Journal of Environmental Biotechnology
Vol. 17, No. 1, 73–81, 2017

Original paper (regular paper)

Complete Degradation of Ethyl tert-Butyl Ether by Co-culture of Rhodococcus
erythropolis ET10 and Pseudonocardia benzenivorans No. 8
Norio Ohkubo1, Shun Tsuboi2, Shigeki Yamamura2, Kazuhiro Iwasaki2 and Osami Yagi3*
1

2

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

Center for Regional Environmental Research, 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

3* TEL: +81–(0)47–474–2574* E-mail: yagi.osami@nihon-u.ac.jp(Received: 4 April, 2017/Accepted: 26 April, 2017)
Rhodococcus erythropolis ET10 isolated from soil degraded ethyl tert-butyl ether (ETBE) and tert-butyl acetate (TBAc)
and produced tert-butyl alcohol (TBA). However, metabolite TBA was not further degraded. We also isolated a TBAdegrading bacterium from andosol, which was identified as Pseudonocardia benzenivorans No. 8 (strain No. 8). Strain
No. 8 degraded 20 mM TBA. Organic substances such as glucose, glycerol and sucrose enhanced the specific TBA degradation rates of strain No. 8. Glucose showed the highest TBA degradation effect. The co-culture of strains ET10 and No. 8
was effective for complete ETBE degradation.
Key words: biodegradation, ETBE, TBA, Rhodococcus erythropolis, Pseudonocardia benzenivorans

1. Introduction
For prevention of global warming, ETBE synthesized froma mixture of ethanol and isobutene is added to automotive gasoline to enhance its octane number 5,7). This ETBE
mixture gasoline is sold as a biological petroleum. ETBEassociated hepatotoxicity in mice is well known 20). Moreover,
ETBE contamination of water resource in the Netherlands
and groundwater in Germany has been reported 28,34). ETBE
exhibits low bio-degradability 1,5,6,9,17,26). TBA is also foundat many sites around the world as a metabolite of ETBE.
Therefore, cleanup of ETBE-contaminated soil and groundwater is difficult. The use of ETBE-degrading bacteria is
expected to remove ETBE from polluted sites 1,26). There isconcern that TBA increases the incidence of renal tubule
adenoma in male rats 2,19).
Previously, we isolated an ETBE-degrading bacterial strain
from soil in Ibaraki Prefecture, Japan. The soil was placed
into a vial containing mineral medium supplemented with
ETBE. ETBE-degraded cultures were streaked onto mineralagar plates with ETBE. Growing colony was selected, andwas purified. The strain was identified as R. erythropolis bymorphological, physiological and 16S rRNA gene analyses
and named as R. erythropolis ET10 (strain ET10) 24).
In the present study, we isolated a TBA-degrading bacterium identified as Pseudonocardia benzenivorans No. 8(strain No. 8). The characteristics of TBA degradation
by strain No. 8 were examined. Moreover, we studied thecomplete degradation of ETBE by co-cultures of strain No. 8
and Rhodococcus erythropolis ET10 (strain No. 10), which
was able to partially degrade ETBE.

2. Materials and methods
2.1	Medium and chemicals
MM liquid medium 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.
The medium was sterilized by autoclaving at 121°C for
20 min. MM agar plates were made by adding 3% agar to
MM liquid medium.
ETBE and TBAc were purchased from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan). TBA and lactose
were purchased from Junsei Chemical Co., Ltd. (Tokyo,
Japan). Reagent-grade agar, starch, glycerol, and glucose
were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan).

74

Ohkubo et al.

