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Applied and Environmental Microbiology, February 2004, p. 1023-1030, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.1023-1030.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Induction of Methyl Tertiary Butyl Ether (MTBE)-Oxidizing Activity in Mycobacterium vaccae JOB5 by MTBE

Erika L. Johnson,1 Christy A. Smith,1 Kirk T. O'Reilly,2 and Michael R. Hyman1*

Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695,1 ChevronTexaco Energy Research and Technology Co., Richmond, California 948022

Received 4 June 2003/ Accepted 31 October 2003


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ABSTRACT
 
Alkane-grown cells of Mycobacterium vaccae JOB5 cometabolically degrade the gasoline oxygenate methyl tertiary butyl ether (MTBE) through the activities of an alkane-inducible monooxygenase and other enzymes in the alkane oxidation pathway. In this study we examined the effects of MTBE on the MTBE-oxidizing activity of M. vaccae JOB5 grown on diverse nonalkane substrates. Carbon-limited cultures were grown on glycerol, lactate, several sugars, and tricarboxylic acid cycle intermediates, both in the presence and absence of MTBE. In all MTBE-containing cultures, MTBE consumption occurred and tertiary butyl alcohol (TBA) and tertiary butyl formate accumulated in the culture medium. Acetylene, a specific inactivator of alkane- and MTBE-oxidizing activities, fully inhibited MTBE consumption and product accumulation but had no other apparent effects on culture growth. The MTBE-dependent stimulation of MTBE-oxidizing activity in fructose- and glycerol-grown cells was saturable with respect to MTBE concentration (50% saturation level = 2.4 to 2.75 mM), and the onset of MTBE oxidation in glycerol-grown cells was inhibited by both rifampin and chloramphenicol. Other oxygenates (TBA and tertiary amyl methyl ether) also induced the enzyme activity required for their own degradation in glycerol-grown cells. Presence of MTBE also promoted MTBE oxidation in cells grown on organic acids, compounds that are often found in anaerobic, gasoline-contaminated environments. Experiments with acid-grown cells suggested induction of MTBE-oxidizing activity by MTBE is subject to catabolite repression. The results of this study are discussed in terms of their potential implications towards our understanding of the role of cometabolism in MTBE and TBA biodegradation in gasoline-contaminated environments.


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INTRODUCTION
 
Methyl tertiary butyl ether (MTBE) is an oxygenating compound that is currently added to gasoline to reduce automobile emissions of carbon monoxide and smog-related air pollutants. Approximately 30% of the gasoline sold in the United States contains MTBE, and its widespread use has led to concerns over the human health effects resulting from chronic exposure to this compound through gasoline contamination of drinking water supplies (19, 35). The U.S. Environmental Protection Agency currently classifies MTBE as a possible human carcinogen and has issued a drinking water advisory for MTBE of 20 to 40 ppb (37).

Several recent studies have shown MTBE can be biodegraded under anaerobic conditions (2, 3, 9, 34, 40). However, like most other gasoline components, the fastest rates of MTBE biodegradation are observed under aerobic conditions (4, 11, 15, 33, 36). Several aerobic bacteria have been isolated that can use MTBE as a sole source of carbon and energy for growth (10, 13, 15, 26). Various other aerobic MTBE-degrading organisms have also been identified that are unable to grow on MTBE but can cometabolically degrade this compound after growth on a variety of hydrocarbons. Like MTBE, some of these hydrocarbons are also present at high concentrations in gasoline and include alkanes (11, 14, 23, 31, 33, 36), aromatics (18, 20), and alicyclics (5, 31). Cometabolic degradation of MTBE has been most extensively studied in propane- (33, 36) and n-pentane-oxidizing bacteria (11). In the case of propane-grown cells of Mycobacterium vaccae JOB5, MTBE is initially oxidized to tertiary butyl formate (TBF) through the sequential activities of an alkane-inducible alkane monooxygenase and a putative hemiacetal-oxidizing alcohol dehydrogenase (33). The subsequent abiotic and biotic hydrolysis of TBF yields tertiary butyl alcohol (TBA), which is then further oxidized by the same monooxygenase responsible for initiating MTBE oxidation. Further steps in the oxidation of MTBE have been proposed but have not been extensively characterized (36).

