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Applied and Environmental Microbiology, October 1999, p. 4693-4696, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Aerobic Degradation of 1,1,1-Trichloroethane by
Mycobacterium spp. Isolated from Soil
Osami
Yagi,1,*
Akiko
Hashimoto,2
Kazuhiro
Iwasaki,1 and
Mutsuyasu
Nakajima3
National Institute for Environmental
Studies1 and
CREST,2 Japan Science and Technology
Corporation, Tsukuba, Ibaraki 305-0053, and Department of
Bioresource Science, Nihon University, Fujisawa, Kanagawa
252-8510,3 Japan
Received 22 February 1999/Accepted 25 June 1999
 |
ABSTRACT |
Two strains of 1,1,1-trichloroethane (TCA)-degrading bacteria, TA5
and TA27, were isolated from soil and identified as
Mycobacterium spp. Strains TA5 and TA27 could degrade 25 and 75 mg · liter of TCA
1 cometabolically in the
presence of ethane as a carbon source, respectively. The compound
2,2,2-trichloroethanol was produced as a metabolite of the degradation process.
| |
TEXT |
Volatile aliphatic chlorinated
compounds such as 1,1,1-trichloroethane (TCA), trichloroethylene (TCE),
and tetrachloroethylene have been detected in groundwater throughout
Japan. These compounds are suspected carcinogens and hepatotoxins.
Bioremediation is one of the most promising new technologies for
cleaning up groundwater contamination because of its low cost and its
potential for the complete destruction of pollutants. There are many
reports on the degradation of TCE by aerobic bacteria (4, 8, 10, 12, 13, 16-18, 22-26) and some reports on anaerobic TCA
degradation (1, 5, 9, 27), but there are few reports of
aerobic TCA degradation. The methane-oxidizing bacterium
Methylosinus trichosporium OB3b can degrade TCA
(18), as can the ammonia-oxidizing bacterium
Nitrosomonas europaea (7, 26), the
propane-oxidizing bacteria Rhodococcus rhodochrous and
Rhodococcus sp. strain Sm-1 (12), and
Pseudomonas putida G786 containing cytochrome
P450CAM (11). However, TCA biodegradation has
not been studied in detail. In this study, we isolated the
TCA-degrading bacterial strains TA5 and TA27 and determined their
biodegradation characteristics.
Isolation and identification of TCA-degrading bacteria.
We
used an enrichment culture method to isolate TCA-degrading bacteria
from various polluted soils under aerobic conditions. Small samples of
soil (0.5 g each) were added to 69-ml serum bottles containing 15 ml of
mineral salt medium each (22). The serum bottles were sealed
with butyl rubber caps and crimped with aluminum rings. Ten milliliters
of air were withdrawn from each bottle with a syringe, and 5 ml of
methane, ethane, propane, or ethylene (as the carbon source) and 5 ml
of oxygen were injected to replace the withdrawn air. TCA-saturated
aqueous solution was added to each serum bottle by syringe to make a
final concentration of 0.6 mg · liter1. The
cultures were incubated at 30°C while being shaken at 120 rpm. After
each week of culture, 0.3 ml of culture broth was transferred to a new
serum bottle containing 15 ml of fresh medium. After at least four
transfers, the culture broth was spread onto an agar plate.