2.2	Measurement of ETBE, TBA and TBACETBE and TBA were evaluated by the headspace gas
method using a gas chromatography-mass spectrometry(GCMS) system (GC-2010/GCMS-QP2010 Ultra; Shimadzu
Co., Kyoto, Japan) equipped with an Rtx®-5MS capillarycolumn (0.25 mm i.d.×30 m; Restek Co., PA). Before injection of the sample to the GCMS, the sample vial was kept at
25°C. For measurement of ETBE and TBA concentrations,
50 μL of headspace gas was injected into the GCMS in all
experiments. ETBE and TBA concentrations were calculatedusing a calibration curve as the amount per bottle.
2.3	Isolation of TBA-degrading bacteria
A total of 9.3 g of andosol (Kanuma City, Tochigi
Prefecture, Japan) was suspended in 10 mL of axenic Milli-Q
water, and 1 μL of the suspension was streaked onto an MM
agar plate. The plate, with 200 μL of TBA, was sealed with
Parafilm M® film (Structure Probe, Inc. and SPI Supplies,PA) and incubated statically for 7 day at 30°C. Growingcolonies were collected by using a 1-μL culture loop andinoculated into 60-mL vials containing 10 mL of MM liquidmedium supplemented with 10 μmol of TBA (10 μmol TBA
bottle–1). These vials were sealed with butyl rubber caps
and crimped with aluminum rings and then incubated at
30°C with shaking at 120 rpm for 16 days. TBA degradation was evaluated using the GCMS headspace gas method.
A TBA-degrading culture was selected. The culture wasstreaked on an MM ager plate with 200 μL of TBA andcultured at 30°C for 5 days. A white-colored colony (No. 8)
was isolated. To confirm TBA degradation, 0.03 mg of strain
No. 8 was inoculated into a 60-mL vial containing 10 mLof MM medium with 10 μmol of TBA. The culture wasincubated at 30°C with shaking at 120 rpm for 16 days. The
TBA-degradation ability of strain No. 8 was determined by
the headspace GCMS method.
2.4	ETBE, TBAc and TBA degradation
The growth of strain ET10 was measured turbidometricallyat 660 nm (OD660) using a spectrophotometer (UVmini-1240,
Shimadzu Co., Kyoto, Japan) and converted to dry weight(mg L–1). Approximately 0.4 mg of strain ET10 was inoculated into 60-mL vials containing 10 mL of MM medium
with 10 to 100 μmol of ETBE (10–100 μmol ETBE bottle–1).
The vials were sealed with butyl rubber stoppers and crimped
with aluminum caps. These cultures were incubated at 30°C
with shaking at 120 rpm for 32 days. The concentrationsof ETBE, TBA, tert-butyl formate (TBF) and TBAc were
measured by the headspace GCMS method.
2.5	TBAc degradation and TBA production
in MM medium
Approximately 0.2 mg of strain ET10 was inoculated into
60-mL vials containing 10 mL of MM medium with 10 or
100 μmol of TBAc (10 or 100 μmol TBAc bottle–1). These
cultures were incubated at 30°C with shaking at 120 rpm

for 26 days. TBAc degradation and TBA production wereconfirmed by GCMS using the headspace gas method.
2.6	Identification of TBA-degrading bacteria
Identification of strain No. 8 was performed biochemically using the oxidation-fermentation (OF) test, oxidase test,
catalase test, assessment of motility and spore formation,
Gram staining, morphological colony type, and 16S rRNA
gene analyses.
Genomic DNA from an isolated bacterium was extractedusing a FastDNA® for soil kit (Q-biogene) according to the
manufacturer’s protocol. The nearly full-length 16S rRNA
gene of the isolated bacterium was PCR amplified withprimers 27f (5’-AGAGTTTGATCMTGGCTCAG-3’) and
1492r (5’-TACGGYTACCTTGTTACGACTT-3’) using a
Takara Thermal Cycler Dice Gradient (Takara Bio, Otsu,
Japan). The PCR mixture consisted of PCR buffer for KOD-plus-, 0.2 U KOD -plus- DNA polymerase (TOYOBO,
Osaka, Japan), 1.0 mM MgSO4, 0.2 mM of each dNTP,
0.3 μM of each primer, 2 μL of template DNA, andnuclease-free water to a final volume of 10 μL. The PCR
conditions were as follows: initial denaturation at 95°C
for 5 min, followed by 30 cycles of denaturation at 94°C
for 30 s, annealing at 55°C for 30 s, extension at 68°C
for 90 s, and then final extension at 68°C 10 min. Due
to TA cloning, deoxyadenine triphosphate was attachedto the 3’ end of the amplicons using 10× A-attachment
mix (TOYOBO) according to the manufacturer’s protocol.
The “a”-attached amplicons were cloned into the pMD20-T
vector using a Mighty TA cloning kit (Takara Bio) according
to the manufacturer’s protocol. The constructed vectors
were transformed into Escherichia coli JM109 competentcells (Takara Bio). Transformed E. coli JM109 was cultured
on Luria-Bertani plates containing 100 μg mL–1 ampicillin,
5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal)and isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C
overnight and then characterized by blue-white selection. The
white colonies were checked using direct PCR with the vector
primers M13 primer M4 (5’-GTATTTCCCAGTCCGAC-3’)
and M13 primer RV (5’-CAGGAAACAGCTATGAC-3’) andQuick Taq HS DyeMix (TOYOBO, Osaka, Japan) to determine whether they had an insert fragment of the correct size.
Twelve E. coli JM109 colonies with a PCR fragment of thecorrect size were picked and used for further sequencing analysis. The positive fragments were sequenced using a BigDye
Terminator kit v.3.1 (Applied Biosystems), with selected primers,on an Applied Biosystems 3730 DNA Analyzer (Applied
Biosystems). The selected primers used for sequencing wereM13 primer M4, 8f (5’-AGAGTTTGATCCTGGCTCAG-3’),
533f(5’-CAGCAGCCGCGGTAA-3’),1114f(5’-GCAACGAGCGCAACCC-3’),529r(5’-CGCGGCTGCTGGCAC-3’),907r(5’-CCGTCAATTCMTTTRAGTTT-3’),
1392r(5’-ACGGGCGGTGTGT-3’) and M13 primer RV. The
obtained sequence fragments were assembled using ATGC
sequence assembly software, ver. 7 (GENETYX, Tokyo,

