Journal of Environmental Biotechnology
Vol. 9, No. 2, 113–122, 2009

Original paper (regular paper)

Biodegradation of Methyl tert-Butyl Ether by Mycobacterium spp.
NORIO OHKUBO1, AKIKO H. HASHIMOTO2, KAZUHIRO IWASAKI2 and OSAMI YAGI3*
1

Department of Water and Sewage Works, Enterprise Bureau of Hitachi City, 4–4–1 Moriyama, Hitachi, Ibaraki 316–0025, Japan
2
3

National Institute for Environmental Studies, 16–2 Onogawa, Tsukuba, Ibaraki 305–8506, Japan

Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, 1–2–1, Izumi-cho,Narashinio-shi, Chiba 275–8575, Japan* TEL: +81–047–474–2574* E-mail: yagi.osami@nihon-u.ac.jp

Ethane-utilizing Mycobacterium duvalii TA5 and Mycobacterium gilvum TA27 and propane-utilizing Mycobacterium
chlorophenolicum TCE 28 isolated from soils were able to degrade methyl tert-butyl ether (MTBE) cometabolically withvarious organic acids and carbohydrates as a carbon source. Among these strains, M. duvalii TA5 showed the highest MTBE
degradation activity. M. duvalii TA5 degraded 45% of 22.7 mmol MTBE L–1 in 7 d cometabolically with 500 mg L–1 ofglucose. The maximal MTBE degradation rate (Vmax) and the half-saturation constant (Km) of MTBE by resting cells of M.
duvalii TA5 were 1,030 nmol min–1 mg dry cells–1 and 6.8 mM, respectively. Among the various carbohydrates, glucose was
the most eﬃcient carbon source for MTBE degradation by M. duvalii TA5. tert-Butyl formate (TBF), tert-butyl alcohol(TBA), 2-hydroxy isobutyric acid (HIBA), 2-propanol, acetone, and hydroxyacetone (HA) were identified as metabolites of
MTBE degradation by M. duvalii TA5. M. duvalii TA5 utilized both TBF and 2-propanol as sole energy and carbon sources.
M. duvalii TA5 was able to degrade MTBE in the soil. The present study demonstrates that M. duvalii TA5 is eﬀective indegrading MTBE and may be a useful tool for the bioremediation of MTBE-contaminated soil.
Key words: MTBE, TBA, Mycobacterium, Vmax, Km

1. Introduction
In the United States (U.S.), methyl tert-butyl ether(MTBE) has been used as a reformulated gasoline oxygenate
additive, instead of lead, since the late 1970s. Because of its
high usage, MTBE is found as a contaminant of soil andgroundwater. The MTBE concentration of potable well waterin several locations in the U.S. ranged from 20 to morethan 200 mg L–1 25). In Southeastern Brazil the groundwater
concentration of MTBE at a former industrial landfill sitewas 1.10 to 159 mg L–1 17). The minimal, average, and maximal groundwater concentrations of MTBE at a leaking underground storage tank site in Iowa exceeded 15 μg L–1,
613 mg L–1, and 99,400 mg L–1, respectively1). MTBE wasfound at the relatively high concentration of 1.8 μg L–1 inhuman blood after heavy exposure to motor vehicle exhaustand gasoline fumes in Fairbanks, Alaska18). Within 10 h of
exposure, approximately 0.9% of inhaled MTBE was excreted unchanged in human urine and 2.4% was excreted asurinary TBA13). MTBE may be carcinogenic25), and MTBE
contamination of drinking water in the U.S. has beenfound26). For this reason, the U.S. EPA established a drinking water advisory concentration of MTBE in the range of
20 to 40 μg L–1 26). MTBE has a high water solubility(51.3 g L–1 at 25°C), which complicates separating MTBE
from water25).