We isolated two strains of TCA-degrading bacteria, TA5 and TA27, from
tetrachloroethylene-polluted soils. Both cell types were rod shaped,
nonmotile, non-spore forming, gram positive, catalase positive, oxidase
negative, and of the menaquinone-9 (H2) type. We found that
both strains could utilize ethane, ethanol, and various other carbon
compounds as their energy sources. Phylogenetic analysis was carried
out with the genomic DNA of strains TA5 and TA27 being extracted by the
method of Mizuguchi et al. and Sambrook et al., respectively (15,
20). The primers used for the PCR amplification of the 16S rRNA
gene were 8FPL and 1492RPL (19). The PCR products were
sequenced with a Big Dye Terminator cycle sequence kit (Applied
Biosystems, Perkin-Elmer). The 16S rRNA gene sequences were compared
with the 16S rRNA gene database from the DDBJ. Strains TA5 and TA27 had
the highest levels of 16S rRNA gene similarity with Mycobacterium
duvalii (level of similarity, 98.0%) (21) and
Mycobacterium gilvum (level of similarity, 99.2%), respectively. From these physiological characteristics, it seemed that
these strains were Mycobacterium spp. It has been reported that fast-growing mycobacteria can degrade various hazardous compounds. Mycobacterium chlorophenolicum can degrade pentachlorophenol
(2, 14). Mycobacterium aurum can utilize vinyl
chloride, the key enzyme for that degradation being alkene
monooxygenase (6). Mycobacterium vaccae JOB-5 can
degrade a number of the major compounds involved in groundwater
pollution: benzene, toluene, propylbenzene, styrene,
cis-1,2-dichloroethylene, and TCE (3, 24, 25). However, these strains could not degrade TCA. Ours is the first report
of substantial TCA degradation by Mycobacterium sp. Isolates TA5 and TA27 could assimilate ethane and ethanol. Moreover, strain TA27
could assimilate propane and glucose. Both TA5 and TA27, grown on
various carbon substrates, could degrade TCA. It seems that the
TCA-degrading enzyme is constitutively produced.
TCA degradation characteristics.
The TCA degradation
experiments were carried out as follows. Either strain TA5 or strain
TA27 was added to a 69-ml serum bottle containing 15 ml of mineral salt
medium (22), and the bottle was sealed with a butyl rubber
cap and aluminum ring. Various concentrations of TCA and ethane were
added to each serum bottle, and the cultures were incubated at 30°C
while being shaken at 120 rpm. The TCA degradation was monitored by
using gas chromatography to measure the concentration of gases in the
headspace of the serum bottle. The water and gaseous concentrations of
the TCA were calculated using Henry's constant. A gas chromatograph
(model GC-7AG; Shimadzu Co., Kyoto, Japan) equipped with a flame
ionization detector using a glass column packed with Silicone DC550 (GL
Science Co., Tokyo, Japan) was used for quantitative determination of the TCA and ethane. Oxygen in the gas phase was analyzed on a Molecular
Sieve 5A packed column equipped with a thermal conductivity detector.
Figure 1 shows the effects of initial
ethane concentrations of 3, 5, and 10% on the degradation of TCA at an
initial concentration of 0.6 mg · liter1. An
initial ethane concentration of 3% yielded inadequate growth of both
strains for effective TCA degradation (Fig. 1A and D). An initial
ethane concentration of 10% yielded the highest growth rate of strain
TA5, maximizing TCA degradation; 60% of the TCA was degraded within 5 days, after which the degradation almost stopped (Fig. 1C). It seemed
that the cessation of TCA degradation was due to a lack of oxygen or
ethane. Good growth of strain TA27 was observed at initial ethane
concentrations of both 5 and 10% (Fig. 1E and F). At an initial ethane
concentration of 5%, about 90% of the initial TCA was degraded within
7 days by strain TA27. However, TCA degradation was inhibited at an
initial ethane concentration of 10% (Fig. 1F). A high concentration of
ethane may competitively inhibit the degradation of TCA by strain TA27.
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FIG. 1.
Effect of initial ethane concentration on TCA
degradation by strains TA5 and TA27. (A to C) Strain TA5; (D to F)
strain TA27. Initial ethane concentrations were 3% (A and D); 5% (B
and E); and 10% (C and F). Initial pH: strain TA5, 6.8; strain TA27,
5.7. Symbols:  , residual TCA;  , bacterial growth (optical density
at 660 nm [OD660nm]);  , residual ethane concentration;  ,
residual O2 concentration. Error bars indicate the ranges
for duplicate samples.
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|
The relative degradation of TCA by strains TA5 and TA27 was affected by
the initial concentration of TCA. The rate of TCA degradation by strain
TA5 was high at low initial TCA concentrations: at an initial TCA
concentration of between 0.05 and 5 mg · liter1,
more than 50% of the TCA was degraded within 7 days (Fig.
2A), after which the cell growth stopped.
However, at an initial TCA concentration of 25 mg · liter1, the growth rate decreased, and only 40% of the
TCA was degraded within 14 days. The degradation discontinued when cell
growth stopped due to the lack of oxygen. It seemed that the
degradation of TCA by strain TA5 was associated with cell growth.