ETBE and TBA Biodegradation by R. erythropolis and P. benzenivorans

Japan).
The determined 16S rRNA gene sequence of strain No. 8
was compared with reference sequences using BLASTN, andclosely related sequences were obtained from the GenBank
databases. Multiple alignments and the calculation of
distance matrices for the aligned sequences were carriedout using ClustalW, and a phylogenetic tree was constructedusing the neighbor-joining method with the MEGA6 software
package 33). Bootstrap re-sampling analysis (1000 replicates)
was carried out to estimate the confidence of tree topologies(Fig. 4).
2.7	Effect of TBA concentration on TBA degradation
To examine the effect of TBA concentration on TBA
degradation by strain No. 8, ca. 0.5 to 20 μmol bottle–1 of
TBA was added to 10 mL of MM medium with or without
0.1 mg of strain No. 8 in 60-mL vials. These cultures wereincubated at 30°C for 20 days on a rotary shaker at 120 rpm.
TBA degradation was measured by the headspace GCMS
method.
2.8	Effect of co-culture of R. erythropolis ET10 and P.
benzenivorans No. 8
The effect of co-culture of strains ET10 and No. 8 on ETBE

75

degradation and TBA production and degradation was also
examined. Approximately 0.2 mg of strain No. 8 and 0.4 mgof strain ET10 were inoculated into 60-mL vials containing
10 mL of MM medium with 10 μmol ETBE bottle–1. These
cultures were maintained at 30°C with shaking at 120 rpm.
The concentrations of ETBE and TBA were measured by theheadspace GCMS method.
2.9	Effect of organic substances on TBA degradation by
P. benzenivorans No. 8
The effect of organic substances on TBA degradation bystrain No. 8 was determined. A total of 5 mg of glucose,lactose, sucrose, succinic acid, glycerol or acetic acid wasadded to 60-mL vials containing 10 mL of MM liquidmedium with 10 μmol bottle–1 of TBA. Cultures were incubated at 30°C with shaking at 120 rpm for 21 days. TBA
degradation was measured by the headspace GCMS method.
The degradation rate constant K (day–1) was calculated in
the logarithmic phase using the following equation (1):K=(1/t-t0)·ln (C0/C)(1),C: Concentration of TBA at time t (μmol·10 mL–1);
C0: Concentration of TBA at time t0 (μmol·10 mL–1);
t: Cultivation time (days);t0: Starting time of logarithmic phase (day).

Fig. 1. Effect of concentration on ETBE degradation and TBA production by strain ET10 in MM medium. Values are means (n=3), anderror bars represent standard deviation. ◇ , ETBE; ◯ , TBA; △ , TBF; □ , TBAc

Ohkubo et al.