The role of cometabolism in the environmental fate of MTBE is currently unclear. For instance, addition of both propane and oxygen to gasoline-contaminated groundwater has been shown to promote MTBE oxidation (1). However, it is not known whether cometabolic processes supported by gasoline hydrocarbon cocontaminants represent an important natural attenuation process for MTBE under aerobic conditions. Recent field studies have shown MTBE biodegradation can be stimulated when anaerobic, gasoline-impacted ground water is oxygenated, either through engineered approaches (30, 41) or through natural ground water transport mechanisms (4, 21). However, as the currently recognized growth substrates thought to be required for cometabolic MTBE biodegradation are often reported to be absent from these environments, these effects of oxygenation have been generally interpreted in terms of a stimulation of growth-related microbial metabolism of MTBE. Nonetheless, a recent report (17) noted that MTBE biodegradation occurred in samples taken from oxygenated environments, both in the absence as well as the presence of organisms similar to the MTBE-metabolizing strain PM-1.

Studies of microbial cometabolic degradation processes for important pollutants such as trichloroethylene (TCE) and MTBE have often focused on identifying substrates that support high rates of biodegradation of these compounds. These substrates are of interest because they not only support microbial growth but also lead to high levels of the key catabolic enzyme activities required for cosubstrate (e.g., TCE and MTBE) degradation. However, as studies of cometabolic TCE degradation have repeatedly demonstrated (8, 16, 22, 24, 29, 32), it is also important to recognize the potential inducing effects of the target pollutant on the expression of enzymes required for its own biodegradation. In the present study we have examined the effect of MTBE on the MTBE-oxidizing activity of M. vaccae JOB5 during carbon-limited growth on diverse nonalkane substrates. Our results demonstrate that cells grown on a wide range of substrates in the presence of MTBE and other oxygenates express the enzyme activities required for the degradation of these gasoline additives. The results of this study have been interpreted in terms of their potential impact on our understanding of the underlying physiology of MTBE cometabolism and the potential role of cometabolism in the environmental fate of MTBE.


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MATERIALS AND METHODS
 
Materials.
M. vaccae JOB5 (ATCC 29678) was obtained from the American Type Culture Collection (Manassas, Va.). Galactose (99% purity), glucose (99.5% purity), fructose (99% purity), pyruvic acid (99+% purity), succinic acid (99% purity), lactic acid (98% purity), glycerol (99% purity), sodium propionate (99% purity), sodium butyrate (98% purity), valeric acid (99+% purity), caproic acid (99.5+% purity), heptanoic acid (99% purity), isovaleric acid (99% purity), 2-methylbutyric acid (98% purity), 2-methylvaleric acid (98% purity), 3-methylvaleric acid (97% purity), 2-methylhexanoic acid (99% purity), TBF (97% purity), TBA (99+% purity), tertiary amyl alcohol (TAA; 99+%), MTBE (99.8% purity), ethyl tertiary butyl ether (ETBE; 99% purity), tertiary amyl methyl ether (TAME; 97% purity), 1-propanol (99.5% purity), 2-propanol (99.5% purity), and rifampin, chloramphenicol, and calcium carbide (technical grade, ~80% purity; for acetylene generation) were obtained from Sigma Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Sodium acetate (99.5% purity) was obtained from Fisher Scientific (Pittsburgh, Pa.). 2-Methyl-1,2-propanediol (2M12PD)was a gift from Lyondell Chemical Co. (Houston, Tex.). Compressed gases used for gas chromatography (GC) (H2, N2, and air) were obtained from local industrial vendors.

Growth experiments.
Most of the experiments described in this study used cells of M. vaccae JOB5 grown in batch culture in glass serum vials (160 ml) sealed with Teflon-lined Mininert valves (Alltech Associates Inc., Deerfield, Ill.). The vials contained mineral salts medium (25 ml) (39), and unless otherwise stated all growth substrates were added from filter-sterilized aqueous solutions to give an initial concentration of 2.5 mM. The culture vials were inoculated (initial optical density at 600 nm [OD600] of <=0.02) with a suspension of cells obtained from axenic cultures of M. vaccae JOB5 previously grown on casein-yeast extract-dextrose (CYD) agar plates (Difco plate count agar). When required, MTBE, ETBE, TAME, TBA, or TAA was added to the sealed vials from a saturated aqueous solution using sterile glass microsyringes. Acetylene (5 ml) was added to the sealed vials as required using sterile disposable plastic syringes fitted with sterile Acrodisc 0.1-µm filters (Pall Corp., Ann Arbor, Mich.). The culture vials were incubated at 30°C in the dark in an Innova 4900 environmental shaker (New Brunswick Scientific Co., Inc., Edison, N.J.) operated at 150 rpm. Losses of MTBE from abiotic control incubations containing no added cells were less than 3% over 7 days under these conditions. Culture growth was measured by determining the OD600 with a Shimadzu 1601 UV/Vis spectrophotometer (Kyoto, Japan). In every experiment, a sample (50 µl) was streaked on CYD plates to subsequently confirm the purity of the culture.

