Environ. Sci. Technol. 2001, 35, 312-317
Substrate Interactions in BTEX and MTBE Mixtures by an MTBE-Degrading Isolate R U L A A . D E E B , §,† H O N G - Y I N G H U , §,‡ JESSICA R. HANSON,# KATE M. SCOW,# AND L I S A A L V A R E Z - C O H E N * ,§ Department of Civil and Environmental Engineering, University of California, 631 Davis Hall, Berkeley, California 94720-1710, and Department of Land, Air and Water Resources, University of California, One Shields Avenue, Davis, California 95616-8627
Groundwater contaminant plumes from recent accidental gasoline releases often contain the fuel oxygenate MTBE (methyl tert-butyl ether) together with BTEX (benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene) compounds. This study evaluates substrate interactions during the aerobic biotransformation of MTBE and BTEX mixtures by a pure culture, PM1, capable of utilizing MTBE for growth. PM1 was unable to degrade ethylbenzene and two of the xylene isomers at concentrations of 20 mg/L following culture growth on MTBE. In addition, the presence of 20 mg/L of ethylbenzene or the xylenes in mixtures with MTBE completely inhibited MTBE degradation. When MTBE-grown cells of PM1 were exposed to MTBE/ benzene and MTBE/toluene mixtures, MTBE degradation proceeded, while the degradation of benzene and toluene was delayed for several hours. Following this initial lag, benzene and toluene were degraded rapidly, while the rate of MTBE degradation slowed significantly. MTBE degradation did not increase to previous rates until benzene and toluene were almost entirely degraded. The lag in benzene and toluene degradation was presumably due to the induction of the enzymes necessary for BTEX degradation. Once these enzymes were induced, sequential additions of benzene or toluene were degraded rapidly, and growth on benzene and toluene was observed. The results of this study suggest that BTEX and MTBE degradation occurs primarily via two independent and inducible pathways. If subsurface microbial communities behave similarly to the culture used in this study, the observed severe inhibition of MTBE degradation by ethylbenzene and the xylenes and the partial inhibition by benzene and toluene suggest that the biodegradation of MTBE in subsurface environments would most likely be delayed until MTBE has migrated beyond the BTEX plume.
Introduction The practice of adding methyl tert-butyl ether, MTBE, to gasoline started in the late 1970s and increased dramatically in the 1990s in an effort to increase combustion efficiency and reduce air pollution. Like other gasoline components, MTBE is released into the environment during the production, distribution, storage and use of MTBE-blended fuels. Following an accidental release of MTBE-blended gasoline, 312
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MTBE is typically the first compound to be detected in groundwater due to its high solubility and low sorption potential (1). While MTBE is likely to dominate at leading edges of gasoline-impacted groundwater plumes, it can be expected to coexist with the monoaromatic components of gasoline near contaminant sources and at lagging edges of contaminant plumes. The major aromatic constituents of gasoline, collectively known as BTEX compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene), have high water solubilities relative to the aliphatic constituents of gasoline. A recent survey reported the cooccurrence of MTBE and BTEX compounds at a large number of gasoline-contaminated sites in California (2). Three years following the elimination of contaminant point sources at these sites, the detection frequencies of concurrent BTEX compounds and MTBE decreased only from 80% to 60% in monitoring wells (2). However, given more time and sufficient distance from point sources, dissolved plumes of MTBE are expected to migrate beyond BTEX plumes. While biodegradation is recognized as the major attenuation mechanism for BTEX compounds in subsurface environments (3, 4), there is no convincing evidence to date that MTBE biodegradation occurs rapidly in the field under natural conditions. Several studies, however, have demonstrated the biodegradability of MTBE in laboratory settings (5-13; and others). Because BTEX compounds and MTBE can co-occur in groundwater, it is important to determine whether the presence of MTBE negatively impacts BTEX attenuation rates leading to the elongation of BTEX plumes, or whether the ease of degradability of BTEX compounds causes the repression of MTBE degradation in mixed contaminant plumes. Few studies have evaluated the effect of substrate interactions on the biodegradation rates of MTBE and BTEX compounds in contaminant