Fuel-contaminated Site Research Articles

Hydrocarbon-Contaminated Sites: Is There Something More Than Exophiala xenobiotica? New Insights into Black Fungal Diversity Using the Long Cold Incubation Method.

Human-made hydrocarbon-rich environments are important reservoirs of microorganisms with specific degrading abilities and pathogenic potential. In particular, black fungi are of great interest, but their presence in the environment is frequently underestimated because they are difficult to isolate. In the frame of a biodiversity study from fuel-contaminated sites involving 30 diesel car tanks and 112 fuel pump dispensers (52 diesel and 60 gasoline, respectively), a total of 181 black fungal strains were isolated. The long cold incubation (LCI) of water-suspended samples, followed by plating on Dichloran Rose Bengal Chloramphenicol Agar (DRBC), gave isolation yields up to six times (6.6) higher than those of direct plating on DRBC, and those of enrichment with a phenolic mix. The sequencing of ITS and LSU-rDNA confirmed the dominance of potentially pathogenic fungi from the family Herpotrichiellaceae and Exophiala xenobiotica. Moreover, other opportunistic species were found, including E. opportunistica, E. oligosperma, E. phaeomuriformis, and Rhinocladiella similis. The recurrent presence of E. crusticola, Knufia epidermidis, Aureobasidium melanogenum, Cladosporium spp., and Scolecobasidium spp. was also recorded. Interestingly, 12% of total isolates, corresponding to 50% of taxa found (16/32), represent new species. All the novel taxa in this study were isolated by LCI. These findings suggest that black fungal diversity in hydrocarbon-rich niches remains largely unexplored and that LCI can be an efficient tool for further investigations.

Combined Use of Diagnostic Fumarate Addition Metabolites and Genes Provides Evidence for Anaerobic Hydrocarbon Biodegradation in Contaminated Groundwater.

The widespread use of hydrocarbon-based fuels has led to the contamination of many natural environments due to accidental spills or leaks. While anaerobic microorganisms indigenous to many fuel-contaminated groundwater sites can play a role in site remediation (e.g., monitored natural attenuation, MNA) via hydrocarbon biodegradation, multiple lines of evidence in support of such bioremediation are required. In this study, we investigated two fuel-contaminated groundwater sites for their potential to be managed by MNA. Microbial community composition, biogeochemical indicators, fumarate addition metabolites, and genes diagnostic of both alkane and alkyl-monoaromatic hydrocarbon activation were assessed. Fumarate addition metabolites and catabolic genes were detected for both classes of hydrocarbon biodegradation at both sites, providing strong evidence for in situ anaerobic hydrocarbon biodegradation. However, relevant metabolites and genes did not consistently co-occur within all groundwater samples. Using newly designed mixtures of quantitative polymerase chain reaction (qPCR) primers to target diverse assA and bssA genes, we measured assA gene abundances ranging from 105–108 copies/L, and bssA gene abundances ranging from 105–1010 copies/L at the sites. Overall, this study demonstrates the value of investigating fuel-contaminated sites using both metabolites and genes diagnostic of anaerobic hydrocarbon biodegradation for different classes of hydrocarbons to help assess field sites for management by MNA.

Estimation of Remediation Cost for Reducing Cancer and Non-cancer Risk of a Fuel-contaminated Site

Estimation of Remediation Cost for Reducing Cancer and Non-cancer Risk of a Fuel-contaminated Site

Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production.

Paraffinic n-alkanes (>C17) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffin n-octacosane (C28H58). Compared with that in controls, the consumption of n-octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. Smithella was postulated to be an important C28H58 degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified assA gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other Smithella organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for assA demonstrated significant increases in the abundance and expression of this gene in C28H58-degrading cultures compared with that in controls, suggesting n-octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C28H58-degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery.IMPORTANCE Understanding the methanogenic biodegradation of different classes of hydrocarbons has important applications for effective fuel-contaminated site remediation and for improved recovery from oil reservoirs. Previous studies have clearly demonstrated that short-chain alkanes (<C17) can be biodegraded anaerobically, but less is understood regarding the biodegradation of longer chain waxy alkanes (>C17) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkane n-octacosane can be biodegraded under methanogenic conditions by a presumed Smithella phylotype. Compared with that of controls, we show an increased abundance and expression of the assA gene, which is known to be important for anaerobic n-alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only in n-octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments.