76

3. Results
3.1	ETBE degradation and TBA and TBAc production bystrain ET10
ETBE degradation and TBAc and TBA production bystrain ET10 were investigated. ETBE was degraded over 12 to
32 days at 10 to 100 μmol bottle–1 of ETBE addition. TBAc
was not detected, but TBA was produced in conjunction with
ETBE degradation. The concentration of TBA reached amaximum at 20 days. TBAc was not detected over 30 daysof incubation (Fig. 1).
3.2	Degradation of TBAc by strain ET10
It is known that the metabolism of ETBE (in Rhodococcus
ruber IFP 2001) proceeds from ETBE to TBA via TBAc 9).
We could not detect TBAc as a metabolite from ETBE instrain ET10 culture. Therefore, we examined the productionof TBA from TBAc by strain ET10 at high and low concentrations. TBAc was completely degraded in 5 days at the
low concentration, whereas 80% of TBAc was converted to
TBA in 14 days at the high concentration. Removed TBAc
was stoichiometrically changed to TBA (Fig. 2). From theseresults, we suggested that strain ET10 expressed esterase
which transformed TBAc to TBA shown in Fig.6.

3.5	Effect of co-culture of strains ET10 and No. 8 on
ETBE and TBA degradation
ETBE degradation was determined by the co-culture ofstrains ET10 and No. 8. Strain ET10 degraded ETBE andproduced TBA. The produced TBA was not degraded bystrain ET10. The co-culture of strains ET10 and No. 8
resulted in complete degradation of ETBE in 25 days, and
TBA was not detected (Fig. 5).
3.6	Effect of organic substances on the degradation of
TBA by strain No. 8
Table 1 shows the effect of organic substances on the
degradation rate constant (day–1) of TBA for strain No. 8.
The highest degradation rate constant was 0.33 day–1 in

3.3	Isolation and characterization of a TBA-degrading
bacterium
Three TBA-degrading bacteria, strain No. 8, strain No. 3Sand strain No. 3M were isolated from andosol. Degradation
activities of strain No. 3S and strain No. 3M were relatively
low. Therefore, we adopted the strain No. 8 as an experimental strain.
Strain No. 8 was gram positive, non-motile, non–spore
forming, and rod shaped. Formed colonies were smooth, andtheir color was pale white. Strain No. 8 was non-fermenting
in the OF test. Oxidase test was negative, and catalase test
was positive. A phylogenetic tree was constructed based on
the 16S rRNA sequences of strain No. 8 and the most closely
related bacterial species. Strain No. 8 displayed the highest
homology to Pseudonocardia benzenivorans B5, 99.8%;
P. dioxanivorans CB1190, 99.4%; P. hydrocarbonoxydans
IMSNU22140, 99.1%; P. sulfidoxydans DSM44248,
99.1%; P. tetrahydrofuranoxydans K1, 98.5%. From thesecharacteristics, it was determined that strain No. 8 was P.
benzenivorans (Fig. 3).
3.4	Effect of concentration on TBA degradation by P.
benzenivorans No. 8
TBA degradation was initiated at 0.5 to 5.0 μmol bottle–1.
TBA was completely degraded in 10 days at less than 1 μmol
bottle–1. At the high concentration of 20 μmol bottle–1, TBA
was degraded in 20 days. The high concentration thus showeda longer lag degradation time (Fig. 4).

Fig. 2. Effect of TBAc concentration on TBA production in MM
medium by strain ET10.
Values are means (n=3), and error bars represent standard deviation.
● ; TBAc, ◯ ; TBA, ■ ; TBAc without strain ET10, □ ;TBA without strain ET10

ETBE and TBA Biodegradation by R. erythropolis and P. benzenivorans

77

Fig. 3. Neighbour-joining tree derived from 16S rRNA gene sequences showing the relationships between strain No. 8 and related Pseudonocardia species.
Bootstrap percentages (based on 1000 replications) are given at branching points.
Bar, 0.01 substitutions per nucleotide position. Amycolatopsis azurea NRRL11412T was used as the outgroup to root the tree. Accession
numbers are given in parentheses. T; type strain.

Fig. 4. Effect of concentration on TBA degradation by strain
No. 8 in MM medium.
◇ ; 20 μmol bottle–1 TBA without strain, ◯ ; 0.5 μmol bottle–1
TBA, △ ; 1 μmol bottle–1 TBA, ■ ; 5 μmol bottle–1 TBA, ● ;10 μmol bottle–1 TBA, ▲ ; 20 μmol bottle–1 TBA

Fig. 5. Effect of ETBE degradation, TBA production and TBA
degradation by combination of strain ET10 and strain No. 8
in MM medium.
□ ; ETBE, with strain No. 8, △ ; ETBE, with strain ET10, ◇ ;ETBE, with strain ET10 and strain No. 8, ◯ ; ETBE withoutstrain, ■ ; TBA production, strain No. 8, ▲ ; TBA production, strain ET10, ◆ ; TBA production, with strain ET10 andstrain No. 8, ● ; TBA production, without strain.