mixtures. In most of the reported studies, the presence of MTBE at concentrations comparable to those reportedly detected in groundwater at gasoline-contaminated sites did not have a negative effect on BTEX degradation rates by cultures that were incapable of MTBE degradation (14-16). The results of these studies suggest that the presence of MTBE does not affect the cell viability and activity of BTEX-degrading cultures. The effect of BTEX compounds on MTBE biodegradation rates has also been evaluated in a number of studies (17-20). In one study, the impact of toluene on MTBE degradation was examined in an MTBE-degrading pilot-scale biofilter (18, 19). While low concentrations of toluene were removed by the biofilter, increasing the concentration of toluene in the influent stream caused both MTBE and toluene removal efficiencies to decrease presumably due to nitrogen limitations at higher carbon loading rates. In another study, the biodegradation of MTBE was evaluated in laboratory columns packed with aquifer material from four gasoline-contaminated sites (17). MTBE was shown to degrade in the columns only in the absence of BTEX compounds. Finally, at a field site where the activity of aquifer microorganisms was stimulated using Oxygen Release Compounds (ORC), MTBE degradation was shown to occur only after BTEX concentrations were significantly reduced (20). In microcosm studies conducted * Corresponding author phone: (510)643-5969; fax: (510)642-7483; e-mail:
[email protected]. †Present address: Malcolm Pirnie, Inc., 180 Grand Ave., Ste. 1000, Oakland, CA 94612. ‡Present address: Department of Environmental Science and Engineering, Tsinghua University, Beijing, 10084 China. § University of California, Berkeley. # University of California, Davis. 10.1021/es001249j CCC: $20.00
2001 American Chemical Society Published on Web 12/07/2000
in parallel with that field study, the presence of xylene together with MTBE caused a 43% reduction in the extent of MTBE degradation (20). Finally, a pure culture designated PELB201 was shown to cometabolically degrade MTBE following growth on benzene (20). However, MTBE degradation was severely inhibited in the presence of benzene at concentrations as low as 0.15 mg/L presumably due to competitive inhibition. In the presence of 0.3 mg/L of benzene, the time required to degrade 3.4 mg/L of MTBE increased from 12 to 50 h (20). Substrate interactions during the biodegradation of gasoline components in mixtures by a pure culture capable of growth on MTBE and of BTEX degradation have not yet been evaluated. Pure culture studies can be useful for characterizing the nature of inhibition or stimulation phenomena observed during the biodegradation of contaminant mixtures. The objectives of this study are therefore as follows: (1) to investigate the biodegradability of MTBE and BTEX compounds by a pure culture capable of utilizing MTBE for growth and (2) to characterize substrate interactions during the biodegradation of MTBE and BTEX compounds in mixtures. Evaluating the impact of contaminant mixtures on the individual biotransformation rates of MTBE and BTEX compounds is useful for predicting the behavior of these compounds in both in situ and ex situ biological treatment systems and could contribute to improved approaches for implementing engineered bioremediation.
Materials and Methods Culture Growth and Conditions. Bacterial strain PM1 was isolated from an MTBE-degrading mixed culture enriched from a compost biofilter at the Los Angeles County Joint Water Pollution Control Plant in Carson City, CA (9). Complete 16S rDNA sequence analysis revealed that PM1 is a member of the Leptothrix branch of β-Proteobacteria (21). PM1 was grown at 25 °C in 250 mL bottles containing 50 mL of a mineral salts medium, MSM (22), using either MTBE or benzene as a sole source of carbon and energy. When grown on MTBE, MTBE was initially added at aqueous concentrations of 20 to 100 mg/L. When some growth was observed in the bottles as indicated by an increase in turbidity, the concentration of MTBE was increased to 500 mg/L. Once MTBE was almost all degraded (typically within a period of 4 days), cells were harvested by centrifuging the culture suspension at 2800 rpm for 20 min using an MSE GT-2 centrifuge (VWR Scientific, West Chester, PA). The cells were then resuspended in MSM for use in biotransformation experiments. When grown on benzene, benzene was initially added at an aqueous concentration of 40 mg/L. Following benzene depletion, the bottles were amended with oxygen and subsequent benzene additions at 40 mg/L. PM1 was checked for purity on a regular basis by streaking the cultures on R2A agar plates.