Field data and numerical simulation of btex concentration trends under water table fluctuations: Example of a jet fuel-contaminated site in Brazil

Mass transfer of light non-aqueous phase liquids (LNAPLs) trapped in porous media is a complex phenomenon. Water table fluctuations have been identified as responsible for generating significant variations in the concentration of dissolved hydrocarbons. Based on field evidence, this work presents a conceptual model and a numerical solution for mass transfer from entrapped LNAPL to groundwater controlled by both LNAPL saturation and seasonal water table fluctuations within the LNAPL smear zone. The numerical approach is capable of reproducing aqueous BTEX concentration trends under three different scenarios – water table fluctuating within smear zone, above the smear zone and partially within smear zone, resulting in in-phase, out-of-phase and alternating in-phase and out-of-phase BTEX concentration trend with respect to water table oscillation, respectively. The results demonstrate the model's applicability under observed field conditions and its ability to predict source zone depletion.

Toxicity and biofilm-based selection for methyl tert-butyl ether bioremediation technology

Extractive membrane biofilm reactor (EMBFR) technology offers productive solutions for volatile and semi-volatile compound removal from water bodies. In this study, the bacterial strains Paenibacillus etheri SH7T (CECT 8558), Agrobacterium sp. MS2 (CECT 8557) and Rhodococcus ruber strains A5 (CECT 8556), EE6 (CECT 8612) and EE1 (CECT 8555), previously isolated from fuel-contaminated sites, were tested for adherence on tubular semipermeable membranes in laboratory-scale systems designed for methyl tert-butyl ether (MTBE) bioremediation. Biofilm formation on the membrane surface was evaluated through observation by field-emission scanning electron microscope (FESEM) as well as the acute toxicity (as EC50) of the bacterial growth media. Moreover, extracellular polymeric substance (EPS) production for each strain under different MTBE concentrations was measured. Strains A5 and MS2 were biofilm producers and their adherence increased when the MTBE flowed through the inner tubular semipermeable membrane. No biofilm was formed by Paenibacillus etheri SH7T, nevertheless, the latter and strain MS2 exhibited the lowest toxicity after growth on the EMBFR. The results obtained from FESEM and toxicity analysis demonstrate that bacterial strains R. ruber EE6, A5, P. etheri SH7T and Agrobacterium sp. MS2 could be excellent candidates to be used as selective inocula in EMBFR technology for MTBE bioremediation.

In-situ determination of field-scale NAPL mass transfer coefficients: Performance, simulation and analysis

Better estimates of non-aqueous phase liquid (NAPL) mass, its persistence into the future, and the potential impact of source reduction are critical needs for determining the optimal path to clean up sites impacted by NAPLs. One impediment to constraining time estimates of source depletion is the uncertainty in the rate of mass transfer between NAPLs and groundwater. In this study, an innovative field test is demonstrated for the purpose of quantifying field-scale NAPL mass transfer coefficients (klN) within a source zone of a fuel-contaminated site. Initial evaluation of the test concept using a numerical model revealed that the aqueous phase concentration response to the injection of clean groundwater within a source zone was a function of NAPL mass transfer. Under rate limited conditions, NAPL dissolution together with the injection flow rate and the radial distance to monitoring points directly controlled time of travel. Concentration responses observed in the field test were consistent with the hypothetical model results allowing field-scale NAPL mass transfer coefficients to be quantified. Site models for groundwater flow and solute transport were systematically calibrated and utilized for data analysis. Results show klN for benzene varied from 0.022 to 0.60d−1. Variability in results was attributed to a highly heterogeneous horizon consisting of layered media of varying physical properties.