In some experiments, concentrated washed cells were used. In these cases the cells were grown in batch culture with the required substrate, as described above. The cells were then harvested from the culture medium by centrifugation (10,000 x g; 10 min), and the resulting cell pellet was resuspended in buffer (10 ml; 50 mM sodium phosphate; pH 7). The washed cells were sedimented again by centrifugation (as above), and the resulting cell pellet was finally resuspended with buffer (1.0 ml, as above) to a final protein concentration of ~2.5 mg of total cell protein ml-1.

Analytical methods.
In some experiments the concentrations of MTBE and its oxidation products (TBA and TBF) were determined by GC using aqueous samples (2 µl) taken directly from the culture vessels. In experiments that followed the time course of organic acid consumption as well as MTBE oxidation, aqueous samples (0.5 ml) were taken from the sealed culture vials at the indicated times using disposable sterile plastic syringes (1 ml) and needles. The samples were transferred to flat-top polypropylene microcentrifuge tubes (1.5 ml), and aqueous samples (2 µl) of the media were then immediately injected into a gas chromatograph. In all experiments the samples were analyzed using Shimadzu GC-8A or GC-14A gas chromatographs fitted with flame ionization detectors and stainless steel columns (0.3 by 183 cm) filled with Porapak Q (60 to 80 mesh; Waters Associates, Framingham, Mass.). The analysis of MTBE, TBF, TBA, ETBE, TAME, TAA, and 2M12PD was conducted at 160°C, while the quantification of valeric acid in the presence of MTBE, TBF, and TBA was conducted at 150°C. In both analyses the injection and detector temperatures were 200 and 220°C, and nitrogen was used as the carrier gas at a flow rate of 15 ml/min. The gas chromatographs were interfaced to Hewlett Packard HP3395 integrators (Palo Alto, Calif.) for data collection. The minimum detection limits for TBF and TBA were ~20 and ~3 nmol ml-1, respectively.

Cell protein concentrations were determined using the Biuret assay (12) after solubilizing cell material for 30 min at 65°C in 3 N NaOH and sedimentation of insoluble material by centrifugation (14,000 x g; 5 min). Bovine serum albumin was used as the standard. The concentration of MTBE in saturated aqueous solution at room temperature (23°C) was taken as 0.544 M (33). The dimensionless Henry's constant (Hc) for MTBE at 30°C was taken as 0.0255 (25). The kinetic constants were derived by computer fitting of the data by nonlinear regression to a single substrate-binding model [Y = Vmax · X/(Ks + X)] using GraphPad Prism version 3.0a for Macintosh (GraphPad Software, San Diego, Calif.).


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RESULTS
 
Effects of nonhydrocarbon growth substrates on MTBE-oxidizing activity.
Cells of M. vaccae JOB5 were grown under carbon-limited conditions on a variety of sugars, tricarboxylic acid cycle intermediates, lactate, and glycerol either without MTBE, with MTBE (14 µmol [~450 µM dissolved MTBE]), or with MTBE (14 µmol) and acetylene (3.7% [vol/vol] gas phase), a potent irreversible inactivator of MTBE-oxidizing activity in this organism (33). After 7 days, culture growth was determined (OD600) and the culture medium was analyzed by GC to determine the extent of MTBE consumption and the accumulation of TBA and TBF. No detectable growth or MTBE consumption occurred when cells were incubated with MTBE alone (data not shown). In contrast, the organism grew, to varying degrees, on all of the other substrates tested, and neither MTBE nor acetylene had any consistent effect on the final culture density (OD600) for any of these substrates (Fig. 1A). The GC analysis (Fig. 1B) revealed variable but often extensive consumption of MTBE had occurred in all of the cultures that contained MTBE alone, whereas little or no MTBE consumption had occurred in cultures grown in the presence of both MTBE and acetylene. For instance, >=70% of the added MTBE (~10 µmol) was consumed by cells grown on either glucose, pyruvate, or fructose in the presence of MTBE. In contrast, <=7% (1 µmol) of the MTBE was consumed when cells were grown on the same substrates in the presence of acetylene. In all cultures where MTBE consumption was observed, both TBA and TBF were also detected. With the exception of cells grown on succinate, the molar ratio of MTBE consumed to total products (TBA plus TBF) detected was low (1:0.21) but close to constant (standard deviation [SD] = 4.4%).



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  		FIG. 1. Growth of M. vaccae JOB5 on nonalkane substrates and concurrent oxidation of MTBE. Cultures of M. vaccae JOB5 were grown for 7 days under carbon-limited conditions in batch culture on the indicated substrates, as described in Materials and Methods. All substrates other than glycerol (7.5 mM) were added to an initial concentration of 2.5 mM. Acetylene (3.75% [vol/vol] gas phase) and MTBE (14 µmol) were added to the cultures as required. (A) Average final culture density (OD600) for three replicate cultures grown under the indicated conditions. The error bars indicate the range of values for all three cultures. (B) Average amounts of MTBE consumed (white bars) and TBA (black bars) and TBF (gray bars) generated after 7 days for the three replicate cultures. The error bars indicate the range of values for MTBE, TBA, and TBF for all three cultures.

