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Applied and Environmental Microbiology, July 1999, p. 2977-2986, Vol. 65, No. 7
0099-2240/99/$04.00+0
Biotransformation of 2,4,6-Trinitrotoluene with
Phanerochaete chrysosporium in Agitated Cultures at pH
4.5
Jalal
Hawari,1,*
Annamaria
Halasz,1
Sylvie
Beaudet,1
Louise
Paquet,1
Guy
Ampleman,2 and
Sonia
Thiboutot2
Biotechnology Research Institute, National
Research Council, Montreal, PQ H4P 2R2,1 and
Defence Research Establishment Valcartier, National Defence,
Val Bélair, PQ G3J 1X5,2 Canada
Received 14 December 1998/Accepted 14 April 1999
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ABSTRACT |
The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 µM) by
Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared
completely, but mineralization (liberated
14CO2) did not exceed 1%. A time study
revealed the presence of several intermediates, marked by the initial
formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT)
followed by their successive transformation to several other products,
including monoaminodinitrotoluenes (ADNT). A group of nine acylated
intermediates were also detected. They included
2-N-acetylamido-4,6-dinitrotoluene and its p
isomer, 2-formylamido-4,6-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene
and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated
DANT), 4-N-acetylhydroxy-2,6-dinitrotoluene and
4-N-acetoxy-2,6-dinitrotoluene (as acetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene.
Furthermore, a fraction of HADNTs were found to rearrange to their
corresponding phenolamines (Bamberger rearrangement), while another
group dimerized to azoxytoluenes which in turn transformed to azo
compounds and eventually to the corresponding hydrazo derivatives.
After 30 days, all of these metabolites, except traces of 4-ADNT and
the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The
biotransformation of TNT was accompanied by the appearance of manganese
peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP
activity was observed almost immediately after TNT disappearance, which
was the period marked by the appearance of the initial metabolites
(HADNT and ADNT), whereas the LiP activity was observed after 8 days of
incubation, corresponding to the appearance of the acyl derivatives.
Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.
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INTRODUCTION |
Contamination of soils by explosives
such as 2,4,6-trinitrotoluene (TNT), generated as waste from the
munitions and defense industries, is a significant worldwide
environmental problem. It is estimated that TNT alone is produced in
amounts close to 2 million pounds a year (19) and threatens
human life through the food chain (51). The compound is
mutagenic and toxic and has a tendency to persist in the environment
(34, 44, 49, 51). There have been several attempts to
biodegrade TNT, but thus far the compound has been found to undergo
biotransformation rather than mineralization (5, 9, 13, 16, 28,
41, 48), giving in most cases the initial products
4-amino-2,6-dinitrotoluene (4-ADNT), 2-amino-4,6-dinitrotoluene
(2-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT), and
2,6-diamino-4-nitrotoluene (2,6-DANT) (11, 24).
Several other studies on the degradation of TNT by Phanerochaete
chrysosporium have been reported, and in most cases mineralization amounts larger than those normally obtained with bacteria were observed
(8, 14, 29, 40, 41, 43). The degree of TNT mineralization
varies and depends on whether ligninolytic (nitrogen-limiting) or
nonligninolytic (nitrogen-sufficient) conditions are used in the
culture medium. For example, Fernando et al. (14) reported 85% degradation of TNT in both water (100 ppm) and soil (10,000 ppm),
with 18.4 and 19.6% mineralization in stationary ligninolytic culture
medium, respectively. This suggested that TNT is not toxic to the fungi
at high concentrations. No products were identified to account for the
remaining TNT that was degraded. Spiker et al. (40)
demonstrated that a TNT concentration of greater than 15 ppm inhibited
mineralization, resulting in 1 to 3% of 14CO2
being liberated. Furthermore, Michels and Gottschalk (30) have reported that high concentrations of TNT inhibit lignin peroxidase (LiP) of the fungi.
The sluggish mineralization that is frequently observed for TNT despite
its efficient transformation is attributed to the formation of dead-end
products that act to deroute the process of mineralization. The
identities of these transformed products remained, in most cases,
unknown due to the absence of rapid and sensitive analytical techniques
suitable for direct detection of transient species during the course of
the reaction. We have recently reported that despite the almost
complete disappearance of TNT with an anaerobic sludge, negligible
amounts of 14CO2 were detected (20).
