Intrinsic Bioremediation Research Articles

Molecular analysis of microbial community in a groundwater sample polluted by landfill leachate and seawater

Seashore landfill aquifers are environments of special physicochemical conditions (high organic load and high salinity), and microbes in leachate-polluted aquifers play a significant role for intrinsic bioremediation. In order to characterize microbial diversity and look for clues on the relationship between microbial community structure and hydrochemistry, a culture-independent examination of a typical groundwater sample obtained from a seashore landfill was conducted by sequence analysis of 16S rDNA clone library. Two sets of universal 16S rDNA primers were used to amplify DNA extracted from the groundwater so that problems arising from primer efficiency and specificity could be reduced. Of 74 clones randomly selected from the libraries, 30 contained unique sequences whose analysis showed that the majority of them belonged to bacteria (95.9%), with Proteobacteria (63.5%) being the dominant division. One archaeal sequence and one eukaryotic sequence were found as well. Bacterial sequences belonging to the following phylogenic groups were identified: Bacteroidetes (20.3%), beta, gamma, delta and epsilon-subdivisions of Proteobacteria (47.3%, 9.5%, 5.4% and 1.3%, respectively), Firmicutes (1.4%), Actinobacteria (2.7%), Cyanobacteria (2.7%). The percentages of Proteobacteria and Bacteroides in seawater were greater than those in the groundwater from a non-seashore landfill, indicating a possible influence of seawater. Quite a few sequences had close relatives in marine or hypersaline environments. Many sequences showed affiliations with microbes involved in anaerobic fermentation. The remarkable abundance of sequences related to (per)chlorate-reducing bacteria (ClRB) in the groundwater was significant and worthy of further study.

Modeling Intrinsic Bioremediation for Interpret Observable Biogeochemical Footprints of BTEX Biodegradation: The Need for Fermentation and Abiotic Chemical Processes

The intrinsic bioremediation of BTEX must be documented by the stoichiometric consumption and production of several other compounds, called 'footprints' of the biodegradation reaction. Although footprints of BTEX biodegradation are easy to identify from reaction stoichiometry, they can be confounded by the stepwise nature of the biodegradation reactions and by several abiotic chemical reactions that also produce or consume the footprints. In order to track the footprints for BTEX biodegradation, the following reactions need to be considered explicitly: (1) fermentation and methanogenesis as separate processes, (2) precipitation and dissolution of calcite, (3) precipitation and dissolution of amorphous iron monosulfide (FeS), (4) conversion of FeS into the thermodynamically stable pyrite (FeS2) with loss of sulfide and abiotic formation of H2, and (5) reductive dissolution of solid iron(III) by oxidation of sulfide. We critically review the research that underlies why these mechanisms must be included and how to describe them quantitatively. A companion manuscript develops and applies a mathematical model that includes these reactions.

Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated acquifers

The assessment of biodegradation in contaminated aquifers has become an issue of increasing importance in the recent years. To some extent, this can be related to the acceptance of intrinsic bioremediation or monitored natural attenuation as a means to manage contaminated sites. Among the few existing methods to detect biodegradation in the subsurface, stable isotope fractionation analysis (SIFA) is one of the most promising approaches which is pronounced by the drastically increasing number of applications. This review covers the recent laboratory and field studies assessing biodegradation of contaminants via stable isotope analysis. Stable isotope enrichment factors have been found that vary from no fractionation for dioxygenase reactions converting aromatic hydrocarbons over moderate fractionation by monooxygenase reactions ( ɛ=−3‰) and some anaerobic studies on microbial degradation of aromatic hydrocarbons ( ɛ=−1.7‰) to larger fractionations by anaerobic dehalogenation reactions of chlorinated solvents ( ɛ=between −5‰ and −30‰). The different isotope enrichment factors can be related to the respective biochemical reactions. Based on that knowledge, we discuss under what circumstances SIFA can be used for a qualitative or even a quantitative assessment of biodegradation in the environment. In a steadily increasing number of cases, it was possible to explain biodegradation processes in the field based on isotope enrichment factors obtained from laboratory experiments with pure cultures and measured isotope values from the field. The review will focus on the aerobic and anaerobic degradation of aromatic hydrocarbons and chlorinated solvents as the major contaminants of groundwater. Advances in the instrumental development for stable isotope analysis are only mentioned if it is important for the understanding of the application.