Isolation and characterization of biosurfactant producing Bacillus sp. from diesel fuel-contaminated site

Among hydrocarbon pollutants, diesel oil is a complex mixture of alkanes and aromatic compounds which are often encountered as soil contaminants leaking from storage tanks and pipelines or as result of accidental spillage. One of the best ecofriendly approaches is to restore contaminated soil by using microorganisms able to degrade those toxic compounds in a bioremediation process. In the present study, nineteen bacteria were isolated by enrichment culture technique from diesel spilled soil collected from electric generator shed of NBAIM, Mau. All the isolates were subjected to screening for lipase production and twelve isolates were found to be positive for lipase. When the isolates were screened for biosurfactant production using CTAB-methylene blue agar plates, only one isolate viz. 2NBDSH3 was found positive which was found to be phylogenetically closely related with Bacillus flexus. Despite having low emulsification index, the bacterium could degrade 88.6% of diesel oil in soil. Biosurfactant from the isolate was extracted and characterized through infra-red spectroscopy which indicated its possible lipopeptide nature which was further supported by strong absorption in UV range in the UV-Vis spectrum. The results of the present study indicated that the isolate either does not produce any bioemulsifier or produces very low amount of emulsifier rather it produces a lipopeptide biosurfactant which helps in degradation of diesel oil by lowering the surface tension. The bacterium thus isolated and characterized can serve as a promising solution for ecofriendly remediation of bacterium diesel contaminated soils.

Gas chromatographic retention index as a basis for predicting evaporation rates of complex mixtures

Models that predict the fate of petroleum fuels in the environment are often required for effective remediation of fuel-contaminated sites. In this research, an environmental fuel spill was simulated by means of a diesel/water microcosm, in which the temporal changes in composition were assessed during evaporation by gas chromatography–mass spectrometry (GC–MS). First-order kinetic rate constants were calculated for 51 selected compounds and utilized to develop predictive models for evaporation rate constants, using GC retention indices on a nonpolar stationary phase. Models were initially developed to predict rates of evaporation of compounds from individual classes (normal alkane, branched alkane, alkyl benzene, and polycyclic hydrocarbon) and then expanded to include all compounds (comprehensive model). Using the comprehensive model, the rate constants were predicted with a mean absolute percent error (MAPE) of 10%, whereas the class-specific models resulted in less error (4–8%). These models were employed to predict the fraction remaining of the total fuel (6% error) as well as the fraction remaining of individual compounds (13% MAPE). Accurate models such as these will facilitate remediation of environmental releases of petroleum products.

Polycyclic Aromatic Hydrocarbon-degrading Bacteria from Aviation Fuel Spill Site at Ibeno, Nigeria

Polycyclic aromatic hydrocarbon (PAH)–degrading bacteria were isolated from aviation fuel contaminated soil at Inua Eyet Ikot in Ibeno, Nigeria. PAH-degrading bacteria in the contaminated soil were isolated by enrichment culture technique. Isolates with high PAH degrading potential characterized by their extensive growth on PAH-supplemented minimal salt medium were screened for their naphthalene, phenanthrene and chrysene degradability. The screening medium which contained selected PAHs as the sole source of carbon and energy showed that Micrococcus varians AFS-2, Pseudomonas putida AFS-3 and Alcaligenes faecalis AFS-5 exhibited a concentration–dependent growth in all the PAH–compounds tested. There were visible changes in the color of growth medium suggesting the production of different metabolites. Their acclimation to different PAH substrates was also evident as A. faecalis AFS-5 isolated from chrysene grew well on other less complex aromatic compounds. The isolate exhibited best growth (0.44 OD600) when exposed to 10 ppm of chrysene for 5 days and could utilize up to 90 ppm of chrysene. This isolate and others with strong PAH-degrading potentials are recommended for bioremediation of PAHs in aviation fuel-contaminated sites in the tropics.

Groundwater Geochemical Characterization of a Fuel-Contaminated Fractured Bedrock in a Permafrost Environment

AbstractRemediation of a contaminated site to site-specific cleanup standard requires a good understanding of the geochemical characteristics of the site. In this study, the groundwater hydrochemical characteristics of a fuel-contaminated mine site in the Northwest Territories (Canada) was evaluated to understand the active geochemical processes and assess the groundwater quality in comparison to guidelines for the protection of freshwater aquatic life. Groundwater samples were taken from installed monitoring wells across the site and analyzed by field and laboratory methods for dissolved metals; ions; and benzene, toulene, ethylbenzene, and xylenes (BTEX) organic constituents. The results showed that the groundwater is Ca-SO4 type because of ubiquitous occurrence of gypsum dissolution and carbonate weathering. The last sampling at the site showed that the BTEX constituents of interest were above the guideline limits in some locations, but other geochemical indicators showed biodegradation is occurring at...