Liquid chromatography-mass spectrometry (LC-MS) and SPME gas
chromatography-MS studies revealed the predominant formation of
triaminotoluene (80%), which subsequently polymerized or was
transformed to other phenolic products. The latter compounds were
formed through hydrolytic cleavage of the NH2 group
(20) rather than through Bamberger rearrangement, which is
encountered in the formation of phenolamines from hydroxylamino
aromatic compounds (10, 15, 21).
One objective of the present study was to apply LC-MS in an attempt to
identify all possible transformed products involved in the
transformation of TNT with the fungus P. chrysosporium in
agitated cultures at pH 4.5. A time course study to help understand the
fate of these products and their effect on the mineralization process
will also be discussed.
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MATERIALS AND METHODS |
Reagents and materials.
TNT was obtained from Centre de
Recherche pour la Défense (Valcartier, Quebec, Canada) with a
chemical purity, as measured by high-pressure liquid chromatography
(HPLC), of 9.5%. [U-14C]TNT was synthesized to a
radiochemical purity that exceeded 95%, as described by HPLC by
Ampleman et al. (2). 2-ADNT, 4-ADNT, 2,4-DANT, 2,6-DANT,
2-hydroxylamino-4,6-dinitrotoluene (2-HADNT), 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT),
2,2',6,6'-tetranitro-4,4'-azoxytoluene (TN-4,4'-AzoxyT),
4,4',6,6'-tetranitro-2,2'-azoxytoluene (TN-2,2'-AzoxyT), 2,2',6,6'-tetranitro-4,4'-azotoluene (TN-4,4'-AzoT), and
4,4',6,6'-tetranitro-2,2'-azotoluene (TN-2,2'-AzoT) were obtained from
AccuStandard Inc. (New Haven, Conn.). The molasses used was a cane
sugar which was analyzed by HPLC and found to contain 36% sucrose, 6%
glucose, and 7% fructose. This type of molasses is also known to
contain pantothenic acid (25 ppm) and only 0.1% nitrogen
(25).
Microcosms for degradation of TNT.
In a typical setup, a
serum bottle (100 ml) was charged with 40 ml of the mineral salt medium
used in the procedure described by Greer et al. (18) [13 mM
KH2PO4, 6.4 mM Na2HPO4,
0.395 mM MgSO4 · 7H2O, 1 µM
AlK(SO4)2 · 12H2O, 2 µM
FeSO4 · 7H2O, 10 µM ZnSO4 · 7H2O, 10 µM
MnSO4 · H2O, 1 µM
CuSO4 · 7H2O, 1 µM
Co(NO3)2 · 6H2O, 10 µM
Ca(NO3)2 · 2H2O, and 2 µM
NaMoO4 · 2H2O], followed by the
addition of molasses (2.65 g/liter) as a carbon source and citric acid
(2.5 g/liter) to maintain a pH of 4.5. The mixture was then autoclaved
at 120°C for 40 min. The fungal strain used in the study was P. chrysosporium BKM-F-1767 (ATCC 24725) and was kept on malt agar
slants (20 g of agar, 20 g of malt extract, and 1 g of yeast
extract/liter). Spore solution (1-ml aliquots; 5 × 106 spores/ml) was added to each microcosm; this was
followed by the addition of TNT, taken from an acetone stock solution
(39,380 ppm), to a final concentration of 40 ppm. The microcosms were then sealed with Teflon-coated serum caps for incubation at 37°C in a
rotary shaker (Brunswick, Edison, N.J.) at 135 rpm. Some serum bottles
(microcosms) were supplemented with [U-14C]TNT (100,000 dpm) and then fitted with a small test tube containing 1.0 ml of 0.5 M
KOH to trap liberated carbon dioxide (14CO2).
The headspace in each microcosm was flushed with oxygen gas to maintain
aerobic conditions and then sealed with butyl rubber septa and aluminum
crimp seals to prevent the loss of 14CO2 and
other volatile metabolites. Control microcosms were prepared by using
the fungus and culture medium without TNT, and a second control
contained an autoclaved fungus medium to which TNT but no fungi were
added. Each microcosm was wrapped with aluminum foil to protect the
mixture against photolysis. Microcosms with 14C-labeled TNT
were routinely sampled (daily or every 2 days) for the determination of
14CO2 in the KOH trap by using a Tri-Carb 4530 liquid scintillation counter (model 2100 TR; Packard Instrument
Company, Meriden, Conn.). Microcosms that did not receive
14C-labeled TNT were reserved for LC-MS analysis of
residual TNT and its metabolites in the aqueous phase after filtration.