Aqueous and Mineral Intrinsic Bioremediation Assessment: Natural Attenuation

An approach to evaluating intrinsic bioremediation, aqueous and mineral intrinsic biodegradation assessment (AMIBA) is described. AMIBA is based on the microbial reduction of Fe\U3+\N and SO\U2-\N\D4\N, forming reduced Fe and S mineral species in amounts stoichiometrically equivalent to the contaminant mass oxidized by microbial processes. Mineral data from sediment cores are emphasized rather than the aqueous data used in existing protocols. AMIBA was demonstrated at Westover Air Force Base, Chicopee, Massachusetts. Over 300 times more reduced Fe\U2+\N was in mineral form, compared to aqueous. The distribution of mineral Fe\U3+\N and Fe\U2+\N marked the historic extent of the plume, confirming plume retreat. Aqueous SO\U2-\N\D4\N reduction resulted in the deposition of equivalent amounts of iron sulfide minerals near the source area. The total mass of fuel degraded by intrinsic bioremediation and the rate of source depletion was estimated. Thus, the past and future performance of intrinsic bioremediation was assessed using one sampling event rather than relying on protracted monitoring, as is the current practice.

Determination of alkylbenzene metabolites in groundwater by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Benzylsuccinate (BSA), methylbenzylsuccinate (methylBSA), and ethylbenzylsuccinate (ethylBSA) are unambiguous anaerobic biotransformation products from toluene, xylenes, and ethylbenzene decay, respectively, and may be used to indicate intrinsic bioremediation is occurring at hydrocarbon-contaminated sites. In order to improve upon current methods that detect and quantify anaerobic hydrocarbon metabolites in field samples, solid-phase extraction (SPE) and direct sample injection methods coupled with liquid chromatography–tandem mass spectrometry (LC–MS–MS) were evaluated. In laboratory studies, recoveries of authentic standards of non-deuterated or deuterated benzylsuccinates and toluates ranged from 80 to 106% with relative standard errors ranging from 2 to 4%. The method detection limits for these analytes using SPE–LC–MS–MS ranged from 0.006 to 0.029 μg/L whereas those for direct injection-LC–MS–MS ranged from 0.61 to 1.5 μg/L. Given the increased sensitivity of using SPE coupled with LC–MS–MS, this technique was then used to analyze for the presence of putative anaerobic alkylbenzene metabolites in groundwater from a hydrocarbon-contaminated site where single-well push–pull tests were conducted using deuterated aromatic hydrocarbons. Both deuterated and non-deuterated benzylsuccinates and toluates were successfully detected and quantified in field samples using this method.

Impact of transverse and longitudinal dispersion on first-order degradation rate constant estimation

A two-dimensional analytical model is employed for estimating the first-order degradation rate constant of hydrophobic organic compounds (HOCs) in contaminated groundwater under steady-state conditions. The model may utilize all aqueous concentration data collected downgradient of a source area, but does not require that any data be collected along the plume centerline. Using a least squares fit of the model to aqueous concentrations measured in monitoring wells, degradation rate constants were estimated at a former manufactured gas plant (FMGP) site in the Midwest U.S. The estimated degradation rate constants are 0.0014, 0.0034, 0.0031, 0.0019, and 0.0053 day −1 for acenaphthene, naphthalene, benzene, ethylbenzene, and toluene, respectively. These estimated rate constants were as low as one-half those estimated with the one-dimensional (centerline) approach of Buscheck and Alcantar [Buscheck, T.E., Alcantar, C.M., 1995. Regression techniques and analytical solutions to demonstrate intrinsic bioremediation. In: Hinchee, R.E., Wilson, J.T., Downey, D.C. (Eds.), Intrinsic Bioremediation, Battelle Press, Columbus, OH, pp. 109–116] which does not account for transverse dispersivity. Varying the transverse and longitudinal dispersivity values over one order of magnitude for toluene data obtained from the FMGP site resulted in nearly a threefold variation in the estimated degradation rate constant—highlighting the importance of reliable estimates of the dispersion coefficients for obtaining reasonable estimates of the degradation rate constants. These results have significant implications for decision making and site management where overestimation of a degradation rate may result in remediation times and bioconversion factors that exceed expectations. For a complex source area or non-steady-state plume, a superposition of analytical models that incorporate longitudinal and transverse dispersion and time may be used at sites where the centerline method would not be applicable.

