Journal of Environmental Biotechnology
Vol. 12, No. 1, 39–46, 2012

Original paper (regular paper)

Biodegradation of Ethyl tert-Butyl Ether (ETBE)
by Rhodococcus erythropolis ET10
Norio Ohkubo1 and Osami Yagi2*
Department of Water and Sewage Works, Enterprise Bureau of Hitachi City,
4–4–1 Moriyama, Hitachi, Ibaraki 316–0025, Japan2
Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University,
1–2–1, Izumi-cho, Narashino-shi, Chiba 275–8575, Japan1* TEL: +81–(0)47–474–2574* E-mail: yagi.osami@nihon-u.ac.jp(Received; 24 April, 2012/Accepted; 28 June, 2012)
We isolated an ethyl tert-butyl ether (ETBE)-degrading soil bacterium, designated strain ET10, which was identified as
Rhodococcus erythropolis based on morphological, physiological, and 16S rRNA gene analyses. Rhodococcus erythropolis
ET10 was able to utilize ETBE as a sole source of growth energy and carbon. Twenty micromoles of ETBE in a 60-mL
bottle containing 10 mL growth medium was completely degraded by strain ET10 in 14 days. Approximately 65% of ETBE
was degraded after 19 days in cultures containing 50 μmol of ETBE bottle-1. We determined that tert-butyl alcohol (TBA)
was the main metabolite in these cultures. The maximum degradation rate (Vmax) and half-saturation constant (Km) of ETBE
were 7.80 μmol mg dry cells–1 h–1 and 2.5 mM, respectively. Strain ET10 was also able to degrade ETBE in four types of soil.
Our results suggest that strain ET10 may be useful for the bioremediation of ETBE-contaminated soil.
Key words: ETBE, TBA, Rhodococcus erythropolis, Vmax, Km

1. Introduction
Ethyl tert-butyl ether (ETBE) is an oxygenate additivethat increases the octane rating of gasoline and has beenused as a substitute for methyl tert-butyl ether (MTBE) in
France, Spain, and Germany. ETBE is manufactured from
isobutylene and ethanol, which is often derived from plants,
including corn and sugar cane9,10). The use of ETBE derived
from bioethanol contributes to reducing the levels of carbon
dioxide in the exhaust of gasoline vehicles and is expected
to help limit global warming. However, ETBE has toxic
eﬀects on the livers of male and female mice16), although thedata of acute toxicity and carcinogenicity are not enoughin the present state of aﬀair15), and ETBE contamination
has been detected in the drinking water of several areasin the Netherlands21) and in groundwater in Düsseldorf,
Germany19). As ETBE has poor biodegradation characteristics, it takes long time to clean up the ETBE-contaminatedsoil and underground water. There are several reports on the
ETBE assimilating-bacteria belonging to Gordonia terrae4,8),
Comamonas testosteroni12), Rhodococcus equi4), Rhodococcus aetherivorans1), and Aquincola tertiaricarbonis18).
Therefore, the mineralization of ETBE by bacteria represents
an eﬀective method for the elimination of ETBE1). However,there is little report on the application of microorganisms toclean up the ETBE contaminated environment.
In the present study, we isolated and characterized an

