Journal of Environmental Biotechnology
Vol. 6, No. 1, 33–39, 2006

Original paper (regular paper)

Characterization of 2,4,6-Trinitrotoluene (TNT)-Metabolizing Bacteria
Isolated from TNT-Polluted Soils in the Yamada Green Zone,
Kitakyushu, JapanTOSHINARI MAEDA1, KIWAO KADOKAMI1,2 and HIROAKI I. OGAWA1*
Department of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering,
Kyushu Institute of Technology, 2–4 Hibikino, Wakamatsu-ku, Kitakyushu 808–0196, Japan
2
Aqua Research Center, Graduate School of Environmental Engineering, The University of Kitakyushu, 1–2–1 Shinike,
Tobata-ku, Kitakyushu 804–0082, Japan1* TEL: +81–93–695–6059 FAX: +81–93–695–6012* E-mail: ogawahi@life.kyutech.ac.jp(Received; 23 November, 2005/Accepted; 20 April, 2006)
Some soils in the Yamada Green Zone, Kitakyushu City, Japan, have been polluted by 2,4,6-trinitrotoluene (TNT), one of
explosives. In order to study the biodegradation and behavior of TNT in this zone, we screened the soils for TNT-biodegrading bacteria. We found 6 strains. Pseudomonas sp. strains TM15 and TM30, and Sphingomonas sp. strain TM22 could
biotransform TNT much better than P. putida strain TM38, P. fluorescens strain TM42, and Pseudomonas sp. strain
TM55. Their siblings possessing resemblances with these 3 strains were specially isolated from the TNT-contaminated soils inthis zone. Six strains converted TNT into reduction products, 2-hydroxylamino-4,6-dinitrotoluene (2HADNT), 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 2-amino-4,6-dinitrotoluene (2ADNT) and 4-amino-2,6-dinitrotoluene (4ADNT).
Strain TM38 also produced 2,4-dinitrotoluene (24DNT). TNT has a strong cytotoxicity although its metabolites were slighteﬀectives. The mutagenicity of 2HADNT and 4HADNT was high at the same level or more as that of TNT. The mutagenicity of 2ADNT, 4ADNT and 24DNT remained high, but were less than that of TNT.
Key words: 2,4,6-trinitrotoluene, soil microorganisms, biotransformation, mutagenicity, deformed frogs

1. Introduction
Since the Industrial Revolution, massive amounts ofchemicals have been produced and released into the environment around the world1,17). Some chemical compounds
such as polychlorinated dioxins and polychlorinated biphenyls(PCBs) resist degradation because of their stable and tightstructures, and remain in the environment, presenting
significant risks of toxicity, mutagenicity and carcinogenicity
to animals, especially mammals such as human being6,11,18,24,27,29,30,38). Such xenobiotics are degraded primarily
by soil microorganisms possessing species and biodegradability of the marvelous of diversities, and partly by ultraviolet
light. Thus, understanding the role of microorganisms inthese biotransformations is essential for understanding the
behavior of these materials in the environment.
The present Yamada Green Zone in Kitakyushu City (atlatitude 33° north and longitude 130° east) was once used
for packing 2,4,6-trinitrotoluene (TNT) and as an ammunition bunker15). Those activities ceased in 1972 and the
Yamada Green Zone was declared as the wide-area park in
1995. Since 1995, frogs (Rana ornativentris) have beenfound with superfluous forelegs in this zone. Mating experi-ments by Kashiwagi et al.20) revealed that these malformations are heritable. This finding suggests that the deformities
are caused by genetic mutations. Several questions thus
arise: What caused the decisive and fatal mutations? What
are the mutations? How are the deformities caused? Why
haven’t these deformed frogs disappeared though natural
selection? These questions are interesting from the viewpointsof environmental microbiology, genetics, population genetics
and embryology. Chemical analysis of the soils in the Yamada
Green Zone revealed a high concentration of TNT21),
but only background concentration of polychlorinateddibenzo-p-dioxins, dibenzofurans and PCBs as well as thosein other general sites19). Therefore, TNT and/or its breakdown products might aﬀect the frog’s genes. TNT is one of
the toxic chemicals in explosives because of the symmetriclocation of the nitro groups on the aromatic ring, which
limits attacks by enzymes involved in the metabolism ofaromatic compounds14). Also, TNT generates the frog’s
chromosomal aberration21). Because of this special feature,
TNT is persistent in the environment, presenting the riskof the toxicity, mutagenicity and carcinogenicity to animals34,37).
It is not clear whether soil bacteria in the Yamada Green

MAEDA et al.