Experimental and Analytical Procedures Single and multiple substrate biotransformation studies were conducted in 250 mL clear glass bottles sealed with Teflonlined Mininert valves and incubated at 25 °C in the dark with shaking at 150 rpm. Bottles containing 50 mL MSM and 6 glass beads were sterilized by autoclaving for 20 min at 121 °C prior to use. Neat MTBE and BTEX compounds were added to the bottles using high precision 5-10 µL syringes (Hamilton Co., Reno, NV). Since Henry’s Law constant of MTBE is low [0.018 at 20 °C (23)], all of the MTBE added to the bottles was assumed to be present in the aqueous phase. In contrast, BTEX concentrations were calculated using dimensionless Henry’s constants with liquid and gas volumes as described previously (24). Following cell inoculation, the disappearance of MTBE and BTEX compounds was moni-
tored by headspace analysis using gas chromatographs (GC) equipped with either flame ionization (FID) or photonionization detectors (PID). GC-FID and GC-PID models, column types, operating parameters and flow rates have been described previously (24, 9). Headspace samples (50-200 µL) were withdrawn from the bottles using Hamilton CR-700 Series constant rate gastight syringes. Sampling proceeded at frequent intervals until BTEX and MTBE concentrations in the bottles dropped below detection limits of the gas chromatographs (1-50 µg/L). Abiotic controls containing BTEX compounds or MTBE but no cells were used to monitor nonbiological losses of volatile compounds from the bottles. Killed controls prepared using sodium azide (1% solution) were also used. In most cases, experiments were performed using duplicate or triplicate bottles, and the experimental error was indicated by the range of duplicate samples or calculated as the standard deviation of triplicate samples. Critical experiments were repeated several times to establish reproducibility of results. In most cases, culture densities (mg/L of dry weight) in the bottles were measured by adding specific volumes of culture suspensions to aluminum weighing dishes and by taking the difference of sample weights after drying at 105 °C for 8 h and after combustion at 550 °C for 30 min. Chemicals. MTBE (>99% ACS reagent grade; >99.9% HPLC grade) was obtained from Aldrich Chemical Co. Inc., Milwaukee, WI or from Fisher Scientific Co., Fair Lawn, NJ. Benzene (>99% ACS reagent) was obtained from Mallinckrodt, Inc., Paris, KY. Toluene (>99.5% ACS reagent grade), ethylbenzene (99.9% certified grade) and p-xylene (99.8% certified grade) were obtained from Fisher Scientific. o-Xylene (spectro grade) was obtained from J. T. Baker, Inc., Phillipsburg, NJ. m-Xylene (spectro grade) was obtained from Eastman Kodak, Rochester, NY. All other chemicals were of the highest purity commercially available and were obtained from standard sources.