Effect of MTBE concentration on MTBE-oxidizing activity.
Two growth substrates characterized in Fig. 1, glycerol and fructose, were used to investigate the effect of MTBE concentration on the level of MTBE oxidation. Cells were grown for 7 days on either glycerol (7.5 mM) or fructose (2.5 mM) in the presence of various amounts of MTBE (0 to ~140 µmol; 0 to ~5 mM in solution). A plot of the total MTBE consumed versus initial dissolved MTBE concentration appeared to be saturable for both growth substrates (Fig. 2). These data were fitted to a hyperbolic, single substrate-binding curve, and good fits (r2 >= 0.99) were obtained in both cases. Half-saturation values (S50) for glycerol (2.4 mM; standard error [SE] = 0.45) and fructose (2.75 mM; SE = 0.53) were obtained from these analyses. Both TBF and TBA were detected in the culture medium, and these products accounted for ~20% of the MTBE consumed in each culture. A plot of total products (TBA and TBF) detected versus initial dissolved MTBE concentration (Fig. 2 inset) also provided comparable S50 values of 3.2 mM (r2 = 0.98; SE = 0.88) and 2.8 mM (r2 = 0.98; SE = 0.38) for cells grown on glycerol and fructose, respectively.



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  		FIG. 2. Effect of MTBE concentration on MTBE oxidation and production of TBA and TBF. Cultures of M. vaccae JOB5 were grown for 7 days under carbon-limited conditions in batch culture on either glycerol (7.5 mM [{blacksquare}]) or fructose (2.5 mM [{blacktriangleup}]) in the presence of a range of initial MTBE concentrations (0 to 140 µmol; 0 to 4.9 mM dissolved MTBE), as described in Materials and Methods. The amount of MTBE consumed in each culture after 7 days was plotted versus the initial amount of MTBE added to each culture. Inset: combined amounts of TBA and TBF detected after 7 days for the same cultures. In all cases the curves drawn are the computer fits to a single substrate-binding model, as described in Materials and Methods.

The results described in Fig. 1 and 2 suggest that the presence of MTBE during growth on diverse nonalkane substrates led to the production of enzyme systems capable of degrading MTBE. However, these results did not address the possibility that MTBE-oxidizing activity was also present in cells grown in the absence of MTBE. We conducted two experiments to investigate this possibility. First, we attempted to determine the specific MTBE-oxidizing activity of concentrated cell suspensions grown on either glycerol (7.5 mM) or fructose (2.5 mM) in the presence and absence of MTBE (initially ~2 mM in solution). After growth for 7 days, the cells were harvested by centrifugation, washed, and finally resuspended at a protein concentration of ~5 mg of total protein ml-1. Samples of the concentrated cell suspension (0.2 ml) were incubated at 30°C in buffer (0.8 ml; 50 mM sodium phosphate [pH 7.0]) in stoppered glass serum vials (10 ml) in the presence of MTBE (1 µmol). After 2 h, the reaction media were analyzed by GC to quantify accumulation of TBA and TBF. We did not detect MTBE consumption or TBA- or TBF-generating activity for cells grown either in the presence or absence of MTBE in these short-term assays. In the second experiment, cells were initially grown on glycerol (35 mM) and then harvested and concentrated by centrifugation. These cells were then incubated with MTBE (~2 mM dissolved MTBE) and a low concentration (1 mM) of glycerol as an energy source. The time course of TBA and TBF production was then determined by GC analysis of the reaction medium. The results (Fig. 3) showed there was a lag phase of 4 h before TBA was first detected in the reaction medium. Although TBF also accumulated during the reaction time course, the concentration of detected TBF never exceeded 20% of the total TBA detected (data not shown). Over the next 6 to 8 h there was a progressive increase in the rate of TBA accumulation, after which the rate of TBA accumulation remained almost constant. When cells were incubated with MTBE in the presence of either chloramphenicol or rifampin (50 µg ml-1 each), the production of both TBA and TBF (data not shown) was strongly or completely inhibited relative to that in the incubation containing MTBE alone. Complete inhibition of both TBA and TBF accumulation was also observed when cells were incubated with MTBE and acetylene (10% [vol/vol] gas phase).



