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.     

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.     

Potential for anaerobic bioremediation of BTEX in petroleum-contaminated aquifers

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

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

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

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

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

Anaerobic Benzene Oxidation by Geobacter Species

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

Microbial mineralization of VC and DCE under different terminal electron accepting conditions

Production of 14CO2 from [1,2-14C] dichloroethene (DCE) or [1,2-14C] vinyl chloride (VC) was quantified in aquifer and stream-bed sediment microcosms to evaluate the potential for microbial mineralization as a pathway for DCE and VC biodegradation under aerobic, Fe(III)-reducing, SO4-reducing, and methanogenic conditions. Mineralization of [1,2-14C] DCE and [1,2-14C] VC to 14CO2 decreased under increasingly reducing conditions, but significant mineralization was observed for both sediments even under anaerobic conditions. VC mineralization decreased in the order of aerobic > Fe(III)-reducing > SO4-reducing > methanogenic conditions. For both sediments, VC mineralization was greater than DCE mineralization under all electron-accepting conditions examined. For both sediments, DCE mineralization was at least two times greater under aerobic conditions than under anaerobic conditions. Although significant microbial mineralization of DCE was observed under anaerobic conditions, recovery of 14CO2 did not differ substantially between anaerobic treatments.     

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.     

油藏铁还原微生物的研究进展

地下深部油藏通常为高温、高压以及高盐的极端环境,含有非常丰富的本源嗜热厌氧微生物,按代谢类群可分为发酵细菌、硫酸盐还原菌、产甲烷古菌和铁还原菌。从油田环境已经分离出90株铁还原微生物,如热袍菌目、热厌氧杆菌目、脱铁杆菌目、δ-变形菌纲脱硫单胞菌目、γ-变形菌纲希瓦氏菌属和广古菌门栖热球菌属等,这些菌株生长温度范围为4-85°C,生长盐度范围为0.1%-10.0%NaCl,还未见到文献报道油藏铁还原菌的耐压性研究。在油藏环境中存在微生物、矿物和流体(油/水)三者之间的相互作用,油藏中的粘土矿物能够作为微生物生命活动的载体,也能为微生物代谢作用提供电子受体。本文综述了油藏铁还原菌分离和表征的研究进展,简述了油藏铁还原菌的环境适用性,并展望了铁还原菌在提高原油采收率方面的应用前景。     

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

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

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

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

Anaerobic biodegradation of 1,4-dioxane by sludge enriched with iron-reducing microorganisms

The potential on anaerobic biodegradation of 1,4-dioxane was evaluated by use of enriched Fe(III)-reducing bacterium sludge from Hangzhou municipal wastewater treatment plant. The soluble Fe(III) supplied as Fe(III)-EDTA was more available for the Fe(III)-reducing bacterium in the sludge compared to insoluble Fe(III) oxide. The addition of humic acid (HA) further stimulated the anaerobic degradation of 1,4-dioxane accompanying with apparent reduction of Fe(III) which is believed that HA could stimulate the activity of Fe(III)-reducing bacterium by acting as an electron shuttle between Fe(III)-reducing bacterium and Fe(III), especially for insoluble Fe(III) oxides. After 40-day incubation, the concentration of 1,4-dioxane dropped up to 90% in treatment of Fe(III)-EDTA+HA. Further study proved that more than 50% of the carbon from 1,4-dioxane was converted to CO2 and no organic products other than biomass accumulated in the growth medium. The results demonstrated that, under the appropriate conditions, 1,4-dioxane could be biodegraded while serving as a sole carbon substrate for the growth of Fe(III)-reducing bacterium. It might be possible to design strategies for anaerobic remediation of 1,4-dioxane in contaminated subsurface environments.     

Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

Dehalogenation and biodegradation of brominated phenols and benzoic acids under iron-reducing, sulfidogenic, and methanogenic conditions.