Bioremediation using MTBE-degrading bacteria has been
proposed as a remedy for MTBE-contaminated environments.
MTBE-degrading bacteria, which assimilate MTBE as thesole source of carbon and energy, include Proteobacteria
sp. PM16), Hydrogenophaga flava ENV7358), Methylibium
petroleiphilum PM112), Methylobacterium mesophilicum
Isolate 2416), Rhodococcus sp. Isolate 3316), Arthrobacter
ilicis Isolate 4116), and the mixed bacterial culture BC-1
(Corynebacterium sp., Pseudomonas sp., Achromobactersp.)20). Graphium sp. ATCC 584007) and Arthrobacter sp.
ATCC 2777814) cometabolically degrade MTBE with n-butane, whereas Mycobacterium vaccae10), Xanthobactersp.10), Nocardia sp. ENV42124), Nocardia sp. ENV425
ATCC 5579824) and Mycobacterium vaccae JOB5 ATCC
2967824) cometabolically degrade MTBE with propane.
Possible pathways for MBTE degradation have been proposed by Hardison et al.7) for the metabolism of MBTE to
TBA and TBF by Graphium sp. ATCC 58400 and by Steﬀan
et al.24) for the metabolism of MTBE to pyruvic acid. However, few reports address the application of MTBE-degrading bacteria for the clean-up of contaminated environments.
Whole-gene analysis of Methylibium petroleiphilum identified a megaplasmid carrying genes with an essential role in
MBTE degradation12). An independent MTBE-regulating geneof degradation enzymes has also been identified8). A further
investigation revealed the protein profile of Mycobacterium

OHKUBO et al.

114

austroafricanum IFP 2012 in the presence of MTBE andglucose by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE)15). These genetic approaches to investigate the mechanisms underlying degradation of MBTE
are continuing.
In this study, we investigated the characteristics of MTBE
degradation by Mycobacterium spp. with the ability to assimilate ethane or propane. We also examined the pathways of
MBTE degradation and the application of MBTE-degrading
bacteria as a tool for the bioremediation of MTBE-contaminated soil.