ETBE-degrading bacterium from soil. Following the identification of this strain, its ETBE degradation products andactivities were evaluated. Moreover, ETBE degradation andtert-butyl alcohol (TBA) production were investigated in fourtypes of soil amended with this strain.
2. Materials and methods
2.1. MediaMM medium was used for culturing bacteria present in
the soil samples and contained the following components(in mg L–1): NH4Cl, 2,140; K2HPO4, 1,170; KH2PO4, 450;
MgSO4·7H2O, 120; FeSO4·7H2O, 28; Ca (NO3)2·4H2O, 4.8;
CaCO3, 100; MnSO4·4-6H2O, 0.6; H3BO3, 0.05; ZnSO4·7H2O,
0.1; (NH4)6Mo7O24·4H2O, 0.01; Co(NO3)2·6H2O, 0.6;
NiSO4·7H2O, 0.06; and CuSO4·5H2O, 0.06; in Milli-Q water.
Prepared MM medium was sterilized by autoclaving at 121°C
for 20 min.
2.2. Chemicals
ETBE was purchased from Tokyo Chemical Industry Co.,
Ltd. (Tokyo, Japan). TBA and lactose were purchased from
Junsei Chemical Co., Ltd. (Tokyo, Japan). Reagent-gradeagar, lactose, starch, sodium acetate trihydrate, sodium
succinate, glycerol, sucrose, and glucose were purchasedfrom Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
TaKaRa Ex Taq® HS was purchased from (TaKaRa Bio

40

Ohkubo and Yagi

Inc., Siga, Japan). Purification of PCR products was used by
ExoSAP-IT (GE Healthcare Tokyo, Japan). BigDye® Terminators v1.1 Cycle Sequencing Kit was used as sequencing
reagent (Applied Biosystems, Tokyo Japan). Purificationof the sequencing reaction was carried out by illustraTM
AutoSeq G-50 Dye Terminator Removal Kit (GE Healthcare, Tokyo Japan).
2.3. Isolation and identification of ETBE-degrading
bacteria
Ten soil samples were collected from various locations in
Ibaraki Prefecture, Japan. For each sample, 0.1 g of soil wasplaced into a 60-mL vial containing 10 mL MM medium
supplemented with 5 μmol ETBE and 5 mg glucose. The
vials were sealed with butyl rubber caps and crimped withaluminum rings, and then incubated at 30°C with shaking
at 120 rpm for approximately 10 to 40 days. ETBE degradation was evaluated using the headspace gas method anda gas chromatography-mass spectrometry (GC-MS) system(GC-2010/GCMS-QP2010; Shimadzu Co., Kyoto, Japan)
equipped with a Rtx®-5MS capillary column (0.25 mm
i.d.×30 m; Restek Co., PA, USA). For measurement of ETBE
or TBA concentration, 50 μl of headspace gas was injected
into GC-MS in all experiments. ETBE or TBA-concentration
was calculated by use of calibration curve as a concentration
per bottle. ETBE concentration in water was calculated byusing Henry’s constant of 1.62 L atm mol–1 (at 30°C).
Following the long-term incubation period, 0.1 mL of thegrowth cultures was streaked onto MM agar plates (2% agar)
by use of a spreader. Ten microliters of ETBE (undiluted solution) was added to the surface of the agar plate. The plates
were then sealed with Parafilm M® film (Structure Probe,
Inc. and SPI Supplies, PA, USA) and incubated statically for
3 days at 30°C. Growing individual colonies were selected
and inoculated into 60-mL vials containing 10 mL MM
medium supplemented with 5 μmol ETBE (5 μmol ETBE
bottle–1). The vials were incubated at 30°C with shaking at
120 rpm for 7 days. ETBE degradation was evaluated using
the GC-MS headspace gas method, as described above. To
isolate individual strains, one μL of ETBE-degrading culture
was streaked onto an MM agar plate to which 10 μL ETBE
was added. The purified colonies were then maintained onagar slant cultures. From this process, one strain was purified
and was named as ET10. Identification of the microorganism
was performed by testing for oxidation/fermentation (OF)of glucose, oxidase, catalase, motility, and spore formation,
and Gram staining, colony morphology, and 16S rRNA gene
analyses.
Genomic DNA was extracted by the method of Molecular Cloning of Sambrook et al. For the sequence analysis, the 16SrRNA gene was amplified by PCR using 27FP(GAGTTTGATCMTGGCTCAG), 518RFP (CCAGCAGC
CGCGGTAAT) and 1392RP (ACGGGCGGTGTGTRC) as
the primers. The PCR mixture (50 μl total volume) consisted of 200 μM of each primer, 200 μM of deoxynucleotide
phosphate, TaKaRa Ex Taq® HS DNA polymerase 0.25 μl,

