Sponsored by American Petroleum Institute  

Predicting MTBE Legacy Impacts to the Sole Source Aquifer of Long Island , NY
Kristy A. Salafrio, New York State Department of Environmental Conservation, Stony Brook,NY
Joseph E. Haas, II, New York State Department of Environmental Conservation, Stony Brook, NY

Microbial Production and Consumption of tertiary Butyl Alcohol
Michael R. Hyman, North Carolina State University , Raleigh , NC  

Anaerobic MTBE & TBA Biodegradation - Microbial Respiratory Processes versus Extent of Biodegradation
Kevin T. Finneran, University of Illinois at Urbana/Champaign, Urbana , IL  

Stable Isotope Probing with 13C-MTBE-amended Bio-Sep® Beads in MTBE-degrading Microcosms
Kerry Sublette, University of Tulsa , Tulsa , OK  

Stable Isotope Fractionation Resulting from Biotic and Abiotic MTBE Attenuation Processes
Paul Philp, University of Oklahoma , Norman , OK  

Predicting MTBE Legacy Impacts to the Sole Source Aquifer of Long Island , NY

Kristy A. Salafrio, New York State Department of Environmental Conservation, Region One Headquarters, SUNY @Stony Brook, 50 Circle Road, Stony Brook, NY 11790-3409, Tel: 631-444-0323, Fax: 631-444-0328, Email: kasalafr@gw.dec.state.ny.us
Joseph E. Haas, II, New York State Department of Environmental Conservation, Region One Headquarters, SUNY @Stony Brook, 50 Circle Road, Stony Brook, NY 11790-3409, Tel: 631-444-0323, Fax: 631-444-0328, Email: jehaas@gw.dec.state.ny.us

T he New York State Department of Environmental Conservation (NYSDEC) conducted a pilot study to better define the extent of MTBE contamination stemming from previously unidentified and/or unreported MTBE blended gasoline releases throughout the Long Island aquifer system. The study yielded sufficient data to project the potential MTBE impacts upon the sole source of drinking water for the residents of Nassau and Suffolk Counties . The study also yielded information that provided insight into the potential costs and work load associated with managing the MTBE legacy impacts to Long Island ’s drinking water supply.

During the study, 52 gasoline retail stations in Nassau and Suffolk Counties (approximately 4.7% of the total number of stations) that had no known prior release of oxygenated gasoline underwent petroleum bulk storage inspections and fuel oxygenates water quality impact assessments. The study found that MTBE was non-detect or less than 10 micrograms/liter (µg/L) at approximately 60% of sites investigated and that the MTBE concentrations in groundwater ranged from non-detect up to 240,000 µg/L in Nassau County and up to 63,000 µg/L in Suffolk County. Additionally, MTBE was found to have exceeded the drinking and groundwater standards of 10 µg/L at 34% and 53% of sites investigated in Suffolk and Nassau Counties , respectively.

Microbial Production and Consumption of tertiary Butyl Alcohol

Michael Hyman, Department of Microbiology, North Carolina State University, Raleigh, NC 27695, Tel: 919-515-7814, Fax: 919-515-7867, Email: michael_hyman@ncsu.edu

Identifying the sources and sinks of tertiary butyl alcohol (TBA) is key to any comprehensive understanding of the environmental impacts of fuel oxygenates. Our recent studies have focused on several aspects of the microbial production and consumption of TBA. In one study we examined the potential for TBA production from other important gasoline components besides ether oxygenates. Isobutane (2-methylpropane) is a universal gasoline component found at its highest concentrations (~4% v/v) in gasoline used in colder climates. Aerobic metabolism and cometabolism of isobutane by pure and mixed hydrocarbon-oxidizing microorganism consistently leads to substantial TBA accumulation. Production of TBA during isobutane metabolism is strongly favored under the O₂-limited conditions typically encountered in gasoline-impacted environments. These results suggest microbial production of TBA might occur even during the biodegradation of gasoline that does not contain ether oxygenates such as MTBE or ETBE. In our studies of TBA consumption we have also isolated two organisms that appear to have different TBA-oxidizing pathways. One organism, strain S1B1, does not grow on n-alkanes and oxidizes TBA through a conventional pathway involving 2-methyl-1,2-propanediol and 2-hydroxyisobutyric acid. Rapid growth of this strain has been achieved in a mixed culture with the alkane-dependent, MTBE-cometabolizing, TBA-generating strain, Pseudomonas mendocina KR1. In the presence of n-octane and MTBE both organisms grow and full mineralization of MTBE is achieved even though neither organism can grow on MTBE alone as a sole source of carbon and energy. Our most recent studies have been directed at identifying the key enzymes responsible for TBA oxidation in strain S1B1 and other TBA-utilizing strains. The most current results of our various studies on microbial TBA production and consumption will be presented and their significance will be discussed.

