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

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2004, p. 1023–1030 Vol. 70, No. 2 0099-2240/04/$08.00ϩ0 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 and ChevronTexaco Energy Research and Technology Co., Richmond, California 948022

Received 4 June 2003/Accepted 31 October 2003

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 glyc- ,(to 2.75 mM 2.4 ؍ erol-grown cells was saturable with respect to MTBE concentration (50% saturation level 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.

Methyl tertiary butyl ether (MTBE) is an oxygenating com- pane-grown cells of Mycobacterium vaccae JOB5, MTBE is pound that is currently added to gasoline to reduce automobile initially oxidized to tertiary butyl formate (TBF) through the emissions of carbon monoxide and smog-related air pollutants. sequential activities of an alkane-inducible alkane monooxy- Approximately 30% of the gasoline sold in the United States genase and a putative hemiacetal-oxidizing alcohol dehydro- contains MTBE, and its widespread use has led to concerns genase (33). The subsequent abiotic and biotic hydrolysis of over the human health effects resulting from chronic exposure TBF yields tertiary butyl alcohol (TBA), which is then further to this compound through gasoline contamination of drinking oxidized by the same monooxygenase responsible for initiating water supplies (19, 35). The U.S. Environmental Protection MTBE oxidation. Further steps in the oxidation of MTBE have Agency currently classifies MTBE as a possible human carcin- been proposed but have not been extensively characterized ogen and has issued a drinking water advisory for MTBE of 20 (36). to 40 ppb (37). The role of cometabolism in the environmental fate of Several recent studies have shown MTBE can be biode- MTBE is currently unclear. For instance, addition of both graded under anaerobic conditions (2, 3, 9, 34, 40). However, propane and oxygen to gasoline-contaminated groundwater like most other gasoline components, the fastest rates of has been shown to promote MTBE oxidation (1). However, it MTBE biodegradation are observed under aerobic conditions is not known whether cometabolic processes supported by gas- (4, 11, 15, 33, 36). Several aerobic bacteria have been isolated oline hydrocarbon cocontaminants represent an important nat- that can use MTBE as a sole source of carbon and energy for ural attenuation process for MTBE under aerobic conditions. growth (10, 13, 15, 26). Various other aerobic MTBE-degrad- Recent field studies have shown MTBE biodegradation can be ing organisms have also been identified that are unable to grow stimulated when anaerobic, gasoline-impacted ground water is on MTBE but can cometabolically degrade this compound oxygenated, either through engineered approaches (30, 41) after growth on a variety of hydrocarbons. Like MTBE, some or through natural ground water transport mechanisms (4, of these hydrocarbons are also present at high concentrations 21). However, as the currently recognized growth substrates in gasoline and include alkanes (11, 14, 23, 31, 33, 36), aro- thought to be required for cometabolic MTBE biodegradation matics (18, 20), and alicyclics (5, 31). Cometabolic degradation are often reported to be absent from these environments, these of MTBE has been most extensively studied in propane- (33, effects of oxygenation have been generally interpreted in terms 36) and n-pentane-oxidizing bacteria (11). In the case of pro- 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 * Corresponding author. Mailing address: Department of Microbi- ology, Gardner Hall, North Carolina State University, Raleigh, NC environments, both in the absence as well as the presence of 27695-7615. Phone: (919) 515-7814. Fax: (919) 515-7867. E-mail: organisms similar to the MTBE-metabolizing strain PM-1. [email protected]. Studies of microbial cometabolic degradation processes for