Field Note: Successful Establishment of a Phytoremediation System at a Petroleum Hydrocarbon Contaminated Shallow Aquifer: Trends, Trials, and Tribulations

We report the establishment of a mixed hybrid poplar (Populus spp.) and willow (Salix spp.) phytoremediation system at a fuel-contaminated site. Several approaches were used to balance competing goals of cost-effectiveness yet successful tree establishment without artificial irrigation or trenching. Bare root and unrooted cuttings were installed using either: (1) 1.2 m deep holes excavated with an 8 cm diameter auger using a direct-push rig and backfilled with the excavated, in situ soil; (2) 1.2 m deep holes created with a 23 cm diameter auger attached to a Bobcat rig and backfilled with clean topsoil from offsite; and (3) shallow holes between 15–30 cm deep that were created with a 1.3 cm diameter rod and no backfill. Tree mortality from initial plantings indicated contaminated zones not quantified in prior site investigations and remedial actions. Aquifer heterogeneity, underground utilities, and prior remediation infrastructure hampered the ability of the site to support a traditional experimental design. Total stem length and mortality were measured for all planted trees and were incorporated into a geographic information system. Planting early in the growing season, augering a larger diameter hole, and backfilling with clean, uncontaminated topsoil was cost effective and allowed for greater tree cutting growth and survival.

Carbon and Hydrogen Isotope Fractionation during Anaerobic Toluene Oxidation by Geobacter metallireducens with Different Fe(III) Phases as Terminal Electron Acceptors

Microbial oxidation of BTEX compounds under iron-reducing conditions is an important attenuation process for fuel-contaminated sites. We evaluated the use of compound-specific isotope analysis for the identification and quantification of anaerobic toluene oxidation by Geobacter metallireducens. 13C and 2H enrichment of toluene was measured in laboratory batch systems and varied significantly for a solid vs a dissolved Fe(III) phase provided as terminal electron acceptor. 13C enrichment factors (epsilonC) in suspensions of a solid Fe(III) phase were between -1.0 and -1.3% per hundred, whereas epsilonC-values were significantly higher in solutions of Fe(lll) citrate (-2.9 to -3.6% per hundred. The same trend was observed for 2H fractionation. Solid phase reduction resulted in an epsilonH-value of -34.6 +/- 0.9% per hundred, compared to -98.4 +/- 3.0% per hundred for the reduction of dissolved Fe(lll). The linear correlation of delta(2)H vs. delta(13)C during toluene oxidation resulted in nearly identical slopes for both systems, confirming that the reaction mechanism, that is enzymatic methyl-group oxidation, was the same. We hypothesize that smaller 2H and 13C fractionation in suspensions is due to toluene transport limitations to cells of G. metallireducens at surfaces of solid Fe(III) phases. Enrichment factors determined in Fe(III) mineral suspensions should be more representative for anaerobic toluene degradation owing to the abundance of solid Fe(III) in soils and aquifers.

Comparison of MTBE concentrations in groundwater of urban and nonurban areas in Germany

The occurrence of the gasoline oxygenate methyl tert-butyl ether (MTBE) in groundwater samples from known fuel-contaminated sites ( n=29 samples), nonurban ( n=74) and urban sites ( n=67) in Germany was investigated. The analyses revealed detection frequencies of 58% (contaminated sites), 24% (nonurban sites) and 63% (urban sites) at a detection limit of 0.01 μg L −1. Median (maximum) MTBE concentrations were calculated for nonurban and urban samples as 0.18 μg L −1 (2.2 μg L −1) and 0.06 μg L −1 (48 μg L −1). The data from nonurban samples revealed MTBE detections mainly at public supply wells with higher pumping rates than monitoring wells. MTBE was more frequently detected in urban samples, most probably due to the higher atmospheric input and direct liquid emissions from motorways or gas stations. Higher concentrations above 1.0 μg L −1 in urban areas were found in wells located at industrial sites, where also a MTBE plume was accidentally detected during the study. The prevalence of MTBE in shallow aquifers was comparable to those in the USA.

