INTRODUCTION

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The prevalent use of methyl t-butyl ether (MTBE) for gasoline oxygenation has led to its introduction into groundwater from spills and leaky underground storage tanks. MTBE is poorly adsorbed, chemically and biologically stable, and very soluble in water, making it very mobile and persistent in the environment. The U.S. Environmental Protection Agency has recently proposed scaling back the use of MTBE in gasoline in light of the increasing frequency with which MTBE is found as a groundwater contaminant nationwide (16; http://www.epa.gov /swerust1/mtbe/browner.pdf). Concentrations of MTBE in groundwater have been reported to range from 0.5 µg/liter to >10 mg/liter. At least 20 states have established MTBE groundwater cleanup levels ranging from 20 to 400 µg/liter for groundwater for potable use (http://www.epa.gov/OUST/mtbe /sumtable.htm). The U.S. Environmental Protection Agency has established a health advisory level of 20 to 40 µg/liter for MTBE in drinking water (19).

Efforts to develop bioremediation processes to help combat MTBE contamination of groundwater have been hampered by the recalcitrance of MTBE. The highly branched nature of MTBE resists most bacterial enzymatic attacks. Only a few pure and mixed bacterial cultures that are able to biodegrade MTBE have been identified (4, 6, 7, 8, 17).

One potential MTBE biodegradation pathway involves the demethylation of MTBE to form tert-butyl alcohol (TBA) and formaldehyde (11, 17; K. L. Hurt, J. T. Wilson, and J. S. Cho, 5th Int. In Situ On-Site Bioremediation Symp., 1999), although the formation of tert-butyl formate from MTBE has also been observed (8). tert-Butyl formate is then hydrolyzed into TBA. In general, TBA is the first stable metabolite of MTBE, regardless of the type of bacterial cultures used. Hyman et al. (8) presented evidence that TBA is degraded by the same enzyme that degrades MTBE, although a soil microcosm was identified that was able to biodegrade TBA but not MTBE (M. J. Zenker, R. C. Borden, and M. A. Barlaz, 5th Int. In Situ On-Site Bioremediation Symp., 1999). TBA is biodegraded at a slower rate than MTBE (8, 17); thus, it tends to build up over time. If TBA is indeed degraded by the same enzyme that degrades MTBE, then the accumulation of TBA can have a detrimental effect on the MTBE biodegradation rate as a result of enzyme competition. Consequently, the effects of TBA on MTBE biodegradation need to be examined.

Most bacteria cannot utilize MTBE as a sole growth substrate. Consequently, many MTBE biodegradation studies have been conducted under cometabolic conditions, using growth substrates such as simple hydrocarbons (6, 8, 17). In addition, these studies have focused mainly on the degradation of moderate-to-high concentrations of MTBE (tens to hundreds of milligrams per liter), at which the observed biodegradation rates would likely be maximal. However, a large quantity of contaminated water contains MTBE at concentrations below 1 mg/liter. No work has been reported regarding the biodegradation of MTBE at low concentrations.

In this work, we examined the abilities of several microorganisms to cometabolize MTBE at low concentrations (range, 100 to 800 µg/liter). It was determined that Arthrobacter bacteria readily degraded MTBE. The degradation kinetics were measured, and the effects of growth substrates and TBA buildup on bacterial cometabolism of MTBE were explored.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. Nocardia (ENV425) and Arthrobacter (ATCC 27778) cultures were grown on basal salt medium (BSM) (17) with propane or lighter fluid butane (Colibri Premium Butane Fuel, 78% mixed butane), respectively, as the sole carbon and energy source. Both cultures were batch grown at room temperature with continuous shaking in 250-ml amber glass bottles sealed with screw caps fitted with Teflon-lined silicone septa (60 ml of growth medium). A 45-ml volume of propane or butane was added as an overpressure. Bottle headspaces were periodically purged with 200 ml of pure oxygen, and additional propane or butane was added. Methylosinus trichosporium OB3b PP358 (12) was maintained as a continuous culture in a chemostat as described by Aziz et al. (3). Streptomyces griseus (ATCC 13273) was maintained on a glucose-enriched medium, and its P-450 enzyme system was induced by soy flour as described by Sariaslani et al. (13).