1023 1024 JOHNSON ET AL. APPL.ENVIRON.MICROBIOL. important pollutants such as trichloroethylene (TCE) and with buffer (1.0 ml, as above) to a final protein concentration of ϳ2.5 mg of total Ϫ1 MTBE have often focused on identifying substrates that sup- cell protein ml . Analytical methods. In some experiments the concentrations of MTBE and its port high rates of biodegradation of these compounds. These oxidation products (TBA and TBF) were determined by GC using aqueous substrates are of interest because they not only support micro- samples (2 ␮l) taken directly from the culture vessels. In experiments that bial growth but also lead to high levels of the key catabolic followed the time course of organic acid consumption as well as MTBE oxida- enzyme activities required for cosubstrate (e.g., TCE and tion, aqueous samples (0.5 ml) were taken from the sealed culture vials at the MTBE) degradation. However, as studies of cometabolic TCE indicated times using disposable sterile plastic syringes (1 ml) and needles. The samples were transferred to flat-top polypropylene microcentrifuge tubes (1.5 degradation have repeatedly demonstrated (8, 16, 22, 24, 29, ml), and aqueous samples (2 ␮l) of the media were then immediately injected 32), it is also important to recognize the potential inducing into a gas chromatograph. In all experiments the samples were analyzed using effects of the target pollutant on the expression of enzymes Shimadzu GC-8A or GC-14A gas chromatographs fitted with flame ionization required for its own biodegradation. In the present study we 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, have examined the effect of MTBE on the MTBE-oxidizing TBF, TBA, ETBE, TAME, TAA, and 2M12PD was conducted at 160°C, while activity of M. vaccae JOB5 during carbon-limited growth on the quantification of valeric acid in the presence of MTBE, TBF, and TBA was diverse nonalkane substrates. Our results demonstrate that conducted at 150°C. In both analyses the injection and detector temperatures cells grown on a wide range of substrates in the presence of 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 MTBE and other oxygenates express the enzyme activities integrators (Palo Alto, Calif.) for data collection. The minimum detection limits Ϫ required for the degradation of these gasoline additives. The for TBF and TBA were ϳ20 and ϳ3 nmol ml 1, respectively. results of this study have been interpreted in terms of their Cell protein concentrations were determined using the Biuret assay (12) after potential impact on our understanding of the underlying phys- solubilizing cell material for 30 min at 65°C in 3 N NaOH and sedimentation of insoluble material by centrifugation (14,000 ϫ g; 5 min). Bovine serum albumin iology of MTBE cometabolism and the potential role of co- was used as the standard. The concentration of MTBE in saturated aqueous metabolism in the environmental fate of MTBE. solution at room temperature (23°C) was taken as 0.544 M (33). The dimension-

less 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 ϭ ⅐ ϩ MATERIALS AND METHODS regression to a single substrate-binding model [Y Vmax X/(Ks X)] using GraphPad Prism version 3.0a for Macintosh (GraphPad Software, San Diego, Materials. M. vaccae JOB5 (ATCC 29678) was obtained from the American Calif.). Type Culture Collection (Manassas, Va.). Galactose (99% purity), glucose ϩ (99.5% purity), fructose (99% purity), pyruvic acid (99 % purity), succinic acid (99% purity), lactic acid (98% purity), glycerol (99% purity), sodium propionate ϩ (99% purity), sodium butyrate (98% purity), valeric acid (99 % purity), caproic RESULTS ϩ acid (99.5 % purity), heptanoic acid (99% purity), isovaleric acid (99% purity), 2-methylbutyric acid (98% purity), 2-methylvaleric acid (98% purity), 3-methyl- Effects of nonhydrocarbon growth substrates on MTBE- valeric acid (97% purity), 2-methylhexanoic acid (99% purity), TBF (97% pu- oxidizing activity. Cells of M. vaccae JOB5 were grown under ϩ ϩ rity), TBA (99 % purity), tertiary amyl alcohol (TAA; 99 %), MTBE (99.8% carbon-limited conditions on a variety of sugars, tricarboxylic purity), ethyl tertiary butyl ether (ETBE; 99% purity), tertiary amyl methyl ether acid cycle intermediates, lactate, and glycerol either without (TAME; 97% purity), 1-propanol (99.5% purity), 2-propanol (99.5% purity), and ␮ ϳ ␮ ϳ MTBE, with MTBE (14 mol [ 450 M dissolved MTBE]), rifampin, chloramphenicol, and calcium carbide (technical grade, 80% purity; ␮ for acetylene generation) were obtained from Sigma Aldrich Chemical Co., Inc. or with MTBE (14 mol) and acetylene (3.7% [vol/vol] gas (Milwaukee, Wis.). Sodium acetate (99.5% purity) was obtained from Fisher phase), a potent irreversible inactivator of MTBE-oxidizing Scientific (Pittsburgh, Pa.). 