the DNA sample and PCR buﬀer (10 mM Tris-HCl, pH 8.3,
50 mM KCl and 1.5 mM MgCl2). The DNA amplification
was conducted by use of thermal cycler TaKaRa MP3000(TaKaRa Bio Inc., Siga, Japan), at one cycle 94°C 3 min., 30
cycle 94°C 30 sec. 55°C 1 min. 72°C 1 min. and last one cycle 72°C 7 min. DNA sequence was measured by ABI Prism®
3100 Genetic Analyzer (Applied Biosystems, Tokyo Japan).
Phylogenetic tree was plotted by a NCBI BLAST search.
2.4. Determination of ETBE degradation products
To investigate the ETBE degradation products produced
by strain ET10, approximately 0.1 mg of strain ET10 was
inoculated into a 60-mL vial containing 10 mL MM medium
supplemented with 5 μmol ETBE. The culture was incubatedat 30°C with shaking at 120 rpm for 6 days, and ETBE and
its degradation products were determined by the headspace
GC-MS method.
2.5. Eﬀect of ETBE concentration on its degradation
Approximately 0.1 mg of strain ET10 was inoculated into
a 60-mL vial containing 10 mL MM medium supplemented
with 2.5 to 50 μmol ETBE. After sealing the vials withbutyl rubber caps and crimping with aluminum rings, thecultures were incubated at 30°C with shaking at 120 rpm for
19 days. ETBE degradation and TBA production were evaluated by the headspace GC-MS method. Bacterial growth
was measured turbidimetrically at 660 nm (OD660) using aspectrophotometer (UV Mini-1240; Shimadzu Co., Kyoto,
Japan) and was converted to dry weight (mg L–1).
2.6. Eﬀect of carbon substrate on specific growth rate
and maximum yield
Approximately 0.3 mg of strain ET10 was inoculated into
a 60-mL vial containing 10 mL MM medium supplemented
with 5 μmol ETBE and 500 mg L–1 of one of the following
test organic substrates: glucose, lactose, sucrose, starch, acetic
acid, succinic acid, or glycerol. The culture was incubated at
30°C with shaking at 120 rpm for 9 days. Bacterial growth
was measured turbidimetrically at 660 nm (OD660) using aspectrophotometer.
Conversion eﬃciency (C) from substrate concentration(S) to maximum cell yield (Y) was calculated as follows.
C (%)=Y (mg l–1)/S (mg l–1)×100
2.7. Kinetics of ETBE degradation
Strain ET10 was pre-cultured in a 60-mL vial containing
10 mL MM medium supplemented with 50 μmol ETBE at
30°C for 7 days. The culture was centrifuged at 1,400×g for
30 min and then washed five times with phosphate buﬀer(pH 7.2). Kinetic experiments were performed using resting
cells. Five milligrams of washed strain ET10 cells was inoculated in 10 mL phosphate buﬀer containing 0.5 to 100 μmol
ETBE (0.5 to 100 μmol bottle–1), and the resulting culture
was incubated at 30°C for 7 h with shaking at 120 rpm. To
stop the ETBE degradation reaction, 200 μL of 1,000 mg L–1
hydrazine was added to the culture, and ETBE level was thenBiodegradation of ETBE by R. erythropolis

41

Fig. 1. 16S rRNA dendrogram showing the phylogenetic position of the ETBE-degrading soil isolate, strain ET10. The numbers above thebranching points represent the bootstrap values (expressed as percentages). The bar equals 0.002 inferred sequence divergency.