After LC-MS analysis, these filtrates were extracted with acetonitrile to account for any insoluble TNT metabolites. Certain microcosms were
sacrificed to determine the metabolite concentrations in the mycelium
mat. The separated mycelia were sonicated (Blackstone Ultrasonics,
Jamestown, N.Y.) with acetonitrile (10 ml) at 10°C for 16 h. The
decanted acetonitrile layer was filtered through a 0.45-µm-pore-size
Millex-HV filter for subsequent LC-MS analysis.
Enzyme assays.
LiP activity was determined by monitoring the
conversion of veratryl alcohol to veratryl aldehyde by hydrogen
peroxide at 310 nm as described by Tien and Kirk (45). The
Mn(II)-dependent peroxidase (MnP) activity was determined by monitoring
the disappearance of vanillyl acetone at 334 nm as described by
Paszcynski et al. (33).
LC-MS.
LC-MS was performed on a Micromass Platform II
benchtop single-quadrupole mass detector fronted by a Hewlett-Packard
1100 series HPLC system. The chromatographic conditions used were a C8 LC column (25 cm by 4.6 mm; 5-µm-diameter particles)
and acetonitrile-water gradient programmed from 30 to 80% (vol/vol),
using a flow rate of 1 ml/min with a postcolumn split of 5:95. Analyte
ionization, a process which produces mainly the deprotonated molecular
mass ion M 
H, was achieved in the negative electron spray
ionization mode by using a probe tip potential of 3.0 kV and a skimmer
voltage of 30 V. The temperature of the electron spray ionization
capillary was maintained at 90°C. The mass spectrum was typically
scanned at a rate of 1 s/100 Da. The total ion current was acquired
between 40 and 500 Da, which was followed by extracting the
deprotonated molecular mass ion [M H]
of the
suspected metabolite. In the case of DANT, analyte ionization was
achieved by using positive electron spray ionization, a process which
produces mainly the protonated molecular mass ion M + H.
Further confirmation of the identities of targeted metabolites was
accomplished by comparison with commercially available reference
compounds. Alternatively, in the case of acetylated metabolites, the
standards were synthesized starting from the corresponding amine by
using the acetic anhydride-bicarbonate method (4). Briefly,
1-ml aqueous aliquots (1 mM) of either the monoamine ADNT, the diamine
DANT, or the hydroxylamine HADNT were treated with acetic anhydride (or
formic acid) and stirred at room temperature for 30 min. The mixture
was neutralized with sodium bicarbonate for subsequent direct analysis
by LC-MS.
Analysis of nitrite and ammonium ions.
The aqueous layer was
analyzed for NO2 ions with an SP 8100 HPLC
with a 25- by 0.46-cm PRP-X 100 Hamilton column and a Waters 431 conductivity detector. Methanol (10%) buffered (pH 8.5) with a
solution of p-hydroxybenzoic acid was used as the mobile
phase at flow rate of 2 ml/min. Analytical grade sodium nitrite was used as the standard. Ammonium ions were analyzed by using the same
system but with a PRP-X 200 Hamilton column with 30% methanol in a 6 mM nitric acid solution at a flow rate of 0.75 ml/min.
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RESULTS AND DISCUSSION |
Metabolite identification.
Figure
1 shows a typical representation of TNT
(40 mg/liter) transformation profiles at three time points, i.e., after
0, 3, and 25 days of incubation with the fungus P. chrysosporium. Several LC-MS signals, representing TNT
intermediate products, were detected during the first 3 days of
incubation, which in turn, as the time progressed, transformed to other
products. Despite TNT disappearance during the first 10 days of
incubation, mineralization did not exceed 1% as measured by liberated
14CO2. After 30 days,
14CO2 liberation reached its maximum value of
10% of the original TNT amount, since no more mineralization was
observed even after the incubation period was extended to 120 days. As
mentioned earlier, high concentrations of TNT have been reported to be
toxic to the fungus (40) and to inhibit LiP production
(30). In the present study, we found that a minimum
concentration of 50 mg of TNT per liter was needed before TNT's toxic
effect could be observed (data not shown).
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FIG. 1.
Typical time course profile of the disappearance of TNT
(40 mg/liter) and the appearance of its intermediate products after
treatment with the fungus P. chrysosporium in an agitated
culture with molasses at pH 4.5. Profiles were obtained at three
different time points (top, 0 days; middle, 3 days; bottom, 25 days).