2-Methyl-1,2-propanediol (2M12PD)was a gift from activity in this organism (33). After 7 days, culture growth was Lyondell Chemical Co. (Houston, Tex.). Compressed gases used for gas chro- determined (OD600) and the culture medium was analyzed by matography (GC) (H2,N2, and air) were obtained from local industrial vendors. Growth experiments. Most of the experiments described in this study used GC to determine the extent of MTBE consumption and the cells of M. vaccae JOB5 grown in batch culture in glass serum vials (160 ml) accumulation of TBA and TBF. No detectable growth or sealed with Teflon-lined Mininert valves (Alltech Associates Inc., Deerfield, Ill.). MTBE consumption occurred when cells were incubated with The vials contained mineral salts medium (25 ml) (39), and unless otherwise MTBE alone (data not shown). In contrast, the organism grew, stated all growth substrates were added from filter-sterilized aqueous solutions to to varying degrees, on all of the other substrates tested, and give an initial concentration of 2.5 mM. The culture vials were inoculated (initial Յ neither MTBE nor acetylene had any consistent effect on the 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- final culture density (OD600) for any of these substrates (Fig. dextrose (CYD) agar plates (Difco plate count agar). When required, MTBE, 1A). The GC analysis (Fig. 1B) revealed variable but often ETBE, TAME, TBA, or TAA was added to the sealed vials from a saturated extensive consumption of MTBE had occurred in all of the aqueous solution using sterile glass microsyringes. Acetylene (5 ml) was added to cultures that contained MTBE alone, whereas little or no the sealed vials as required using sterile disposable plastic syringes fitted with sterile Acrodisc 0.1-␮m filters (Pall Corp., Ann Arbor, Mich.). The culture vials MTBE consumption had occurred in cultures grown in the were incubated at 30°C in the dark in an Innova 4900 environmental shaker (New presence of both MTBE and acetylene. For instance, Ն70% of Brunswick Scientific Co., Inc., Edison, N.J.) operated at 150 rpm. Losses of the added MTBE (ϳ10 ␮mol) was consumed by cells grown on MTBE from abiotic control incubations containing no added cells were less than either glucose, pyruvate, or fructose in the presence of MTBE. 3% over 7 days under these conditions. Culture growth was measured by deter- In contrast, Յ7% (1 ␮mol) of the MTBE was consumed when mining the OD600 with a Shimadzu 1601 UV/Vis spectrophotometer (Kyoto, Japan). In every experiment, a sample (50 ␮l) was streaked on CYD plates to cells were grown on the same substrates in the presence of subsequently confirm the purity of the culture. acetylene. In all cultures where MTBE consumption was ob- In some experiments, concentrated washed cells were used. In these cases the served, both TBA and TBF were also detected. With the ex- cells were grown in batch culture with the required substrate, as described above. ception of cells grown on succinate, the molar ratio of MTBE The cells were then harvested from the culture medium by centrifugation (10,000 ϫ consumed to total products (TBA plus TBF) detected was low g; 10 min), and the resulting cell pellet was resuspended in buffer (10 ml; 50 ϭ mM sodium phosphate; pH 7). The washed cells were sedimented again by (1:0.21) but close to constant (standard deviation [SD] centrifugation (as above), and the resulting cell pellet was finally resuspended 4.4%). VOL. 70, 2004 MTBE OXIDATION INDUCTION IN MYCOBACTERIUM VACCAE JOB5 1025