measured by GC-MS. The maximum ETBE degradation rate
(Vmax; μmol mg dry cells–1 min–1) and half-saturation constantof ETBE (Km; mM) were determined from Lineweaver-Burkplots.
2.8. ETBE degradation and TBA production in four soil
types
ETBE degradation and TBA production by strain ET10
was evaluated using four types of soil: fine sand (Hokota
City, Ibaraki Prefecture, Japan), clay (Mashiko Town,
Tochigi Prefecture, Japan), Andosol (Kanuma City, Tochigi
Prefecture, Japan) and Kanuma soil (Kanuma City, Tochigi
Prefecture, Japan). For each sample, 11.6 g of soil, 5 μmol
ETBE, and approximately 2.8 mg of strain ET10 were added
to each 60-mL vial. The soil microcosms were incubated at
30°C with shaking at 120 rpm for 21 days. ETBE degradation and TBA production were determined by the headspace
GC-MS method.
2.9. Eﬀect of organic substances on ETBE degradation
and TBA production in soil
The eﬀects of organic substances on ETBE degradation
and TBA production by strain ET10 were investigated using
Andosol soil collected in Kanuma City, Tochigi Prefecture,
Japan. For the analysis, 11.6 g of soil sample, 5 μmol ETBE,
10 mL of a 500 mg L–1 solution of either glucose, starch,glycerol, or lactose, and 0.7 mg of strain ET10 were added to
60-mL vials. These soil microcosms were incubated at 30°C
with shaking at 120 rpm for 19 days, and ETBE degradation
and TBA production were then determined by the headspace
GC-MS method.
3. Results
3.1. Isolation and identification of an ETBE-degrading
bacterium
An ETBE-degrading bacterium was isolated from soil
and designated strain ET10. The isolated strain was Gram
positive, non-motile and non-spore forming, and appeared
coccoid to rod-shaped. Formed colonies were smooth andpale white in color. Strain ET10 was negative for oxidationand fermentation in the OF test. Tests for oxidase and cata-Fig. 2. ETBE and its degradation products produced by strain ET10.

lase were negative and positive, respectively.
A phylogenetic tree was constructed based on the 16S
rRNA sequences of strain ET10 and the most closely related
bacterial species (Fig. 1). Strain ET10 displayed the highest
homology to Rhodococcus erythropolis DSM 43066T(X79289) (99.9%) (Fig. 1). From these characteristics, it wasdetermined that strain ET10 was a R. erythropolis.
3.2. ETBE degradation products
Fig. 2 shows the gas chromatogram of headspace gas after
culturing strain ET10 for 21 days in medium supplemented
with ETBE. The retention time of TBA standard was similar
to that of the main ETBE degradation product (4.2 min).
Moreover, typical MS peaks at 29, 31, 41, 43, and 59 m/z
for TBA standard were observed for the TBA metabolite
produced by strain ET10 (data not shown). Therefore, it
was confirmed that the main ETBE degradation product was
TBA. In addition to the main ETBE degradation product,a peak that eluted at a retention time of approximately
8.4 min was observed (Fig. 2); however, this substance was
not identified.

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Ohkubo and Yagi

3.3. Eﬀect of ETBE concentration on ETBE degradation
The eﬀects of ETBE concentration on its degradation,
TBA production, and cell yield were next examined (Fig. 3).
ETBE at 2.5, 5, 10, and 20 μmol bottle–1 was completelydegraded by strain ET10 in 6, 14, 19, and 14 days, respectively. At 50 μmol ETBE bottle–1, approximately 65% of
ETBE was degraded in 19 days. The degradation rate decreased with increasing ETBE concentration.
TBA production increased with the increase of incubation time. Maximum TBA production by strain ET10 was

observed after 6 days at low ETBE concentrations (2.5, 5,
and 10 μmol ETBE bottle–1) and after 14 and 19 days at thehighest examined ETBE concentrations (20 and 50 μmol
ETBE bottle–1) (Fig. 3). The levels of TBA increased withincreasing ETBE concentration. However, TBA was notdegraded by strain ET10. At 2.5, 5, 10, 20, and 50 μmol
ETBE bottle–1, maximum cell yields of 53, 56, 69, 88, and
104 mg L–1, respectively, were observed. Bacterial cell yield
increased with increasing concentration of ETBE added tothe cultures (Fig. 3).