Ohkubo et al.

78

culture supplemented with glucose. The addition of otherorganic substances slightly increased the degradation rate
constant compared with no addition (Table 1).

Table 1.

4. Discussion
Table 2 summarizes the ETBE-degrading Rhodococcus
species. Various Rhodococcus spp. and R. erythropolis ET10
are able to degrade ETBE but not TBA. A mixed culture of
R. wratislaviensis IFP 2016 and R. aetherivorans IFP 2017
completely degraded 13 different compounds. However, TBA
was not degraded 1). A mixed culture of R. erythropolis ET10
and P. benzenivorans No. 8 degraded ETBE, and metabolite
TBA was completely degraded, as shown in Figure 5.
It is known that TBA is not easily degraded by microorganisms. Table 3 summarizes current data regarding
TBA-degrading microorganisms and the degradation rates ofvarious bacteria. Some of these bacteria, including Aquincola
tertiaricarbonis L108 21,29), Comamonas testosteroni E1 17),
Pseudonocardia sp. 3), Pseudonocardia benzenivorans No. 8,
Methylibium petroleiphilum PM1 25,29,30), Bradyrhizobium
sp. IFP 2049 4), Mycobacterium austroafricanum IFP 2015 10)

Effect of organic substances on the specific TBA
degradation rate by strain No. 8 in MM medium with
10 μmol bottle–1 of TBA

Organic substance

Degradation rate constant (day–1)

No organic substance
GlucoseGlycerolLactoseSucroseSuccinic acidAcetic acid

0.04
0.33
0.14
0.08
0.10
0.09
0.06

Complete degradation was observed in 13 days.
Lag time is three days. Each organics are 5 mg bottle–1.

Table 2.

Summary of ETBE degrading Rhodococcus spp.
Degradation of substrate
ETBE
MTBE
TBA

Organisms
Rhodococcus erythropolis ET10

+

ND

–

Rhodococcus ruber IFP 2001
Rhodococcus ruber E10
Rhodococcus equi IFP 2002
Rhodococcus aetsherivora IFP 2017
Rhodococcus wratislaviensis IFP 2016
Rhodococcus sp. IFP 2042

+
+
+
+
+
+

–
+
–
+
+
ND

–
ND
–
–
–
–

Reference
This study
24)
6, 9)
26)
6)
1)
1)
4)

+; degradation, –; no degradation, ND; not determined,

Table 3.
Organisms

Type ofmetabolism

Aquincola tertiaricarbonis L108 Assimilation

Summary of TBA degradation rates by various bacteria
Metabolic or The highest TBA
cometabolic concentration incarbon sources the experiments
TBA

Comamonas testosteroni E1
Assimilation TBA
Pseudonocardia sp.
Assimilation TBA
Pseudonocardia benzenivorans Assimilation TBA
No. 8
Methylibium petroleiphilum
Assimilation TBA
PM1
Bradyrhizobium sp. IFP 2049
Assimilation TBA
Mycobacterium austroafricanum Assimilation TBA
IFP 2015
Mycobacterium austroafricanum Assimilation TBA
IFP 2012
Mycobacterium austroafricanum Cometabolism Hexane
IFP 2012
Propane,Hexadecane
Mycobacterium vaccae JOB5
Cometabolism Propane
Mycobacterium austroafricanus Cometabolism 2-Methylbutane
JOB5
Mycobacterium duvalii TA5
Cometabolism Glucose
Methylibium sp. R8Cometabolism MTBE+; Degradation, –; No degradation, ND; not determined,

5700 mg l–1

TBA degradation rate

Degradation
MTBE ETBE

Reference

2.82 mmol g biomass–1 h–1(208 mg g biomass–1 h–1)
0.017 mmol l–1 h–1
0.008 mmol l–1 h–1++

21, 29)

+
+

+
+

17)
3)

0.008 mmol l–1 h–1

ND–This study

45 mgl–1

14.4 mmol l–1 g protein–1 h–1+–

29, 30, 25)

198 mg l–1
316 mg l–1

0.022 mmol l–1 h–1
0.81 mmol g biomass–1 h–1

ND
+

–
+

4)
10)

10.4 mg l–1

0.003 mmol l–1 h–1

+

ND

12)

