Petroleum-contaminated Aquifer Research Articles

Metabolite formation and toxicity measurements in evaluating bioremediation of a jet-fuel-contaminated aquifer.

The metabolic capabilities of subsurface, jet-fuel-contaminated, aquifer microbial communities were characterized using an ecological approach to biotreatment assessment. A multifaceted experimental design was used that incorporated quantification of metabolite formation and toxicity screening along with the typical microbial activity measurements and pollutant degradation measurements used for assessing bioremediation potential. For select experiments, dissolved oxygen levels and pH in microcosm systems were also monitored. Results suggest that a sizable, metabolically active microbial community exists in both contaminated and uncontaminated areas of the study site. Time course metabolism analyses indicated that the microbial communities were capable of degrading all three test compounds (amino acids, decane, and toluene) without any apparent adaptation period. Measurements of mineralization, cellular uptake, and metabolite formation indicated that metabolite formation was the predominant fate of the target pollutants in the microcosms. The results of toxicity screening time courses indicated that under oxygen-limiting conditions, the potential for the accumulation of toxic, acidic metabolites that could adversely affect the rates and extent of bioremediation existed. The experimental results indicate that the microbial communities at the site possess the metabolic potential for in situ biodegradation of the jet fuel. Care must be taken in the design of an in situ biodegradation treatment system (for this site and perhaps other petroleum-contaminated aquifers) to avoid the development of microaerophilic or oxygen-depleted zones, which could result in possible self-poisoning owing to acidic metabolite accumulation.

Environmental Risk Assessment for Underground Storage Tanks Through an Interval Parameter Fuzzy Relation Analysis Approach

In this study, an interval parameter fuzzy relation analysis (IPFRA) model is proposed for environmental risk assessment of petroleum-contaminated aquifers due to leakage from underground storage tanks. The model can effectively incorporate effects of different pollutants and different remediation techniques within a general framework. Also, it can directly reflect uncertainties presented as inexact intervals for a number of modeling inputs. Results of a case study indicate that reasonable solutions for risk assessment under different system conditions have been generated. When no remediation action is undertaken, environmental conditions at the study site may be ''highly risky'' in terms of their relation to drinking water safety. Four potential site remediation strategies are analyzed. They have different environmental and economic characteristics, with lower risks generally correspond ing to higher costs. Trade-offs between environmental and economic objectives are then analyzed. In general, the IPFRA approach is useful for comprehensively evaluating risks within a system containing many factors with complicated interrela tionships. The modeling results have provided bases for determining desirable site remediation actions.

Biodegradation during Contaminant Transport in Porous Media. 2. The Influence of Physicochemical Factors

The biodegradation of contaminants in the subsurface has become a topic of great interest. In systems wherein biodegradation is coupled with transport, the magnitude and rate of biodegradation is influenced not only by microbial properties but also by physicochemical properties. The purpose of this work is to systematically evaluate the impact of coupled physicochemical factors (residence time, substrate concentration, and electron-acceptor concentra tion) on the biodegradation of contaminants during transport in porous media. A suite of miscible-displacement experiments was conducted with different residence times and initial contaminant concentrations, using a petroleum-contaminated aquifer material and benzoate as a model compound. The results were evaluated using a framework developed from a mathematical analysis of the nondimensional parameters that control biodegradation during transport. The results show that the type of transport behavior observed is dependent upon system conditions and is predict...

Determining Anaerobic BTEX Decay Rates in a Contaminated Aquifer

Intrinsic biodegradation of petroleum hydrocarbons in the subsurface has gained increased acceptance as a remedial alternative where the risk of exposure is within acceptable standards. However, methods to predict reliably the rate and extent of biodegradation at contaminated sites have not been established. Laboratory microcosm and \Iin-situ\N column experiments were conducted in a petroleum-contaminated aquifer undergoing intrinsic biodegradation to (1) document that anaerobic biodegradation of benzene, toluene, ethylbenzene, and xylene (BTEX) isomers occurred, (2) determine the rate and pattern of BTEX biodegradation, and (3) compare measured decay rates in laboratory and in situ microcosm results with contamination concentrations along the length of the plume. Both methods verified that indigenous microorganisms have the capability to biodegrade anaerobically BTX under ambient conditions. However, microcosms constructed with aquifer material from different areas of the plume showed significant variability in rate and extent of contaminant biodegradation. In experiments conducted in the same area of the plume, the biodegradation rate for the individual compounds measured in both \Iin situ\N columns and microcosms was over 10 times higher than the rate calculated from field monitoring. Thus, these methods should not be used to infer field-scale decay rates.

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.