Fig. 3. Eﬀect of ETBE concentration on the degradation of ETBE, production of TBA, and growth of strain ET10.
○ , ETBE; △ , TBA; and □ , Yield
Values are presented as means (n=3), and error bars represent standard deviation.

Biodegradation of ETBE by R. erythropolis

43

Table 1. Eﬀect of carbon substrate on the specific growth rate and maximum yield of Rhodococcus erythropolis ET10.
Substrates

Specific growth rate (day–1)

Maximum cell yield (mg L–1)

Conversion eﬃciency (%)

0.10
0.08
0.13
0.14
0.15
0.15
0.18
0.25

104
9
94
53
223
274
236
117

20.4
1.8
18.8
10.6
44.6
54.8
47.2
23.4

ETBE (50 μmol 10 mL =ca. 510 mg L )Lactose (500 mg L–1)
Succinic acid (500 mg L–1)
Starch (500 mg L–1)
Glucose (500 mg L–1)
Glycerol (500 mg L–1)
Sucrose (500 mg L–1)
Acetic acid (500 mg L–1)
–1

–1

3.4. Specific growth rate and yield
Table 1 shows the eﬀects of organic substances on thespecific growth rate (day–1) and maximum cell yield (mgL–1) of strain ET10. The highest specific growth rate was
0.25 day–1 in cultures supplemented with acetic acid, while
the highest cell yield of 274 mg L–1 was observed with glycerol. Cultures of strain ET10 supplemented with glycerol
also displayed the highest conversion eﬃciency of cell yield(54.8%). The conversion eﬃciency of ETBE (20.8%) was
similar in the presence of acetic acid (23.4%) and succinicacid (18.8%).
3.5. Vmax and Km of strain ET10
Fig. 4 shows a Lineweaver-Burk plot determined by
the ETBE degradation rate (μmol mg dry cells–1 min–1)–1of strain ET10 at various ETBE concentrations (mM)–1.
The maximum degradation rate (Vmax) and half-saturation
constant (Km) of ETBE were 0.13 μmol mg dry cells–1 min-1(7.80 μmol mg dry cells–1 h–1) and 2.5 mM, respectively.
3.6. ETBE degradation and TBA production in soil
ETBE degradation and TBA production by strain ET10
were examined in four types of soil (Fig. 5). Strain ET10
completely degraded the 5 μmol ETBE added to the finesand, clay, Andosol, and Kanuma soils after 4 days. TBA
production increased with ETBE degradation and reached amaximum at 7 days for four soil types. TBA was completelydegraded in the fine sand, clay, and Kanuma soils by 21 days.
In the absence of strain ET10, a slight reduction of ETBE
was observed in the four soil types; however, TBA production was not detected. The reduction of ETBE was likely
caused by its absorption to soil particles.
Fig. 6 shows the eﬀect of organic substances on ETBE
degradation by strain ET10 in Andosol. In the absence oforganic substances, ETBE was degraded within 13 days.
ETBE was degraded in 2, 4, 9, and 9 days in Andosol supplemented with glycerol, glucose, lactose, and starch, respectively(Fig. 6A). The ETBE degradation rate by strain ET10 clearly
increased following the addition of organic substances. TBA
generated as a metabolite was also degraded (Fig. 6B).

Fig. 4. Determination of Vmax and Km for ETBE degradation bystrain ET10.

