Degradation pathways of trichloroethylene and 1,1,1-trichloroethane by Mycobacterium sp. TA27

Article abstract of DOI:10.1271/bbb.66.385

We analyzed the kinetics and metabolic pathways of trichloroethylene and 1,1,1-trichloroethane degradation by the ethane-utilizing Mycobacterium sp. TA27. The apparent V_max and K_m of trichloroethylene were 9.8 nmol min^-1 m

Full text of DOI:10.1271/bbb.66.385

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Degradation Pathways of Trichloroethylene and
1,1,1-Trichloroethane by Mycobacterium sp. TA27
Akiko HASHIMOTOa, Kazuhiro IWASAKIa, Naou NAKASUGIa, Mutsuyasu NAKAJIMAb & Osami
YAGIc
a National Institute for Environmental Studies and CREST, Japan Science and Technology
Corporation 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
b College of Bioresource Sciences, Nihon University 1866 Kameino, Fujisawa, Kanagawa
252-8510, Japan
c Research Center for Water Environment Technology, the University of Tokyo 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
Published online: 22 May 2014.
To cite this article: Akiko HASHIMOTO, Kazuhiro IWASAKI, Naou NAKASUGI, Mutsuyasu NAKAJIMA & Osami YAGI
(2002) Degradation Pathways of Trichloroethylene and 1,1,1-Trichloroethane by Mycobacterium sp. TA27, Bioscience,
Biotechnology, and Biochemistry, 66:2, 385-390, DOI: 10.1271/bbb.66.385
To link to this article: http://dx.doi.org/10.1271/bbb.66.385
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Biosci. Biotechnol. Biochem., 66 (2), 385–390, 2002
Degradation Pathways of Trichloroethylene and 1,1,1-Trichloroethane
by Mycobacterium sp. TA27
1
Akiko HASHIMOTO,1 Kazuhiro IWASAKI,1 Naou NAKASUGI
,
3,
†
Mutsuyasu NAKAJIMA,2 and Osami YAGI
1National Institute for Environmental Studies and CREST, Japan Science and Technology Corporation,
16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
2College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan
3Research Center for Water Environment Technology, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8656, Japan
Received August 31, 2001; Accepted October 19, 2001
We analyzed the kinetics and metabolic pathways of
trichloroethylene and 1,1,1-trichloroethane degradation
by the ethane-utilizing Mycobacterium sp. TA27. The
apparent Vmax and Km of trichloroethylene were
groundwater. Bioremediation, which is the practice
of clean-up of contaminated sites by using microbial
degradation activities for the pollutants, is one of the
most promising and cost-eŠective new technologies
for cleaning up groundwater contamination. For
eŠective bioaugmentation, which is the practice of
clean-up of contaminated sites by amending cultured
microorganisms which are able to degrade the
pollutants, it is important to measure the kinetic
parameters and degradation products of TCE and
TCA by the microbial inocula. TCE degradation
products can be oxidized by aerobic bacteria that are
able to utilize methane,1–6) toluene,7,8) phenol,9)
propane,10–12) propylene,13) and ammonia.14) There
have been few reports on the metabolites of TCE and
TCA. Methylosinus trichosporium OB3b is known to
metabolize TCE through production of TCE epoxide
and chloral by a soluble methane monooxygenase.4)
Chloral is oxidized to trichloroacetic acid (TCAA) or
reduced to 2,2,2-trichloroethanol (TCAol), while
TCE oxide is hydrolyzed to dichloroacetic acid
(DCAA), glyoxylic acid, formic acid, and carbon
monoxide. As the degradation products of TCE by
toluene dioxygenase are formic acid and glyoxylic
acid, the degradation pathways might be diŠerent
from the monooxygenase pathway.7)
9.8 nmol min„1 mg of cells„1 and 61.9
The apparent max and
0.11 nmol min„1 mg of cells„1 and 3.1
m
M
, respectively.
m of 1,1,1-trichloroethane were
, respectively.