34

Zone convert TNT into metabolites toxic to DNA. We began
our research into the biodegradation and dynamics of TNTby the microorganisms present in the Yamada Green Zone
by screening soil from that area for TNT-biodegrading
bacteria. In this paper, we report on the identification andcharacterization of some of the strains we isolated, on thebiodegradability of TNT by these bacteria, and on the
mutagenicities of TNT and its metabolites produced bythese strains. We also briefly discuss the possibility of a
relationship between the mutagenicities of TNT-degradation
compounds and deformed frogs in the Yamada Green Zone.

2. Materials and Methods
2.1.

Materials and chemicals

Soil samples were collected from 4 sites contaminatedwith TNT and 3 clean sites, and then screened for bacteria.
As chemical standards we used TNT (a gift of Chugoku
Kayaku Co. Ltd., Japan), 4-hydoroxylamino-2,6-dinitrotoluene (4HADNT) (a gift of Dr. Y. Kumagai, Tsukuba University, Japan), 2-hydroxylamino-4,6-dinitrotoluene (2HADNT)(a gift of Dr. R. Spanggord, SRI International, USA),
2-amino-4,6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT) (AccuStandard Inc., USA), 2,4-dinitrotoluene (24DNT) and 2,6-dinitrotoluene (26DNT) (Tokyo
Kasei Kogyo Co. Ltd., Japan). All chemicals were of thehighest purity commercially available. TNT solution(100 mg/L) was autoclaved (110°C , 10 min).
2.2.

Screening and identification of TNT-metabolizing
bacteria

M8 medium was identical to the M9 medium25), exceptthat NH4Cl was omitted. TNT solution (100 mL) with M8
minimal medium containing TNT (100 mg/L) and acetate(10 mM) as nitrogen and carbon sources was mixed with20 g of soil, and then the mixture was aerobically incubatedat 30 °C in the dark with shaking (120 rpm). After 1-weekincubation, serial dilutions were spread on M8 minimal
medium agar plates with TNT and acetate. Colonies wereisolated and characterized using standard procedures including the NF-18 Quick Identification Kit (Nissui, Japan) andAPI20NE bacterial identification system (BioMerieux,
Japan), and by homology search of 16S ribosomal RNA
genes22,23). Independent 200 clones from 1 soil sample wereisolated and examined. Sequences of 16S rDNA werecompared to the known DNA sequences in the composite
nonredundant database (including GenBank, EMBL, DDBJ
and PDB) by using the BLAST search program.
2.3.

Culture conditions

All isolated bacteria were aerobically grown until the latelogarithmic phase in Luria-Bertani (LB) broth (10 g Bactotryptone, 5 g Bacto yeast extract and 5 g sodium chloride/L-distilled water) containing 100 mg/L of TNT at 30°C in
the dark. Cells were washed twice with M9 buﬀer, and thenwere resuspended in 0.5 mL of the same buﬀer. Cell suspen-

sions were mixed into the TNT solution.
2.4. TNT biodegradation by isolated bacteria
Cells (about 5×106 cells/mL) were incubated in TNT
solution (100 mL) with M8 minimal medium containing
TNT (100 mg/L) and acetate (10 mM) at 30°C in the darkwith shaking (120 rpm). Percentage biotransformation was
calculated from the disappearance of TNT. We measured
the degrading of TNT and the production of nitrite from
TNT to see whether these bacteria could utilize TNT ascarbon and nitrogen sources. Cells were removed by centrifugation (5000×g, 10 min). The concentration of TNT wasdetermined by measuring the absorbance at 447 nm of thesupernatant (1.5 mL) supplemented with 240 µL 1 M
NaOH26). Biodegradation of the aromatic ring of TNT was
calculated from the disappearance of the aromatic ring as
measured by absorbance at 230–280 nm. Nitrite was determined by the method of Snell et al.31). The absorbance wasmeasured with a UV/VIS Spectrophotometer V-530 (Jasco,
Japan).
2.5.