The anaerobic biodegradation of monobrominated phenols and benzoic acids by microorganisms enriched from marine and estuarine sediments was determined in the presence of different electron acceptors [i.e., Fe(III), SO4(2-), or HCO3-]. Under all conditions tested, the bromophenol isomers were utilized without a lengthy lag period whereas the bromobenzoate isomers were utilized only after a lag period of 23 to 64 days. 2-Bromophenol was debrominated to phenol, with the subsequent utilization of phenol under all three reducing conditions. Debromination of 3-bromophenol and 4-bromophenol was also observed under sulfidogenic and methanogenic conditions but not under iron-reducing conditions. In the bromobenzoate-degrading cultures, no intermediates were observed under any of the conditions tested. Debromination rates were higher under methanogenic conditions than under sulfate-reducing or iron-reducing conditions. The stoichiometric reduction of sulfate or Fe(III) and the utilization of bromophenols and phenol indicated that biodegradation was coupled to sulfate or iron reduction, respectively. The production of phenol as a transient intermediate demonstrates that reductive dehalogenation is the initial step in the biodegradation of bromophenols under iron- and sulfate-reducing conditions.     

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

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

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.     

A Hydrogen-Oxidizing, Fe(III)-Reducing Microorganism from the Great Bay Estuary, New Hampshire

A dissimilatory Fe(III)- and Mn(IV)-reducing bacterium was isolated from bottom sediments of the Great Bay estuary, New Hampshire. The isolate was a facultatively anaerobic gram-negative rod which did not appear to fit into any previously described genus. It was temporarily designated strain BrY. BrY grew anaerobically in a defined medium with hydrogen or lactate as the electron donor and Fe(III) as the electron acceptor. BrY required citrate, fumarate, or malate as a carbon source for growth on H₂ and Fe(III). With Fe(III) as the sole electron acceptor, BrY metabolized hydrogen to a minimum threshold at least 60-fold lower than the threshold reported for pure cultures of sulfate reducers. This finding supports the hypothesis that when Fe(III) is available, Fe(III) reducers can outcompete sulfate reducers for electron donors. Lactate was incompletely oxidized to acetate and carbon dioxide with Fe(III) as the electron acceptor. Lactate oxidation was also coupled to the reduction of Mn(IV), U(VI), fumarate, thiosulfate, or trimethylamine n-oxide under anaerobic conditions. BrY provides a model for how enzymatic metal reduction by respiratory metal-reducing microorganisms has the potential to contribute to the mobilization of iron and trace metals and to the immobilization of uranium in sediments of Great Bay Estuary.     

Evidence for syntrophic butyrate metabolism under sulfate-reducing conditions in a hydrocarbon-contaminated aquifer

The importance of syntrophy in the degradation of butyrate in an aquifer where sulfate reduction was shown to be an important terminal electron-accepting process was assessed. Hydrocarbon-contaminated aquifer sediments coupled butyrate degradation to sulfate reduction and methane production. Butyrate degradation in methanogenic microcosms was inhibited by the addition of 2-bromoethanesulfonic acid, and was restored by the addition of 10 mM sulfate and a hydrogen- and formate-using sulfate reducer, but not by the addition of 10 mM sulfate alone. Molybdate addition inhibited butyrate degradation in sulfate-reducing microcosms. The addition of CO, which inhibits hydrogenases, to sulfate-reducing microcosms inhibited butyrate metabolism and caused the hydrogen partial pressure to increase to levels that would make syntrophic butyrate degradation via sulfate reduction energetically unfavorable (-5 to +3 kJ mol(-1) ). DNA extracted from the most probable number cultures and contaminated sediments contained sequences related to members of the families Syntrophomonadaceae and Syntrophaceae, whose members are known to syntrophically degrade fatty acids, as well as sequences related to uncultured Firmicutes, Desulfobulbaceae, Desulfobacteriaceae, and Desulfovibrionaceae. These data show that contaminated sediments degraded butyrate syntrophically coupled to methane production and sulfate reduction.