V
K
m
M
2,2,2-trichloroethanol, trichloroacetic acid, chloral, and
dichloroacetic acid were detected as metabolites of
trichloroethylene. 2,2,2-trichloroethanol, trichloroacet-
ic acid, and dichloroacetic acid were also detected as
metabolites of 1,1,1-trichloroethane. The amounts of
2,2,2-trichloroethanol, trichloroacetic acid, chloral, and
dichloroacetic acid derived from the degradation of
3.60
0.11
m
m
mol trichloroethylene were 0.16
mol (3.1 ), 0.02 mol (0.6 ), and 0.02
), respectively. The amounts of 2,2,2-tri-
chloroethanol, trichloroacetic acid and dichloroacetic
acid derived from the degradation of 1.73 mol 1,1,1-
trichloroethane were 1.48 mol (85.5 ), 0.22 mol
(12.7 ), and 0.02 mol (1.2 ), respectively. More
than 90 of theoretical total chloride was released in
m
mol (4.4
%
mol
),
%
m
%
m
(0.6
%
m
m
%
m
%
m
%
%
trichloroethylene degradation. Chloral and 2,2,2-
trichloroethanol were transformed into each other, and
were ˆnally converted to trichloroacetic acid, and
dichloroacetic acid. Trichloroacetic acid and dichloroa-
cetic acid were not degraded by strain TA27.
In earlier studies we isolated the ethane-utilizing
bacterium, Mycobacterium sp. TA27, which can co-
metabolically degrade TCE and TCA.15–17) Only a few
bacteria can degrade both TCE and TCA.5,18) In this
study, we investigated the kinetic parameters and
degradation pathways of TCE and TCA by strain
TA27.
Key words: biodegradation; Mycobacterium; kinetics;
pathway
Volatile chlorinated compounds such as
trichloroethylene (TCE) and 1,1,1-trichloroethane
(TCA), which have been widely used as organic sol-
vents and degreasers, occur widely in wastewater and
†
To whom correspondence should be addressed. Osami YAGI, Fax: +81-3-5841-8528; E-mail: yagi
＠
env.t.u-tokyo.ac.jp
Abbreviations: TCE, trichloroethylene; TCA, 1,1,1-trichloroethane; TCAA, trichloroacetic acid; TCAol, 2,2,2-trichloroethanol; DCAA,
dichloroacetic acid; OD, optical density; GC-FID, gas chromatography with a ‰ame ionization detector

386
A. HASHIMOTO et al.
measured by GC-MS.20,21) The reaction mixture in the
Materials and Methods
×
serum bottle was centrifuged for 10 min at 10,000
g
Chemicals. TCE, TCA, TCAA, DCAA, chloral,
and acidiˆed to pH 1.7. The supernatant was trans-
ferred into a 100-ml separating funnel. One gram of
NaCl was added to the funnel, which was shaken to
and TCAol were purchased from Wako Pure Chemi-
cals (Osaka, Japan). [1,2-14C] TCE (45 mCi mmol„1
,
DuPont, Bostone, MA) was dissolved in methanol to
make a stock solution of 1.58 mg of [1,2-14C] TCE
ml„1. ACSII (Amersham Pharmacia Biotech AB,
Uppsala, Sweden) was a scintillation ‰uid used to
measure radioactivity.
dissolve the NaCl. Then 0.4 ml of
di‰uoroaniline in ethyl acetate, 0.4 ml of 1
1
M
M
2,4-
dicyclo-
hexylcarbodiimide in ethyl acetate, and 15 ml of ethyl
acetate were added, and the funnel was shaken for
40 min on a reciprocal shaker. After 5 g of NaCl had
been added and dissolved, the aqueous layer was
separated and extracted again with 5 ml of ethyl
acetate. The combined ethyl acetate extract was suc-
Kinetic experiments on TCE and TCA oxidation.