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 ␮mol) 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. ␮mol). After 2 h, the reaction media were analyzed by GC to Two growth substrates characterized in Fig. 1, glycerol and quantify accumulation of TBA and TBF. We did not detect fructose, were used to investigate the effect of MTBE concen- MTBE consumption or TBA- or TBF-generating activity for tration on the level of MTBE oxidation. Cells were grown for cells grown either in the presence or absence of MTBE in these 7 days on either glycerol (7.5 mM) or fructose (2.5 mM) in the short-term assays. In the second experiment, cells were initially presence of various amounts of MTBE (0 to ϳ140 ␮mol; 0 to grown on glycerol (35 mM) and then harvested and concen- ϳ5 mM in solution). A plot of the total MTBE consumed trated by centrifugation. These cells were then incubated with versus initial dissolved MTBE concentration appeared to be MTBE (ϳ2 mM dissolved MTBE) and a low concentration (1 saturable for both growth substrates (Fig. 2). These data were mM) of glycerol as an energy source. The time course of TBA fitted to a hyperbolic, single substrate-binding curve, and good and TBF production was then determined by GC analysis of fits (r2 Ն 0.99) were obtained in both cases. Half-saturation the reaction medium. The results (Fig. 3) showed there was a ϭ values (S50) for glycerol (2.4 mM; standard error [SE] 0.45) lag phase of 4 h before TBA was first detected in the reaction and fructose (2.75 mM; SE ϭ 0.53) were obtained from these medium. Although TBF also accumulated during the reaction analyses. Both TBF and TBA were detected in the culture time course, the concentration of detected TBF never ex- medium, and these products accounted for ϳ20% of the ceeded 20% of the total TBA detected (data not shown). Over MTBE consumed in each culture. A plot of total products the next 6 to 8 h there was a progressive increase in the rate of (TBA and TBF) detected versus initial dissolved MTBE con- TBA accumulation, after which the rate of TBA accumulation centration (Fig. 2 inset) also provided comparable S50 values of remained almost constant. When cells were incubated with 3.2 mM (r2 ϭ 0.98; SE ϭ 0.88) and 2.8 mM (r2 ϭ 0.98; SE ϭ MTBE in the presence of either chloramphenicol or rifampin Ϫ 0.38) for cells grown on glycerol and fructose, respectively. (50 ␮gml 1 each), the production of both TBA and TBF (data The results described in Fig. 1 and 2 suggest that the pres- not shown) was strongly or completely inhibited relative to that ence of MTBE during growth on diverse nonalkane substrates in the incubation containing MTBE alone. Complete inhibition led to the production of enzyme systems capable of degrading of both TBA and TBF accumulation was also observed when MTBE. However, these results did not address the possibility cells were incubated with MTBE and acetylene (10% [vol/vol] that MTBE-oxidizing activity was also present in cells grown in gas phase). the absence of MTBE. We conducted two experiments to in- Effects of other ethers and tertiary alcohols. We also exam- vestigate this possibility. First, we attempted to determine the ined whether other ether oxygenates and their tertiary alcohol specific MTBE-oxidizing activity of concentrated cell suspen- oxidation products behaved similarly to MTBE. Cells were sions grown on either glycerol (7.5 mM) or fructose (2.5 mM) grown on glycerol (7.5 mM) in the presence of either TAME, in the presence and absence of MTBE (initially ϳ2mMin ETBE, TAA, or TBA. Additional cultures were also grown solution). After growth for 7 days, the cells were harvested by using the same growth substrate and oxygenate-alcohol com- centrifugation, washed, and finally resuspended at a protein binations in the presence of acetylene (3.7% [vol/vol] gas Ϫ concentration of ϳ5 mg of total protein ml 1. Samples of the phase). After growth for 5 days, the reaction media were an- concentrated cell suspension (0.2 ml) were incubated at 30°C alyzed by GC to determine the extent of oxygenate consump- in buffer (0.8 ml; 50 mM sodium phosphate [pH 7.0]) in stop- tion and product accumulation. The results (Table 1) showed pered glass serum vials (10 ml) in the presence of MTBE (1 that substantial consumption of TAME but not ETBE oc- 1026 JOHNSON ET AL. APPL.ENVIRON.MICROBIOL.

