EPA Grant Number: R827015-01-0
Title: Continuation of an Investigation into the Anaerobic Intrinsic Bio
remediation of Whole Gasoline
Investigators: Joseph Suflita, G. Todd Townsend
Institution: University of Oklahoma
EPA Project Officer: Bala Krishnan
Project Period: March 1, 2001 to February 28, 2002 (N/C Ext. to August
28, 2002)
Project Amount: $111,344
Research Category: Intrinsic bioremediation/natural attenuation
We have initiated a long-term experiment designed to monitor the fate of a whole gasoline in the environment with an improved experimental design. Anaerobic incubations were constructed containing 50 gm of contaminated sediment and 75 ml of pristine site water. These incubations are held under methanogenic and sulfate reducing conditions and have been amended with one µl of a defined API test gasoline as substrate. Besides autoclaved controls, parallel control incubations also include incubations amended with molybdate to inhibit sulfate reduction, incubations amended with bromoethanesulfonic acid to inhibit methanogenesis, and incubations amended with both molybdate and bromoethanesulfonic acid to inhibit both terminal electron accepting processes. All incubations are performed in triplicate.
We have developed an analytical method that is simple to perform but has the sensitivity and resolving ability to measure the headspace concentrations of over seventy gasoline components. Using a GC equipped with a capillary column specifically designed for gasoline analysis and a well-characterized whole gasoline as a standard, we have tentatively identified over fifty individual compounds based upon their retention time. We are presently confirming these compounds' identity by GC-MS analysis. Substrate concentrations in experimental incubations are calculated relative to water standards containing 1.0, 0.8, 0.6, 0.4, 0.2, and 0.1 ul of gasoline. All substrate losses are compared to triplicate sterile and inhibited incubations. The following data presents the preliminary results of the incubations held under sulfate-reducing conditions after 90 days of incubation. In order to condense the data for this report, data from sterile and inhibited incubations are not shown. Typical abiotic loss was less than 10% during the 90-day course of incubation. All hydrocarbon concentrations are given relative to that individual hydrocarbon's concentration at time zero.
All of the predominant n-alkanes present in gasoline appear to be readily biodegradable with the biodegradation rate related to the chain length. Octane is the most rapidly biodegraded while butane is the slowest with 15% loss compared to the inhibited and sterile controls.
The predominant n-alkenes found in gasoline appear to be even more susceptible to biotransformation under sulfate-reducing conditions than the n-alkanes. The relative rates of biodegradation of C5 and C6 alkenes appear to be quite similar, regardless of the location or conformation of the double bond
Of the fourteen branched alkanes that we have monitored, we have found 2- methyloctane, 2-and 3-methylhexane, and 2-methylpentane to demonstrate varying degrees of biodegradability. Although branching does result in reduced biodegradability compared to these substrate's straight chain analogs, it does not preclude their biodegradation.
The branched alkenes demonstrate much greater biodegradability in comparison to their saturated branched alkane counterparts.
As shown in previous studies with this and other sites, toluene and m- xylene are the most readily biodegradable of the BTEX hydrocarbons. We have some evidence for o-xylene biodegradation at this time.
Notable among the alicyclic biodegradation results is the recalcitrance of both cyclopentane and ethylcyclopentane relative to the unsaturated cylcopentene and the methylcyclopentane.
To date, most biodegradation studies have involved a single substrate at relatively high concentrations. This experiment differs in its use a complex mixture of hydrocarbons, whole gasoline, as substrate at a relatively low concentration , ca. 10 ppm. These preliminary results have demonstrated that the Ft. Lupton aquifer microbiota harbor a diverse range of hydrocarbon-degrading metabolic activities. We have observed the rapid depletion of a variety of alkanes, alkenes, and alicyclic hydrocarbons relative to the more resistant aromatic BTEX compounds. Furthermore, we have found that though branching does lead to increased recalcitrance for the alkane fraction, certain branched alkanes are subject to anaerobic decay while branched alkenes are biodegraded quite rapidly. It should be noted that though unsaturated alkenes and alicyclic compounds were rapidly consumed, the natural gas condensate to which the microbiota has been previously exposed does not contain appreciable amounts of unsaturated hydrocarbons. While BTEX hydrocarbons have been the most studied fraction of gasoline, our results indicate that loss of other fractions of whole gasoline may be precede BTEX depletion. The loss of this nonaromatic fraction in situ may serve as evidence of the initial phase of intrinsic bioremediation of this complex hydrocarbon mixture.