Mycobacterium sp. TA27 isolated from soil was used
throughout this experiment. This strain was grown in
a mineral-salt medium in an ethane-air (2:8, vol
cessively washed with 3
M
HCl, saturated NaHCO3,
and saturated NaCl, then dehydrated by anhydrous
Na2SO4. The solution was evaporated to dryness and
dissolved in 2 ml of benzene twice. The solution was
analyzed by GC-MS. Detection limits of TCAA,
DCAA, TCAol, and chloral were 0.2, 0.6, 10, and
vol„1) atmosphere at 30
9
C for 3 days, as described in
a previous paper.15) The cells were harvested at late-
log phase and washed twice with 0.05 phosphate
M
buŠer (pH 7.0). The kinetic parameters of TCE and
TCA oxidation were measured with resting cells of
strain TA27. Five milliliters of reaction mixture con-
10 m
g l„1, respectively. The amount of chloride
released by TCE degradation was measured by using
a chloride electrode (MA235 pH, Mettler Toledo,
Osaka, Japan).
taining strain TA27 cells and TCE or TCA in 0.05
M
phosphate buŠer at pH 7.0 were added to each 27-ml
bottle, which was crimped with a butyl rubber stop-
per and an aluminum cap. The ˆnal cell concentra-
Radiolabeled TCE biodegradation. For the
tion was 0.64 mg ml„1 (OD660nm 1.0). The oxidation
radioisotope-labeled TCE degradation experiment,
＝
rate was measured at 5 concentrations between 0.5
and 50 mg l„1 of TCE and TCA. The water concen-
trations of the TCE and TCA were calculated by us-
ing Henry's constant.19) Reactions were carried out
5.6 m
Ci of [1,2-14C] TCE and 9 mg l„1 of unlabeled
TCE were added to a 155-ml serum bottle containing
30 ml of 0.05 phosphate buŠer (pH 7.0) with strain
TA27 (OD660nm
1.0). Measurement of 14C activity in
M
＝
with shaking at 120 rpm at 25
9
C. The amounts of
various fractions from reaction mixtures after 24 h
was based on the method of Little et al.1) The fractio-
nation of CO2, residual TCE, water-soluble, and cell
fractions was carried out as follows. After the reac-
TCE and TCA degraded were measured by the head-
space gas method using gas chromatography with a
‰ame ionization detector (GC-FID). Kinetic
parameters were calculated by the graphical methods
of a Lineweaver-Burk plot using the values of oxida-
tion rate (V) and substrate concentration (S). All
kinetic tests were measured in duplicate.
tion mixture was alkaliˆed with 1
was started and residual TCE was removed by the ad-
dition of 30 ml of -hexane 3 times. Next, the water
fraction was acidiˆed with 6 HCl and the CO2
evolved was adsorbed into 5 ml of 1 NaOH solu-
tion (CO2 fraction). The reaction mixture was alkali-
ˆed with 1 NaOH, and 1 ml of head-space gas was
N
NaOH, aeration
n
N
N
Measurements of degradation products of TCE
and TCA. Experiments on TCE, TCA, and their
degradation products (TCAol, chloral, TCAA, and
DCAA) were conducted in serum bottles (155 ml),
N
transferred to a vial with 5 ml of scintillation solution
(residual TCE gas fraction). The reaction mixture
each containing 30 ml of 0.05
M
phosphate buŠer
was acidiˆed with 1
N
HCl, and then centrifuged at
(pH 7.0) with strain TA27. The cell concentration
was 0.64 mg ml„1 in the degradation experiments of
TCE and its degradation products, and was 3.2 mg
ml„1 in the case of TCA degradation experiments.
The bottle was crimped with a butyl rubber stopper
and an aluminum cap. After the addition of TCE
7,000 rpm for 10 min. The radioactivity of the super-
natant was measured (water-soluble fraction). The
cells were washed with phosphate buŠer (pH 7.0)
twice, and their radioactivity was measured (cell frac-
tion).
(6.83
TCAol (2.02
(1.84 mol), the reaction mixture was incubated at
30 C for 24 h. All of the data are means of triplicate
samples.