Combined application of stable carbon isotope analysis and specific metabolites determination for assessing in situ degradation of aromatic hydrocarbons in a tar oil-contaminated aquifer.

To evaluate the intrinsic bioremediation potential in an anoxic tar oil-contaminated aquifer at a former gasworks site, groundwater samples were qualitatively and quantitatively analyzed by compound-specific isotope analysis (CSIA) and signature metabolites analysis (SMA). 13C/12C fractionation data revealed conclusive evidence for in situ biodegradation of benzene, toluene, o-xylene, m/p-xylene, naphthalene, and 1-methylnaphthalene. In laboratory growth studies, 13C/12C isotope enrichment factors for anaerobic degradation of naphthalene (epsilon = -1.1 +/- 0.4) and 2-methylnaphthalene (epsilon = -0.9 +/- 0.1) were determined with the sulfate-reducing enrichment culture N47, which was isolated from the investigated test site. On the basis of these and other laboratory-derived enrichment factors from the literature, in situ biodegradation could be quantified for toluene, o-xylene, m/p-xylene, and naphthalene. Stable carbon isotope fractionation in the field was also observed for ethylbenzene, 2-methylnaphthalene, and benzothiophene but without providing conclusive results. Further evidence for the in situ turnover of individual BTEX compounds was provided by the presence of acetophenone, o-toluic acid, and p-toluic acid, three intermediates in the anaerobic degradation of ethylbenzene, o-xylene, and p-xylene, respectively. A number of groundwater samples also contained naphthyl-2-methylsuccinic acid, a metabolite that is highly specific for the anaerobic degradation of 2-methylnaphthalene. Additional metabolites that provided evidence on the anaerobic in situ degradation of naphthalenes were 1-naphthoic acid, 2-naphthoic acid, 1,2,3,4-tetrahydronaphthoic acid, and 5,6,7,8-tetrahydronaphthoic acid. 2-Carboxybenzothiophene, 5-carboxybenzothiophene, a putative further carboxybenzothiophene isomer, and the reduced derivative dihydrocarboxybenzothiophene indicated the anaerobic conversion of the heterocyclic aromatic hydrocarbon benzothiophene. The combined application of CSIA and SMA, as two reliable and independent tools to collect direct evidence on intrinsic bioremediation, leads to a substantially improved evaluation of natural attenuation in situ.

Intrinsic Aromatic Hydrocarbon Biodegradation for Groundwater Remediation

Intrinsic biodegradation, representing the key process in natural attenuation, is increasingly considered for the remediation of contaminated sites as an alternative to more active measures. In this paper, intrinsic biodegradation is discussed with respect to BTEX and PAH. In the first part, an overview is given summarizing the current understanding of microbial aromatic hydrocarbon degradation and the methods available for the assessment of intrinsic bioremediation. In the second part, the concept and selected results of a case study are presented. Both aerobic and anaerobic biodegradation of aromatic hydrocarbons contribute to pollutant elimination at contaminated sites such as former manufactured gas plants and tar-oil polluted disposal sites. Intrinsic biodegradation processes usually result in a sequence of redox zones (methanogenic, sulfate-reducing, Fe(III)-reducing, denitrifying, aerobic) in the groundwater plume downgradient the source of contamination. Methods to assess redox zonation include hydro- and geochemical analysis, measurement of the redox potential, and determination of hydrogen. Biodegradation of target pollutants can be demonstrated by alterations in the pollutant profiles, isotopic fractionation, specific metabolic products, and by microcosm studies with authentic field samples. Microcosm studies in particular are a useful tool to identify degradation mechanisms and to understand the role of specific electron acceptors and redox conditions. In a case study, intrinsic biodegradation was examined at a tar-oil polluted disposal site. Due to the low sorption capacity of the aquifer, decreasing pollutant concentrations with increasing plume length were attributed predominantly to biodegradation. Sulfate reduction and Fe(III) reduction were the most important redox processes in the anaerobic core of the groundwater plume. Changing pollutant profiles with increasing plume length indicated active biodegradation processes, e.g. biodegradation of toluene and naphthalene in the anaerobic zones. In microcosms amended with model pollutants, biodegradation of toluene and ethylbenzene was observed under sulfate-reducing conditions. Degradation of toluene, ethylbenzene, benzene and naphthalene occurred in the presence of Fe(III). Under aerobic conditions, all BTEX and PAH were rapidly degraded.