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  		FIG. 3. Effects of chloramphenicol, rifampin, and acetylene on MTBE-oxidizing activity in glycerol-grown cells of M. vaccae JOB5. Cells of M. vaccae JOB5 were grown in batch culture for 5 days on glycerol (35 mM) in the absence of MTBE. The cells were harvested, washed, and stored at 4°C, as described in Materials and Methods. The reactions were conducted in glass serum vials (10 ml) sealed with butyl rubber stoppers and aluminum crimp seals. The reaction vials contained buffer (50 mM sodium phosphate; pH 7; ~900 µl), MTBE (2.8 µmol), and glycerol (1 µmol). The reactions were initiated by the addition of an aliquot (100 µl) of concentrated cell suspension (0.21 mg of total protein), and the vials were incubated at 30°C in a shaking water bath (150 rpm). At the indicated times, aqueous samples (2 µl) were removed and analyzed by GC for the accumulation of TBA and TBF, as described in Materials and Methods. The time course for TBA accumulation is shown for cells incubated with MTBE alone ({blacksquare}), MTBE plus acetylene (10% [vol/vol] gas phase) ({square}), MTBE plus chloramphenicol (50 µg ml-1) (•), and MTBE plus rifampin (50 µg ml-1) ({circ}).

Effects of other ethers and tertiary alcohols.
We also examined whether other ether oxygenates and their tertiary alcohol oxidation products behaved similarly to MTBE. Cells were grown on glycerol (7.5 mM) in the presence of either TAME, ETBE, TAA, or TBA. Additional cultures were also grown using the same growth substrate and oxygenate-alcohol combinations in the presence of acetylene (3.7% [vol/vol] gas phase). After growth for 5 days, the reaction media were analyzed by GC to determine the extent of oxygenate consumption and product accumulation. The results (Table 1) showed that substantial consumption of TAME but not ETBE occurred during growth on glycerol. TAA (~1 µmol) was detected as a product of TAME oxidation, although TAA accumulation only represented ~10% of the TAME consumed. Consumption of TAA (~6 µmol) also occurred when cells were grown in the presence of TAA, and both TAME and TAA consumption was inhibited by acetylene. These results suggest that the low recovery of TAA in the cultures grown with TAME was most likely due to concurrent oxidation of both TAME and TAA. Similar results were also observed for cultures grown in the presence of TBA. Approximately 50% (~23 µmol) of the added TBA was consumed during growth on glycerol, and a single high-boiling-point product that coeluted with 2M12PD was detected. This accounted for ~50% (~12 µmol) of the TBA consumed by glycerol-grown cells. Both the consumption of TBA and the production of 2M12PD were inhibited by the presence of acetylene.


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  		TABLE 1. Oxygenate consumption and product accumulation by cells grown on glycerola

Effects using organic acids as growth substrates.
In addition to conventional growth substrates described above, we also examined whether MTBE-oxidizing activity was stimulated by MTBE in cells grown on more environmentally relevant compounds. Volatile organic acids were chosen for study because they are frequently found in gasoline-impacted ground water environments as products of anaerobic biodegradation of gasoline hydrocarbons (6, 7). In one experiment, cells were grown in the presence of MTBE (14 µmol) using carbon-limiting concentrations (2.5 mM) of several branched acids (isovaleric, 2-methylbutyric, 2-methylvaleric, 3-methylvaleric, and 2-methylhexanoic acids). After growth for 7 days, both TBA and TBF were detected in the incubations containing acids and MTBE, whereas neither product was observed in the same incubations conducted in the presence of acetylene (3.7% [vol/vol] gas phase). The average consumption of MTBE in these cultures was 7.4 µmol (SD = 2.1), and the average molar yield of TBF and TBA combined was 68% of the MTBE consumed. We were unable to quantify cell growth in these and subsequent acid-grown cultures described later, due to clumping of cells.

Cells were also grown under carbon-limited conditions on equimolar concentrations (2.5 mM) of a series (C2 to C7) of straight-chain acids, either in the presence of MTBE (14 µmol) or MTBE (14 µmol) plus acetylene (3.7% [vol/vol] gas phase). The time course of TBA and TBF production was then determined by GC analysis of the culture medium. In all cultures containing MTBE alone, neither product was detected until at least 24 h after the culture was initiated (Fig. 4). The chain length of the acid substrate had two distinct effects. First, in general the longer the acid carbon chain length, the longer the lag phase before MTBE oxidation products (TBA and TBF) were observed. For example, products were observed with acetate-grown cells after 24 h, while cells grown on heptanoic acid did not show product accumulation until ~50 h. Second, in general the longer the acid chain length, the greater the amounts of MTBE oxidation products that were observed. For example, acetate-grown cells generated <2 µmol of products, while caproic acid-grown cells generated ~4 µmol of products. The average molar ratio of MTBE consumed to total products (TBA plus TBF) detected for the range of acids tested was 1:0.62. However, this ratio progressively decreased from 1:0.84 with acetate-grown cells to 1:0.37 with cells grown on heptanoic acid. In a duplicate series of incubations, acetylene fully inhibited the production of both TBA and TBF in all cases (data not shown).