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To help understand the fate of TNT, particularly that of the
transformed fraction that escaped mineralization, key products
were
first identified, subgrouped according to their functional
groups, and
then fitted in a pathway by using a time course study.
In most cases,
the TNT metabolites were identified by using a
combination of their
mass data as represented by the deprotonated
molecular mass ion and the
retention time (M
H [in daltons],
retention time [in
minutes]) and, when available, by comparison
with reference materials.
In a few cases the identities of the
products had to be predicted based
on their mass data and their
estimated location in the transformation
process.
The first such group of LC-MS signals (peaks 4 to 7) were identified by
comparison with commercially available reference materials
as 2-HADNT
(peak 4) (212, 15.3), 4-HADNT (peak 5) (212, 16.3),
2-ADNT (peak 6)
(196, 19.8), and 4-ADNT (peak 7) (196, 20.6).
The two suspected nitroso
derivatives 2-nitroso-4,6-dinitrotoluene
(2-NsT) (peak 2) (210, 25.1)
and 4-nitroso-2,6-dinitrotoluene
(4-NsT) (peak 3) (210, 25.1) severely
overlapped with TNT and
could be identified only by extracting their
M
H at
m/z 210
Da. The mass spectrum of TNT alone
did not show this characteristic
mass ion. These initial products have
been frequently detected
under both aerobic and anaerobic conditions
(
8,
12).
A second group of LC-MS peaks (peaks 8 to 16) were identified as the
acyl (formyl and/or acetyl) derivatives of ADNTs (
ortho and
para), HADNTs (
ortho and
para),
2,4-DANT, and 2-hydroxylamino-4-amino-6-nitrotoluene.
Peaks 8 (224, 12.0) and 9 (224, 15.7), matching a molecular formula
of
C
8H
6N
3O
5, were
identified as 2-formamido-4,6-dinitrotoluene
(2-N-FmDNT) and
4-formamido-2,6-dinitrotoluene (4-N-FmDNT), respectively.
Their
identities were confirmed by comparison with reference materials
prepared by reacting 2- and 4-ADNT separately with formic acid
and
sodium bicarbonate. Likewise, peaks 10 and 11 both showed
their
[M
H]
at
m/z 238 Da, which
represented a molecular formula of
C
9H
8N
3O
5,
and were
identified as
ortho-acetylamido-4,6-dinitrotoluene
(2-
N-AcDNT)
and
para-acetylamido-2,6-dinitrotoluene (4-
N-AcDNT),
respectively.
Their identities were confirmed by comparison with
reference materials
prepared by acetylating 2- and 4-ADNT separately
with acetic anhydride
and sodium
bicarbonate.
Similarly, peaks 12 (254, 9.1) and 13 (254, 12.8) showed the same
[M
H]
at
m/z 254 Da, which matched a
molecular formula of
C
9H
8N
3O
6.
The two peaks
were tentatively identified as
2,6-dinitro-4-
N-acetylamidohydroxytoluene
(4-
N-AcHDNT) (peak 12) and
2,6-dinitro-4-
N-acetoxytoluene (4-
N-AcoxyDNT)
(peak 13). We presumed that isomer 12, which is expected to be
more
polar because of a free OH group, eluted before isomer 13.
No acetyl
derivatives of 2-HADNT were observed, presumably because
their
formation was inhibited by the steric effects of the
ortho-CH
3 group (
10,
29). In an
attempt to prepare the two suspected
products by acetylating the HADNT,
we obtained instead two products
with [M
H]
also occurring at 254 Da but with retention times different from
those
for peaks 12 and 13. These two chemically generated products
were
presumed to be the acetyl derivatives of the phenolamines
formed via
the acid-catalyzed Bamberger rearrangement of HADNT.
This result might
be taken as indirect evidence that a selective
enzymatic acetylation of
4-HADNT occurred to produce products
12 and 13 in the fungus-treated
TNT
culture.
On the other hand, LC-MS peaks 14 to 16, which appeared between 5.0 and
7.0 min, became visible only after 20 days of incubation
(Fig.
1).