Identification of TNT metabolites

The cells were aerobically grown for diﬀerent periods(indicated times in Table 2) at 30°C and were removed bycentrifugation at 5000×g for 10 min. The culture fluid was
extracted 3 times with 100 mL ethyl ether (pH 7.0 or 2.0),
then the extracts were dried over anhydrous sodium sulfate,and excess solvent was removed by rotary evaporation
under pressure at 30°C. Gas chromatography-mass spectrometry (GC-MS) analyses of these samples were carriedout with a HP6890 Series GC system/5973 Mass selective
Detector (Hewlett Packard, USA). TNT metabolites wereidentified by comparison of retention times and mass spectra between samples and standard chemicals. Also, detection of 2HADNT and 4HADNT were carried out by highperformance liquid chromatography (HPLC). HPLC
analyses were performed on an Inertsil ODS-2 column (GLSciences Inc., Japan) with acetonitrile-water (40 : 60) as themobile phase, with a flow rate of 0.4 mL/min. These chemicals were detected at 254 nm with a Shimazu SPD-10AVPUV-VIS detector (Japan).
2.6.

Cytotoxicity and mutagenicity

Cytotoxicity was assayed as follows. Frozen stock ofEscherichia coli K-12 strain AB11572) was streaked onto
LB plates and grown overnight at 37°C. A single colony wasthen suspended in 1.5 mL M8 buﬀer. Cell suspension (10 µL)
and LB medium (5 mL) containing TNT, 2ADNT, 4ADNT,
24DNT, 2HADNT or 4HADNT (100 parts LB medium to
1 part chemical in dimethylsulfoxide (DMSO); chemicals
tested at 0, 55, 110, 220 and 440 µM) were mixed and incubated for 12 h at 37°C. Serial dilutions were spread on LB
agar plates. These plates were incubated for 24 h at 37°C.
Survival rate was calculated as the ratio of viable cells ineach concentration of TNT, 2ADNT, 4ADNT, 24DNT,
2HADNT and 4HADNT to those in control. We also used

Pseudomonas and Sphingomonas from TNT polluted soils

the umu test with bioluminescent bacteria (Salmonella
typhimurium strain TA1535/pTL210, harboring the transcriptional fusion between the S. typhimurium umuC geneand the lux photoprotein genes from Vibrio fischeri (a giftof Toyota Central R&D Laboratories, Japan) to examine
the mutagenicity of TNT and its metabolites33,35). In thisstrain, expression of the lux operon is under the controlof the umuC promoter, from which transcription is induced
by DNA-damaging agents such as TNT. The intensity wascalculated from the integration value per second per well at
maximum luminescence with a JNR AB-2100 Luminescencer (ATTO, Japan).

3.
3.1.

Results

Dynamics of TNT-metabolizing bacteria

TNT-biodegrading clones were found in both TNTpolluted and unpolluted soils. However, all clones obtained
from unpolluted soils produced smaller colonies on
M8 minimal medium with TNT and acetate than those35

from polluted soils (data not shown). All pure clones wereGram-negative, non-glucose-fermenting rod bacteria. Characterization using the NF-18 and API20NE systems andhomology searches of 16S ribosomal RNA genes classified
these clones into 5 strains in the genus Pseudomonas and1 strain in Sphingomonas. Four strains, designated as Pseudomonas sp. strain TM15, TM30 and TM55, and Sphingomonas sp. strain TM22, may be new species, judging
from the 16S rDNA results. The DNA homologies ofTM15, TM22, TM30 and TM55 were 95.4% in Pseudomonas olevorans, 91.8% in Sphingomonas subractica, 96.2%
in Pseudomonas putida and 95.9% in Pseudomonas aeruginosa, respectively. The remaining strains were designatedas P. putida strain TM38 and Pseudomonas fluorescens
strain TM42. Pseudomonas sp. strains TM15 and TM30,
and Sphingomonas sp. strain TM22 were able to eﬃciently
biotransform TNT (Fig. 1). Isolates with biochemical
characteristics and 16S rDNA sequences similar to those of
TM15, TM22 and TM30, were also obtained from some
TNT-polluted soils (Site A, B, C and D indicated in Table 1)

Table 1. TNT biotransforming bacteria isolated from the soils of the Yamada Green Zone and their composition.
Site

TNT
concentration(µg/kg dry-soil)

A

120

B

67

C

13

D

7.5

E

N.D.c

F

N.D.