2. Materials and Methods
2.1. Microorganisms and degradation experiments
For degradation experiments, we used the ethane-utilizing
bacteria Mycobacterium duvalii TA5 and Mycobacterium
gilvum TA27 isolated from tetrachloroethylene polluted
soils. Both strains were able to utilize ethane, ethanol, andvarious other carbon compounds as their energy sources27).
A propane-utilizing bacterium, Mycobacterium chlorophenolicum TCE 28, was isolated from field soil11). M. duvalii
TA5, M. gilvum TA27, and M. chlorophenolicum TCE 28 areunable to utilize trichloroethylene (TCE) as the sole carbon
source but can degrade TCE cometabolically with ethane.
The basal MM medium (in mg L–1) was as follows: NH4Cl,
2,140; K2HPO4, 1,170; KH2PO4, 450; MgSO4·7H2O, 120;FeSO 4·7H 2O, 28; Ca(NO 3) 2·4H 2O, 4.8; CaCO 3, 100;
MnSO 4·4-6H 2O, 0.6; H 3BO 3, 0.05; ZnSO 4·7H 2O, 0.1;
( N H 4 ) 6 M o 7 O 2 4 · 4 H 2 O, 0 . 0 1 ; C o ( N O 3 ) 2 · 6 H 2 O, 0 . 6 ;NiSO4·7H2O, 0.06; CuSO4·5H2O, 0.06; and H2SeO4, 0.04 inwater. The final pH of the medium was adjusted to 6.9.
MTBE degradation experiments were carried out as follows. M. duvalii TA5, M. gilvum TA27, or M. chlorophenolicum TCE 28 was inoculated into 60 mL serum bottles
containing 12 mL MM medium with MTBE and various
carbon sources. Propane, glucose, acetic acid, and succinicacid were used as carbon sources. Propane was injected into
the headspace of the culture bottles to obtain a 10% (vol/vol) propane atmosphere. Glucose, acetic acid, and succinicacid were used at 500 mg L–1.
In the experiment for the eﬀect of glucose and MTBE
concentration on MTBE degradation, glucose concentrationfrom 100 to 2,000 mg L–1 and MTBE concentration from15 to 300 μmol bottle–1 were used. MTBE concentration
was calculated by using Henry’s law constant of 0.0225 (atm,25°C)25). MTBE concentration of 100 mg L–1 is corresponding to 13.6 μmol 12 ml–1 or 15.0 μmol bottle–1. The inoculums were precultured in MM medium with glucose at
500 mg L–1 and harvested by centrifugation. The isolated
cells were washed three times with phosphate buffer(pH 7.0). The initial optical density (OD) at 660 nm of
each strain was 0.02.
The MBTE-degrading ability of M. duvalii TA5 was assessed by using MTBE and the MTBE metabolites tertbutyl alcohol (TBA), tert-butyl formate (TBF), hydroxyace-tone (HA), 2-propanol, and acetone. The culture bottles
were sealed with butyl rubber caps and crimped by aluminum rings. The bacterial cultures were incubated at 30°C
with shaking at 120 rpm. Bacterial growth was monitored
by measuring the OD660 of the cultures.
2.2. Analysis of MTBE and MTBE metabolites
MTBE and the MTBE metabolites TBF, TBA, 2-propanol, and acetone were measured by means of a headspacegas chromatography-mass spectrometry (GC-MS) system(GC-17A/GCMS-QP5000, Shimadzu Co., Kyoto, Japan)
equipped with a DB-1 capillary column (0.32 mm i.d.×30 m,
J & W Scientific, California, USA) . MTBE concentration
was caluculated by using Henry’s law constant25). The degradation products generated by the metabolism of MTBE by
M. duvalii TA5 were determined by the following procedure. M. duvalii TA5 was cultured in 12 mL MM medium
containing 500 mg L–1 of glucose and 75 μmol MTBE in
60-mL serum bottles at 30°C, with shaking at 120 rpm, for
3 or 5 d. Culture broth was loaded and adsorbed onto a
Waters Se-Pak Plus PS-2 cartridge column (Nihon Waters
Corporation, Tokyo, Japan). The metabolites in the column
were eluted with 10 mL 99.8% acetone and concentrated to
1 mL with a stream of nitrogen (N2) gas. MTBE degradation and MBTE metabolites in the samples were analyzed
by headspace GC-MS.
2.3. Kinetics of MTBE degradation
The kinetics of MTBE degradation were determined byusing resting cells of M. duvalii TA5. Preparation of resting
cells was as follows. Strain TA5 was cultured in basal MM
medium containing 500 mg L–1 glucose at 30°C, with shaking at 120 rpm, for 5 d. The cells were harvested by centrifugation, washed with 0.1 M phosphate buﬀer (pH 7.0).
MTBE degradation rates were determined as follows.
MTBE and resting cells were added into 60 mL serum
bottles containing 10 mL phosphate buﬀer (pH 7.0), andincubated at 30°C. Final resting cell concentration was
400 mg dry cells L–1. MTBE was used at concentrations
ranging from 0.1 mM to 30 mM. To determine the initialdegradation rate, we measured the MTBE degradation
amount at 0, 120, 300 and 450 minutes, periodically. MTBE
degradation was stopped by the addition of 100 mg L–1
aminobenzotoriazole. The maximal rate of MTBE degradation (Vmax; nmol min–1 mg dry cells–1) and the half-saturation constant for MTBE (Km; mM) were determined froma Lineweaver-Burk plot. The cell concentration of M. duvalii
TA5 was converted into the dry weight equivalent by measuring the OD660, where an OD value of 1 was equivalent to a
dry weight of 580 mg dry cells L–1.
2.4. MTBE degradation in soil
MTBE at 6 μmol and resting cells at 4.4 mg(9.8×109 cells) were added to a to 60-mL serum bottle with
13 g soil, with a water content of 38% and incubated at
30°C with shaking at 120 rpm for 20 d. Resting cells were