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 [■]) or fructose (2.5 mM [Œ]) in the presence of a range of initial MTBE concentrations (0 to 140 ␮mol; 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. curred during growth on glycerol. TAA (ϳ1 ␮mol) was de- 2-methylbutyric, 2-methylvaleric, 3-methylvaleric, and 2-meth- tected as a product of TAME oxidation, although TAA accu- ylhexanoic acids). After growth for 7 days, both TBA and TBF mulation only represented ϳ10% of the TAME consumed. were detected in the incubations containing acids and MTBE, Consumption of TAA (ϳ6 ␮mol) also occurred when cells whereas neither product was observed in the same incubations were grown in the presence of TAA, and both TAME and conducted in the presence of acetylene (3.7% [vol/vol] gas TAA consumption was inhibited by acetylene. These results phase). The average consumption of MTBE in these cultures suggest that the low recovery of TAA in the cultures grown was 7.4 ␮mol (SD ϭ 2.1), and the average molar yield of TBF with TAME was most likely due to concurrent oxidation of and TBA combined was 68% of the MTBE consumed. We both TAME and TAA. Similar results were also observed for were unable to quantify cell growth in these and subsequent cultures grown in the presence of TBA. Approximately 50% acid-grown cultures described later, due to clumping of cells. (ϳ23 ␮mol) of the added TBA was consumed during growth Cells were also grown under carbon-limited conditions on on glycerol, and a single high-boiling-point product that coe- equimolar concentrations (2.5 mM) of a series (C2 to C7)of luted with 2M12PD was detected. This accounted for ϳ50% straight-chain acids, either in the presence of MTBE (14 ␮mol) (ϳ12 ␮mol) of the TBA consumed by glycerol-grown cells. or MTBE (14 ␮mol) plus acetylene (3.7% [vol/vol] gas phase). Both the consumption of TBA and the production of 2M12PD The time course of TBA and TBF production was then deter- were inhibited by the presence of acetylene. mined by GC analysis of the culture medium. In all cultures Effects using organic acids as growth substrates. In addition containing MTBE alone, neither product was detected until at to conventional growth substrates described above, we also least 24 h after the culture was initiated (Fig. 4). The chain examined whether MTBE-oxidizing activity was stimulated by length of the acid substrate had two distinct effects. First, in MTBE in cells grown on more environmentally relevant com- general the longer the acid carbon chain length, the longer the pounds. Volatile organic acids were chosen for study because lag phase before MTBE oxidation products (TBA and TBF) they are frequently found in gasoline-impacted ground water were observed. For example, products were observed with ac- environments as products of anaerobic biodegradation of gas- etate-grown cells after 24 h, while cells grown on heptanoic oline hydrocarbons (6, 7). In one experiment, cells were grown acid did not show product accumulation until ϳ50 h. Second, in the presence of MTBE (14 ␮mol) using carbon-limiting in general the longer the acid chain length, the greater the concentrations (2.5 mM) of several branched acids (isovaleric, amounts of MTBE oxidation products that were observed. For VOL. 70, 2004 MTBE OXIDATION INDUCTION IN MYCOBACTERIUM VACCAE JOB5 1027

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 M. vaccae glycerol (35 mM) in the absence of MTBE. The cells were harvested, FIG. 4. Time course of TBA production by cells of JOB5 M. vaccae washed, and stored at 4°C, as described in Materials and Methods. The during growth on linear organic acids. A series of cultures of JOB5 were grown on linear organic acids (C2 to C7) (initial concen- reactions were conducted in glass serum vials (10 ml) sealed with butyl ϭ ␮ rubber stoppers and aluminum crimp seals. The reaction vials con- tration 2.5 mM) in the presence of MTBE (14 mol), as described tained buffer (50 mM sodium phosphate; pH 7; ϳ900 ␮l), MTBE (2.8 in Materials and Methods. The time course of TBA accumulation is ␮ ␮ shown for cultures grown on acetic (Œ), propionic (‚), butyric (■), mol), and glycerol (1 mol). The reactions were initiated by the ᮀ F E addition of an aliquot (100 ␮l) of concentrated cell suspension (0.21 valeric ( ), caproic ( ), and heptanoic ( ) acids. The symbols repre- mg of total protein), and the vials were incubated at 30°C in a shaking sent the average for two replicate cultures, and the error bars show the water bath (150 rpm). At the indicated times, aqueous samples (2 ␮l) range of values for both cultures combined. 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 (■), MTBE plus acetylene (10% [vol/vol] gas phase) (ᮀ), MTBE plus chloram- Effect of acid concentration of MTBE-oxidizing activity. The Ϫ Ϫ phenicol (50 ␮gml 1)(F), and MTBE plus rifampin (50 ␮gml 1)(E). 