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  		FIG. 4. Time course of TBA production by cells of M. vaccae JOB5 during growth on linear organic acids. A series of cultures of M. vaccae JOB5 were grown on linear organic acids (C2 to C7) (initial concentration = 2.5 mM) in the presence of MTBE (14 µmol), as described in Materials and Methods. The time course of TBA accumulation is shown for cultures grown on acetic ({blacktriangleup}), propionic ({triangleup}), butyric ({blacksquare}), valeric ({square}), caproic (•), and heptanoic ({circ}) acids. The symbols represent the average for two replicate cultures, and the error bars show the range of values for both cultures combined.

Effect of acid concentration of MTBE-oxidizing activity.
The results described in Fig. 4 have features that might be expected if the expression of the enzymes involved in MTBE oxidation were subject to catabolite repression. To investigate this further we quantified the time course of TBA and TBF production in relation to both MTBE and valeric acid consumption for cells grown either with or without MTBE (7.5 µmol) in the presence of two different initial amounts of valeric acid (70 and 35 µmol). These culture conditions were also duplicated in incubations containing acetylene (3.7% [vol/vol] gas phase). In the cultures containing the lower initial concentration of valeric acid, the acid was fully consumed within 40 h (Fig. 5). The consumption of MTBE and the production of both TBA and TBF were first detected in the growth medium when ~10 µmol of valeric acid remained. The time course of both TBA and TBF accumulation continued to reflect MTBE consumption over the next 40 h. However, the MTBE oxidation reaction was not sustainable, and after ~80 h the rates of both MTBE consumption and product accumulation steadily declined to close to zero. After 120 h the molar ratio of MTBE consumed to total products (TBF plus TBA) detected was 1:0.72. The corresponding control experiment (valeric acid plus MTBE plus acetylene) showed acetylene had no discernible effect on the rate of valeric acid consumption but completely inhibited both MTBE consumption and production of both TBA and TBF over the entire reaction time course. A substantially similar pattern of biodegradation was observed when the experiment was repeated with twofold-higher initial amounts of valeric acid. The onset of both MTBE consumption and production of TBA and TBF was delayed by ~20 h relative to that for the cultures grown with lower initial amounts of valeric acid. However, the onset of both of these activities still occurred when ~10 µmol of valeric acid remained in the culture medium. The total amounts of MTBE degraded and TBA and TBF generated were greater in these cultures than in those with lower initial amounts of valeric acid. However, the molar ratio of MTBE consumed to total products detected (TBA plus TBF) after 120 h was 1:0.80 and was comparable to the results obtained with the lower valeric acid concentration. As a final component of this study, we also conducted a similar experiment to that described in Table 1 using valeric acid rather than glycerol as a growth substrate. Substantially similar activities to those observed with glycerol were observed and included the oxidation of TBA, TAA, and TAME but not ETBE by valeric acid-grown cells (data not shown).



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  		FIG. 5. Time course of MTBE and valeric acid oxidation during growth of M. vaccae JOB5. Cultures of M. vaccae JOB5 were grown in batch culture on valeric acid in the presence of MTBE (7.5 µmol), as described in Materials and Methods. The time course for valeric acid consumption is shown for cultures grown with 35 µmol (squares) or 70 µmol (circles) of valeric acid, either in the presence (open symbols) or absence (closed symbols) of acetylene (3.75% [vol/vol] gas phase). Also shown is the corresponding time course for MTBE consumption for cultures grown with 35 µmol (inverted triangles) or 70 µmol (upright triangles) of valeric acid, either in the presence (open symbols) or absence (closed symbols) of acetylene (3.75% [vol/vol] gas phase). In addition, the combined amount of TBA and TBF generated from MTBE is shown for cultures grown with 35 µmol (asterisks) or 70 µmol (filled diamonds) of valeric acid. No TBF or TBA was observed for cultures grown in the presence of acetylene (3.75% [vol/vol] gas phase), and these data were not plotted, to aid in the clarity of the figure. The data plotted are the averages of two replicates for cultures grown in the absence of acetylene and a single replicate for all cultures that contained acetylene. The error bars show the range of values obtained for the replicate cultures.