Biodegradation of MTBE by Mycobacterium

prepared by the same method as the kinetic experiment.
The headspace gas was measured by GC-MS to analyze
MTBE degradation and the production of TBA.
2.5. Chemicals
Acetone, glucose, acetic acid, succinic acid, and hydroxyacetone (HA) were purchased from Wako Pure Chemical
Industries Ltd. (Osaka, Japan). Pyruvic acid, 2-propanol,and 2-hydroxy isobutyric acid (HIBA) were purchased from
Kanto Chemical Co. Inc. (Tokyo, Japan). MTBE and TBA
were purchased from Merck (Darmstadt, Germany) and
Junsei Chemical Co. Ltd. (Tokyo, Japan), respectively. TBF
and 1-amonobenzotoriazol were purchased from Sigma-Aldrich (St. Louis, MO, USA), respectively. Propane (99.5%)
was purchased from GL Sciences Inc. (Tokyo, Japan). All
chemicals were purified reagent grade.

3. Results

1151A). In the presence of 10% propane in the air phase,about 50%, 20%, and 20% of initial MTBE were degradedby, M. chlorophenolicum TCE 28, M. duvalii TA5, and M.
gilvum TA27 in 6 d, respectively (Fig. 1 B). The highest level of MTBE degradation (80%) was observed by M. duvalii
TA5 by using a mixture of glucose, acetic acid, and succinicacid, each at 500 mg L–1, as the carbon and energy source.
Under the same culture conditions, ca. 70% and 60% of
MTBE were degraded by M. gilvum TA27 and M. chlorophenolicum TCE 28, respectively (Fig. 1C).
Fig. 2 shows the eﬀect of 10% propane, acetic acid, succinic acid, and glucose as carbon sources on the degradationof MTBE by M. duvalii TA5. MTBE at 15.0 μmol bottle–1
was degraded by 20%, 30%, 30%, and 87% in 7 days with
10% propane, acetic acid, succinic acid, or glucose. This result demonstrates that glucose is the best carbon source forthe degradation of MTBE by M. duvalii TA5.

3.1. Effects of carbon sources on MTBE degradation

3.2. Eﬀect of glucose and MTBE concentration on
MTBE degradation

The eﬀects of diﬀerent carbon sources on the ability of M.
duvalii TA5, M. gilvum TA27, and M. chlorophenolicum
TCE 28 to degrade MTBE are shown in Fig. 1. None ofthese three strains degraded MTBE in MM medium with
15.0 μmol MTBE bottle–1 (100 mg MTBE L–1) nor could
any utilize MTBE as the sole carbon and energy source (Fig.

Fig. 3 shows the eﬀect of glucose (100–2,000 mg L–1) and
MTBE concentration (15, 75 and 300 μmol bottle–1) on
MTBE degradation, cell yield (OD660) and pH by M. duvalii
TA5 in 7 d incubation. The growth of M. duvalii TA5 increased with the increase of glucose concentration. The
yield was about OD660 1.4 at 2,000 mg L–1 of glucose in 7 d.

Fig. 1. Effect of carbon sources on MTBE degradation by Mycobacterium duvalii TA5, Mycobacterium gilvum TA27, and Mycobacterium
chlorophenolicum TCE 28.
A, MM medium; B, MM medium with propane (10% in gas phase); C, MM medium with 500 mg L–1 each of glucose, acetic acid, andsuccinic acid. Symbols: ◆ , without microorganism; ● , M. duvalii TA5; ▲ , M. gilvum TA27; □ , M. chlorophenolicum TCE 28
Values are means (n=3), and error bars represent standard deviation.

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116

Good MTBE degradation was observed more than
1,000 mg L–1 of glucose. M. duvalii TA5 degraded 45% of
22.7 mmol MTBE L–1 (corresponding to 2,000 mg MTBE
L–1 or 300 μmol bottle–1) in 7 d with 500 mg L–1 of glucose,
while MTBE was completely degraded at low concentrationof 15 μmol bottle–1 with 1,000 mg L–1 of glucose.
The reason was not clarified why MTBE was not completely degraded at the high concentration.
3.3. Analysis of MTBE and MTBE metabolites

total ion chromatogram of MTBE and the MTBE metabolites resulting from MTBE degradation by M. duvalii TA5.
Peaks A, B, and C appeared after 3 and 4 d of degradation,
whereas peaks D and E occurred after 5 d. Peak B represents MTBE, and peak A was defined as TBA by the m/z
of 39, 41, and 59 (data not shown). Peak C was determined
to be TBF by the m/z of 41, 43, 56, 57, 59, and 87 (datanot shown). Peak E was determined to be HIBA by the m/z
of 40, 51, 69, 91, and 92 (data not shown); and peak D was
identified as HA by the m/z of 43, 45, 58 and 74 (data not