Anaerobic MTBE & TBA Biodegradation – Microbial Respiratory Processes versus Extent of Biodegradation

Kevin T. Finneran, PhD, University of Illinois - Urbana Champaign, Dept of Civil and Environmental Engineering, NCEL 205 N. Mathews, Urbana, IL, 61801, Tel: 217-333-1514, Fax: 217-333-6967, Email: finneran@uiuc.edu

Methyl tert butyl ether (MTBE) and tert-butyl alcohol (TBA) are groundwater contaminants of concern arising from use in fuel as oxygenates.  TBA is also problematic because it can accumulate during MTBE biodegradation.  These compounds are often contaminants in anaerobic subsurface environments, where degradation rate and extent are influenced by different microbial processes and shifting microbial communities.  Unlike aerobic respiration which is limited to oxygen, numerous anaerobic electron acceptors can be present simultaneously, which alters MTBE and TBA biodegradation in a positive or negative manner.  The data to date have been primarily empirical, and this has led to great variability amongst the data sets.  The major issue arising is “what conditions lead to TBA degradation versus accumulation”?  The research presented here describes MTBE and TBA biodegradation studies in contaminated sediment under shifting electron accepting processes.  In addition, we describe the first anaerobic, Fe(III)-reducing enrichment culture that degrades MTBE, strain NW1.

Sediment incubations were constructed with MTBE- and/or TBA-contaminated aquifer material from several sites located throughout the U.S.   The dominant terminal electron accepting process (TEAP) was identified for each site, which was the starting or baseline condition for the experiment.  Electron acceptors (nitrate, Fe(III), electron shuttles to promote Fe(III) reduction, fumarate, or sulfate) were added depending on the initial conditions at concentrations that would allow multiple TEAPs to overlap.  This was to create what we refer to as “shifting” TEAP conditions.  We compared this to conditions in which the TEAP was stable over time.  Uniformly radiolabeled [¹⁴C]-MTBE or [¹⁴C]-TBA were added and ¹⁴CO₂ and/or ¹⁴CH₄ were quantified over time.  Liquid enrichment cultures were also initiated in freshwater media with the same electron acceptors and MTBE or TBA as the sole electron donors.

While degradation was site specific the greatest extent of mineralization has been quantified in sediment amended with Fe(III) or Fe(III) plus electron shuttles, despite the initial conditions.  Nearly 80% of TBA has been mineralized to CO₂ in Fe(III) plus AQDS amended sediment.  Approximately 25% has been mineralized in sulfate amended sediment.  Interestingly, MTBE is not oxidized at all at this site, which is unique.  Microbial community composition will be monitored to determine why MTBE is not degraded while TBA is.  Fumarate has promoted MTBE and TBA degradation in a variety of incubations; fumarate is an alternate electron acceptor for Fe(III)-reducing microbes.  Shifting conditions to higher redox processes such as nitrate did not stimulate activity.  In fact – all TBA degradation ceased when nitrate was added.  This argues against broad assumptions that “higher redox potential” acceptors stimulate activity.  Finally, a liquid enrichment culture, strain NW1, was obtained from one site.  This culture oxidizes MTBE as the sole carbon and energy source coupled to Fe(III) and AQDS reduction.  It accumulates stoichiometric TBA, but MTBE is completely degraded.  This culture is critical as it provides the first Fe(III)-reducing model culture to investigate MTBE degradation (and TBA accumulation) at the cellular level.