4. Discussion
Numerous microorganisms, including Gordonia terrae IFP
20014,8), Comamonas testosteroni E112), Centre for Disease
Control group A-5 E212), a microbial consortium13), Rhodococcus equi IFP 20024), Rhodococcus aetherivorans IFP
20171), Aquincola tertiaricarbonis L10818), and a mixture offilamentous microorganisms11), are able to assimilate ETBE
as an energy and carbon source (Table 2). Here, we isolated
an ETBE-assimilating bacterium, strain ET10, which was
identified as R. erythropolis. Among strains of R. erythropolis identified to date, ET10 is the first that is capableof assimilating ETBE. It is well known that members of
the genus Rhodococcus have high metabolic versatility andcatabolize a wide range of compounds. Thus, Rhodococcus
spp., such as strain ET10, may be useful for the bioremediation of ETBE and other harmful chemicals in contaminatedenvironments.
The degradation rates of ETBE by G. terrae IFP 2001 andR. equi IFP 20024) are 3.72 and 1.37 μmol mg protein–1 h–1,
respectively. We determined that the degradation rate of
ETBE by strain ET10 is 7.80 μmol mg dry cells–1 h–1(corresponding to 15.6 μmol mg protein–1 h–1), a value that
represents the highest ETBE degradation rate among pure

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Ohkubo and Yagi

Fig. 5. ETBE degradation and TBA production by strain ET10 in four types of soil.
○ , ETBE concentration with strain ET10; ● , TBA concentration with strain ET10; △ , ETBE concentration without strain ET10; ▲ ,TBA concentration without strain ET10. Values are presented as means (n=3), and error bars represent standard deviation.

Fig. 6. Eﬀect of organic substances on the degradation of ETBE (A) and production of TBA (B) by strain ET10 in Andosol.
◆ , Without organic substances; ■ , glucose; ▲ , starch; ◇ , glycerol; □ , lactose

cultures of ETBE-assimilating bacteria reported to date
(Table 2).
It has been demonstrated that Mycobacterium vaccae
JOB5 co-metabolically degrades ETBE with propane or
2-propanol at rates of 1,250 and 1,000 μg L–1 h–1, respec-tively20). Moreover, propane-oxidizing strain ENV425
co-metabolically degrades ETBE with propane, 2-propanol,
ethanol or acetone at rates of 324, 174, 174, and 50 μg L–1 h–1,
respectively20). Another propane-oxidizing strain, ENV420, is
capable of co-metabolically degrading ETBE in the presenceBiodegradation of ETBE by R. erythropolis

45

Table 2. Summary of ETBE degradation rates of various bacteria.
Microorganism
Gordonia terrae IFP 2001
Gordonia terrae IFP 2001
Comamonas testosteroni E1
Centre for disease control group A-5 E2Microbial consortium
Rhodococcus equi IFP 2002
Rhodococcus aetherivorans IFP 2017
Aquincola tertiaricarbonis L108
Mixture of filamentous microorganisms(batch tests)
Mixture of filamentous microorganisms(upflow fixed-bed reactor)
Rhodococcus erythropolis ET 10
Mycobacterium vaccae JOB5

Type ofmetabolism

Metabolic or
cometabolic
carbon sources

Assimilation
Assimilation
Assimilation
Assimilation
Assimilation
Assimilation
Assimilation
Assimilation
Assimilation

ETBE
ETBE
ETBE
ETBE
ETBE
ETBE
ETBE
ETBE
ETBE

Vmax=3.72 μmol mg protein–1 h–1

0.12 μmol mg dry cells–1 h–1
Vmax=1.11 μmol mg dry cells–1 h–1
Vmax=0.21 μmol mg dry cells–1 h–1

4)
8)
12)
12)
13)
4)
1)
18)
11)

Assimilation

ETBE

Vmax=0.06 μmol mg dry cells–1 h–1

11)Assimilation

ETBE

Vmax=7.80 μmol mg dry cells–1 h–1
100% of 167 mg L–1 for 14 days (497 μg L–1 h–1)

This study

Propane2-Propanol
Propane2-Propanol
Ethanol
AcetoneEthanol
Nutrient broth
Nutrient broth
Nutrient brothn-pentane