G

N.D.

StrainIsolation ratea

Pseudomonas sp. TM15
Sphingomonas sp. TM22
Pseudomonas sp. TM30
Pseudomonas sp. TM55
P. putida TM38
Pseudomonas sp. TM15 likeb
Sphingomonas sp. TM22 like
Pseudomonas sp. TM30 like
Pseudomonas sp. TM55 likeP. putida TM38 likeP. fluorescens TM42
Pseudomonas sp. TM15 like
Sphingomonas sp. TM22 like
Pseudomonas sp. TM30 like
Pseudomonas sp. TM55 likeP. putida TM38 like
Sphingomonas sp. TM22 like
Pseudomonas sp. TM30 like
Pseudomonas sp. TM55 likeP. putida TM38 like
Sphingomonas sp. TM22 like
Pseudomonas sp. TM55 likeP. putida TM38 like
Pseudomonas sp. TM55 likeP. putida TM38 like
Pseudomonas sp. TM55 likeP. putida TM38 likeP. fluorescens TM42 like

0.27
0.25
0.21
0.12
0.15
0.20
0.24
0.14
0.13
0.13
0.16
0.18
0.31
0.31
0.09
0.11
0.28
0.31
0.19
0.22
0.07
0.43
0.5
0.46
0.54
0.40
0.36
0.24

Two hundreds of independent clones were tested as described in Material and Methods from each soil samples.
Isolation rate indicates the ratio of isolated bacteria from each soil.
These strains have the sample characterization as beacteria isolated from soil A.
c
Not detected.
a

b

36

MAEDA et al.

Fig. 1. Biotransformation of TNT by the bacteria isolated fromthe TNT-polluted soils. The cells were incubated in TNT solution (100 mL) with M8 minimal medium containing TNT(100 mg/L) and acetate (10 mM) at 30°C in the dark withshaking (120 rpm). ○ , Pseudomonas sp. TM15. △ , Sphingomonas sp. TM22. ● , Pseudomonas sp. TM30. ▲ , Pseudomonas sp. TM55. □ , P. putida TM38. ■ , P. fluorescens
TM42.

Fig. 2. Release of nitrite by the bacteria isolated from the TNTpolluted soils. The cells were incubated in TNT solution(100 mL) with M8 minimal medium containing TNT (100 mg/
L) and acetate (10 mM) at 30°C in the dark with shaking(120 rpm). ○ , Pseudomonas sp. TM15. △ , Sphingomonas
sp. TM22. ● , Pseudomonas sp. TM30. ▲ , Pseudomonas sp.
TM55. □ , P. putida TM38. ■ , P. fluorescens TM42.

Fig. 3. Products resulting from TNT metabolism by isolated bacteria. 2ADNT, 4ADNT and 24DNT were identified by GC-MS analyses.
2HADNT and 4HADNT are detected by HPLC analyses. TNT, 2,4,6-trinitrotoluene. 2ADNT, 2-amino-4,6-dinitrotoluene. 4ADNT,
4-amino-2,6-dinitrotoluene. 24DNT, 2,4-dinitrotoluene. 2HADNT, 2-hydroxylamino-4, 6-dinitrotoluene. 4HADNT, 4-hydroxylamino-2,
6-dinitrotoluene.

Pseudomonas and Sphingomonas from TNT polluted soils

37

Table 2. Time course of TNT degradation to 2ADNT, 4ADNT and 24DNT by isolated bacteria.
strainchemicalsa

Reaction time (day)
0

1

3

7

TM15

TNT
2ADNT
4ADNT
24DNT

b

440.0
N.D.c
N.D.
N.D.

164.4
7.5
9.3
N.D.

25.2
1.8
2.9
N.D.

1.8
0.2
0.2
N.D.

TM22

TNT
2ADNT
4ADNT
24DNT

441.3
N.D.
N.D.
N.D.

173.9
15.7
20.6
N.D.

37.8
2.4
2.9
N.D.

2.4
0.4
0.7
N.D.