Intrinsic bioremediation of a petroleum-impacted wetland

Following the 1994 San Jacinto River flood and oil spill in southeast Texas, a petroleum-contaminated wetland was reserved for a long-term research program to evaluate bioremediation as a viable spill response tool. The first phase of this program, presented in this paper, evaluated the intrinsic biodegradation of petroleum in the contaminated wetland. Sediment samples from six test plots were collected 11 times over an 11-month period to assess the temporal and spatial petroleum concentrations. Petroleum concentrations were evaluated using gas chromatography–mass spectrometer analyses of specific target compounds normalized to the conservative biological marker, C 3017α,21β(H)-hopane. The analyses of specific target compounds were able to characterize that significant petroleum biodegradation had occurred at the site over the one-year period. Total resolved saturate and total resolved aromatic hydrocarbon data indicated the petroleum was degraded more than 95%. In addition, first-order biodegradation rate constants were calculated for the hopane-normalized target compounds and supported expected biodegradation patterns. The rapid degradation rates of the petroleum hydrocarbons are attributed to conditions favorable to biodegradation. Elevated nutrient levels from the flood deposition and the unconsolidated nature of the freshly deposited sediment possibly provided a nutrient rich, oxic environment. Additionally, it is suggested that an active and capable microbial community was present due to prior exposure to petroleum. These factors provided an environment conducive for the rapid bioremediation of the petroleum in the contaminated wetland.

Naphthalene and anthracene mineralization linked to oxygen, nitrate, Fe(III) and sulphate reduction in a mixed microbial population.

A microbial consortium from a mixture of garden soil and an enrichment of a coal-tar contaminated sediment mineralized naphthalene and anthracene when oxygen, nitrate, Fe(III) (soluble and insoluble) or sulphate were provided as terminal electron acceptors (TEAs). Rates of polyaromatic hydrocarbon disappearance and mineralization were similar in the presence of oxygen and nitrate, and slower with the other TEAs. A maximum mineralization of 37.5% naphthalene and 8.5% anthracene occurred in 30 and 160 days respectively when oxygen was provided as the TEA. On the other hand, only 9.5% naphthalene and 3.2% anthracene were mineralized in 42 and 160 days respectively with FeOOH. Mineralization occurred only when a TEA was provided and ceased when the naphthalene concentration decreased to non-detectable levels (less than 0.008 micromoles/L), as measured by fluorescence spectroscopy. CH4 was not detected in the headspace of any microcosm. These results showed that mineralization of polyaromatic hydrocarbons such as naphthalene and anthracene can be linked to wide range of TEAs demonstrating that intrinsic polyaromatic hydrocarbon bioremediation is possible if any of these TEAs were available.

Visualizing reductive dechlorination in support of intrinsic bioremediation using multivariate plots and GIS

AbstractDemonstrating intrinsic bioremediation requires not only that the right types of evidence be gathered, but also that those data be analyzed at an appropriate level of detail and presented in a manner that clearly illustrates the key trends to the target audience. The goal of this study was to develop one type of multivariate pattern diagram, the pie diagram, for clearly and efficiently presenting data demonstrating the occurrence of reductive dechlorination at sites contaminated by chlorinated ethenes. The pie diagrams were created using normalized ethenes molar concentrations to estimate and illustrate the changes in the concentrations of contaminants and metabolic intermediates that could be attributed to biodegradative processes. Spatial pie‐diagram maps illustrating the normalized chlorinated‐ethenes data were produced using geographic information system (GIS) software. Application of this visualization tool is demonstrated using an example data set and is compared with a conventional x‐y graph of the data. The trends elucidated on the basis of the pie diagrams, coupled with additional site evidence of natural attenuation (e.g., electron donor and acceptor data), are shown to provide a consistent interpretation of the site data. © 2002 Wiley Periodicals, Inc.

