobic biodegradation of benzene in the presence of ethylic and methylic biodiesel. Biodiesel was produced by methanolysis and ethanolysis of virgin sunflower oil and ethanolysis of waste frying oil, and further characterized according to the European Biodiesel Standard EN 14214 for water content, fatty acid esters content, acid value, kinematic viscosity at 40°C, and oxidation stability at 110°C. Microcosms were set up in order to assess the biodegradation of the different biodiesel types and their impacts on benzene biodegradation. Methane production and benzene biodegradation were monitored for 85 days. Denaturing gradient gel electrophoresis was used to assess impacts on archaeal and bacterial community fingerprints. The results showed that the three types of biodiesel were readily biodegraded, even in the presence of benzene. In microcosms with only biodiesel, the highest biodegradation rate and extent was observed for sunflower methylic biodiesel followed by sunflower ethylic biodiesel and waste frying oil ethylic biodiesel. Similar results were observed in the presence of benzene. The presence of any biodiesel type negatively impacted benzene biodegradation with soybean ethylic biodiesel impacting rates the least. Microbial community fingerprint analyses showed that biodiesel composition selected distinct communities according to the alcohol employed in the transesterification reaction.

Effect of Biofuels on Biodegradation of Benzene and Toluene at Gasoline Spill Sites.

The risk that benzene and toluene from spills of gasoline will impact drinking water wells is largely controlled by the natural anaerobic biodegradation of benzene and toluene. Benzene and toluene, as well as ethanol and other biofuels, are degraded under anaerobic conditions to the same pool of degradation products. Biodegradation of biofuels may produce concentrations of degradation products that make the thermodynamics for degradation of benzene and toluene infeasible under methanogenic conditions and produce larger plumes of benzene and toluene. This study evaluated the concentrations of fuel alcohols that are necessary to inhibit the anaerobic degradation of benzene and toluene under methanogenic conditions. At two ethanol spill sites, concentrations of ethanol greater ≥42 mg/L inhibited the anaerobic degradation of toluene. The pH and concentrations of acetate, dissolved inorganic carbon, and molecular hydrogen were used to calculate the Gibbs free energy for the biodegradation of toluene. In general, the anaerobic biodegradation of toluene was not thermodynamically feasible in water with ≥42 mg/L ethanol. In a microcosm study, when the concentrations of ethanol were ≥14 mg/L or the concentrations of n-butanol were ≥16 mg/L, the biodegradation of the alcohols consistently produced concentrations of hydrogen, dissolved inorganic carbon, and acetate that would preclude natural anaerobic biodegradation of benzene and toluene by syntrophic organisms. In contrast, iso-butanol and n-propanol only occasionally produced conditions that would preclude the biodegradation of benzene and toluene.

Cultivating microbial dark matter in benzene-degrading methanogenic consortia.

The microbes responsible for anaerobic benzene biodegradation remain poorly characterized. In this study, we identified and quantified microbial populations in a series of 16 distinct methanogenic, benzene-degrading enrichment cultures using a combination of traditional 16S rRNA clone libraries (four cultures), pyrotag 16S rRNA amplicon sequencing (11 cultures), metagenome sequencing (1 culture) and quantitative polymerase chain reaction (qPCR; 12 cultures). An operational taxonomic unit (OTU) from the Deltaproteobacteria designated ORM2 that is only 84% to 86% similar to Syntrophus or Desulfobacterium spp. was consistently identified in all enrichment cultures, and typically comprised more than half of the bacterial sequences. In addition to ORM2, a sequence belonging to Parcubacteria (candidate division OD1) identified from the metagenome data was the only other OTU common to all the cultures surveyed. Culture transfers (1% and 0.1%) were made in the presence and absence of benzene, and the abundance of ORM2, OD1 and other OTUs was tracked over 415 days using qPCR. ORM2 sequence abundance increased only when benzene was present, while the abundance of OD1 and other OTUs increased even in the absence of benzene. Deltaproteobacterium ORM2 is unequivocally the benzene-metabolizing population. This study also hints at laboratory cultivation conditions for a member of the widely distributed yet uncultivated Parcubacteria (OD1).

Influence of the Anaerobic Biodegradation of Different Types of Biodiesel on the Natural Attenuation of Benzene

In the present research work, different types of biodiesel were produced by a homogeneous alkali transesterification reaction using soybean oil, pork lard, and castor bean oil as raw materials, to evaluate how their different compositions may affect the biodegradability, namely, in the presence of benzene. Biodiesel was characterized according to the European standard EN 14214. The anaerobic biodegradation of the different types of biodiesel was examined as well as its influence on the biodegradation of benzene. Analyses were performed to determine the volume of methane (directly related to the anaerobic biodegradation of biodiesel), the concentration of benzene over time, and the production of organic acids. The results showed methane production resulting from the anaerobic degradation of all biodiesel types. The differences between the degradation behavior of each fuel were negligible, contrary to what was expected; however, the amount of methane produced was low due to nutrient limitations. This fact was confirmed by the organic acid analysis as well as by the addition of new media. Anaerobic benzene biodegradation was found to be negatively impacted by the presence of all biodiesel types on average; therefore, the results of this study may impact management of sites that contain biodiesel and fuel hydrocarbon contamination.