Results The culture used in this study, PM1, is capable of degrading MTBE to CO2 and of growing on it as a single source of carbon and energy (9). In addition, PM1 has been shown to effectively utilize tert-butyl alcohol, TBA, for growth (25). In this study, the biodegradation of MTBE by PM1 was evaluated when MTBE was present alone or in bisubstrate mixtures with each of the BTEX compounds. Both MTBE and benzene were individually degraded by MTBE-grown cells of PM1 at maximum degradation rates of 5.0 and 1.1 mg L-1 h-1, respectively (Figure 1a). MTBE degradation commenced without a lag, while the degradation of benzene proceeded after a lag period of approximately 20 h. In a mixture with MTBE, the lag phase for benzene degradation diminished considerably to approximately 4 h, and the maximum degradation rate increased to 3.5 mg L-1 h-1 (Figure 1a). Furthermore, when benzene and MTBE were degraded in a mixture, a 3 phase pattern for MTBE degradation was observed (Figure 1b). Initially, the degradation of MTBE proceeded at a fast rate without a lag period (time 0-3 h). Once benzene degradation accelerated to significant levels (time 3-7.5 h), the rate of MTBE degradation in the mixture slowed significantly. Only when benzene was almost all degraded did the rate of MTBE degradation increase again. This pattern was repeatedly observed in multiple experiments (e.g., see Figure 5a). To evaluate the nature of the observed benzene degradation enhancement in the presence of MTBE, MTBE-grown cells of PM1 were exposed to benzene in mixtures with either MTBE, a rich carbon source such as pyruvate, or an easily degradable substrate such as ethanol. As illustrated in Figure 2a,b, the presence of MTBE, pyruvate or ethanol at concentrations of 20 mg/L promoted benzene degradation with VOL. 35, NO. 2, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Biotransformations by MTBE-grown cells of PM1: (a) benzene (B) alone, MTBE alone, and benzene in a mixture with MTBE; (b) MTBE alone and in a mixture with benzene, and benzene in a mixture with MTBE. The initial culture density in each bottle was 112 mg/L. Error bars indicate the standard deviation of triplicate samples. much shorter lag periods and more rapid rates than when benzene was present alone. Moreover, rates of benzene degradation in the presence of higher concentrations of pyruvate (50 and 100 mg/L) were similar to rates of benzene degradation in mixtures with 20 mg/L MTBE, ethanol or pyruvate. While pyruvate and ethanol enhanced benzene degradation, their presence in mixtures with MTBE had a negligible effect on the degradation rate of MTBE, while the presence of benzene retarded MTBE degradation (Figure 2c). To determine whether the lag period observed during benzene degradation by MTBE-grown cells was due to induction or some other phenomenon, additional 20 mg/L benzene supplements were added to the bottles after the initial 20 mg/L of benzene was entirely degraded. Sequential additions of benzene were degraded by the cells without a lag period at approximate rates of 6.7 to 9.5 mg L-1 h-1 (Figure 3). Furthermore, culture densities in the bottles during the degradation of benzene increased significantly as evidenced by increases in turbidity suggesting that PM1 is capable of utilizing benzene for growth. In fact, PM1 was shown to effectively utilize benzene as the sole source of carbon and energy when benzene was supplied to cells grown either in liquid or on solid medium. Experiments conducted with benzene-grown cells of PM1 revealed that benzene, toluene and ethylbenzene were rapidly degraded without a lag, while MTBE was slowly degraded following a lag period (>10 h) (Figure 4a,b). PM1 loses MTBE and benzene induction during short incubations (10 h). To evaluate the longevity and nature of benzene and MTBE induced metabolic activities, sequential additions of substrates were made following induction with benzene. After the depletion of benzene followed by that of MTBE in a mixture by MTBE-grown cells, the degradation of a sequential addition of benzene proceeded without a lag almost 8 h after the cells were last exposed to benzene (Figure 5a). Immediately following the depletion of benzene in a mixture with pyruvate, an addition 314
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of toluene was degraded without a lag (Figure 5b). Furthermore, following the depletion of toluene, the degradation of MTBE proceeded without an obvious lag almost 23 h after the MTBE-grown cells were last exposed to MTBE. In general, the degradation trends observed with toluene/ MTBE mixtures were similar to those with benzene/MTBE mixtures. That is, a 3 phase pattern was observed for the degradation of 20 mg/L MTBE in the presence of 20 mg/L toluene (data not shown). Furthermore, benzene-grown cells of PM1 were capable of degrading toluene without a lag and were capable of utilizing it for growth as evidenced by increases in turbidity with repeated 20 mg/L toluene additions. The major difference in the biodegradation behavior of PM1 toward toluene and benzene was that MTBE-grown cells of PM1 were generally unable to degrade toluene in the absence of MTBE, especially at low culture inoculum densities (