Analytical techniques. MTBE and TBA concentrations were measured using a gas chromatograph (GC). A 4-µl sample was injected into a Hewlett-Packard 5890A GC equipped with a 30-m Megabore DB-Wax column (J&W Scientific) with a 5-m guard column and a flame ionization detector. The injector and detector were maintained at 150 and 250°C, respectively. The initial column temperature was set at 40°C and maintained for 6 min after sample injection. The column temperature was then increased at a rate of 20°C/min to a final temperature of 150°C and maintained for 3 min. Hydrogen and air flow to the flame ionization detector was set at 35 and 400 ml/min, respectively. Helium was used as the carrier gas, and the column head pressure was set at 5 lb/in². Nitrogen was used as makeup gas. The retention times of MTBE and TBA were 2.5 and 4.3 min, respectively. Concentrations were quantified against primary standard curves.

MTBE biodegradation assays and kinetic determination. Using the headspace-free syringe method described by Aziz et al. (3), a harvested cell suspension was injected into a 60-cm³ disposable plastic syringe (Becton Dickinson) containing 40 ml of aerated growth medium, spiked with MTBE, and capped. At timed intervals, samples were collected by ejecting aliquots from the syringe through a disposable syringe filter (0.2-µm pore size; cellulose acetate) and directly into GC autosampler vials. A new syringe filter was used with each sample collected. Abiotic tests showed no loss of MTBE with time using the headspace-free syringe method. Inhibition assays with Arthrobacter bacteria were also conducted using the headspace-free syringe method described above. BSM was aerated and amended with butane, acetylene, or TBA prior to use. The dissolved oxygen concentration was measured at the end of every experiment to ensure that it did not fall below 3.5 mg/liter. Degradation and inhibition assays were conducted in duplicate.

Radiolabeled-MTBE assay. Arthrobacter bacteria were tested with radiolabeled MTBE to confirm the mineralization of MTBE. The assay was conducted using the headspace-free syringe method. The syringe was filled with 55 ml of aerated BSM, 0.9 µCi of uniformly labeled [¹⁴C]MTBE (10.1 mCi/mmol; lot no. 3048-175B; Dupont New England Nuclear Products) in 12 µl of ethanol, and unlabeled MTBE. Harvested Arthrobacter bacteria were then added to the syringe. The resulting biomass concentration was 1,100 mg of TSS/liter, and the total initial MTBE concentration was 670 µg/liter. At timed intervals, aliquots were collected from the syringe and analyzed for MTBE and TBA concentrations using GC and total radioactivity and ¹⁴CO₂ production using a liquid scintillation counter. ¹⁴CO₂ was separated by trapping the volatile components in solvents and base as previously described (9).

Transformation capacity. A cell suspension was injected into a 250-ml amber glass bottle containing 200 ml of BSM and 2.15 µmol of MTBE to provide a TSS concentration of 1,100 mg/liter. The bottle was capped with a screw top Mininert valve. Aliquots of 2 to 3 ml were withdrawn from the bottle at timed intervals and syringe filtered directly into GC autosampler vials for MTBE and TBA measurement. When the MTBE concentration had decreased by 90% of the initial concentration, the experiment was paused to remove any remaining TBA. The cell suspension was centrifuged, rinsed with BSM, and resuspended with 10 ml of BSM. The suspended cells were injected into a new amber glass bottle containing enough BSM and MTBE to bring the total liquid volume to 80 to 90% of the volume of the cell suspension before centrifugation, and the experiment was continued. The process was repeated until the degradation rate fell to approximately 10% of that at the beginning of the experiment. Progressively smaller amber glass bottles were used as the total volume of the cell suspension decreased, such that the headspace volume was never greater than 25% of the total volume of the bottle. The biomass concentration was determined for each dosing period by measuring the optical density at 550 nm. The MTBE concentrations for each dosing ranged from 950 to 4,100 µg/liter.