TM30

TNT
2ADNT
4ADNT
24DNT

440.8
N.D.
N.D.
N.D.

242.2
21.2
21.4
N.D.

121.1
39.3
39.8
N.D.

38.0
18.3
20.1
N.D.

TM38

TNT
2ADNT
4ADNT
24DNT

439.6
N.D.
N.D.
N.D.

373.7
14.9
6.2
6.0

329.3
34.5
11.7
2.5

327.3
16.8
6.0
1.1

TM42

TNT
2ADNT
4ADNT
24DNT

437.1
N.D.
N.D.
N.D.

415.2
7.5
2.0
N.D.

393.6
16.1
3.3
N.D.

358.5
11.93
1.5
N.D.

TM55

TNT
2ADNT
4ADNT
24DNT

448.8
N.D.
N.D.
N.D.

394.9
16.7
N.D.
N.D.

353.8
39.1
N.D.
N.D.

353.4
39.9
N.D.
N.D.

TNT, 2ADNT, 4ADNT and 24DNT indicated the compounds of 2,4,6-trinitrotoluene, 2-amino-4,6-dinitrotoluene,
4-amino-2,6-dinitrotoluene and 2,4-dinitrotoluene, respectively.
µM
c
Not detected.
abwhile isolates similar to the remaining strains were obtained
from polluted and unpolluted both soils. Nitrite was transiently accumulated and decreased in all bacterial cultures
except that of strain TM42 (Fig. 2), indicating that thesebacteria can release nitrite from TNT or the TNT metabolites and utilize it as a sole nitrogen source.
3.2.

TNT biotransformation by isolated bacteria

HPLC analysis detected 2HADNT and 4HADNT, and
GC/MS analysis identified 2ADNT, 4ADNT and 24DNT
as the metabolites from TNT. All strains except Pseudomonas sp. strain TM55 biotransformed TNT into 2HADNT,4HADNT, 2ADNT and 4ADNT. TM55 accumulated only
2ADNT (Fig. 3). P. putida strain TM38 also converted
TNT into 24DNT. The accumulation of 2,6-dinitrotoluene
was not observed in all bacteria. The production of thesecompounds by each strain is shown in Table 2. 2ADNT
and/or 4ADNT in the cultures of 6 strains and 24DNT in
the culture of TM38 were transiently accumulated, and thengradually decreased with time. Interestingly, strains TM38,
TM42 and TM55, which degraded TNT slowly, apparently
biotransformed TNT into 2ADNT in preference to 4ADNT,

which was predominant in 3 strains that biodegraded TNT
faster than TM38, TM42 and TM55.
3.3.

Cytotoxicity and mutagenicity of TNT and its
initial metabolites

We tested whether the 2ADNT, 4ADNT, 24DNT,
2HADNT and 4HADNT produced from TNT by the soil
microorganisms are cytotoxic to E. coli K-12 strain AB1157.
Increasing concentrations of TNT dramatically decreased
survival, whereas 2ADNT, 4ADNT, 24DNT, 2HADNT and
4HADNT had only a slight eﬀect (Fig. 4A). The mutagenicities of 2HADNT and 4HADNT were similar to or
higher than that of TNT. The mutagenicities of 2ADNT,
4ADNT and 24DNT were lower than that of TNT,
although still substantial (Fig. 4B). In the results of TNT
mutagenicity, luminescence disappeared above 71 pmol of
TNT, but remained in the presence of the other chemicals.
The reason is probably that the luminescent bacteria were
killed by a high concentration of TNT, which has a strong
cytotoxicity.

MAEDA et al.

38

Fig. 4. Cytotoxicity and mutagenicity of TNT and its metabolites,
2HADNT, 4HADNT, 2ADNT, 4ADNT and 24DNT,
produced by isolated bacteria. (A) Survival of E. coli cells
exposed to TNT (white bars), 2ADNT (black bars), 4ADNT(banded bars), 24DNT (hatched bars), 2HADNT (gray bar)and 4HADNT (grille bars). Data are averages of 5 independent experiments; error bars indicate the standard errors ofthe means. (B)Assay of mutagenicity of TNT ( ● ), 2HADNT( ▲ ), 4HADNT ( △ ), 2ADNT ( ■ ), 4ADNT ( □ ) and 24DNT
(×).