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 fur- Ͻ ␮ example, acetate-grown cells generated 2 mol of products, ther we quantified the time course of TBA and TBF produc- ϳ ␮ while caproic acid-grown cells generated 4 mol of products. tion in relation to both MTBE and valeric acid consumption The average molar ratio of MTBE consumed to total products for cells grown either with or without MTBE (7.5 ␮mol) in the (TBA plus TBF) detected for the range of acids tested was presence of two different initial amounts of valeric acid (70 and 1:0.62. However, this ratio progressively decreased from 1:0.84 35 ␮mol). These culture conditions were also duplicated in with acetate-grown cells to 1:0.37 with cells grown on hep- incubations containing acetylene (3.7% [vol/vol] gas phase). In tanoic acid. In a duplicate series of incubations, acetylene fully the cultures containing the lower initial concentration of va- inhibited the production of both TBA and TBF in all cases leric acid, the acid was fully consumed within 40 h (Fig. 5). The (data not shown). consumption of MTBE and the production of both TBA and TBF were first detected in the growth medium when ϳ10 ␮mol of valeric acid remained. The time course of both TBA and TABLE 1. Oxygenate consumption and product accumulation TBF accumulation continued to reflect MTBE consumption by cells grown on glycerola over the next 40 h. However, the MTBE oxidation reaction was ϳ Amt of oxygenate Product Amt of product not sustainable, and after 80 h the rates of both MTBE Oxygenate consumed (␮mol) generated (␮mol) ␮ detected, consumption and product accumulation steadily declined to ( mol added) Ϫ b Ϫ C2H2 ϩ Ϫ Abiotic C2H2 C2H2 C2H2 close to zero. After 120 h the molar ratio of MTBE consumed TBA (50) 0.3 22.9 (2.1)c 2M12PD NDd 11.6 (1.9) to total products (TBF plus TBA) detected was 1:0.72. The ETBE (20) Յ0.1 0.5 (0.3) ND corresponding control experiment (valeric acid plus MTBE TAME (25) 1.4 9.5 (0.5) TAA ND 1.0 (0.1) plus acetylene) showed acetylene had no discernible effect on TAA (50) 0.6 6.5 (1.05) ND the rate of valeric acid consumption but completely inhibited a Glycerol was supplied as the growth substrate at an initial concentration of both MTBE consumption and production of both TBA and 7.5 mM. TBF over the entire reaction time course. A substantially b Abiotic control incubations contained all substrates but no cells. Losses of each substrate in abiotic controls were indistinguishable from cultures containing similar pattern of biodegradation was observed when the ex- all substrates, cells, and acetylene (3.75% [vol/vol] gas phase). periment was repeated with twofold-higher initial amounts c The values reported are the averages of two replicate cultures for each of valeric acid. The onset of both MTBE consumption and condition. The values in parentheses indicate the range of values around the ϳ mean for the combined data for both cultures. production of TBA and TBF was delayed by 20 h relative d ND ϭ none detected. to that for the cultures grown with lower initial amounts of 1028 JOHNSON ET AL. APPL.ENVIRON.MICROBIOL.

tions, 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 FIG. 5. Time course of MTBE and valeric acid oxidation during this activity was strongly inhibited by both chloramphenicol growth of M. vaccae JOB5. Cultures of M. vaccae JOB5 were grown in and rifampin, evidence that indicates this response involves ␮ batch culture on valeric acid in the presence of MTBE (7.5 mol), as both transcription and de novo protein synthesis. Our failure to described in Materials and Methods. The time course for valeric acid consumption is shown for cultures grown with 35 ␮mol (squares) or 70 detect MTBE-oxidizing activity in cells previously grown for 7 ␮mol (circles) of valeric acid, either in the presence (open symbols) or days in the presence of MTBE probably reflects the consistent absence (closed symbols) of acetylene (3.75% [vol/vol] gas phase). postinduction decline in MTBE-oxidizing activity we observed Also shown is the corresponding time course for MTBE consumption ␮ ␮ in several experiments (Fig. 4 and 5) conducted over an equiv- for cultures grown with 35 mol (inverted triangles) or 70 mol (up- alent period of time. right triangles) of valeric acid, either in the presence (open symbols) or absence (closed symbols) of acetylene (3.75% [vol/vol] gas phase). In Second, cells grown on valeric acid did not show a progres- addition, the combined amount of TBA and TBF generated from sive increase in MTBE oxidation rate throughout the time MTBE is shown for cultures grown with 35 ␮mol (asterisks) or 70 course of acid consumption (Fig. 5), an effect that would be ␮ mol (filled diamonds) of valeric acid. No TBF or TBA was observed predicted if MTBE-oxidizing activity was constitutive and in- 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 creased consistently with increases in cell density. In contrast, figure. The data plotted are the averages of two replicates for cultures these cells only showed evidence for both MTBE consumption grown in the absence of acetylene and a single replicate for all cultures and TBA and TBF production once the residual valeric acid that contained acetylene. The error bars show the range of values had been depleted to ϳ10 ␮mol (Fig. 5). This effect occurred obtained for the replicate cultures. 