Nitrate Addition Has Minimal Effect on Anaerobic Biodegradation of Benzene in Coastal Saline (salt), Brackish and Freshwater Marsh Sediments

In response to the 2010 British Petroleum (BP) Deepwater Horizon Macondo well oil spill, saline (salt), brackish and freshwater marsh sediments were used to study whether nitrate addition could stimulate petroleum biodegradation. Benzene was selected as a model petroleum hydrocarbon, and its biodegradation was assumed to follow first-order kinetics. The results show that the intrinsic benzene degradation activities in the marsh sediments were inversely correlated (R 2 = 0.83, n = 9) with the sediment salinity, with following order of benzene degradation rate regardless of nitrate treatments, freshwater > brackish > salt (P < 0.05). Addition of nitrate shows a minimal potential to stimulate the benzene degradation. Increase of redox potentials by addition of nitrate even shows a slight inhibition effect on benzene degradation in these coastal marsh sediments without a clear mechanism. Excessive loading of nitrate has been causing frequent occurrence of coastal eutrophication and hypoxia in the Gulf of Mexico. Therefore, application of additional nitrate to the coastal marsh sediments for the remediation of the 2010 BP oil spill is not recommended.

Anaerobic biodegradation of benzene in salt marsh sediment of the Louisiana Gulf coast

The 2010 BP oil spill became the worst environmental disaster in US history with unprecedented damages to the environment and human health. Ecosystem recovery from the oil spill largely depends on the microbial degradation of oil contaminants. In this study benzene was selected as a representative of petroleum hydrocarbons to assess its anaerobic biodegradation potential in salt marsh sediment from the Louisiana Gulf coast. The experiment was conducted in two separate phases of incubation of the sediment slurries to determine the benzene biodegradation kinetics (phase-1), and to determine if any stimulation of benzene degradation occurred after benzene exposure (phase-2). Benzene biodegradation was assumed to follow Michaelis–Menten kinetics, and the Vmax was determined to be 1.66mgg−1 week−1 and the Km to be 1.17mgg−1. According to this model, it would take 11 weeks for 1m2 of the salt marsh to degrade 1L of benzene. Benzene degradation rates were found to have a minimum increase after the sediment had been exposed to benzene for 4 weeks, which suggests that the sediment has almost reached its natural maximum biodegradation potential.

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.

Anaerobic benzene biodegradation by a pure bacterial culture of Bacillus cereus under nitrate reducing conditions

A pure culture using benzene as sole carbon and energy sources was isolated by screening procedure from gasoline contaminated soil. The analysis of the 16S rDNA gene sequence, morphological and physiological characteristics showed that the isolated strain was a member of genus Bacillus cereus. The biodegradation performance of benzene by B. cereus was evaluated, and the results showed that benzene could be efficiently biodegraded when the initial benzene concentration was below 150 mg/L. The metabolites of anaerobic nitrate-dependent benzene oxidation by strain B. cereus were identified as phenol and benzoate. The results of substrate interaction between binary combinations for benzene, phenol and benzoate showed that the simultaneous presence of benzene stimulated the degradation of benzoate, whereas the addition of benzene inhibited the degradation of phenol. Benzene degradation by B. cereus was enhanced by the addition of phenol and benzoate, the enhanced effects were more pronounced at higher concentration. To our knowledge, this is the first report that the isolated bacterial culture of B. cereus can efficiently degraded benzene under nitrate reducing conditions.

Field metabolomics and laboratory assessments of anaerobic intrinsic bioremediation of hydrocarbons at a petroleum‐contaminated site

SummaryField metabolomics and laboratory assays were used to assess the in situ anaerobic attenuation of hydrocarbons in a contaminated aquifer underlying a former refinery. Benzene, ethylbenzene, 2‐methylnaphthalene, 1,2,4‐ and 1,3,5‐trimethylbenzene were targeted as contaminants of greatest regulatory concern (COC) whose intrinsic remediation has been previously reported. Metabolite profiles associated with anaerobic hydrocarbon decay revealed the microbial utilization of alkylbenzenes, including the trimethylbenzene COC, PAHs and several n‐alkanes in the contaminated portions of the aquifer. Anaerobic biodegradation experiments designed to mimic in situ conditions showed no loss of exogenously amended COC; however, a substantive rate of endogenous electron acceptor reduction was measured (55 ± 8 µM SO4 day−1). An assessment of hydrocarbon loss in laboratory experiments relative to a conserved internal marker revealed that non‐COC hydrocarbons were being metabolized. Purge and trap analysis of laboratory assays showed a substantial loss of toluene, m‐ and o‐xylene, as well as several alkanes (C6–C12). Multiple lines of evidence suggest that benzene is persistent under the prevailing site anaerobic conditions. We could find no in situ benzene intermediates (phenol or benzoate), the parent molecule proved recalcitrant in laboratory assays and low copy numbers of Desulfobacterium were found, a genus previously implicated in anaerobic benzene biodegradation. This study also showed that there was a reasonable correlation between field and laboratory findings, although with notable exception. Thus, while the intrinsic anaerobic bioremediation was clearly evident at the site, non‐COC hydrocarbons were preferentially metabolized, even though there was ample literature precedence for the biodegradation of the target molecules.