Strain TA27 degraded more than 95 and 40% of the TCA within 7 days
when the initial TCA concentrations were 0.5 and 25 mg · liter1, respectively, while 10% of the TCA was degraded
within 14 days when the initial TCA concentration was 75 mg · liter1 (Fig. 2B). The percentage of degradation decreased
with increasing initial TCA concentrations. This may be due to the
production of toxic metabolites.
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FIG. 2.
Effect of TCA concentration on TCA degradation by
strains TA5 and TA27. (A) Strain TA5; (B) strain TA27. Initial pH:
strain TA5, 6.8; strain TA27, 5.7. Initial ethane concentrations:
strain TA5, 10%; strain TA27, 5%. Initial TCA concentrations (in
mg · liter1):  , 75;  , 50;  , 25;  , 5;
, 2.5; , 0.5; , 0.25; , 0.05. Error bars indicate the
ranges for duplicate samples.
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|
Temporal pattern of TCA degradation products.
To isolate and
identify water-soluble TCA metabolites, after 7 days the culture broth
was acidified to pH 2 and extracted three times with the same volume of
diethyl ether (16). The organic phase was concentrated to
approximately 2 ml at reduced pressure and was methylated with
diazomethane; this was followed by gas-chromatographic mass
spectrometry. Two peaks (peaks A and B) were detected in both strains.
Peak B was identified as 2,2,2-trichloroethanol. We were unable to
identity the compound represented by peak A, but we determined that it
was not a chlorinated compound.
Figure 3 shows the temporal pattern of
residual TCA, 2,2,2-trichloroethanol production, residual oxygen and
ethane concentrations, cell growth, and the change of pH at an initial
TCA amount of 0.23 µmol. This experiment was carried out at the
favorable initial pH levels of 6.8 and 5.7 for TCA degradation of
strains TA5 and TA27, respectively. The concentrations of TCA, oxygen,
and ethane decreased with the increasing growth of strain TA5, while
cell growth reached a maximum after 3 days and when 55% of the initial TCA had been degraded.
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FIG. 3.
Temporal pattern of TCA degradation by strains TA5 and
TA27. (A) Strain TA5; (B) strain TA27. Initial ethane concentrations:
strain TA5, 10%; strain TA27, 5%. Initial pH: strain TA5, 6.8; strain
TA27, 5.7. Symbols: , residual TCA amount; ,
2,2,2-trichloroethanol production; , residual ethane concentration;
, residual O2 concentration; , bacterial growth
(optical density at 660 nm [OD660nm]); , pH. Error bars indicate
the ranges for duplicate samples.
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The growth of strain TA27 reached a maximum after 2 days and when 22%
of the initial TCA had been degraded; by 5 days, 99% of the TCA had
been degraded and the pH had decreased. A decrease of more than 2 U of
pH revealed the production of acidic metabolites and the capability to
degrade TCA at low pH levels. The major product of TCA degradation by
both strains was 2,2,2-trichloroethanol. The 0.13 and 0.23 µmol of
TCA consumed within 7 days was converted to 0.06 and 0.09 µmol of
2,2,2-trichloroethanol by strains TA5 and TA27, respectively; the rates
of TCA conversion to 2,2,2-trichloroethanol by strains TA5 and TA27
were 46 and 39%, respectively. These low percentages may have resulted
from other conversion products being made, or they may be due to
strains TA5 and TA27 degrading the 2,2,2-trichloroethanol. The
degradation products of 2,2,2-trichloroethanol need to be identified.
In summary, we isolated ethane-utilizing Mycobacterium spp.
that could degrade TCA at an initial concentration as high as 75 mg · liter1. 2,2,2-Trichloroethanol was a TCA
metabolite. We believe that these data will be useful in the
development of bioremediation techniques for TCA-contaminated sites.
Nucleotide sequence accession number.
The nucleotide sequences
of strains TA5 and TA27 have been deposited in the DDBJ, EMBL, and
GenBank databases under accession no. AB028483 and AB028482, respectively.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National
Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki
305-0053, Japan. Phone: 81-298-50-2542. Fax: 81-298-50-2571. E-mail:
yagiosa{at}nies.go.jp.
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Applied and Environmental Microbiology, October 1999, p. 4693-4696, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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