The MTBE degradation products were separated by solidphase extraction and analyzed by GC-MS. Fig. 4 shows theFig. 2. Effect of carbon sources on MTBE degradation by Mycobacterium duvalii TA5. Symbols: □ , without carbon source;△ , 10% of propane in gas phase; ▲ , 500 mg L–1 glucose; ○ ,500 mg L–1 acetic acid; ● , 500 mg L–1 succinic acid.
Values are means (n=3), and error bars represent standard
deviation.

Fig. 3. Effect of glucose and MTBE concentration on MTBE
degradation, yield and pH by Mycobacterium duvalii TA5 at
7 day incubation.
Symbols: MTBE (μmol bottle–1) ○ , 15; △ , 75; □ , 300

Fig. 4. Total ion chromatogram of MTBE degradation products produced by Mycobacterium duvalii TA5.

Biodegradation of MTBE by Mycobacterium

shown). Both TBF and TBA are products from the earlystage of MTBE degradation, whereas HIBA and HA are
products from the late stage of MTBE degradation.

117

3.6. Degradation of MTBE metabolites and theirdegradation products

Fig. 5 shows the time course of MTBE degradation andthe formation of MTBE metabolites, including TBF, TBA,
2-propanol, and acetone, by M. duvalii TA5 with glucose.
MTBE at 13.6 μmol bottle–1 was degraded by approximately
10% in 8 d. In this experiment, the MTBE degradation rate
was low. It seemed that the activity of inoculated M. duvalii
TA5 might be low. However, several metabolites were produced. After 2 d, 0.48 μmol bottle–1 TBF was produced andafter 2 more days, the production of TBA increased andreached 1.56 μmol bottle–1 after 8 d. Small amounts of
2-propanol (0.24 μmol bottle–1; m/z=45, 41, 27, and 59;
data not shown) and acetone (0.10 μmol bottle–1 ; m/z=15,
39, 43, and 58; data not shown) were present after 2 d.

To determine degradation of MTBE metabolites andtheir degradation products, metabolite spike tests were performed by using TBF, TBA, 2-propanol, and acetone(12 μmol bottle–1, respectively) in MM medium wituout glucose (Fig. 7). The production of TBA from TBF occurred,
and the slight growth of M. duvalii TA5 (DO660 nm=0.030)
suggested that M. duvalii TA5 assimilated TBF (Fig. 7A).
In the case of TBA, a small amount of acetone was produced (Fig. 7B). In the case of 2-propanol, slight degradation of 2-propanol and acetone production accompanied
slight growth of M. duvalii TA5 (DO660=0.012, data notshown) (Fig. 7C). Acetone degradation was not clearely observed (Fig. 7D). From resuits of Fig. 6 and 7, it seems thatM. duvalii TA5 is able to assimilate TBF, TBA, 2-propanol
and acetone without glucose.

3.5. Degradation of MTBE metabolites

3.7.

Fig. 6 shows the degradation of TBF, TBA, 2-propanol,
and acetone (12 μmol bottle–1, respectively) in MM medium
with and without glucose by M. duvalii TA5. A decrease in
the level of TBF of approximately 38% was observed without M. duvalii TA5 after 6 d (Fig. 6A). This finding is consistent with a report by Smith et al. (2003) that TBF is degraded by abiotic hydrolysis22). However, degradation of
TBF by 75% and 99% was observed by M. duvalii TA5
alone and with glucose, respectively, after 6 d (Fig. 6A). Although TBA was not degraded by M. duvalii TA5 in theabsence of glucose, about 50% of the TBA was degradedby M. duvalii TA5 in the presence of glucose after 4 d (Fig.
6B). M. duvalii TA5 degraded 2-propanol by ca. 82% in
the presence of glucose but only by ca. 14% in the absenceof glucose after 6 d (Fig. 6C). Also in the presence of glucose,
M. duvalii TA5 degraded acetone by ca. 33% (Fig. 6D).