	RESULTS AND DISCUSSION

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Bacterial strain selection. Three bacterial strains were tested for the ability to biodegrade MTBE at low concentrations. The methanotroph M. trichosporium OB3b PP358 was selected because of its production of a nonspecific soluble methane monooxygenase that is capable of degrading various chlorinated solvents (3, 5, 15). S. griseus, which has long been exploited for its ability to degrade a diverse array of structurally complex xenobiotics (13, 14, 18), was studied in order to evaluate a P-450 enzyme system for the biodegradation of MTBE (17). The last organism tested was Arthrobacter sp. strain ATCC 27778. This strain was isolated from a natural- and domestic- gas-contaminated site and has been shown to utilize butane and butanol as growth substrates (10). Because most of the identified MTBE-cometabolizing bacteria utilize alkane as a feed substrate (6, 8, 17), we hypothesized that a culture that had been previously exposed to a mixture of hydrocarbons, including branched butane, would have the ability to produce the necessary enzymes for MTBE degradation. The MTBE degradation activity of the test strains was compared to that of ENV425, a Nocardia strain that utilizes propane as an energy and carbon source and is a known MTBE degrader (17).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   MTBE degradation and TBA production by Arthrobacter and ENV425.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Cometabolism of MTBE by Arthrobacter, ENV425, M. trichosporium OB3b, and S. griseus.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Formation of [14C]TBA and 14CO2 from degradation of uniformly radiolabeled MTBE by Arthrobacter.

MTBE degradation kinetics. The Monod kinetic parameters K_S (apparent half-saturation coefficient) and k_c (maximum specific rate of substrate utilization) for MTBE degradation by Arthrobacter and ENV425 were determined for cultures grown with nitrate or ammonia as the nitrogen source (Table 1). For each bacterial strain tested, five or six degradation assays were performed with a range of initial MTBE concentrations (100 to 22,000 µg/liter). The data from these degradation assays were then simultaneously fitted to the Monod equation to obtain a unique set of Monod parameters (3). The calculated pseudo-first-order rate constant (k₁) values based on the measured Monod parameters were closely bracketed by the k₁ measured from independent degradation assays (Table 1).

Factors affecting MTBE degradation. The growth substrates played an important role in the ability of Arthrobacter bacteria to biodegrade MTBE. Arthrobacter bacteria were grown in batches on butane, 1-butanol, glucose, and glucose plus butane and in TPB. Cells that were grown with butanol, glucose, or TPB had no detectable MTBE degradation activity (Table 2). Slight MTBE degradation activity was observed in cultures that were fed a combination of butane and glucose (k₁ = 0.01 liters/mg-day), while cultures given only butane had the highest rate constant (k₁=0.20 liters/mg-day). The type of butane used as a growth substrate did not have any apparent effect on the k₁ values (Table 2). Arthrobacter bacteria grown on either n-butane (99.0% purity) or lighter fluid butane were able to degrade MTBE at the same rate (k₁ = 0.20 liters/mg-day), with no initial lag period (data not shown).

MTBE transformation capacity. The transformation capacity, T_c, defined as the maximum mass of substrate that can be transformed per unit mass of cells (1), was measured for butane-grown Arthrobacter bacteria. Transformation capacity was determined by consecutively dosing a culture with MTBE until the MTBE degradation rate decreased to approximately 10% of the initial value. In order to minimize the inhibition effect of TBA on MTBE degradation, cells were centrifuged and resuspended in fresh medium before each additional dosing of MTBE. This process prevented excessive accumulation of TBA in the culture medium but did not prevent simultaneous degradation of MTBE and TBA. Mass balance calculations from GC measurements showed that approximately 35% of the TBA formed from MTBE was thus degraded. The T_c for butane-grown Arthrobacter was determined to be 20 µg of MTBE/mg of biomass (Fig. 4). This value is relatively small compared to the cometabolism of other chemicals. For example, the T_c of OB3b cometabolizing TCE ranged from 26 to 108 µg/mg of TSS (5). Since TBA was removed from the culture before each MTBE dosage, the bacterial cells were not being significantly inhibited by a buildup of TBA. Because the enzyme was not being inhibited, then the decrease in the MTBE degradation rate with time is due to either the consumption of reducing power within the cells or the production of toxic oxidation products from MTBE degradation. Although TBA was periodically removed from the cell culture, some of the TBA was still degraded. Consequently, it is possible that a metabolite further down in the degradation chain is toxic and thus partly caused the observed decrease in the MTBE degradation rate with time. Alternatively, a low T_c value can be indicative of the fact that Arthrobacter lacks the ability to store excess reducing power under the conditions under which it was grown. Additional research into the effect of adding reducing power to the culture medium during degradation assays would help clarify this matter.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Transformation capacity of Arthrobacter degrading MTBE. Cells were centrifuged and resuspended in fresh medium before each addition of MTBE.