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DISCUSSION
 
The results of this study provide strong and consistent evidence showing MTBE oxidation occurs during growth of M. vaccae JOB5 on a wide range of nonalkane substrates (Fig. 1, 3, and 4). We have also shown acetylene inhibits the production of both TBA and TBF (Fig. 1B and 5) from MTBE, as well as consumption of MTBE (Fig. 5). Based on these observations, we conclude that the enzyme responsible for MTBE oxidation in this study is the same alkane-inducible, acetylene-sensitive, MTBE- and TBA-oxidizing monooxygenase we have previously characterized in propane-grown cells of this bacterium (33). The remaining sections of the Discussion expand on this conclusion and the broader implications of our findings.

Evidence for an inductive effect of MTBE.
Three lines of evidence suggest the MTBE-oxidizing activity characterized in this study is due to a specific inducing effect of MTBE on the expression of alkane monooxygenase, rather than due to constitutive, low-level expression of this enzyme during growth on nonhydrocarbon substrates. These lines of evidence are as follows.

First, MTBE-oxidizing activity was not detected immediately after glycerol-grown cells were harvested (Fig. 3). However, these cells showed a time-dependent acquisition of MTBE-oxidizing activity after exposure to MTBE. The appearance of this activity was strongly inhibited by both chloramphenicol and rifampin, evidence that indicates this response involves both transcription and de novo protein synthesis. Our failure to detect MTBE-oxidizing activity in cells previously grown for 7 days in the presence of MTBE probably reflects the consistent postinduction decline in MTBE-oxidizing activity we observed in several experiments (Fig. 4 and 5) conducted over an equivalent period of time.

Second, cells grown on valeric acid did not show a progressive increase in MTBE oxidation rate throughout the time course of acid consumption (Fig. 5), an effect that would be predicted if MTBE-oxidizing activity was constitutive and increased consistently with increases in cell density. In contrast, these cells only showed evidence for both MTBE consumption and TBA and TBF production once the residual valeric acid had been depleted to ~10 µmol (Fig. 5). This effect occurred with two different acid concentrations. The apparent need for cells to deplete the acid concentration to below a threshold value is strongly suggestive of a catabolite repression effect, a feature that again supports a model involving MTBE-dependent gene induction. This is consistent with a previous study of the growth substrate range of M. vaccae JOB5 that indicated cells grown on acetate, propionate, and butyrate did not have detectable n-alkane (C1 to C8) or primary alcohol (C2 to C8) oxidizing activity (28).

Third, cells grown on either glycerol (Table 1) or valeric acid oxidized both TBA and TAME, but not ETBE. All of these compounds are oxidized by propane-grown cells of M. vaccae JOB5 (33, 36; C. A. Smith and M. R. Hyman, unpublished results). However, our observation that only two of these compounds were oxidized during growth on nonalkane substrates (Table 1) suggests that the oxidation process is determined by features of these compounds as inducers rather than by the substrate range of a constitutively expressed enzyme.

Inductive effects of cometabolites in other organisms.
If the MTBE-oxidizing activity described in this study is due to an inductive effect controlled by catabolite repression, it is important to recognize that these effects would be expected to be most apparent in cultures grown under the carbon-limited conditions used in this study. It is also important to recognize that it is not uncommon for bacterial monooxygenase gene expression to be induced by substrates for these enzymes that themselves do not support cell growth. For instance, alkane hydroxylase activity in Pseudomonas aeruginosa is strongly induced by diethoxymethane and dicyclopropylmethanol. Neither of these compounds supports cell growth, although both compounds are oxidized by induced cells (38). Another relevant example is given by the strong effect of TCE on the diverse organisms that cometabolically degrade this compound. Diverse toluene-oxidizing oxygenases (16, 22, 24, 29, 32) are all induced to various degrees by the presence of TCE. In the case of Pseudomonas mendocina KR-1, the induction of toluene-4-monooxyganease activity in cells grown on glutamate with TCE is ~86% of the level of activity observed with toluene-grown cells (24). The inducing effect of TCE in toluene-oxidizing organisms has been proposed to reflect the structural similarity between the carbon-carbon double bond in TCE and the carbon-carbon bond within the aromatic ring of toluene. A wide range of chlorinated alkenes, including TCE, also induce propylene monooxygenase activity in Xanthobacter sp. strain Py2 (8). In this case there is an even stronger structural resemblance between the growth substrates for this organism and TCE. It is notable that M. vaccae JOB5 was originally isolated from a 2-methyl butane enrichment culture and grows on a wide range of branched alkanes (27). The inductive effects of MTBE may therefore be a reflection of the ability of this organism to respond to more metabolizable branched alkanes that structurally resemble MTBE and other oxygenates.