Vmax and Km values of MTBE degradation for resting
cells of M. duvalii TA5 are shown in Fig. 8. The Vmax and
Km of MTBE degradation by M. duvalii TA5 were1,030 nmol min–1 mg dry cells-1 and 6.8 mM, respectively.

3.4. Time course of MTBE degradation

Vmax and Km values of MTBE degradation

3.8. MTBE degradation and TBA production in soil
Fig. 9 shows MTBE degradation and TBA production by
M. duvalii TA5 in garden soil. The initial amount of MTBE
was 5.6 μmol bottle–1. MTBE was degraded by approximately 7% in the absence of M. duvalii TA5 after 10 d.
However, in the presence of M. duvalii TA5 MTBE was degraded by approximately 60% after 10 d. Furthermore,
TBA production reached the maximum level of 1.9 μmol
bottle–1 in the presence of M. duvalii TA5 after 7 d and ca.
0.48 μmol bottle–1 in the absence of M. duvalii TA5 after
10 d.

Fig. 5. MTBE degradation and metabolite produced by Mycobacterium duvalii TA5 in MM medium with 500 mg L–1 glucose and 12 μmol
bottle–1 MTBE.
Symbols: ■ , MTBE; ▲ , TBF; ◆ , TBA; □ , 2-propanol; ○ , acetone.
Values are means (n=3), and error bars represent standard deviation.

OHKUBO et al.

118

Fig. 6. Degradation of MTBE metabolites TBF, TBA, 2-propanol, and acetone by Mycobacterium duvalii TA5.
Symbols: ○ , with glucose (500 mg L–1); □ , without glucose; △ , without M. duvalii TA5 and glucose.
Values are means (n=3), and error bars represent standard deviation.

4. Discussion
Several strains of bacteria utilize MTBE as an energy andcarbon source 6,8,16,20). Some MTBE-degrading bacteria areunable to use MTBE as an energy and carbon source but
can cometabolically degrade MTBE in the presence of pentane, n-alkane, or propane5,7,24). In the present study, M. duvalii TA5 cometabolically degraded MTBE in the presenceof 10% propane in the headspace of the culture bottle. M.
duvalii TA5 may utilize an alkane-degrading enzyme, suchas propane monooxygenase, to degrade MTBE. Some Mycobacterium has cytochrome P-45019). M. duvalii TA5 isable to degrade MTBE with glucose. In this case, MTBE
degaradation might be caused by the production of cytochrome P-450.
In the present study we determined the rates of MTBE

degradation by various bacteria and calculated the Vmax
and Km (Table 1). Proteobacteria sp. PM1 is reported toassimilate MTBE at a rate of 970 nmol d–1 mL–1 6). Threeother bacterial strains, Methylobacterium mesophilium
isolate 24, Rhodococcus sp. isolate 33, and Artherobacter
ilicis isolate 41, also are capable of assimilating MTBE andwere reported to degrade 200 mg L–1 MTBE by 29%(47.0 nmol d–1 mL–1), 28% (45.4 nmol d–1 mL–1), and 28%(45.4 nmol d–1 mL–1), respectively, after 2 wk16). Here, M.
duvalii TA5 cometabolocally degraded 45% of 2,000 mg
MTBE L–1 (22.7 mmol MTBE L–1) after 7 d (1,460 nmol
day–1 mL–1) in the presence of 500 mg L–1 glucose. Therefore, M. duvalii TA5 degraded MTBE at a rate 1.5 times
higher than that of Proteobacteria sp. PM1 and about 30
times higher than those of M. mesophilium isolate 24,
Rhodococcus sp. isolate 33, and A. ilicis isolate 41. MTBE