In Situ Rhamnolipid Production at an Abandoned Petroleum Refinery

A simple screening method was developed to detect in situ biosurfactant production by exploiting the relationship between surface tension (ST) and surfactant concentration. Filtered groundwater from contaminated wells with ST values of 60 to 70 dynes/cm decreased to 29 dynes/cm after being concentrated 10 to 15 times in a rotary evaporator, indicating that biosurfactants in the sample reached the critical micelle concentration (CMC). Samples from uncon-taminated groundwater concentrated 25 times showed no decrease in ST below 72 dynes/cm, suggesting that biosurfactants were not present. Microorganisms from soil cores were cultured on diesel fuel and identified using fatty acid methyl ester (FAME) analysis. Pseudomonas aeruginosa was found at very low numbers in uncontami-nated soil but was the dominant species in contaminated soil, indicating that hydrocarbon release impacted microbial diversity significantly. High-performance liquid chromatography (HPLC) was used to quantify rhamnolipids, biosurfactants produced by P. aeruginosa, in concentrated ground-water samples. Rhamnolipid concentrations in samples from contaminated soil were observed equal to their CMC (50 mg/L), but were not detected in samples from un-contaminated wells. We conclude that biosurfactant production may be an indicator of intrinsic bioremediation.

Geophysical Investigation of Vadose Zone Conductivity Anomalies at a Hydrocarbon Contaminated Site: Implications for the Assessment of Intrinsic Bioremediation

Electromagnetic (EM-31), dc resistivity, and ground penetrating radar (GPR) methods were used to investigate part of a former refinery site in Kalamazoo, Michigan, USA. The study area is underlain by about [Formula: see text] of heterogeneous glacial drift deposits overlying a sandy outwash unit. Three large above ground storage tanks (ASTs) formerly occupied the study area and were removed by the U.S. Environmental Protection Agency (USEPA) in 1997. EM-31 surveys revealed eight remnants of buried pipe ranging from [Formula: see text] to more than [Formula: see text] in length. Emanating from some of these abandoned pipe segments are extensive near surface conductivity anomalies, with values ranging from [Formula: see text] to greater than [Formula: see text] vs. background values of less than [Formula: see text]. GPR and dipole-dipole resistivity surveys performed along selected profiles with anomalous conductive zones revealed a channel form within the shallow subsurface. Shallow hand augured soil borings into these conductive zones encountered hydrocarbons perched on thin clay lenses, or trapped in low permeability sandy-silt layers, while borings in background conductivity areas showed only clean sand and silt. We also observed that areas underlain by clays in association with perched hydrocarbons exhibited a higher conductivity (greater than [Formula: see text] and up to [Formula: see text]) when compared to areas underlain by uncontaminated clays. Based on these observations and results of our prior work in similar settings, we infer that the conductivity anomalies may be due to the occurrence of intrinsic biodegradation of the hydrocarbons. Thus, the previous conductive model originally proposed by our research group for ‘aged’ hydrocarbon spills at the water table can be extended upward to include vadose zone occurrences of biodegradation of these contaminants. The findings from this study have significant implications for the use of geophysical techniques in the assessment of vadose zone biodegradation.

Detection of anaerobic metabolites of saturated and aromatic hydrocarbons in petroleum-contaminated aquifers.