Quantities of TCAA, DCAA, TCAol and chloral
m
mol), TCA (4.12
m
mol), chloral (2.04
m
mol),
Gas chromatograph and GC-MS conditions. A gas
chromatograph (model GC-14B, Shimadzu Co.,
Kyoto, Japan) equipped with a ‰ame ionization de-
tector and an electron ionization detector was used
for measurement of volatile chlorinated compounds.
m
mol), DCAA (2.33
m
mol), or TCAA
m
9
×
The glass column (0.3 200 cm) was packed with Sili-
in the reaction mixture were extracted into ethyl
acetate as derivatives of 2,4-di‰uoroaniline and
cone DC550 (GL Science Inc., Tokyo, Japan). The
temperatures of the injection port, oven, and detec-

Trichloroethylene Degradation Pathways of Mycobacterium sp.
387
tor were 2509C, 1209C, and 2509C, respectively.
Nitrogen gas was used as a carrier. A QP5050 GC-
MS system (Shimadzu Co, Kyoto, Japan) with an
×
OV-17 capillary column (0.32 mm 30 m; GL
Science, Tokyo, Japan) was used with the following
program: initial temperature 40
9
C for 5 min; temper-
C min„1 up to 100
C; tempera-
C min„1 up to 230
C, and ˆnal
C for 10 min. The temperatures of
injection and the detector were kept at 200 C and
260 C, respectively.
ature gradient of 5
ture gradient of 10
9
9
9
9
temperature 230
9
9
9
Results
Kinetic parameters of TCE and TCA oxidation
The resting cells of strain TA27 degraded 13 and
of 10 mg l„1 TCA and TCE in 4 h, respectively.
The values of 1 V and 1 S were calculated and
z
54
z
W
W
plotted on Fig. 1. The apparent
Vmax and Km of TCE
oxidation were 9.8 nmol min„1 mg cells„1 and
61.9
m , respectively, by the graphical methods
M
of Lineweaver-Burk. The apparent Vmax and Km of
TCA oxidation were 0.11 nmol min„1 mg cells„1 and
3.09
m , respectively. The Vmax Km of 0.033 for TCA
M
W
oxidation was lower than that of 0.158 for TCE oxi-
dation. Both values are on the same order level.
Fig. 1. Lineweaver-Burk Plot Used to Determine TCE (A) and
Identiˆcation of TCE and TCA degradation
products
TCA (B) Degradation Rates (Vmax) and Km
Symbols: , TCE; , TCA. Biodegradation was examined
at 25 C and pH 7.0. Initial TCE concentrations were 1, 5, 10,
30, and 50 mg l„1. Initial TCA concentrations were 0.5, 1, 5, 10,
and 30 mg l„1
.


9
Table 1 shows the degradation products from
TCE, TCA, chloral, TCAol, DCAA, and TCAA. Of
.
the 6.83 mmol of TCE added, 52.7z was degraded by
strain TA27. Small amounts of TCAol, TCAA,
DCAA and chloral were produced from TCE.
Table 2. Chloride Release from TCE and TCA Degradation by
Strain TA27
Eighty-six
z
of degraded TCA was transformed into
TCAol. Small amounts of TCAA and DCAA were
produced from TCA. Recovery of the 3 degradation
Theoretical
amount of
Free
Amount
Chloride
released
chloride
released
Substrate degraded
chloride released
products of TCA was almost 100
z
. It has been
(
m
mol)
(z)
(
mmol)
( mmol)
reported that the degradation product of TCA by
Methylosinus trichosporium OB3b, Nitrosomonas
TCE
TCA
2.38
1.73
7.14
5.19
6.59
0.02
92.1
0.3
europaea
,
and Pseudomonas putida G786 is
TCAol.2,18,22) Ours is the ˆrst study to report that the
TCA degradation products are not only TCAol, but
also TCAA and DCAA. In addition, we examined
the degradation of the 4 metabolites. Chloral and
The initial quantities of TCE and TCA were 6.83
respectively.
