Period Covered by the Report: February 1, 2000 to January 31, 2001
Date of Report: March 31, 2001
EPA Grant Number: R827015-01-0
Title: Anaerobic Intrinsic Bioremediation of Whole Gasoline
Investigators: Joseph Suflita
Institution: University of Oklahoma
EPA Project Officer: Bala Krishnan
Project Period: February 1, 1999 to January 31, 2000 (N/C Ext. to February 28, 2001)
Project Amount: $130,036
Research Category: Intrinsic bioremediation/natural attenuation
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.
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.
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.
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.
G. Todd Townsend, Roger C. Prince, and Joseph M. Suflita. The Anaerobic Biodegradability of Whole Gasoline. IPEC Meeting, Albuquerque, NM (October).
G. Todd Townsend, Roger C. Prince, and Joseph M. Suflita. Anaerobic Biodegradation of Alicyclic Constituents of Gasoline and Natural Gas Condensate by Bacteria from an Anoxic Aquifer. In Preparation.
G. Todd Townsend, Roger C. Prince, and Joseph M. Suflita. The Anaerobic Oxidation of Crude Oil Hydrocarbons by the Resident Microorganisms of a Contaminated Anoxic Aquifer. In Preparation
hydrocarbon, biodegradation, bioremediation, methanogenic, sulfate-reducing, nitrate-reducing, BTEX