Recent investigations have demonstrated that several classes of petroleum hydrocarbons are susceptible to anaerobic decay, including alkanes and mono- and polycyclic aromatic compounds. In previous work, benzylsuccinates were shown to be useful indicators of in situ anaerobic alkylbenzene metabolism. In the present study, we sought to determine whether metabolites of alkanes and naphthalenes could similarly be used as indicators of the intrinsic decomposition of these compounds in petroleum-contaminated aquifers. Such metabolites include succinate derivatives of n-alkanes, cyclic alkanes, and alkylaromatic hydrocarbons as well as naphthoic acids. Using gas chromatography-mass spectrometry (GC-MS), we analyzed trimethylsilyl-derivatized organic extracts from six hydrocarbon-contaminated groundwaters for MS fragment ions indicative of such anaerobic metabolites. Geochemical indicators in these aquifers suggested the prevalence of anaerobic processes. In the groundwaters of the contaminated sites, we found compounds whose MS profiles suggested that they were indeed alkylsuccinic acids, ranging from C3 to C11 succinates. Propyl-, hexyl-, octyl-, and decylsuccinic acids were positively identified in the groundwaters by GC-MS matches with chemical or biologically produced standards. In two of the aquifers, we also detected components whose MS profiles matched with authentic standards of naphthoic acids and tetrahydronaphthoic acids. Metabolites were detected in nanomolar concentrations. The finding of these putative anaerobic metabolites of alkanes and naphthalenes signifies the in situ biodegradation of these hydrocarbons and attests to their value as indicators of intrinsic bioremediation.

Natural attenuation: what does the subsurface have in store?

Throughout the world, organic and inorganic substances leach into the subsurface as a result of human activities and accidents. There, the chemicals pose direct or indirect threats to the environment and to increasingly scarce drinking water resources. At many contaminated sites the subsurface is able to attenuate pollutants which, potentially, lowers the costs of remediation. Natural attenuation comprises a wide range of processes of which the microbiological component, which is responsible for intrinsic bioremediation, can decrease the mass and toxicity of the contaminants and is, therefore, the most important. Reliance on intrinsic bioremediation requires methods to monitor the process. The subject of this review is how knowledge of subsurface geology and hydrology, microbial ecology and degradation processes is used and can be used to monitor the potential and capacity for intrinsic bioremediation in the subsurface and to verify degradation in situ. As research on natural attenuation in the subsurface has been rather fragmented and limited and often allows only conclusions to be drawn of the site under investigation, we provide a concept based on Environmental Specimen Banking which will contribute to further understanding subsurface natural attenuation processes and will help to develop and implement new monitoring techniques.

Microbial aspects of atrazine degradation in natural environments.

The potential toxicity of the s-triazine herbicide atrazine motivates continuous bioremediation-directed research. Several indigenous soil atrazine-catabolizing microbial associations and monocultures have been enriched/isolated from compromised sites. Of these, Pseudomonas sp. strain ADP has become a reference strain and has been used to elucidate sequences of the catabolic enzymes atzA, atzB, atzC and atzD involved in one aerobic degradation pathway and develop probes for the genes which encode these enzymes. Despite this, hitherto unknown or novel microorganisms, with unique sequences and different enzyme-mediated operative pathways, warrant continued investigations for effective site bioremediation. Also, the sustained effectiveness of natural attenuation must be demonstrated continually so regular site evaluations and results analyses, despite the limitations of chemical extraction methodologies, are crucial practices. For both directed and intrinsic bioremediation monitoring, traditional microbial association studies must be complemented by more advanced physiological and molecular approaches. The occurrence of catabolic plasmids, in particular, should be probed with DNA hybridization techniques. Also, PCR-DGGE and subsequent new sequence elucidation should be used prior to developing new primers for DNA sequences encoding novel catabolic enzymes, and for hybridization probe development, to establish the degradative potential of a compromised site, or adoption of FISH to, for example, monitor bioaugmented remediation.

Anaerobic mineralization of toluene by enriched sediments with quinones and humus as terminal electron acceptors.

The anaerobic microbial oxidation of toluene to CO(2) coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [(13)C]toluene were recovered as (13)CO(2) in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [(13)C]toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.

Assessment of natural attenuation of chlorinated aliphatics and BTEX in subarctic groundwater.