Biodegradation of MTBE by Mycobacterium

119Fig. 7. Degradation of MTBE metabolites and their degradation products by Mycobacterium duvalii TA5.
M. duvalii TA5 was cultured in MM medium without glucose and with 12 μmol bottle–1 of metabolites. A, TBF; B, TBA; C, 2-propanol;D, acetone. Symbols: ▲ , TBF; ◆ , TBA; □ , 2-propanol; ○ , acetone.
Values are means (n=3), and error bars represent standard deviation.

was degraded cometabolically by Nocardia spp., Mycobacterium vaccae JOB5 ATCC 29678, Xanthobacter sp.,
Graphium sp. ATCC 58400, and Arthrobacter sp. ATCC
27778 at rates of 4.6, 12.2, 25.5, 0.18, and 3.39 nmol min–1
mg dry cell–1, respectively. By comparison, the rate of
MTBE degradation by M. duvalii TA5 was markedly higher, at 1,030 nmol min–1 mg dry cell–1. The Vmax for MTBE
degradation by M. duvalii TA5 was the highest among thebacterial strains assessed in the present study.
The rate of MTBE degradation by M. duvalii TA5 waslow (3.2 nmol min–1 mg dry cell–1) at a low concentration of
MTBE (0.01 mM). M. duvalii TA5 does not have the ability to degrade MTBE at low concentrations (data notshown). The reason for this phenomenon is related to thedegradation kinetics. The Km value of M. duvalii TA5 for
MTBE is 6.8 mM, which is ca. 7, 5, and 3 times higher thanthe Km values of Mycobacterium vaccae (0.95 mM)10), Mycobacterium vaccae JOB5 ATCC 2967 (1.4 mM)21), and
Xanthobacter sp. (2.4 mM)10) for MTBE. The higher Km
value of M. duvalii TA5 for MTBE compared with otherbacterial strains indicates that M. duvalii TA5 has a lower

Fig. 8. Determination of Vmax and Km on MTBE degradation by
Mycobacterium duvalii TA5.

aﬃnity for MTBE than do other bacteria.
We determined several MTBE metabolites. The MTBE
degradation metabolites TBF, TBA, HIBA, and HA wereanalyzed by solid-phase extraction (Fig. 4). The productionof TBF, TBA, 2-propanol, and acetone was quantified byheadspace GC-MS analyses (Fig. 5). Our proposed pathway
for the degradation of MTBE by M. duvalii TA5 is illustrated in Fig. 10. MTBE is degraded via the degradation intermediates TBF, TBA, HIBA, 2-propanol, acetone, and HA.
The following degradation pathways have been reported
for various strains of bacteria. Gordonia terrae IFP 2007 isable to degrade MTBE to TBA4). Graphium sp.10), Graphium sp. ATCC 584007), and Pseudomonas mendocina KR122) have the ability to degrade MTBE via TBF to TBA.
Mycobacterium vaccae JOB5 degrades MTBE to TBF,
TBA, and 2-methyl-1,2-propanediol21). Mycobacterium
austroafricanum IFP 2012 degrades MTBE via TBF and
TBA to HIBA and CO23). Steﬀan et al. (1997) demonstrated
that the native propane-oxygenating bacteria strain ENV425(ATCC 55798) degraded MTBE via the intermediates TBF,
TBA, 2-methyl-2-hydroxy-1-propanol, HIBA, 2-propanol,
acetone, HA, and pyruvic acid24). Here, we show that M.
duvalii TA5 degrades MTBE via TBF, TBA, HIBA, 2-propanol, acetone, and HA. This MTBE degradation pathway
is similar to the degradation pathway reported for ENV425.
Although we did not identify the degradation intermediates
Resting cell
Growing cell
Resting cell

Arthrobacter sp. ATCC 27778

Mycobacterium duvalii TA5

Mixed bacterial culture BC-1
Graphium sp. ATCC 58400

Growing cell
Growing cell
Growing cell
Growing cell
Resting cell
Resting cell–
Resting cell
Resting cell
Resting cell
Resting cell
Resting cell