Comparison of field-observed and model-predicted plume trends at fuel-contaminated sites: Implications for natural attenuation rates

Subsequent to modeling of natural attenuation processes to predict contaminant trends and plume dynamics, monitoring data were used to evaluate the effectiveness of natural attenuation at reducing contaminant concentrations in groundwater at seven fuel-contaminated sites. Predicted and observed contaminant trends at seven sites were compared in order to empirically assess the accuracy of some fundamental model input parameters and assumptions. Most of the models developed for the study sites tended to overestimate plume migration distance, source persistence, and/or the time required for the benzene, toluene, ethyl benzene, and xylenes (BTEX) plumes to attenuate. Discrepancies between observed and predicted contaminant trends and plume behavior suggested that the influence of natural attenuation process may not have been accurately simulated. The conservatism of model simulations may be attributed to underestimation of natural source weathering rates, overestimation of the mass of contaminant present in the source area, and/or use of overly conservative first-order solute decay rates.

Formation of alkylated aromatic acids in groundwater by anaerobic degradation of alkylbenzenes

Evidence for the presence of previously unreported alkylated aromatic acids in anaerobic groundwater samples from a jet fuel-contaminated site is presented. The redox conditions are dominated by sulfate reduction. A gas chromatography/mass spectrometry screening showed a composition of aromatic hydrocarbons ranging from BTEX (C 0–C 2-substituted benzenes) up to C 7-benzenes. Known metabolites like methylbenzoates (C 1-BA), C 2-BA, C 3-BA as well as methylbenzylsuccinates (C 1-BSA) were detected. Additionally, previously unreported metabolites of higher alkylated benzenes have been identified in the extracts: those are C 2-BSA to C 5-BSA as well as C 4-BA to C 6-BA, which were detected in the μg/l-concentration range. The formation of C 1- and C 2-BSA as well as the C 1- and C 2-BA can be linked to structurally corresponding parent contaminants like the xylene isomers and the trimethylbenzene isomers. The C 3-BSA and C 3-BA represent metabolites of C 4-benzenes. The occurrence of C 4–C 5-BSA and C 4–C 6-BA is attributed to C 5–C 7-benzenes acting as parent compounds. The concentration of total aromatic contaminants decreased along the plume centre line. In contrast, benzoates and benzylsuccinates (BA and BSA) showed constant concentrations over a distance of 84 m. BA concentrations were up to 162 times higher compared to BSA, as indicated by the ratio f BA/BSA. A pronounced transient behaviour of BSA or an overall persistent behaviour of BA can explain this. Hence, along the plume centre line, f BA/BSA was constant over a distance of 128 m. The degradation products detected in a monitoring well are not necessarily tied to contaminants detected in that well. Therefore, the metabolite plume is partly located in front of the contaminant plume, but does not significantly migrate downstream the plume toe.

The catechol 2,3-dioxygenase gene and toluene monooxygenase genes from Burkholderia sp. AA1, an isolate capable of degrading aliphatic hydrocarbons and toluene

sp. AA1 isolated from a diesel fuel-contaminated site degraded toluene, as well as a wide range of alkanes from decane (C8) to pentacosane (C25) as sole carbon and energy sources. This strain also utilized m-toluate, p-toluate, o-toluate, and m-cresol as sole carbon and energy sources. Toluene- and toluate-grown cells showed catechol 2,3-dioxygenase activity and indole oxidation activity that is exhibited by some toluene oxygenation enzymes. The catechol 2,3-dioxygenase gene (catB) was cloned and sequenced. Its deduced amino acid sequence is analogous to the extradiol dioxygenases cloned from a variety of microorganisms. A DNA fragment containing the genes for the indole oxidation activity was cloned and sequenced. A seven-gene cluster designated as tbhABCDEFG was identified. Significant similarities were found with multicomponent monooxygenase systems for toluene, benzene and phenol from different bacterial strains. Journal of Industrial Microbiology & Biotechnology (2000) 25, 127–131.