We examined biogeochemistry and microbiology associated with natural attenuation of trichloroethene (TCE), trichloroethane (TCA), and benzene in a subarctic aquifer. Identification of a predominant terminal electron-accepting process (TEAP) and characterization of typical natural attenuation footprints was difficult. Hydrogen and ferrous iron concentrations suggested that iron reduction was the predominant TEAP; calculated in situ Gibbs free energies for iron reduction were energetically feasible at all wells although a source of ferric iron has not been conclusively determined. The presence of dissolved sulfide and favorable free energies for sulfate reduction provided support of concurrent iron and sulfate reduction. Methanogenesis from H2/CO2 was generally not energetically favorable. The presence of TCE and TCA degradation intermediates suggested that biological reductive dechlorination occurred, although proportions of intermediates relative to parent compounds remained stable. By September 2000, contaminant concentrations were within regulatory standards at most sampling points. However, low rates of microbial activity and incomplete degradation imply that intrinsic bioremediation did not likely represent an important contribution to contaminant removal atthis site, where dilution appeared to be the primary attenuation mechanism.

Stable carbon isotope evidence for intrinsic bioremediation of tetrachloroethene and trichloroethene at area 6, Dover Air Force Base.

Area 6 at Dover Air Force Base (Dover, DE) has been the location of an in-depth study by the RTDF (Remediation Technologies Development Forum Bioremediation of Chlorinated Solvents Action Team) to evaluate the effectiveness of natural attenuation of chlorinated ethene contamination in groundwater. Compound-specific stable carbon isotope measurements for dissolved PCE and TCE in wells distributed throughout the anaerobic portion of the plume confirm that stable carbon isotope values are isotopically enriched in 13C consistent with the effects of intrinsic biodegradation. During anaerobic microbial reductive dechlorination of chlorinated hydrocarbons, the light (12C) versus heavy isotope (13C) bonds are preferentially degraded, resulting in isotopic enrichment of the residual contaminant in 13C. To our knowledge, this study is the first to provide definitive evidence for reductive dechlorination of chlorinated hydrocarbons at a field site based on the delta13C values of the primary contaminants spilled at the site, PCE and TCE. For TCE, downgradient wells show delta13C values as enriched as -18.0/1000 as compared to delta13C values for TCE in the source zone of -25.0 to -26.0/1000. The most enriched delta13C value on the site was observed at well 236, which also contains the highest concentrations of cis-DCE, VC, and ethene, the daughter products of reductive dechlorination. Stable carbon isotope signatures are used to quantify the relative extent of biodegradation between zones of the contaminant plume. On the basis of this approach, it is estimated that TCE in downgradient well 236 is more than 40% biodegraded relative to TCE in the proposed source area.

Characterizing Intrinsic Bioremediation in a Petroleum Hydrocarbon-Contaminated Aquifer by Combined Chemical, Isotopic, and Biological Analyses

Chemical, isotopic, and biological parameters were evaluated over a 1-year period to characterize microbial processes associated with intrinsic bioremediation in a petroleum hydrocarbon-contaminated aquifer located in Studen, Switzerland. Chemical parameters measured included oxidants such as O2, NO3 −, and SO4 2−, reduced species such as Fe2+ and CH4, and dissolved inorganic carbon (DIC). Stable carbon isotope analyses of DIC were used to differentiate between different processes that contribute to DIC production. Microbial populations were identified by sequence analysis of archaeal 16S rDNA and in situ hybridization using a general DNA binding dye (DAPI) and specific probes targeting the domain Archaea (Arch915) and Bacteria (Eub338), as well as the species Methanosaeta concilii (Rotcl1) and Methanospirillum sp. (Rotcl2). Groundwater exhibited reduced conditions and elevated concentrations of DIC within the contaminated zone. Spatially distinct values of δ13C ranging from −16.5l%c to −4.44%o were found, indicating the presence of different ongoing microbial processes. Detected microbial populations (% of DAPI-stained cells) within the contaminated zone belonged to Archaea (9±2% to 31±13%) and Bacteria (13±3% to 32±13%). In wells with methanogenic activity, Methanosaeta concilii accounted for up to 26% of all DAPI-detected microorganisms. These results demonstrate that this novel combination of chemical, isotopic, and biological analysis provides valuable insights that can be used for the characterization of microbial processes in contaminated aquifers.