Peroxygen Mediated Bioremediation of MTBE.

ABSTRACT: Evidence is mounting, from laboratory and field studies, that oxygen plays a role in the bioremediation of methyl tertiary butyl ether (MTBE). Our experience with a number of commercial applications of peroxygen, in the form of Oxygen Release Compound (ORCâ), correlates the provision of enhanced aerobic conditions with enhanced MTBE biodegradation. Subsequent laboratory and field studies have documented this phenomenon while showing it 1) follows Michaelis-Menten kinetics, 2) is subject to interference by background hydrocarbons as represented by benzene and xylene and that, 3) this interference dynamic may be specifically described in terms of classical competitive inhibition. Most recently, evidence for benzene mediated co-oxidation of MTBE has been demonstrated. Given that requisite enzymatic inductions will have already taken place in a co-contaminated aquifer, enhanced aerobic bioremediation with ORC, represents a cost-effective approach to facilitate both the direct bioremediation of MTBE and the inhibitory hydrocarbons that need to be eliminated as well.


MTBE complicates remediation and closure of properties contaminated with BTEX and other fuel hydrocarbons. A number of parties are becoming increasingly concerned about the environmental impact of MTBE. Several factors are responsible for the heightened level of concern as folows: 1) MTBE tends to degrade very slowly, 2) MTBE is highly soluble in water and does not easily sorb onto the aquifer matrix; retardation of MTBE is therefore minimal and plume dimensions are enhanced, 3) Due to its Henry's Law constant, MTBE is slow to volatilize out of groundwater, 4) Taste and odor thresholds for MTBE are very low – approximately 35 ppb, 5) MTBE toxicity and carcinogenicity profiles are largely undetermined.

Some of these characteristics compromise active remediation methods such as air sparging and pump and treat systems. In the latter case, stripping inefficiencies encountered with extracted water have caused many consultants to evaluate other treatment options. One of these options is in-situ aerobic bioremediation and we have been demonstrating for several years that the bioremediation of MTBE is enhanced by ORC.

As early as 1994, Regenesis noticed that MTBE concentrations decreased at an unusually high rate, relative to the literature (Howard, et al., 1991), in monitoring wells containing ORC filter socks. With a series of laboratory experiments eliminating abiotic chemical and physical mechanisms as the cause, emphasis was placed on bioremediation as the operant mechanism. Eventually, more compelling field evidence became available using monitoring wells downgradient of ORC injection fields. Furthermore, there was as suggestion that background hydrocarbon contaminants repressed MTBE degradation; on a majority of the sites investigated MTBE degradation only occurred after decreases in BTEX levels (Koenigsberg, 1997). Currently there are number of examples of ORC-mediated MTBE degradation in Regenesis files; a representative case is presented below. Similar results have been published that involve the sparging of air or oxygen (Javanmardian and Glasser ,1997; Carter et al., 1997).


MTBE Bioremediation Field Results. A service station in Lake Geneva, Wisconsin was contaminated with high levels of MTBE and BTEX due to a leaking underground storage tank (UST). Measurements indicated MTBE and BTEX concentrations reached levels up to 800 ppb and 14,000 ppb, respectively. Though project engineers removed the UST and excavated the contaminated soil, MTBE and BTEX still persisted in the groundwater.

Project geologists injected 1,700 pounds of ORC as a slurry (via direct-push technology) to enhance aerobic degradation in the saturated zone. MTBE degradation results are presented in Figure 1. Over nine months following ORC injection, results from two downgradient monitoring wells indicate that MTBE concentrations degraded to less than two ppb. The site has since been submitted for closure to the state of Wisconsin.

FIGURE 1. MTBE bioremediation field results.

MTBE Bioremediation as a Function of Dissolved Oxygen. An independent laboratory study by Fortin and Deshusses at the University of California, Riverside (supported by Regenesis) investigated the biodegradation of MTBE by respirometry using a mixed culture. In the experiment, oxygen uptake rates at various dissolved oxygen concentrations were used to quantify the influence of dissolved oxygen concentration on the rate of MTBE biodegradation. Results of the experiment, are presented in Figure 2 and demonstrate 1) the rate of MTBE biodegradation was proportional to the concentration of dissolved oxygen in water and 2) MTBE uptake followed a Michaelis-Menten kinetics with respect to dissolved oxygen.

FIGURE 2. MTBE bioremediation as a function of dissolved oxygen.

Does Competitive Inhibition Play a Role in MTBE Bioremediation?

Competitive inhibition is a term used to describe enzymatic activity in which two or more different substrates compete for the same enzyme. One indication that competitive inhibition may be occurring is when the degradation of one substrate is repressed in the presence of another substrate.