mmol and 4.12 mmol,
Table 1. Degradation of TCE, TCA, and Their Products by Strain TA27
Amount
added
Amount
degraded
mol)
Product amount (
DCAA
mmol)
Degradation
Substrate
(
z
)
(
m
mol)
(
m
Chloral
0.02 0.01
TCAol
TCAA
total
TCE
TCA
Chloral
TCAol
DCAA
TCAA
6.83
4.12
2.04
2.02
2.33
1.84
52.7
42.2
95.7
12.5
0.0
3.60
1.73
1.96
0.25
0.00
0.00
±
±
±
±
±
±
0.05
±
×
0.16
1.48
1.59
±
±
±
0.05
0.22
0.20
0.02
±
±
±
±
0.00
0.11
0.22
0.36
0.09
±
±
±
±
0.02
0.02
0.02
0.01
0.33
1.72
2.02
0.27
±
±
±
±
—
—
0.07
0.23
0.22
0.07
0.02
0.02
0.01
0.00
0.00
º
2.04
10„3
0.02
0.06
0.03
0.00
0.01
0.00
a
—
0.15
a
±
0.05
—
a
b
º
º
2.04
2.04
×
10„3
10„3
º
º
2.02
2.02
×
×
10„3
10„3
—
º
3.67
×
10„5
a
b
0.0
×
º
1.40
×
10„4
—
Values are means (standard error). a: Degradation substrate, b: below detection limit. Abbreviations for compounds: TCAol, 2,2,2-trichloroethanol; DCAA,
dichloroacetic acid; TCAA, trichloroacetic acid.

388
A. HASHIMOTO et al.
TCAol were transformed into each other and ˆnally
became TCAA and DCAA. The acidic compounds
such as TCAA and DCAA were not degraded by
strain TA27. The recovery percentages from chloral
and 4 to 10
m
M
for Pseudomonas cepacia G4, P.
mendocina KR1 and P. putida F1.9) Vmax and Km of
509 174 nmol min„1 mg protein„1 and 145 61
m
M
±
±
were obtained for Methylosinus trichosporium OB3b
and TCAol degradation were almost 100
z
. There-
grown in continuous culture under copper stress with
fore, it was conˆrmed that the degradation of TCA
did not result in mineralization.
20 m
M
formate.5,9) The Vmax and Km of strain TA27
for TCE degradation were 9.81 nmol min„1 mg
cells„1 (28.3 nmol min„1 mg protein„1) and 61.9
mM.
Distribution of 14C labeled TCE degradation
products
These values were similar to those for other bacteria,
apart from Methylosinus trichosporium OB3b. It
seems that the high Vmax obtained for OB3b was
based on the addition of formate as an energy source.
We calculated the Vmax and Km of strain TA27 for
TCA degradation (Table 3). The kinetic parameters
of TCA degradation had previously been reported
only for Methylosinus trichosporium OB3b by
[1,2-14C] TCE was used to measure TCE minerali-
zation. Thirty-ˆve per cent of the total amount of
TCE in the gas and liquid fractions remained un-
degraded. The distributions of radioactivity in the
cell, CO2, and water-soluble fractions represented
80.8
(100
z
, 15.6
z z
, and 3.6 of the degraded TCE
z), respectively. Therefore, TCE was mineral-
Oldenhuis.5) The
Vmax for TCA degradation by strain
ized by the ethane-utilizing bacterium, strain TA27.
Also, a compound corresponding to 0.14 of the
formic acid in degraded TCE produced was detected
by HPLC analysis to be a water-soluble product of
TCE degradation (data not shown).