100% of 30 mg L-1 for 24 h (1,250 μg L–1 h–1)
80% of 30 mg L-1 for 24 h (1,000 μg L–1 h–1)
26% of 30 mg L–1 for 24 h (324 μg L–1 h–1)
14% of 30 mg L-1 for 24 h (174 μg L–1 h–1)
14% of 30 mg L-1 for 24 h (174 μg L–1 h–1)
4% of 30 mg L-1 for 24 h (50 μg L–1 h–1)
31% of 30 mg L-1 for 24 h (390 μg L–1 h–1)
18% of 100 mg L-1 for 30 days (25 μg L–1 h–1)
25% of 100 mg L-1 for 30 days (35 μg L–1 h–1)
23% of 100 mg L-1 for 30 days (32 μg L–1 h–1)
Vmax=0.78 μmol mg protein–1 h–1

20)
20)
20)
20)
20)
20)
20)
2)
2)
2)
17)

Cometabolism
Cometabolism
Propane oxidizing bacterial strain ENV425 Cometabolism
Cometabolism
Cometabolism
Cometabolism
Propane oxidizing bacterial strain ENV420 Cometabolism
MTBE degradable consortia mKMS
Cometabolism
MTBE degradable consortia mKGS1
Cometabolism
MTBE degradable consortia mKGS2
Cometabolism
Pseudomonads containing consortium
Cometabolism

ETBE degradation rate

3.54 μmol mg protein–1 h–1
0.03 μmol mg protein–1 h–1
0.02 μmol mg protein–1 h–1
0.93 μmol mg protein–1 h–1
Vmax=1.37 μmol mg protein–1 h–1

Reference

Vmax: Maximum degradation rateof ethanol at a rate of 390 μg L–1 h–1. The ETBE-degrading
consortia mKMS, mKGS1, and mKGS2 co-metabolicallydegrade ETBE with nutrient broth at rates of 25, 35, and32 μg L–1 h–1, respectively2). As the degradation rate of ETBE
by strain ET10 was found to be 497 μg L–1 h–1, it is clearthat strain ET10 has a relatively high ETBE degradation
capacity.
Several studies have investigated ETBE degradation
enzymes. Notably, cytochrome P-450 has been demonstrated
to function as a catalyst for monooxygenase in G. terrae IFP
2001 after induction with ETBE3,8). It is reported that R.
ruber IFP 2001, R. zopfii IFP2005, and Gordonia sp. IFP
2009 are able to degrade ETBE with cytochrome P-450 and
CYP24914). Although cytochrome P-450 appears to play animportant role in ETBE degradation, we could not detect
cytochrome P-450 in strain ET10. Therefore, further investigation of the mechanisms of ETBE oxidation, including therole of cytochrome P-450, are needed.
In MM medium, strain ET10 was able to degrade ETBE
but not to degrade TBA that was derived from ETBE (Fig. 3).
However, in the case of the four examined soil microcosms,
ETBE and its main metabolite TBA were degraded withstrain ET10 (Fig. 5).
R. aetherivorans IFP 2017, G. terrae IFP 2001, and R.
equi IFP 2002 also transform ETBE to TBA, whereas TBA
is not utilized by these bacteria1), as the tertiary alcohol

structure of TBA is resistant to degradation4,5). In contrast,
Mycobacterium austroafricanum IFP 20156) and 20127),
and Aquincola tertiaricarbonis L108 are able to grow on
TBA18). As we observed TBA degradation in soil, it is possiblethat indigenous TBA-degrading bacteria are present in thefour soil types examined here. Notably, glycerol, glucose,lactose, and starch promoted ETBE degradation by strain
ET10 (Fig. 6). Taken together, our results suggest that strain
ET10 may be useful for the clean-up of ETBE-contaminatedenvironments, particularly soil, where organic substances arelimited.

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