Biological souring of crude oil under anaerobic conditions

Seawater injection into oil reservoirs for purposes of secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil-field water are believed to be produced by microbial degradation of crude oil. The objective of this research was to investigate mechanisms of souring, focusing specifically on VFA production via crude oil biodegradation. To this end, a microbial consortium collected from an oil–water separator was suspended in seawater; crude oil or liquid n-alkane mixture was added to the culture medium as the sole carbon source, and the culture was incubated under anaerobic conditions for 190 days. Physicochemical analysis showed that preferential toluene degradation and sulfate reduction occurred concomitantly in the culture containing crude oil. Sulfide concentrations were much lower in the alkane-supplemented culture than in the crude oil-supplemented culture. These observations suggest that SRB are related to the toluene activation and VFA consumption steps of crude oil degradation. Therefore, the electron donors for SRB are not only VFA, but many components of crude oil, especially toluene. Alkanes were also degraded by microorganisms, but did not contribute to reservoir souring.     

Biological souring and mitigation in oil reservoirs

Souring in oilfield systems is most commonly due to the action of sulfate-reducing prokaryotes, a diverse group of anaerobic microorganisms that respire sulfate and produce sulfide (the key souring agent) while oxidizing diverse electron donors. Such biological sulfide production is a detrimental, widespread phenomenon in the petroleum industry, occurring within oil reservoirs or in topside processing facilities, under low- and high-temperature conditions, and in onshore or offshore operations. Sulfate reducers can exist either indigenously in deep subsurface reservoirs or can be “inoculated” into a reservoir system during oilfield development (e.g., via drilling operations) or during the oil production phase. In the latter, souring most commonly occurs during water flooding, a secondary recovery strategy wherein water is injected to re-pressurize the reservoir and sweep the oil towards production wells to extend the production life of an oilfield. The water source and type of production operation can provide multiple components such as sulfate, labile carbon sources, and sulfate-reducing communities that influence whether oilfield souring occurs. Souring can be controlled by biocides, which can non-specifically suppress microbial populations, and by the addition of nitrate (and/or nitrite) that directly impacts the sulfate-reducing population by numerous competitive or inhibitory mechanisms. In this review, we report on the diversity of sulfate reducers associated with oil reservoirs, approaches for determining their presence and effects, the factors that control souring, and the approaches (along with the current understanding of their underlying mechanisms) that may be used to successfully mitigate souring in low-temperature and high-temperature oilfield operations.     

Anaerobic benzene degradation

Although many studies have indicated that benzene persists under anaerobic conditions in petroleum-contaminated environments, it has recently been documented that benzene can be anaerobically oxidized with most commonlyconsidered electron acceptors for anaerobic respiration. These include: Fe(III),sulfate, nitrate, and possibly humic substances. Benzene can also be convertedto methane and carbon dioxide under methanogenic conditions. There is evidencethat benzene can be degraded under in situ conditions in petroleum-contaminatedaquifers in which either Fe(III) reduction or methane production is the predominant terminal electron-accepting process. Furthermore, evidence from laboratory studies suggests that benzene may be anaerobically degraded in petroleum-contaminated marine sediments under sulfate-reducing conditions. Laboratory studies have suggested that within the Fe(III) reduction zone of petroleum-contaminated aquifers, benzene degradation can be stimulated with the addition of synthetic chelators which make Fe(III) more available for microbial reduction. The addition of humic substances and other compounds that contain quinone moieties can also stimulate anaerobic benzene degradation in laboratory incubations of Fe(III)-reducing aquifer sediments by providing an electron shuttle between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides. Anaerobic benzene degradation in aquifer sediments can be stimulated with the addition of sulfate, but in some instances an inoculum of benzene-oxidizing,sulfate-reducing microorganisms must also be added. In a field trial, sulfate addition to the methanogenic zone of a petroleum-contaminated aquifer stimulated the growth and activity of sulfate-reducing microorganisms and enhanced benzene removal. Molecular phylogenetic studies have provided indications of what microorganisms might be involved in anaerobic benzene degradation in aquifers. The major factor limiting further understanding of anaerobic benzene degradation is the lack of a pure culture of an organism capable of anaerobic benzene degradation.     

Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ∼ 10¹⁰ colony forming units g^-1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36 h in vitro incubation. The total VFA concentration was 10.5 mmol dL^-1 at 36 h incubation, which included 55.7 mol% acetate, 18.0 mol% n-butyrate and 15.1 mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

Detection of anaerobic processes and microorganisms in immobilized activated sludge of a wastewater treatment plant with intense aeration

Attached activated sludge from the Krasnaya Polyana (Sochi) wastewater treatment plant was studied after the reconstruction by increased aeration and water recycle, as well as by the installation of a bristle carrier for activated sludge immobilization. The activated sludge biofilms developing under conditions of intense aeration were shown to contain both aerobic and anaerobic microorganisms. Activity of a strictly anaerobic methanogenic community was revealed, which degraded organic compounds to methane, further oxidized by aerobic methanotrophs. Volatile fatty acids, the intermediates of anaerobic degradation of complex organic compounds, were used by both aerobic and anaerobic microorganisms. Anaerobic oxidation of ammonium with nitrite (anammox) and the presence of obligate anammox bacteria were revealed in attached activated sludge biofilms. Simultaneous aerobic and anaerobic degradation of organic contaminants by attached activated sludge provides for high rates of water treatment, stability of the activated sludge under variable environmental conditions, and decreased excess sludge formation.     

Anaerobic degradation of dehydrodiisoeugenol by rumen bacteria

The degradation of dehydrodiisoeugenol (DDIE) by cow rumen bacteria was studied under strictly anaerobic conditions. After two days of cultivation, about 23% of DDIE (1.2 mM) was degraded to volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. The aromatic intermediates were vanillic acid, 5-methylvanillin and 3-methyl-4-hydroxybenzaldehyde, which suggested that the coumaran ring in DDIE was cleaved during degradation. These results indicate that the rumen anaerobes can degrade this lignin-related dimer to monoaromatic compounds and VFA.     

Degradation of benzoate by an anaerobic consortium and some properties of a hydrogenotrophic methanogen and sulfate-reducing bacterium in the consortium

A highly simplified anaerobic consortium which was able to degrade benzoate under mesophilic conditions was obtained from digested sludge acclimatized with benzoate. It converted 5 mM benzoate to methane quantitatively within 3 weeks in the absence of any organic nutrients under an N₂/CO₂ atmosphere. Degradation of benzoate was strictly inhibited by hydrogen. The consortium consisted of at least three microorganisms including an autofluorescent irregular coccus which was identified as Methanogenium sp., a short rod which did not autofluoresce and was considered to be a benzoate degrader, and a filamentous bacterium apparently classified as Methanothrix (= “Methanosaeta”. When sulfate was added to the medium, the methanogens were readily replaced by a sulfate-reducing bacterium, probably belonging to the genus Desulfovibrio, which had still remained in very low number in the consortium in the absence of sulfate, and benzoate was stoichiometrically converted to acetate without methanogenesis. Of various compounds which were expected to be intermediates in the benzoate degradation, only crotonate was degraded by concentrated cells of the consortium.     

Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

A comprehensive overview of bacteria and fungi used for pentachlorophenol biodegradation

Pentachlorophenol (PCP) is an extremely dangerous worldwide pollutant due to its high toxicity towards all organisms. It has been introduced into the environment mainly as a wood preservative, biocides and from the bleaching of paper or tissues. The use of PCP indiscriminate has led to the contamination of water and soil systems. Many countries have specific regulations, guidelines or procedures for the management and disposal of PCP but the most common methods are: adsorption with activate carbons, incineration in an approved and secure area, closed in sealed containers and biological degradation. PCP depletion can occur either by abiotic processes such as: absorption, volatilization and photo degradation or by biotic degradation. One of the main studies focused on remediation using plants, animals and microbial communities. Aerobic and anaerobic microorganisms can degrade PCP under a variety of conditions and at different PCP concentrations. Bacterial strains such as Pseudomonas sp., Sphingomonas sp., Arthrobacter sp., Mycobacterium sp., Flavobacterium sp., Serratia sp. and Bacillus sp., and fungal cultures as Trametes sp., Phanerochaete sp., Anthracophyllum sp., Armillaria sp., Bjerkandera sp., Ganoderma sp., Lentinula sp., Penicillium sp, Trichoderma sp., Rhizopus sp. and Plerotus sp. showed various rates and extent of PCP degradation. This review focuses on PCP degradation by various aerobic and anaerobic microorganisms with emphases on the biological and chemical aspects. Furthermore we will analyze intermediate products, processes and enzymes involved in the degradation of PCP in different environmental conditions and at various PCP concentrations.     

Development of a PCR method for the detection and quantification of benzoyl-CoA reductase genes and its application to monitored natural attenuation

Benzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10–40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.     

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.     

Anaerobic degradation of catechol by Desulfobacterium sp. strain Cat2 proceeds via carboxylation to protocatechuate.