Anaerobic Benzene Oxidation in the Fe(III) Reduction Zone of Petroleum-Contaminated Aquifers

The potential for anaerobic benzene oxidation in the Fe(III)-reduction zone of petroleum-contaminated aquifers was evaluated. Sediments were incubated under strict anaerobic conditions without any amendments in order to simulate in situ conditions. [14C]Benzene was not oxidized to 14CO2 at most sites examined, which is consistent with previous studies that have found that benzene tends to persist in unamended, anaerobic aquifer materials and/or long periods of time are required in order to adapt the microbial population for benzene degradation. However, at one site located in Bemidji, MN, [14C]benzene was oxidized to 14CO2 in unamended sediments without an apparent lag, suggesting that benzene was anaerobically degraded in situ. Benzene was not significantly oxidized in sediments from nearby Fe(III)-reducing sites nor in sediments collected from an uncontaminated background site in the same aquifer. Culturing and 16S rRNA-based molecular studies of the Bemidji aquifer demonstrated that while all sites con...

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 Biodegradation of Alkylbenzenes in Laboratory Microcosms Representing Ambient Conditions

A microcosm study was performed to document the anaerobic biodegradation of benzene, toluene, ethylbenzene, m- xylene, and/or o-xylene in petroleum-contaminated aquifer sediment from sites in Michigan (MI) and North Carolina (NC) and relate the results to previous field investigations of intrinsic bioremediation. Laboratory microcosms, designed to simulate ambient conditions, were constructed under anaerobic conditions with sediment and groundwater from source, mid-plume, and end-plume locations at each site. The general patterns of biodegradation and electron acceptor utilization in the microcosms were consistent with field data. At the MI site, methane was produced after a moderate lag period, followed by toluene degradation in all sets of microcosms. At the NC site, biodegradation of the target compounds was not evident in the source area microcosms. In the mid-plume microcosms, toluene and o-xylene biodegraded first, followed by m-xylene and benzene, a pattern consistent with contaminant decay along the plume length. Chemical extraction of microcosm sediment at the beginning and end of me incubation indicated that iron-reducing conditions were dominant and iron reduction occurred on a sediment fraction not extracted by 0.5N HC1. In the end-plume microcosms, degradation of benzene, toluene, and xylene isomers occurred but was variable between replicates. Consistent with field data, dissolved concentrations of the target contaminant(s) persisted at low but detectable levels (0.05 to 0.25 μM) in microcosms from both sites where biodegradation was measured.

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.

Microbial colonization and weathering of silicates in a petroleum-contaminated groundwater

The influence of native microorganisms on mineral dissolution and precipitation was examined in a petroleum-contaminated aquifer near Bemidji, Minnesota. In-situ microcosms containing clean silicate fragments were established in the contaminated, microbially active groundwater over one-year periods. The recovered minerals were then examined by SEM and ESEM for microbial colonization patterns and weathering features. These experiments reveal distinct patterns of colonization and weathering associated with microbial attachment and growth. Microcline, anorthoclase, and oligoclase were widely colonized, and the colonized surfaces deeply weathered, with secondary clays precipitated on some uncolonized surfaces. Other feldspars, in contrast, were uncolonized and unweathered. Specific mineral removal rate was estimated from etch pit depth, while bulk weathering rate was estimated from the rate of change of silica concentration in the groundwater. In this system the primary control of silicate dissolution is apparently microbial colonization and metabolic activity.

Mechanisms for chelator stimulation of microbial Fe(III)-oxide reduction

The mechanisms by which nitrilotriacetic acid (NTA) stimulated Fe(III) reduction in sediments from a petroleum-contaminated aquifer were investigated in order to gain insight into how added Fe(III) chelators stimulate the activity of hydrocarbon-degrading, Fe(III)-reducing microorganisms in these sediments, and how naturally occurring Fe(III) chelators might promote Fe(III) reduction in aquatic sediments. NTA solubilized Fe(III) from the aquifer sediments. NTA stimulation of microbial WHO reduction did not appear to be the result of making calcium, magnesium, potassium, or trace metals more available to the microorganisms. Stimulation of WHO reduction could not be attributed to NTA serving as a source of carbon or fixed nitrogen for Fe(III)-reducing bacteria as NTA was not degraded in the sediments. Studies with the Fe(III)-reducing microorganism, Geobacter metallireducens, and pure Fe(III)-oxide forms, demonstrated that NTA stimulated the reduction of a variety of Fe(III) forms, including highly crystalline Fe(III)-oxides such as goethite and hematite. The results suggest that NTA solubilization of insoluble Fe(III)-oxide is an important mechanism for the stimulation of Fe(III) reduction by NTA in aquifer sediments.

Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments

We studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. Three Pseudomonas isolates, P. pickettii PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K(m) and Vmax) for catechol 2,3-dioxygenase (C230), a key shared enzyme of the toluene-degradative pathway for these strains, and compared these parameters with those for the analogous enzymes from archetypal toluene-degrading pseudomonads which did not show enhanced, nitrate-dependent toluene degradation. C230 purified from strains W31, PKO1, and CFS215 had a significantly greater affinity for oxygen as well as a significantly greater rate of substrate turnover than found for the analogous enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleotide and deduced amino acid sequences of C23O from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the subfamily of C23Os that preferentially cleave monocyclic substrates. Moreover, deletion analysis of the nucleotide sequence upstream of the translational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of sequences critical for regulated expression of tbuE, including a sequence homologous to the ANR-binding site of Pseudomonas aeruginosa PAO. When present in cis, this site enhanced expression of tbuE under oxygen-limited conditions. Taken together, these results suggest the occurrence of a novel group of microorganisms capable of oxygen-requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and xylenes in hypoxic environments. Strain PKO1, which exemplifies this novel group of microorganisms, compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation.

Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.

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.

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer.

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions for in situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, or p-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10(3) to 10(5) bacteria ml-1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain of Pseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.

Stimulated anoxic biodegradation of aromatic hydrocarbons using Fe(III) ligands.

Contamination of ground waters with water-soluble aromatic hydrocarbons, common components of petroleum pollution, often produces anoxic conditions under which microbial degradation of the aromatics is slow. Oxygen is often added to contaminated ground water to stimulate biodegradation, but this can be technically difficult and expensive. Insoluble Fe(III) oxides, which are generally abundant in shallow aquifers, are alternative potential oxidants, but are difficult for microorganisms to access. Here we report that adding organic ligands that bind to Fe(III) dramatically increases its bioavailability, and that in the presence of these ligands, rates of degradation of aromatic hydrocarbons in anoxic aquifer sediments are comparable to those in oxic sediments. We find that even benzene, which is notoriously refractory in the absence of oxygen, can be rapidly degraded. Our results suggest that increasing the bioavailability of Fe(III) by adding suitable ligands provides a potential alternative to oxygen addition for the bioremediation of petroleum-contaminated aquifers.

In-situ Groundwater Aeration as an Effective Technique for Remediation of Petroleum-Contaminated Aquifers: ABSTRACT

Petroleum contamination of groundwater is a widespread occurrence and is traditionally remediated using groundwater extraction with surface treatment. This remediation scheme is ineffective due to irregular groundwater flow paths, and the low solubility and high soil sorption tendencies of petroleum products in the subsurface. In-situ groundwater aeration, sometimes referred to as air sparging, provides a more effective approach. In-situ groundwater aeration technology takes advantage of the high volatility and biodegradability of many health concerned petroleum constituents. By injecting air into the subsurface, volatile organic compounds readily partition into the vapor phase and are subsequently transported to the vadose zone for collection by a soil vapor extraction system. The system also provides sufficient amounts of oxygen to the groundwater to promote biodegradation of petroleum contaminants. Development of an in-situ groundwater aeration system for petroleum releases within a regulatory framework includes several steps. First, site specific fate and transport mechanisms relevant to petroleum releases must be evaluated. Next, key design parameters, such as injection well construction, well locations, and air injection rates are discussed. Approximate capital, operation, and maintenance costs are given along with typical system remedial time frames. A case history involving a gasoline release from an underground storage tank ismore » presented to illustrate the development and success of an in-situ aeration system.« less

Discussion of: Evaluation of groundwater extraction for remediation of petroleum‐contaminated aquifers, R. C. Borden, C.‐M. Kao, 64, 28 (1992).

Water Environment ResearchVolume 65, Issue 7 p. 899-899 Article Discussion of: Evaluation of groundwater extraction for remediation of petroleum-contaminated aquifers, R. C. Borden, C.-M. Kao, 64, 28 (1992). Thomas S. Soerens, Thomas S. SoerensSearch for more papers by this authorRobert C. Knox, Robert C. KnoxSearch for more papers by this author Thomas S. Soerens, Thomas S. SoerensSearch for more papers by this authorRobert C. Knox, Robert C. KnoxSearch for more papers by this author First published: 01 November 1993 https://doi.org/10.2175/WER.65.7.12Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume65, Issue7November-December 1993Pages 899-899 RelatedInformation

Microbial Control of Silicate Weathering in Organic-Rich Ground Water

An in situ microcosm study of the influence of surface-adhering bacteria on silicate diagenesis in a shallow petroleum-contaminated aquifer showed that minerals were colonized by indigenous bacteria and chemically weathered at a rate faster than theoretically predicted. Feldspar and quartz fragments were placed in anoxic, organic-rich ground water, left for 14 months, recovered, and compared to unreacted controls with scanning electron microscopy. Ground-water geochemistry was characterized before and after the experiment. Localized mineral etching probably occurred in a reaction zone at the bacteria-mineral interface where high concentrations of organic acids, formed by bacteria during metabolism of hydrocarbon, selectively mobilized silica and aluminum from the mineral surface.