Grantee Research Project Results

2001 Progress Report: Anaerobic Intrinsic Bioremediation of Whole Gasoline

EPA Grant Number: R827015C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R827015
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: HSRC (1989) - Northeast HSRC
Center Director: Sidhu, Sukh S.
Title: Anaerobic Intrinsic Bioremediation of Whole Gasoline
Investigators: Suflita, Joseph
Current Investigators: Thoma, Greg , Beyrouty, Craig , Wolf, Duane
Institution: University of Oklahoma
EPA Project Officer: Aja, Hayley
Project Period: February 1, 1999 through January 31, 2000 (Extended to June 30, 2001)
Project Period Covered by this Report: February 1, 2000 through January 31, 2001
Project Amount: Refer to main center abstract for funding details.
RFA: Integrated Petroleum Environmental Consortium (IPEC) (1999) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

The reliance upon intrinsic bioremediation for the removal of spilt gasoline hydrocarbons has gained increased acceptance as our understanding of the underlying microbial processes has evolved. Despite the recently recognized ability of anaerobic microorganisms to metabolize a broad range of hydrocarbons has, the acceptance of anaerobic intrinsic bioremediation as a remedy for petroleum contamination however is largely limited to BTEX compounds. Regulators and site operators are faced with the difficult challenge of determining which sites are amenable to intrinsic bioremediation and which will require a more active and costly remediation effort. The decision-making process is complicated by the inherent difficulty in measuring the biodegradation of hydrocarbon mixtures in the environment. The development of more sophisticated tools to assess intrinsic bioremediation is therefore needed to make sound Risk Based Corrective Action assessment. To this end, we aim to document the anaerobic biodegradation of whole gasoline and identify both patterns of decay and the most recalcitrant compounds of gasoline as unique signatures of anaerobic hydrocarbon biodegradation.

Specifically, the objectives of this research are i) demonstrate the anaerobic removal of BTEX hydrocarbons in the presence of other HC co-contaminants, iii) determine the prospects for the biodegradation of non-BTEX hydrocarbons present in gasoline spills, ii) examine the influence of alternate electron acceptors on these processes, and iv) identify the most recalcitrant components of gasoline as possible biomarkers of anaerobic decay. The project used sediments from a site contaminated by gas condensate that has demonstrated robust anaerobic hydrocarbon biodegradative activities. The removal of over fifty individual components of gasoline was monitored in long term incubations along with the concurrent consumption of electron acceptors and/or production of reduced products.

Progress Summary:

The project used sediments from a site contaminated by gas condensate that has demonstrated robust anaerobic hydrocarbon biodegradative activities. We have examined the biodegradation of two complex petroleum mixtures, whole gasoline and an aritficially-weathered crude oil, under methanogenic, sulfate-reducing, and nitrate-reducing conditions. The removal of over fifty individual components of gasoline was monitored in long term incubations along with the concurrent consumption of electron acceptors and/or production of reduced products.

In unamended incubations, the endogenous rates of sulfate reduction and methanogenesis were high over the entire period of incubation; rates of nitrate reduction, however, are comparatively low in unamended incubations with no significant loss of nitrate. Sulfate reduction and methanogenesis are believed to be the dominant terminal electron-accepting processes at this site and probably in most contaminated anaerobic environments.

As hypothesized in our proposal, the resident microbiota at the Ft. Lupton site have demonstrated broad abilities to metabolize petroleum hydrocarbons under anaerobic conditions. Hydrocarbon analysis of gasoline-amended incubations has demonstrated the biodegradation of several classes of compounds, including alicyclic compounds, short straight chain alkanes, and branched alkanes, classes of hydrocarbons that were widely held to be recalcitrant in the absence of oxygen. Comparing the fate of hydrocarbons under the three different terminal electron-accepting conditions, it is clear that sulfate-reducing conditions are the most advantageous for gasoline degradation at the Ft. Lupton site. As an example, toluene, known to be biodegradable under all three conditions, was completely consumed within 40 days of incubation under sulfate-reducing conditions while less than 30% and 10% were consumed under methanogenic and nitrate-reducing conditions, respectively. Similarly, n- alkane biodegradation was much greater in the presence of sulfate with the C-5 to C-7 alkanes being readily degraded in its presence but not in its absence. A great deal of biodegradation of the gasoline occurred during the first forty days of incubation under sulfate-reducing conditions. Subsequently, hydrocarbon biodegradation has slowed. This suggests an easily degraded, labile fraction of the gasoline was quickly consumed, while an extended incubation will be required for the degradation of the more recalcitrant fraction. It is also possible that the decrease in the rate of degradation was due to toxicity, and this possibility will be investigated. Under sulfate-reducing conditions, compounds of which more than 60% was consumed in the first 40 days include aromatic compounds, alicyclic compounds, and alkanes. The rapid biodegradation of the alkane and alicyclic fraction is surprising and attests to the previously unrecognized metabolic capabilities of the indigenous microorganisms. Among the BTEX compounds, toluene, m-xylene, o-xylene, were degraded, while p- xylene and ethylbenzene appear to be relatively recalcitrant. These results are in contrast to single compound laboratory experiments with these BTEX compounds and may more accurately reflect the in situ process.