The inductive effect of MTBE on the MTBE-oxidizing activity of M. vaccae JOB5 appears to be considerably weaker than the inductive effects of TCE described above. For example, the best estimate of the rate of MTBE oxidation we can derive from our data is from the experiment described in Fig. 3. The maximal rate of TBA production after induction was 0.35 nmol min-1 mg of total protein-1. This is close to the rate we have previously described for MTBE oxidation by 1-propanol-grown cells and is only ~1% of the estimated Vmax value for propane-grown cells (33). However, in this study we have also shown that the molar ratio of products detected to MTBE consumed varies widely depending on which substrate is used to support growth. The rate estimate given above therefore most likely underestimates the true rate of MTBE oxidation, which could be as much as fivefold higher if the combined production of TBA and TBF represents only 20% of the consumed MTBE (Fig. 1 and 2). It should also be recognized that these cells were exposed to concentrations of MTBE below the S50 for MTBE (~2.5 mM) (Fig. 3) and only slightly higher than the Ks for MTBE (1.3 mM) (33). These factors suggest the maximal level of induction that can be achieved under appropriate conditions is likely to be considerably higher than 5% of the maximal activity of propane-grown cells.

Significance to understanding of MTBE cometabolism.
Our results also provide several other interesting observations relevant to our understanding of MTBE oxidation by this organism. For example, our results with acid-grown cells (Fig. 4 and 5) showed MTBE oxidation was unsustainable (Fig. 5). It may be that a delicate balance exists between the maximum concentration of growth substrate that allows for induction of MTBE-oxidizing activity and the minimum concentration of growth substrate needed to supply the anabolic demands for de novo protein synthesis and reductant supply to the newly synthesized MTBE-oxidizing monooxygenase. While these are interesting questions for future studies, our present results certainly add further weight to our previous report (33) that M. vaccae JOB5 does not grow on MTBE when it is supplied as a sole carbon and energy source to this organism.

Another interesting observation was the accumulation of 2M12PD when cells were grown on glycerol in the presence of TBA (Table 1). Oxidation of TBA by propane-grown M. vaccae JOB5 is catalyzed by the same alkane monooxygenase responsible for MTBE oxidation (33, 36), and 2M12PD is the predicted product of this reaction (36). We have not previously observed accumulation of 2M12PD during MTBE oxidation by propane-grown cells. This may reflect our previous focus on oxidation of low MTBE concentrations and the likelihood this product is rapidly further oxidized by alcohol and aldehyde dehydrogenase activities coinduced with alkane monooxygenase activity in alkane-grown cells. Accumulation of 2M12PD in both glycerol-grown and valeric acid-grown cells exposed to TBA may indicate that the effects of TBA lead to the induction of alkane monooxygenase without extensive concurrent coinduction of alcohol dehydrogenase activity. Again, this interpretation is compatible with a previous study of substrate utilization patterns by M. vaccae JOB5 that reported cells grown on fatty acids do not have detectable alcohol-oxidizing activity (28).

Implications for the environmental fate of MTBE and TBA.
Our results also have potential impacts on our understanding of the role of cometabolism in the environmental fate of MTBE. As indicated in the introduction, several studies have demonstrated that oxygenation of anaerobic, gasoline-impacted environments can promote MTBE biodegradation. These studies have typically been conducted in environments that do not contain gasoline-derived alkanes or other substrates that could be argued to support "conventional" cometabolic degradation processes. However, our results with organic acids suggest cometabolic processes could have an unforeseen role in these environments. Organic acids accumulate in gasoline-impacted environments as a result of anaerobic degradation of gasoline hydrocarbons (6, 7). Our present results therefore suggest that if low concentrations of acids were present with MTBE in environments undergoing oxygenation, the physiological conditions could be met for a cometabolic degradation process to occur. These conditions include organic acids as a growth substrate, MTBE as an inducer, and oxygen as both a terminal electron acceptor and a substrate for monooxygenase activity. Our results (Table 1) also suggest a similar effect can be expected with TBA, a compound that is often regarded as an indicator of MTBE biodegradation. Future studies are clearly needed to determine whether the effects described in this study are specific for M. vaccae JOB5 or are generally applicable to organisms capable of MTBE cometabolism. Future studies are also clearly needed to address the possibility that MTBE degradation in oxygenated environments is not solely due to bacterial metabolism of MTBE.


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ACKNOWLEDGMENTS
 
This research was supported by funding to M.R.H. from the American Petroleum Institute.

The opinions expressed are those of the authors and not necessarily those of the funding agency.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Gardner Hall, North Carolina State University, Raleigh, NC 27695-7615. Phone: (919) 515-7814. Fax: (919) 515-7867. E-mail: michael_hyman{at}ncsu.edu. Back


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Applied and Environmental Microbiology, February 2004, p. 1023-1030, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.1023-1030.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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