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 valeric acid. However, the onset of both of these activities feature that again supports a model involving MTBE-depen- still occurred when ϳ10 ␮mol of valeric acid remained in dent gene induction. This is consistent with a previous study of the culture medium. The total amounts of MTBE degraded the growth substrate range of M. vaccae JOB5 that indicated and TBA and TBF generated were greater in these cultures cells grown on acetate, propionate, and butyrate did not have

than in those with lower initial amounts of valeric acid. detectable n-alkane (C1 to C8) or primary alcohol (C2 to C8) However, the molar ratio of MTBE consumed to total prod- oxidizing activity (28). ucts detected (TBA plus TBF) after 120 h was 1:0.80 and Third, cells grown on either glycerol (Table 1) or valeric acid was comparable to the results obtained with the lower va- oxidized both TBA and TAME, but not ETBE. All of these leric acid concentration. As a final component of this study, compounds are oxidized by propane-grown cells of M. vaccae we also conducted a similar experiment to that described in JOB5 (33, 36; C. A. Smith and M. R. Hyman, unpublished Table 1 using valeric acid rather than glycerol as a growth results). However, our observation that only two of these com- substrate. Substantially similar activities to those observed pounds were oxidized during growth on nonalkane substrates with glycerol were observed and included the oxidation of (Table 1) suggests that the oxidation process is determined by TBA, TAA, and TAME but not ETBE by valeric acid-grown features of these compounds as inducers rather than by the cells (data not shown). substrate range of a constitutively expressed enzyme. Inductive effects of cometabolites in other organisms. If the DISCUSSION MTBE-oxidizing activity described in this study is due to an inductive effect controlled by catabolite repression, it is impor- The results of this study provide strong and consistent evi- tant to recognize that these effects would be expected to be dence showing MTBE oxidation occurs during growth of M. most apparent in cultures grown under the carbon-limited con- vaccae JOB5 on a wide range of nonalkane substrates (Fig. 1, ditions used in this study. It is also important to recognize that 3, and 4). We have also shown acetylene inhibits the produc- it is not uncommon for bacterial monooxygenase gene expres- tion of both TBA and TBF (Fig. 1B and 5) from MTBE, as well sion to be induced by substrates for these enzymes that them- as consumption of MTBE (Fig. 5). Based on these observa- selves do not support cell growth. For instance, alkane hydrox- VOL. 70, 2004 MTBE OXIDATION INDUCTION IN MYCOBACTERIUM VACCAE JOB5 1029 ylase activity in Pseudomonas aeruginosa is strongly induced by tainly add further weight to our previous report (33) that M. diethoxymethane and dicyclopropylmethanol. Neither of these vaccae JOB5 does not grow on MTBE when it is supplied as a compounds supports cell growth, although both compounds sole carbon and energy source to this organism. are oxidized by induced cells (38). Another relevant example is Another interesting observation was the accumulation of given by the strong effect of TCE on the diverse organisms that 2M12PD when cells were grown on glycerol in the presence of cometabolically degrade this compound. Diverse toluene-oxi- TBA (Table 1). Oxidation of TBA by propane-grown M. vac- dizing oxygenases (16, 22, 24, 29, 32) are all induced to various cae JOB5 is catalyzed by the same alkane monooxygenase degrees by the presence of TCE. In the case of Pseudomonas responsible for MTBE oxidation (33, 36), and 2M12PD is the mendocina KR-1, the induction of toluene-4-monooxyganease predicted product of this reaction (36). We have not previously ϳ activity in cells grown on glutamate with TCE is 86% of the observed accumulation of 2M12PD during MTBE oxidation by level of activity observed with toluene-grown cells (24). The propane-grown cells. This may reflect our previous focus on inducing effect of TCE in toluene-oxidizing organisms has oxidation of low MTBE concentrations and the likelihood this been proposed to reflect the structural similarity between the product is rapidly further oxidized by alcohol and aldehyde carbon-carbon double bond in TCE and the carbon-carbon dehydrogenase activities coinduced with alkane monooxygen- bond within the aromatic ring of toluene. A wide range of ase activity in alkane-grown cells. Accumulation of 2M12PD in chlorinated alkenes, including