Isotopic Evidence Suggests Different Initial Reaction Mechanisms for Anaerobic Benzene Biodegradation

The initial metabolic reactions for anaerobic benzene biodegradation remain uncharacterized. Isotopic data for carbon and hydrogen fractionation from nitrate-reducing, sulfate-reducing, and methanogenic benzene-degrading enrichment cultures and phylogenic information were used to investigate the initial reaction step in anaerobic benzene biodegradation. Dual parameter plots of carbon and hydrogen isotopic data (deltadelta2H/ deltadelta13C) from each culture were linear, suggesting a consistent reaction mechanism as degradation proceeded. Methanogenic and sulfate-reducing cultures showed consistently higher slopes (m = 29 +/- 2) compared to nitrate-reducing cultures (m = 13 +/- 2) providing evidence for different initial reaction mechanisms. Phylogenetic analyses confirmed that culture conditions were strictly anaerobic, precluding any involvement of molecular oxygen in the observed differences. Using published kinetic data, we explored the possibility of attributing such slopes to reaction mechanisms. The higher slopes found under methanogenic and sulfate-reducing conditions suggest against an alkylation mechanism for these cultures. Observed differences between the methanogenic and nitrate-reducing cultures may not represent distinct reactions of different bonds, but rather subtle differences in relative reaction kinetics. Additional mechanistic conclusions could not be made because kinetic isotope effect data for carboxylation and other putative mechanisms are not available.

Benzene Biodegradation under Anaerobic Conditions Coupled with Metal Oxides Reduction

Anaerobic benzene biodegradation was performed in batch experiments using Rhine River sediment as inoculum and amorphous Mn(IV) or Fe(III) as independent final electron acceptors. Benzene (4.5 μmol) was degraded in 80 and 710 days in batch experiments under Mn(IV) and Fe(III) reducing conditions, respectively. Highest benzene degradation rate, 0.07 μmol/day, was obtained under Mn (IV) reducing conditions, with soluble Mn(II) and CO2 recoveries of 71.5% and 93% regarding to the stoichiometric values, respectively. Likewise, benzene biodegradation was performed in a continuous column coupled to the reduction of Mn(IV). Efficiency of benzene biodegradation was up to 97% under steady state operation in a sediment column operated continuously for more than 160 days. The carbon dioxide and Mn(II) recoveries were 88% and 77%, respectively, of the theoretical ratio according to the stoichiometry for benzene biodegradation.

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.

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.

Metabolites Detected during Biodegradation of 13C6-Benzene in Nitrate-Reducing and Methanogenic Enrichment Cultures

The mechanism for anaerobic metabolism of benzene remains unknown. To date, there have been only a few studies reporting metabolites of anaerobic benzene biodegradation, in part because anaerobic benzene-degrading enrichment cultures are not very common and only two isolates have been characterized to date. Phenol and benzoate, metabolites consistent with benzene hydroxylation or benzene carboxylation, have been identified previously in mixed cultures, and more recently benzene methylation to toluene has been proposed as another possible mechanism for anaerobic benzene degradation. In this study, 13C6-benzene was added to nitrate-reducing and methanogenic enrichment cultures and specific 13C-labeled metabolites were monitored over time. The putative metabolites were detected by gas chromatography/mass spectrometry in ether extracts of 100-mL samples of culture taken at each time point. This method of analysis provided the sensitivity required to accurately quantify low concentrations of these compounds. In addition, benzoate trapping was used in an attemptto increase concentrations of upstream metabolites. In both cultures, in the presence and absence of unlabeled benzoate (trap), [ring-13C]-toluene and [ring-13C]benzoate were detected transiently during degradation. The data strongly support initial methylation of benzene to toluene, followed bytransformation to benzoate. Although benzene methylation has been proposed previously, this is the first direct evidence to supportthis pathway. In the methanogenic culture only, 13C6-phenol was also detected. The transient appearance of phenol, which appeared to be further transformed to benzoate, suggests that a pathway involving hydroxylation to phenol, as proposed in other studies, was also operative.

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.