200 mg l–1

0.16, 0.10, 0.095 mmol gbiomass–1 h–1

+

ND

11)

1480 mg l–1
7.4 mg l–1

0.62 mmol g protein–1 h–1
2.52 mmol g protein–1 h–1

+
+

ND
ND

15, 31)
14)

11.1 mg l–1
500 mg l–1

0.005 mmol l–1 h–1
1.08 mmol g protein–1 h–1

+
+

ND
–

23)
29)

200 mg l–1
29.6 mg l–1
148 mg l–1

ETBE and TBA Biodegradation by R. erythropolis and P. benzenivorans

79

Fig. 6. Proposed pathways for ETBE or MTBE
degradation by microorganisms.
This pathway was modified from following
authors 5,7–9,13,22,23,27,32).

80

Ohkubo et al.

and Mycobacterium austroafricanum IFP 2012 12) are able
to assimilate TBA. Most of the TBA-degrading bacteria areable to degrade methyl tert-butyl ether (MTBE). However,some of them are not able to degrade ETBE.
The highest degradation rate was 14.4 mmol g protein–1 h–1
by M. petroleiphilum PM1 for the protein base 30). Aquincola
tertiaricarbonis L108 showed the highest TBA degradation
rate of 2.82 mmol g biomass–1 h–1 for the biomass base 21).
The degradation rates were very low for L–1 h–1 base. The
highest degradation rate was by Bradyrhizobium sp. IFP
2049 at 0.022 mmol L–1 h–1 for L–1 h–1 base. Pseudonocardia
benzenivorans No. 8 (present study) and Pseudonocardia
sp 3) showed the lowest TBA degradation rate of 0.008 mmol
L–1 h–1. The reason for the low degradation rates was basedon the low cell concentration of L–1.
Mycobacterium co-metabolizes TBA. The degradation
rate of M. austroafricanum IFP 2012 is significantly increasedby the addition of hexane, propane and hexadecane. The
degradation rate increased more than 50-fold with hexane.
The TBA degradation rate increased with 2-methylbutane
and glucose as a co-metabolic substrate by M. vaccae JOB5
(M. austroafricanum ATCC 29678) and M. duvalii.
Figure 6 shows the proposed pathways for microbial ETBE and MTBE degradation. ETBE is degradedthrough tert-butoxy-ethanol (TBE), tert-butyl acetate(TBAc), TBA 9,27), 2-methy-1,2-propanediol (2-M-1, 2-PD),
2-hydroxy-2-methylprop anal, 2-hydroxy isobutyric acid andpyruvic acid 5,7–9,13,22,23,27,32).
Three different enzymes are necessary to degrade ETBE
and MTBE to TBA: oxygenase, dehydrogenase and esterase.
Rhodococcus ruber IFP 2001 9), Comamonas testosteroni
E1 17), M. vaccae JOB5 31) and M. austroafricanum IFP
2012 use monooxygenase for the degradation of MTBE
and ETBE 12). Ferreira et al. (2006) reported that TBAc wasproduced from TBE by dehydrogenase and decomposed to
TBA and acetate by esterase 9).
Produced TBA is decomposed by a monooxygenase anddehydrogenase. TBA is converted to 2-M 1,2-PD by amonooxygenase 22). M. austroafricanus JOB5 degrades TBA
using an alkane-inducible monooxygenase with n-nonane,2-methylbutane and p-xylene as cometabolic substrates 14).
M. vaccae JOB5 degrades TBA with propane as a cometabolic substrate using a short-chain alkane monooxygenase 31).
It is reported that bacteria of the genus Rhodococcus areable to degrade ETBE but not TBA 1,4,6). These results suggestthat the monooxygenase of Rhodococcus species cannot
degrade TBA. Mixed cultures of Rhodococcus sp. IFP 2042
and Bradyrhizobium sp. IFP 2049 degrade ETBE to CO2 4).
Pseudonocardia benzenivorans and P. dioxanivorans mineralize benzene and 1,4-dioxane, respectively 16,18). Digabel and
Guichard (2014) 3) reported that Pseudonocardia sp. degrade
ETBE and TBA. In our research, P. benzenivorans No. 8
assimilated TBA. Therefore, it was hypothesized that these
Pseudonocardia species are promising for the degradationof recalcitrant organic substances such as aromatic hydrocarbons, ether, ETBE and TBA.

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