Stable Isotope Probing with 13C-MTBE-amended Bio-Sep® Beads in MTBE-degrading Microcosms

Xiaomin Yang, BP Corporation North America, Warrenville , IL
John Wilson and Cherri Adair , U.S. EPA, Ada , OK
Jennifer Busch-Harris, Kerry Sublette and Eleanor Jennings, University of Tulsa, Tulsa, OK Tomasz Kuder and Paul Philp, University of Oklahoma, Norman, OK
Greg Davis, Microbial Insights, Inc., Rockford , TN
William E. Holmes, University of California , Davis , CA

The relative concentrations of tert-butyl alcohol ( TBA ) and methyl tert-butyl ether (MTBE) in groundwater samples from a gasoline spill site in Orange County , CA suggested that MTBE was being transformed to TBA .  Stable carbon isotope ratio analysis of MTBE in groundwater also indicated that MTBE had been biologically degraded.  Sediment was collected from the site for an anaerobic microcosm studies.  The removal of MTBE in the microcosms was rapid.  The initial concentration of MTBE (1300 mg/L) was depleted to the analytical detection limit (<3 mg/L) within three months. MTBE removal was accompanied by a stoichiometric accumulation of TBA .  The first-order rate constant for MTBE biodegradation was 25 ± 4 per year at 95% confidence.  As MTBE was removed during the incubation, ¹³C was enriched in the remaining MTBE with d¹³C values increasing from -29.7‰ to +40 ‰. 

MTBE biodegradation was also observed in microcosms that were amended with ¹³C₅-MTBE-loaded Bio-Sep beads using either recycled or original sediments from the study referenced above.  ¹³C-labeling of specific phospholipids indicated that sulfate-reducing bacteria (SRB) played a major role in MTBE biodegradation in these microcosms. However, these microcosms were severely sulfate limited and Fe(III) reduction may well have been a significant, and possibly limiting, electron acceptor for the SRB.  To our knowledge this is the first demonstration of a direct linkage between MTBE biodegradation and a specific group of bacteria.    A mass balance in these microcosms suggested that some TBA degradation may have occurred in that the TBA inventory at the conclusion of the experiment was significantly lower than the amount of MTBE degraded.

This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.

Stable Isotope Fractionation Resulting from Biotic and Abiotic MTBE Attenuation Processes 

Tomasz Kuder, Paul Philp and Jon Allen, School of Geology and Geophysics, University of Oklahoma, Norman, OK,73019

This presentation will discuss the application of compound-specific isotope analysis (CSIA) to MTBE attenuation studies. Results published to date indicate that: (i) it is practical to distinguish between the effects of aerobic and anaerobic MTBE biodegradation by combined carbon + hydrogen CSIA; (ii) the magnitude of carbon isotopic fractionation in the anaerobic process is large and consistent among different anaerobic microbial cultures (carbon isotope enrichment factors reported in literature for three different cultures are between -9.2 ± 5.0 and -15.6 ± 4.1) and (iii) there is little evidence of mineralization of the tert-butyl group of MTBE (tert-butyl alcohol accumulates upon MTBE degradation). This presentation will show new data from anaerobic, MTBE-degrading microcosms, aiming at accurate determination of carbon and hydrogen isotope effects. The values of carbon isotope enrichment factor were obtained from six different methanogenic and sulfate reducing cultures grown in agitated soil to assure uniform medium distribution. The resulting carbon isotope enrichment factors are similar to each other and higher than the previously reported ones, clustering between –17 and –20. It is proposed that calculation of anaerobic biodegradation progress based on Rayleigh model should use the latter value for conservative estimate of the extent of biodegradation. Hydrogen isotope data are consistent with the previously published results obtained from field samples. Hydrogen enrichment factor interpolated from the 2D-CSIA data is approximately –30 (i.e., the net hydrogen effect is similar to that of aerobic MTBE biodegradation). Abiotic in-situ degradation of MTBE is possible either at sites treated by chemical oxidation remedies or due to spontaneous acid hydrolysis. Examples of isotope fractionation will be shown for laboratory experiments on Fenton reagent degradation and acid hydrolysis of MTBE. In both cases, isotope effects are in agreement with the proposed reaction mechanisms. 2D-CSIA trends resulting from both reaction types are identical to those resulting from biological aerobic MTBE degradation. Published data on isotope effects upon phase partitioning, volatilization etc. suggest that these processes result with minor isotope fractionation and should not interfere with the studies of biodegradation. It will be shown that under certain environmental conditions, measurable changes of carbon and hydrogen isotope ratios are likely due to MTBE volatilization from aqueous or hydrocarbon phase. While the magnitude of those changes is low in comparison with those due to anaerobic biodegradation, volatilization and aerobic biodegradation can be difficult to distinguish from each other.

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