Field observations suggest that background hydrocarbons may repress MTBE degradation and vice versa. As presented in Figure 3, data from a site in Michigan show that in the presence of ORC, MTBE degradation occurs after BTEX concentrations subside. This effect has been documented at the majority of MTBE-impacted sites using ORC.

FIGURE 3. Competitive inhibition field results.

Prompted by such field results, a series of laboratory experiments were conducted to test whether background hydrocarbons interfere with MTBE degradation. In an in-vitro experiment with aerobic bacteria (known to be capable of degrading MTBE and BTEX), results suggest that MTBE metabolism is inhibited by background hydrocarbons. MTBE degradation was measured in the presence of 1) MTBE only and 2) MTBE and xylene during a seven day period. Results indicated a 52% reduction of MTBE in the absence of xylene versus a 9% reduction of MTBE with xylene present.

Independent experiments were then performed for Regenesis, by Pelorus EnBiotech Corporation, that explored the hypothesis that MTBE biodegradation is 1) an aerobic co-oxidative process and 2) that competitive inhibition could exist between a primary substrate and MTBE. The most likely primary substrates involved in co-oxidation and competitive inhibition are compounds found at the aerobic fringe of a petroleum hydrocarbon plume. Initial studies, using resting cell transformation tests, demonstrated that substantial removal of MTBE was achieved with cultures that were acclimated to benzene, camphor, o-xylene and cyclohexanone. In those tests a specific benzene acclimated mixed culture, designated PEL-B201, was most efficient in degrading MTBE (58% removal). This established the possibility that a single organism could metabolize both MTBE and alternate substrates and therefore be under the influence of competitive inhibition dynamics. The competitive inhibition hypothesis was bolstered by demonstrating both MTBE inhibition of benzene metabolism and the inhibition of MTBE metabolism with increasing benzene concentrations.

The benzene utilizing culture (PEL-B201) used in the experiments was grown in basal salts media on benzene vapors. Growth and activity experiments were performed to determine optimum conditions for biomass production. MTBE biotransformation experiments were performed in 160 ml Wheaton serum bottles containing oxygen saturated phosphate buffer supplemented with MTBE (3.35 mg/L). The bottles were sealed with Teflon lined serum septa. To evaluate the effects of benzene on MTBE degradation a stock solution of benzene in DMF was added to achieve final concentrations of 1.9 mmM and 3.8 mmM respectively. PEL-B201 acclimated cell suspensions were added to each test reactor to a cell density of approximately 2.0 x 10E8 cells/ml. Controls were inoculated to the same level with un-acclimated PEL-B201 cells grown on succinate. Over a 48 hour test period, samples were removed from each reactor and placed in 2.0 ml GC vials. Headspace samples were analyzed for MTBE by gas chromatography (GC/PID).

Optimum growth conditions established for strain PEL-B201 were developed through growth curve and oxygen uptake studies on benzene. Optimum degradative activity and cell yield were achieved when optical densities reached a nominal value of approximately 1.10 (OD 600). Results of oxygen uptake (OU) tests are shown in Table 1. These tests clearly indicate that MTBE inhibits oxygen uptake associated with benzene metabolism.

Results of the biotransformation experiments with PEL-B201are presented in Figure 4. Benzene induced cell suspensions degrade >99% of the added MTBE. Increasing levels of benzene (1.9 uM and 3.8 uM) result in a significant reduction in the rates of MTBE degradation. No degradation of MTBE was observed with cells grown on the non-inducing substrate succinate. The lack of MTBE degradation on succinate grown cells demonstrates that the MTBE metabolism occurs with an enzyme system associated with benzene metabolism and reaffirms the hypothesis that MTBE is metabolized by co-oxidation.


Howard, P.H., Boethling, R.S., Jarvis, W.F., Meylan, W.M. and E.M. Michaelenko. 1991. Handbook of Environmental degradation Rates. Lewis Publishers, Boca Raton, FL.

Koenigsberg, 1997. 'MTBE Wildcard in Ground Water Cleanup'. Environmental Protection. 8(11): 26-28

Javanmardian, M and H.A. Glasser. 1997. 'In Situ Biodegradation of MTBE Using Biosparging'. In: Proceedings of the American Chemical Society Division of Environmental Chemistry, April 13-17, San Francisco, CA. Pp 424.

Carter, S.R., Bullock, J.M. and W.R. Morse. 1997. 'Enhanced Biodegradation of MTBE and BTEX using Pure Oxygen Injection'. In: B.C. Alleman and A. Leeson (Eds.), In Situ and On Site Bioremediation 4(4):147. Battelle Press,Columbus,OH

TABLE 1. Oxygen Uptake Rates (OUR) with resting cell suspensions of the benzene degrading bacterial culture PEL-B201.

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