Peaks 14 (194 Da, 6.7 min) and 15 (208, 7.1) matched
molecular formulas
of C
8H
8N
3O
3 and
C
9H
10N
3O
3,
respectively. Reference
materials prepared by acylating 2,4-ADNT with
either formic acid
or acetic anhydride showed LC-MS data similar to
those obtained
earlier for the two metabolites 14 and 15 (i.e., 194, 6.7 and
208, 7.1, respectively). Peaks 14 and 15 were eventually
identified
as 4-
N-formamido-2-amino-6-nitrotoluene
(4-
N-FmANT) and
4-
N-acetylamino-2-amino-6-nitrotoluene
(4-
N-AcANT), respectively. The third LC-MS peak, peak 16 (224,
5.52), which appeared at a retention time of 5.52 min and
possessed
an [M
H]
at
m/z 224 Da,
matched a molecular formula of
C
9H
10N
3O
4. Another
relevant mass ion at
m/z 286 Da was also observed and was
attributed
to M
+ CH
3CN + H
2O + 2H
+. This peak was tentatively
identified as
4-
N-acetylamino-2-hydroxylamino-6-noitrotoluene
(4-
N-AcOHANT), since no reference materials could be
obtained.
Further details are shown in Fig.
2. The three acylated derivatives
14 to
16 have been observed recently by Bruns-Nagel et al. (
6)
during a coupled anaerobic-aerobic composting of TNT. In that
case,
however, a positive chemical ionization was used in the
LC-MS, thus
giving the protonated molecular mass ions [M + H]
+
at
m/z 196, 210, and 226 Da instead of the present
deprotonated
[M
H]
values at
m/z
194, 208, and 224 Da, respectively.
Two more LC-MS peaks, designated 17 (212 Da, 25.9 min) and 18 (212, 26.1), both matched a molecular formula of
C
7H
6N
3O
5, which
was
similar to that obtained earlier for the two HADNT isomers
4 and 5. However, when either 2-HADNT or 4-HADNT was treated with
dilute
hydrochloric acid (pH 4.5), a major LC-MS signal was detected
in each
case with the same [M
H] at
m/z 212 Da but with
the
same retention times as those observed for peaks 17 and 18 (i.e.,
25.9 and 26.1 min, respectively). These two peaks were eventually
identified as the two phenolamines
ortho-amino-5-hydroxy-4,6-dinitrotoluene
(2-A-5-OH-4,6-DNT)
(peak 17) and
para-amino-5-hydroxy-2,6-dinitrotoluene
(4-A-5-OH-2,6-DNT) (peak 18). It has been reported that when aromatic
hydroxylamines are generated under acidic conditions the NHOH
group
rearranges to produce the corresponding phenolamine (
3,
26,
37,
42,
50). This acid-catalyzed rearrangement, known
as Bamberger
rearrangement, has recently been found to occur enzymatically
during
TNT degradation under neutral conditions with the anaerobic
microorganism
Clostridium acetobutylicum (
22).
The last group of peaks, designated 19 to 24, with retention times
ranging from 30 to 33 min, were apparently related to a
dimerization
reaction that involved HADT. Peaks 19 and 20 were
identified as the two
azoxy isomers TN-2,2'-AzoxyT and TN-4,4'-AzoxyT,
respectively, by
comparison with commercially available reference
materials, using their
[M
H]
at
m/z 405 and their retention
times at 32.1 and 32.3 min, respectively.
The formation of TNT azoxy
products is frequently observed under
both biotic and abiotic
conditions, and their formation was attributed
to a spontaneous
condensation between 4-HADNT and 4-NsT (
29).
Likewise, the
two peaks 21 and 22 also had different retention
times (i.e., 33.5 and
34.1 min) but the same [M
H]
(
m/z
389 Da). The two signals both had the [M
H]
at
16 mass units (1 O atom) lower than that of the corresponding
azoxy
derivative, indicating their presence as the corresponding
reduced
azoxy isomers. By comparison with commercially available
reference
materials, these two peaks were identified as TN-2,2'-AzoT
(peak 21)
and TN-4,4'-AzoT (peak 22). The remaining pair of LC-MS
signals, peaks
23 and 24, which were not completely resolved,
were also detected at
two different retention times (i.e., 33.3
and 33.7 min) but once again
with the same [M
H]
at
m/z 391 Da,
which was 2 mass units (2 H atoms) higher than
that of the
above-described azo dimers. Peaks 23 and 24 were tentatively
identified
as the reduced forms of the azo derivatives (i.e.,
4',6,6'-tetranitro-2,2'-hydrazotoluene [TN-2,2'-HydrazoT] and
2,2',6,6'-tetranitro-4,4'-hydrazotoluene [TN-4,4'-HydrazoT],
respectively.