4.

Discussion

We isolated TNT-biodegrading bacteria from the soils in
the Yamada Green Zone, where abnormal frogs have appeared and where TNT has been detected. These bacteria
converted TNT into 2HADNT, 4HADNT, 2ADNT,
4ADNT and 24DNT (Fig. 3), in agreement with the resultsof previously studies9,14,32). These bacteria seem to be involved in the degradation of TNT in the Yamada Green
Zone. 2ADNT and 4ADNT are produced from TNT bysix-electron reduction of its nitro group. Barrow et al.4)
suggested that the nitro group in the para position is much
more easily reduced than those in the ortho positions of
TNT because of an electron-donor eﬀect of the methylgroup (namely, accumulation of 4ADNT is expected to behigher than that of 2ADNT). Strains TM15, TM22 andTM30, which were able to eﬃciently degrade TNT, preferentially accumulated 4ADNT, in contrast to strains TM38,
TM42 and TM55, which preferentially accumulated
2ADNT (Table 2). These results may be due to diﬀerencesin substrate specificity of degrading enzymes and/or in thesubsequent biodegradability of 2ADNT and 4ADNT. Strain
TM38 also converted TNT to 24DNT through the elimina-tion of a nitro group (Fig. 3). This result resembles the
TNT biotransformation pathway reported by Duque et al.8).
As shown in Fig. 4-B, 2HADNT and 4HADNT arestrong mutagens as well as TNT and they have reported insome previous reports3,5,28). Homma-Takeda et al. reported
that these compounds caused oxidative DNA damage(formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine) in thepresence of NADH and Cu(II)16). Also, partially reduced
forms of TNT react among themselves under aerobic conditions to form recalcitrant tetranitro-azoxytoluenes12), whichcause a higher rate of mutations than does TNT10). The
mutagenicities of 2ADNT and 4ADNT were less than thatof TNT although still high in our results. This result is
consistent with previous reports7,10,34). 2ADNT and 4ADNT
are more stable in the environment and more mobile, posing
a potential threat to drinking water supplies, human healthand the environment13). On the other hand, cytotoxicity of
2HADNT, 4HADNT, 2ADNT, 4ADNT and 24DNT wasslight compared to that of TNT (Fig. 4A). Kashiwagi etal.20) and Kitakyushu City Agency21) suggested that the frog’s
deformities were caused by genetic mutations induced bysome factors. The results of their mating experiment of four
patterns (normal males×normal females, normal males×
abnormal females, abnormal males×normal females, andabnormal males×abnormal females) demonstrated that
only abnormal oﬀsprings were born from deformed males.
Namely, mutations leading to deformities are transferred todescendants by the sperm. They assumed that the mutations
were induced in the frog’s sperm in vivo or fertilized eggs in
the environment. Because sperm cells have low activities of
DNA repair enzymes36), mutations are expected to be easily
fixed in sperm cells. In this paper, we revealed that TNT
and its metabolites have the DNA damaging activities;
therefore, as one of possibilities, these compounds may relate to generating the frogs with deformities in the Yamada
Green Zone. Developed studies on how these metabolites
have genotoxic eﬀects for frogs or their sperm cells will be
required. Also, we will need to research detail distribution
analyses (dynamics) for accumulation of TNT and its metabolites in the Yamada Green Zone.

Acknowledgments
We are grateful to Chugoku Kayaku Co. Ltd., Japan for
their gift of 2,4,6-trinitrotoluene, to Dr. Yoshito Kumagai,
Institute of Community Medicine, University of Tsukuba
for his gift of 2-hydrodylamino-4,6-dinitrotoluene, to Dr.
Ronald Sapanggord, Chemical Sciences and Technology
Department, SRI International for his gift of 4-hydroxylamino-2,6-dinitrotoluene, and to researchers at Toyota Central R&D Laboratories who supplied strain S. typhimurium
TL210. We thank Kyoko Tanada, Aqua Research Center,
Kitakyushu City Institute of Environmental Sciences, Japanfor her support in the umu test, and Yukiko Koyama andHitoshi Ohara, TOTO Ltd., Japan for their support in
DNA sequencing.

Pseudomonas and Sphingomonas from TNT polluted soils

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