Cometabolic
Cometabolic

Cometabolic

Heterotrophic
Heterotrophic
Heterotrophic
Heterotrophic
Cometabolic
Cometabolic
Cometabolic
Cometabolic
Cometabolic
Cometabolic
Heterotrophic
Cometabolic

Culture condition Type of metabolism

Proteobacteria sp. PM1
Methylobacterium mesophilicum Isolate 24
Rhodococcus sp. Isolate 33
Arthrobacter ilicis Isolate 41
Nocardia sp. ENV421
Nocardia sp. ENV425 ATCC 55798
Mycobacterium vaccae JOB5 ATCC 29678
Mycobacterium vaccae JOB5 ATCC 29678
Mycobacterium vaccae
Xanthobacter sp.

Organism

Fig. 9. MTBE degradation and TBA production by Mycobacterium
duvalii TA5 in soil. Symbols: ○ , with M. duvalii TA5; □ ,without M. duvalii TA5.
Values are means (n=3), and error bars represent standard
deviation.
Glucose or Propane
Glucosen-Butane

MTBE
MTBE
MTBE
MTBE
PropanePropanePropanePropanePropanePropaneMTBE
n-Butane

Metabolic or cometabolic
carbon sources

MTBE degradation rate

6.4 nmol min–1 mg dry cells–1
10.5 nmol h–1 mg dry weight–1 (0.18 nmol min–1 mg dry cells–1)
Vmax=0.43 mg–1 mg suspended solids–1 d–1 (3.39 nmol–1 min–1
mg dry cells–1), Km=2.14 mg L–1 (0.02 mM)
45% of 22.7 mmol L–1 in 7 d (1460 nmol d–1 mL–1)
Vmax=1030 nmol min–1 mg dry cells–1, Km=6.8 mM

970 nmol d–1 mL–1
29% of 200 mg L–1 in 2 wk (47.0 nmol d–1 mL–1)
28% of 200 mg L–1 in 2 wk (45.4 nmol d–1 mL–1)
28% of 200 mg L–1 in 2 wk (45.4 nmol d–1 mL–1)
9.2 nmol min–1 mg protein–1
4.6 nmol min–1 mg protein–1
Not reported
Vmax=24.4 nmol min–1 mg protein–1, Km=1.4 mM
Km=0.95 mM
Vmax=51 nmol min–1 mg protein–1, Km=2.4 mM

Table 1. Summary of MTBE degradation rates of various bacteria

This study

14

19
7

10

20

23

16

6

Reference

120
OHKUBO et al.

Biodegradation of MTBE by Mycobacterium

121

Fig. 10. Proposed degradation pathways of MTBE by Mycobacterium duvalii TA5.

2-methyl-2-hydroxy-1-propanol and pyruvic acid, we didconfirm HA, 2-propanol, and acetone as MTBE degradation products.
Many studies highlight the potential of MTBE-degrading
bacteria for the bioremediation of MTBE-contaminated soil
and groundwater. Charathirakup et al. (2006) reported that
0.3 mg MTBE was degraded in a 10 g soil microcosm after
7 d incubation with the bacterial consortia mKGS1, consisting of 6 bacterial strains2). In an in situ bioaugmentation
study, Hristova et al. (2003) reported that 0.11 mM(1.32 μmol 12 mL–1) MTBE was completely degraded after
25 d by adding the MTBE-degrading bacteria PM1 with ox-ygen to MTBE-polluted groundwater at the Vandenberg Air
Force Base (Lompoc, California)9). Spinnler et al. (2001) investigated the addition of a mixture of aerobic MTBE-degrading bacteria and oxygen for 6 month by means of abiobarrier system to the groundwater of a retail gasoline
station polluted with MTBE in California23).
In this experiment, we showed that M. duvalii TA5 degraded 53% of 5.6 μmol MTBE in bottle containing 13 g
soil after 10 d. This MTBE degradation rate is almost sameas the previously reported rates of MTBE-degrading bacteria.
We demonstrated that M. duvalii TA5 is useful for thebioremediation of MTBE-contaminated soil and groundwater.

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122

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