Under anoxic conditions, most methoxylated mononuclear aromatic compounds are degraded by bacteria, with catechol being formed as an important intermediate. On the basis of our experiments with the sulfate-reducing bacterium Desulfobacterium sp. strain Cat2, we describe for the first time the enzymatic activities involved in the complete anaerobic oxidation of catechol and protocatechuate. Results obtained from experiments with dense cell suspensions of strain Cat2 demonstrated that all enzymes necessary for protocatechuate and benzoate degradation were induced during growth with catechol. In addition, anaerobic oxidation of catechol was found to be a CO2-dependent process. Phenol was not degraded in suspensions of cells grown with catechol. In cell extracts of Desulfobacterium sp. strain Cat2, protocatechuyl-coenzyme A (CoA) was formed from catechol, bicarbonate, and uncombined CoA. This oxygen-sensitive reaction requires high concentrations of both bicarbonate and protein, and only very low levels of enzyme were detected. In a second oxygen-sensitive step, protocatechuyl-CoA was reduced to 3-hydroxybenzoyl-CoA by reductive elimination of the p-hydroxyl group. Further dehydroxylation to benzoyl-CoA was not detectable. Key reactions described for anaerobic degradation of benzoate were catalyzed by cell extracts of strain Cat2, too.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: A review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

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.     

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

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

Microbiological Degradation of Malodorous Substances of Swine Waste under Aerobic Conditions

Phenol, p-cresol, and volatile fatty acids (VFA; acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids) were used as odor indicators of swine waste. Aeration of the waste allowed the indigenous microorganisms to grow and degrade these malodorous substances. The time required for degradation of these substances varied according to the waste used, and it was not necessarily related to their concentrations. Using a minimal medium which contained one of the malodorous compounds as sole carbon source, we have selected from swine waste microorganisms that can grow in the medium. The majority of these microorganisms were able to degrade the same substrate when inoculated in sterilized swine waste but with an efficiency varying from one strain to the other. None of these strains was able to degrade all malodorous substances studied. Within 6 days of incubation these selected strains degraded the following: Acinetobacter calcoaceticus, phenol and all VFA; Alcaligenes faecalis, p-cresol and all VFA; Corynebacterium glutamicum and Micrococcus sp., phenol, p-cresol, and acetic and propionic acids; Arthrobacter flavescens, all VFA. On a laboratory scale, the massive inoculation of swine waste with C. glutamicum or Micrococcus sp. accelerated degradation of the malodorous substances. However, this effect was not observed with all of the various swine wastes tested. These results suggest that an efficient deodorization process of various swine wastes could be developed at the farm level based on the aerobic indigenous microflora of each waste.     

Diversity and Distribution of Sulfate-Reducing Bacteria in Permanently Frozen Lake Fryxell, McMurdo Dry Valleys, Antarctica

The permanently frozen freshwater Lake Fryxell, located in the Dry Valleys of Antarctica, exhibits an ideal geochemistry for microbial sulfate reduction. To investigate the population of sulfate-reducing bacteria in Lake Fryxell, both 16S rRNA gene and metabolic primer sets targeting the dsrA gene for the dissimilatory sulfite reductase alpha subunit were employed to analyze environmental DNA obtained from the water column and sediments of Lake Fryxell. In addition, enrichment cultures of sulfate-reducing bacteria established at 4°C from Lake Fryxell water were also screened using the dsrA primer set. The sequence information obtained showed that a diverse group of sulfate-reducing prokaryotes of the domain Bacteria inhabit Lake Fryxell. With one exception, the enrichment culture sequences were not represented within the environmental sequences. Sequence data were compared with the geochemical profile of Lake Fryxell to identify possible connections between the diversity of sulfate-reducing bacteria and limnological conditions. Several clone groups were highly localized with respect to lake depth and, therefore, experienced specific physiochemical conditions. However, all sulfate-reducing bacteria inhabiting Lake Fryxell must function under the constantly cold conditions characteristic of this extreme environment.     

Anaerobic degradation of No. 2 diesel fuel in the wetland sediments of Barataria-Terrebonne estuary under various electron acceptor conditions

The biodegradation of No. 2 diesel fuel under anaerobic conditions was investigated using sediments collected from wetlands of Barataria-Terrebonne estuary in Louisiana. The results indicated enhanced biodegradation of diesel fuel under sulfate-reducing, nitrate-reducing, methanogenic, and mixed electron acceptor conditions. However, the rate of diesel degradation was the highest under mixed electron acceptor conditions followed in order by sulfate-reducing, methanogenic, and nitrate-reducing conditions. Under mixed electron acceptor condition, 99% removal of diesel fuel was achieved within 510 days, while under sulfate-reducing condition 62% degradation of diesel fuel was observed for the same period. Diesel fuel was also degraded to a smaller extent in the culture condition where electron acceptors were not supplemented (natural attenuation condition). This study showed evidence for enhanced diesel fuel metabolism in a mixed microbial population system similar to any contaminated field site, where a heterogeneous microbial population exists.