Time course profiles of metabolites for mechanism elucidation.
As the preceding discussion indicated, there were several products
formed during TNT degradation with P. chrysosporium. To help
us to understand the various transformations among these intermediates,
LC-MS time course studies were conducted. The LC-MS peak areas of TNT
and/or its intermediate products were measured at various time points
and graphed to produce profiles that can be used to monitor the
appearance and disappearance of related intermediates. To measure
mineralization (liberated 14CO2) a separate set
of micrococosms containing [U-14C]TNT was used.
To help us to understand the physiological state of the fungus during
TNT biotransformation, both LiP and MnP activities were
measured at
various time intervals, as shown in Fig.
3. The fungus
was found to exhibit both
MnP and LiP activities. However, the
LiP activity was found to be lower
than that of MnP by at least
a factor of 10. Also, a lag period of 6 to
8 days was needed before
LiP activity could be observed. In contrast,
no such delay was
observed in the case of MnP. Interestingly, both
enzymes showed
maximum activity at between 10 and 15 days, after which
the activities
of both enzymes declined until they approached zero
after 20 days
of incubation. Stahl and Aust (
41) have
reported similar physiological
behavior from the same fungus spores.
However, the MnP activity
was found to be higher than the LiP activity
by at least a factor
of 5.
Figure
4 shows the disappearance of TNT
together with the appearance of its two prime metabolites, 2- and
4-HADNT (metabolites
4 and 5), and their reduced monoamines
(metabolites 6 and 7).
Both HADNT prime products were transformed
beyond detection after
less than 10 days of incubation, which was also
the time marked
by TNT disappearance under nonligninolytic conditions
(Fig.
3).
The reduction in the concentrations of the two HADNTs was
accompanied
by a gradual buildup of the two monoamines 2-ADNT and
4-ADNT (metabolites
6 and 7). Although the regioselectivity of HADNT
formation seemed
to favor reduction at the
ortho position by
a factor of 2,
para-ADNT
(metabolite 7) was formed in a
yield which was 25% higher than
that of its
ortho isomer
(metabolite 6). This indicated that the
rate of 2-HADNT transformation
is higher than that for its
para isomer.
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FIG. 4.
Time course profile for the disappearance and appearance
of TNT prime metabolites HADNT and ADNT together with mineralization
data following the treatment of TNT (40 mg/liter) with the fungus
P. chrysosporium. A-OH-DNTs (metabolites 17 and 18) are the
summed Bamberger-rearranged products of HADNTs (4 and 5).
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Figure
4 shows that the two HADNTs could be observed only in the
presence of the original substrate TNT and not necessarily
in the
presence of the monoamines, thereby implying the absence
of any
reversible connection between HADNTs (metabolites 4 and
5) and their
reduced monoamines ADNTs (metabolites 6 and 7). The
formation of the
two monoamines peaked at between 10 and 15 days,
but almost complete
transformation occurred after 30 days. Interestingly,
the highest
ligninase activity was observed between 10 and 15
days. After 10 days
of incubation, a period marked by the disappearance
of TNT and its two
prime products 4 and 5, roughly 30% of the
transformed TNT could be
accounted for by the formation of the
two ADNTs 6 and 7. Despite the
disappearance of TNT and its prime
metabolites 4 to 7, less than 10%
of the transformed amount of
TNT was measured as
14CO
2. As Fig.
4 shows, mineralization
commenced after 6 days, which
is the period marked by the appearance of
ligninase activity (Fig.
3). In contrast, TNT disappearance and the
subsequent formation
of its prime metabolites HADNTs and ADNTs were
obviously nonligninolytic
processes.
Another time course study was thus conducted to monitor the formation
and disappearance of the monoamine metabolites (metabolites
6 and 7)
against those of other TNT intermediates, such as Bamberger-rearranged
intermediates (2-A-5-OH-4,6-DNT [metabolite 17] and 4-A-5-OH-2,6-DNT
[metabolite 18]) and the acyl derivatives (Fig.
5). By examining
the time profiles of TNT
biotransformation in Fig.
5, it can be
seen that the metabolites can be
classified into primary and secondary
products. For example, the data
in Fig.