Under methanogenic conditions, it is obvious that the biodegradative process is slower than in the presence of sulfate, and it appears to have a more limited substrate range as. As noted previously, a relative recalcitrance of the short-chain alkanes in methanogenic incubations as compared to sulfate-reducing incubations was observed. Over 90% of the octane and nonane was depleted during the first forty days of incubation while pentane and hexane persisted. Also, the BTEX biodegradation was comparatively limited with only toluene and o-xylene appearing to be readily degraded. Although the rate of biodegradation coupled to methanogenesis is slower than that coupled to sulfate reduction, it is uncertain whether or not the two incubation conditions will ultimately yield similar or different profiles of hydrocarbon depletion.

In incubations amended with artificially-weather Alaska North Slope crude oil (ANS), we found the resident microorganisms were able to completely biodegrade the long-chain n-alkane fraction under both methanogenic and sulfate-reducing conditions. These n-alkanes are much larger than those found in the native contamination, ranging from C-14 to C-34. These findings demonstrate that the indigenous microorganisms harbor biodegradative activity towards hydrocarbons that are not found in the native contamination to which they have been adapted.

We have been continually monitoring the susceptibility of whole gasoline to decay under methanogenic, sulfate- reducing, and nitrate-reducing conditions in ongoing, extended incubations. Although high rates of endogenous methanogenesis and sulfate reduction have been observed in the first calendar year, to date, no stimulation has been seen due to the initial 10 l gasoline amendment. Nitrate-amended microcosms have demonstrated little nitrate reduction or hydrocarbon biodegradation. These and previous findings indicate that this terminal electron acceptor does not play a significant role in petroleum metabolism at this site.

We have begun to analyze the extensive hydrocarbon data from the first six months of these incubations. There are significant differences between the amounts of analytes detected in the aqueous phases under different terminal electron accepting-conditions; this may reflect differential partition to the solids.

Overall, our preliminary findings indicate much greater biodegradation under sulfate-reducing conditions than under methanogenic conditions by the indigenous microbiota at the Ft. Lupton site. A wide range of aromatic, alicyclic, and alkane substrates were degraded under sulfate-reducing conditions, surprisingly with most of the degradation occurring in the first 40 days of incubation. Branched alkanes were much more resistant to biodegradation than straight chain alkanes. Also, BTEX compoundswere selectively degraded under sulfate-reducing and methanogenic conditions. Of the xylene isomers, the meta- and ortho- isomers were more labile than the para-isomerunder sulfate-reducing conditions. Under methanogenic conditions, the para- and meta-isomers were preferred over the orth-isomer. Benzene clearly appears to be the most recalctrant of the BTEX isomers.

Parallel experiments, examining the fate of alkane and alicyclic compounds as sole substrates have continued. As in the gasoline experiment, high rates of endogenous methanogenesis and sulfate-reduction have largely masked any stimulation that may have loccurred due to the substrate amendments. However, we have transferred the original incubations into media, and this has resulted in both lower endogenous rates of methanogenesis and sulfate reduction as well as significant stimulation of these process in pentane-, hexane-, octane-, undecane-, and pentadecane- amended. These experiments reveal the ability of the resident microbiota to biodegraded these components commonly found in gasoline and gas condensate and confirm the results found with gasoline as a complex substrate.

Future Activities:

Using complex petroleum mixtures as substrates, we have demonstrated that the indigenous microbiota at the Ft. Lupton site are able to simlutaneoulsy biodegrade a wide variety of petroleum hydrocarbons. The extent and rates of biodegradation were optimal under sulfate-reducing conditions. Biodegradation proceeded under methanogenic conditions but at a reduced rate; nitrate-reducing conditions greatly inhibited hydrocarbon metabolism. With the exception of benzene, all BTEX compounds were completely degraded under sulfate-reducing conditions. The resident microorganisms also metabolized n-alkanes and to a lesser degree branched alkanes and alicyclic compounds.

This research has revealed previously unrecognized microbial decay of specific fractions of gasoline and crude oil under anaerobic conditions.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Publications Views

Other subproject views:	All 11 publications	1 publications in selected types	All 1 journal articles
Other center views:	All 120 publications	19 publications in selected types	All 16 journal articles

Publications

Type	Citation	Sub Project	Document Sources
Journal Article	Townsend GT, Prince RC, Suflita JM. Anaerobic oxidation of crude oil hydrocarbons by the resident microorganisms of a contaminated anoxic aquifer. Environmental Science & Technology 2003;37(22):5213-5218.	R827015C004 (2001) R827015C017 (2002) R827015C017 (Final)	Abstract from PubMed Abstract: ES&T Exit

Supplemental Keywords:

hydrocarbon, biodegradation, bioremediation, methanogenic, sulfate-reducing, nitrate-reducing, BTEX., RFA, Scientific Discipline, Geographic Area, Waste, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Remediation, State, Chemistry, Northwest, Civil/Environmental Engineering, Environmental Microbiology, Microbiology, Biochemistry, Hazardous Waste, Oil Spills, Bioremediation, Biology, Engineering, Environmental Engineering, Groundwater remediation, Hazardous, risk assessment, gasoline, anaerobic treatment, biodegradation, electron acceptor, anaerobic biodegradation, anaerobic bioconversion, hydrocarbons, risk assessments, Ft. Lupton, CO, biological markers, anaerobic bioremediation, anaerobic biotransformation, Colorado (CO)