TCE, also induce propylene both glycerol-grown and valeric acid-grown cells exposed to monooxygenase activity in Xanthobacter sp. strain Py2 (8). In TBA may indicate that the effects of TBA lead to the induction this case there is an even stronger structural resemblance be- of alkane monooxygenase without extensive concurrent coin- tween the growth substrates for this organism and TCE. It is duction of alcohol dehydrogenase activity. Again, this interpre- notable that M. vaccae JOB5 was originally isolated from a tation is compatible with a previous study of substrate utiliza- 2-methyl butane enrichment culture and grows on a wide range tion patterns by M. vaccae JOB5 that reported cells grown on of branched alkanes (27). The inductive effects of MTBE may fatty acids do not have detectable alcohol-oxidizing activity therefore be a reflection of the ability of this organism to (28). respond to more metabolizable branched alkanes that struc- Implications for the environmental fate of MTBE and TBA. turally resemble MTBE and other oxygenates. Our results also have potential impacts on our understanding The inductive effect of MTBE on the MTBE-oxidizing ac- of the role of cometabolism in the environmental fate of tivity of M. vaccae JOB5 appears to be considerably weaker MTBE. As indicated in the introduction, several studies have than the inductive effects of TCE described above. For exam- demonstrated that oxygenation of anaerobic, gasoline-im- ple, the best estimate of the rate of MTBE oxidation we can pacted environments can promote MTBE biodegradation. derive from our data is from the experiment described in Fig. These studies have typically been conducted in environments 3. The maximal rate of TBA production after induction was Ϫ1 Ϫ1 that do not contain gasoline-derived alkanes or other sub- 0.35 nmol min mg of total protein . This is close to the rate we have previously described for MTBE oxidation by 1-propa- strates that could be argued to support “conventional” cometa- ϳ bolic degradation processes. However, our results with organic nol-grown cells and is only 1% of the estimated Vmax value for propane-grown cells (33). However, in this study we have acids suggest cometabolic processes could have an unforeseen also shown that the molar ratio of products detected to MTBE role in these environments. Organic acids accumulate in gas- consumed varies widely depending on which substrate is used oline-impacted environments as a result of anaerobic degrada- to support growth. The rate estimate given above therefore tion of gasoline hydrocarbons (6, 7). Our present results there- most likely underestimates the true rate of MTBE oxidation, fore suggest that if low concentrations of acids were present which could be as much as fivefold higher if the combined with MTBE in environments undergoing oxygenation, the production of TBA and TBF represents only 20% of the con- physiological conditions could be met for a cometabolic deg- sumed MTBE (Fig. 1 and 2). It should also be recognized that radation process to occur. These conditions include organic these cells were exposed to concentrations of MTBE below the acids as a growth substrate, MTBE as an inducer, and oxygen ϳ as both a terminal electron acceptor and a substrate for mono- 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 oxygenase activity. Our results (Table 1) also suggest a similar maximal level of induction that can be achieved under appro- effect can be expected with TBA, a compound that is often priate conditions is likely to be considerably higher than 5% of regarded as an indicator of MTBE biodegradation. Future the maximal activity of propane-grown cells. studies are clearly needed to determine whether the effects Significance to understanding of MTBE cometabolism. Our described in this study are specific for M. vaccae JOB5 or are results also provide several other interesting observations rel- generally applicable to organisms capable of MTBE cometabo- evant to our understanding of MTBE oxidation by this organ- lism. Future studies are also clearly needed to address the ism. For example, our results with acid-grown cells (Fig. 4 and possibility that MTBE degradation in oxygenated environ- 5) showed MTBE oxidation was unsustainable (Fig. 5). It may ments is not solely due to bacterial metabolism of MTBE. 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 ACKNOWLEDGMENTS growth substrate needed to supply the anabolic demands for de This research was supported by funding to M.R.H. from the Amer- novo protein synthesis and reductant supply to the newly syn- ican Petroleum Institute. thesized MTBE-oxidizing monooxygenase. While these are in- The opinions expressed are those of the authors and not necessarily teresting questions for future studies, our present results cer- those of the funding agency. 1030 JOHNSON ET AL. APPL.ENVIRON.MICROBIOL.

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