TA27 was much lower than that by OB3b. However,
strain TA27 has a strong ability to degrade TCA
under growing conditions.15) Therefore, it seems that
degradation activity is increased by the presence of
an energy source. Strain TA27 had a higher a‹nity
for TCA than did OB3b.
z
Chloride release by TCE degradation
Signiˆcantly high radioactivity of 80.8
Figure 2 shows a hypothetical pathway for TCE
and TCA degradation by strain TA27. Some reports
have described TCE degradation pathways in aerobic
bacteria. TCE is initially converted to chloral and
TCE oxide by methane-oxidizing bacteria.4,21,24,25)
Then, TCE oxide and its metabolites are converted to
carbon dioxide, while chloral is oxidized to TCAA
and DCAA, or reduced to TCAol. The quantities of
metabolites and the pathways of TCE degradation by
strain TA27 were similar to those of degradation by
methane-oxidizing bacteria. TCAol and chloral were
detected as TCE degradation products of a propane-
oxidizing bacterium, Mycobacterium vaccae JOB-5,
z
for
degraded TCE was observed in the cell fraction. We
examined the amount of chloride released by TCE
and TCA degradation (Table 2). If all of the chloride
were to be released from the degraded TCE, the
amount released would be 7.14
mmol. After 24 h of
incubation, 6.59 mol of chloride was measured. The
m
recovery percentage was 92.1
z. In TCA degradation
by strain TA27, little chloride was released.
Discussion
Several reports have supplied kinetic data for aero-
bic TCE-degrading bacteria (Table 3). By varying the
and these products accounted for 25z of the degrad-
ed TCE.12) However, strain TA27, like the metano-
trophs, produced less TCAol and chloral than M.
vaccae JOB-5.
TCE concentration from 5 m
M
to 75 m , the Vmax
M
and Km of toluene- and phenol-oxidizing bacteria
were calculated as 8 to 20 nmol min„1 mg protein„1
TCE mineralization by several bacteria has been
Table 3. Comparison of Michaelis-Menten Kinetic Parameters for TCE and TCA Degradation
Microorganisms
Substrate
Enzyme
Growth substrate
Vmax
Km
(
m
M
)
Reference
Mycobacterium sp. TA27
Methylosinus trichosporium OB3b
TCE
TCE
Ethane monooxygenase
Methane monooxygenase
ethane
methane
28.3a
509a
37.5a,c
16a
62
This study
145
75
116
187
4
10
5
30
3
24
5
9
13
23
9
9
9
Xanthobacter sp. strain Py2
Rhodococcus corallinus B-276
Pseudomonas cepacia G4
Pseudomonas mendocina KR1
Pseudomonas putida F1
Nitrosomonas europaea
Mycobacterium sp. TA27
Methylosinus trichosporium OB3b
TCE
TCE
TCE
TCE
TCE
TCE
TCA
TCA
Alkene monooxygenase
Alkene monooxygenase
Toluene 2-monooxygenase
Toluene 4-monooxygenase
Toluene dioxygenase
Ammonia monooxygenase
Ethane monooxygenase
Methane monooxygenase
propene
NBG medium
phenol
phenol
toluene
ammonia
ethane
methane
2.4a
9a
20a
8a
—
14
This study
5
0.1b
214b
a
(nmol min„1 mg protein„1).
b
(nmol min„1 mg cell„1).
c
Initial V (nmol min„1 mg protein„1).

Trichloroethylene Degradation Pathways of Mycobacterium sp.
389
Fig. 2. Proposed Pathway of TCA and TCE Degradation by Strain TA27.
studied. Of the degradation products of Rhodococ-
cus rhodochrous and Rhodococcus sp. strain Sm-1
with propane monooxygenase, CO2 constituted 30
References
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Lidstrom, M. E., Tyndall, R. L., and Gilmer, P. J.,
z
For
,
10)
the aqueous fraction 50
M. vaccae JOB-5 degradation of TCE, the distribu-
tion of radioactivity was 12 in the CO2 fraction,
86 in the water-soluble fraction, and 3 in the cell-
associated fraction.12) The distribution of radioactivi-
ties was 32 to 41 in the CO2 and 3.7 to 36 in
the cell-associated fractions for methane-oxidizing
bacteria.1,25) With strain TA27, more than 80
of the
z, and biomass 10
z.
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a methane-
z
z
z
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z
z
z
z
z
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