Progress and Final Reports:

Original Abstract

1999

2000 Progress Report

Final Report

Main Center Abstract and Reports:

R827015    HSRC (1989) - Northeast HSRC

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827015C001 Evaluation of Road Base Material Derived from Tank Bottom Sludges
R827015C002 Passive Sampling Devices (PSDs) for Bioavailability Screening of Soils Containing Petrochemicals
R827015C003 Demonstration of a Subsurface Drainage System for the Remediation of Brine-Impacted Soil
R827015C004 Anaerobic Intrinsic Bioremediation of Whole Gasoline
R827015C005 Microflora Involved in Phytoremediation of Polyaromatic Hydrocarbons
R827015C006 Microbial Treatment of Naturally Occurring Radioactive Material (NORM)
R827015C007 Using Plants to Remediate Petroleum-Contaminated Soil
R827015C008 The Use of Nitrate for the Control of Sulfide Formation in Oklahoma Oil Fields
R827015C009 Surfactant-Enhanced Treatment of Oil-Contaminated Soils and Oil-Based Drill Cuttings
R827015C010 Novel Materials for Facile Separation of Petroleum Products from Aqueous Mixtures Via Magnetic Filtration
R827015C011 Development of Relevant Ecological Screening Criteria (RESC) for Petroleum Hydrocarbon-Contaminated Exploration and Production Sites
R827015C012 Humate-Induced Remediation of Petroleum Contaminated Surface Soils
R827015C013 New Process for Plugging Abandoned Wells
R827015C014 Enhancement of Microbial Sulfate Reduction for the Remediation of Hydrocarbon Contaminated Aquifers - A Laboratory and Field Scale Demonstration
R827015C015 Locating Oil-Water Interfaces in Process Vessels
R827015C016 Remediation of Brine Spills with Hay
R827015C017 Continuation of an Investigation into the Anaerobic Intrinsic Bioremediation of Whole Gasoline
R827015C018 Using Plants to Remediate Petroleum-Contaminated Soil
R827015C019 Biodegradation of Petroleum Hydrocarbons in Salt-Impacted Soil by Native Halophiles or Halotolerants and Strategies for Enhanced Degradation
R827015C020 Anaerobic Intrinsic Bioremediation of MTBE
R827015C021 Evaluation of Commercial, Microbial-Based Products to Treat Paraffin Deposition in Tank Bottoms and Oil Production Equipment
R827015C022 A Continuation: Humate-Induced Remediation of Petroleum Contaminated Surface Soils
R827015C023 Data for Design of Vapor Recovery Units for Crude Oil Stock Tank Emissions
R827015C024 Development of an Environmentally Friendly and Economical Process for Plugging Abandoned Wells
R827015C025 A Continuation of Remediation of Brine Spills with Hay
R827015C026 Identifying the Signature of the Natural Attenuation of MTBE in Goundwater Using Molecular Methods and "Bug Traps"
R827015C027 Identifying the Signature of Natural Attenuation in the Microbial Ecology of Hydrocarbon Contaminated Groundwater Using Molecular Methods and "Bug Traps"
R827015C028 Using Plants to Remediate Petroleum-Contaminated Soil: Project Continuation
R827015C030 Effective Stormwater and Sediment Control During Pipeline Construction Using a New Filter Fence Concept
R827015C031 Evaluation of Sub-micellar Synthetic Surfactants versus Biosurfactants for Enhanced LNAPL Recovery
R827015C032 Utilization of the Carbon and Hydrogen Isotopic Composition of Individual Compounds in Refined Hydrocarbon Products To Monitor Their Fate in the Environment
R830633 Integrated Petroleum Environmental Consortium (IPEC)
R830633C001 Development of an Environmentally Friendly and Economical Process for Plugging Abandoned Wells (Phase II)
R830633C002 A Continuation of Remediation of Brine Spills with Hay
R830633C003 Effective Stormwater and Sediment Control During Pipeline Construction Using a New Filter Fence Concept
R830633C004 Evaluation of Sub-micellar Synthetic Surfactants versus Biosurfactants for Enhanced LNAPL Recovery
R830633C005 Utilization of the Carbon and Hydrogen Isotopic Composition of Individual Compounds in Refined Hydrocarbon Products To Monitor Their Fate in the Environment
R830633C006 Evaluation of Commercial, Microbial-Based Products to Treat Paraffin Deposition in Tank Bottoms and Oil Production Equipment
R830633C007 Identifying the Signature of the Natural Attenuation in the Microbial Ecology of Hydrocarbon Contaminated Groundwater Using Molecular Methods and “Bug Traps”
R830633C008 Using Plants to Remediate Petroleum-Contaminated Soil: Project Continuation
R830633C009 Use of Earthworms to Accelerate the Restoration of Oil and Brine Impacted Sites