5 clearly shows two parabolic
curves: one to the left between 2 and 10 days and the second to
the right between 3 and 30 days,
representing the evolution of
their secondary acyl (actyl and formyl)
products (metabolites
8 to 11). Interestingly, the formation of the
prime ADNT metabolites
(6 and 7) started immediately after the
disappearance of TNT under
nonligninolytic conditions, and their
presence was maintained
into the ligninolytic state of the fungus,
whereas the formation
of the acyl secondary products (Fig.
5)
correlated with the LiP
and MnP enzymatic activity profiles of the
fungus (Fig.
3), in
which maximum amounts of these products were
obtained after both
enzymes achieved their maximum activity levels,
i.e., after 10
to 15 days of incubation. Valli et al. (
47)
reported that the
initial amine metabolites formed from the treatment
of 2,4-dinitrotoluene
with the same fungus undergo oxidation by MnP to
produce quinones.
These quinones are then reduced, methylated, and
denitrated by
either LiP and MnP. However, neither nitrire nor nitrate
was found
in the present study.
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|
FIG. 5.
Time course study of the formation and disappearance of
TNT prime metabolites (HADNT and ADNT) together with their secondary
products the phenolamines (A-OH-DNTs) and the acylated derivatives
during TNT degradation with the fungus P. chrysosporium. The
cumulative amounts of the Bamberger-rearranged phenolamines (A-OH-DNTs)
were plotted instead of the discrete isomeric ones. In the case of the
acyl derivatives (8 to 11) the peak area counts instead of the actual
amounts were used to draw the curves.
|
|
Figure
5 shows that the amounts of the formylated metabolites
(Fm-
N-DNT; metabolites 8 and 9) were always larger than
those
of the acetylated ones (Ac-
N-DNT; metabolites 10 and
11) by at
least a factor of 2. Also, the
para isomer in each
case was formed
in a yield which is about 25% higher than that of its
ortho counterpart,
which was possibly caused by steric
inhibitory effects from the
ortho CH
3 group. All
acylated derivatives 8 to 11 could be detected
only while their
suspected precursors, the two monoamines, were
still present in the
system. After 30 days of incubation, both
the monoamines (6 and 7) and
their acylated derivatives (8 to
11) disappeared, but without causing a
dramatic increase in mineralization
(i.e., liberated
14CO
2 did not exceed 10%). This observation
might support the reversible
connection between the acylated
metabolites and their corresponding
precursors, the monoamines. For
instance, this hypothesis was
recently supported by Bruns-Nagel et al.
(
6), who described
the reversible formation of acetylated
TNT intermediates (i.e.,
they do not form dead-end products in the
degradation process).
It has also been reported that formylation and
acetylation processes
could serve as detoxification mechanisms in soil,
as is the case
with aniline (
46). In the present study, the
presence of the
amines and their acyl derivatives together may support
the view
of their coexistence in a reversible
relationship.
The acylated derivatives 12 to 16 were not included in the time course
study. However, both acetyl derivatives of HADNT, 12
and 13, could be
detected only as long as HADNT was present in
the system, also implying
the presence of a reversible reaction
between them. The acylated
metabolites 4-
N-FmANT (metabolite 14),
4-
N-AcANT
(metabolite 15), and 4-
N-AcHANT (metabolite 16) were
all
detected in trace amounts and could not be quantified for
inclusion in
the time course
study.
Since no significant increase in CO
2 was observed and all
of these acylated intermediates disappeared, then one may ask what
became of them. Although we cannot provide an answer to this question
at this time, we can presume that some of these acylated derivatives,
particularly 12 and 13, are reduced to give ADNT in a reaction
similar
to the one that occurred for the reduction of HADNT to
produce ADNT.
For instance, by the end of the incubation period,
which lasted 30 days, 4-ADNT was the only prime metabolite that
could be detected
(although in trace amounts). Nonetheless, none
of the detected acylated
TNT intermediates accumulated in the
system.
It was also presumed that 4-
N-AcANT (metabolite 15) was a
derivative of 2,4-DANT and not the reverse. The 2,4-DANT itself
was
detected only occasionally and in trace amounts. Furthermore,
4-
N-AcANT was encountered in soil samples that had been
contaminated
with 2,4-DANT and also in experiments designed to
biodegrade the
diamine (
36). On the other hand, Gilcrease
and Murphy (
17)
have reported that under nitrate-reducing
conditions, TNT can
be transformed by
Pseudomonas
fluorescens to 2,4-DANT, which subsequently
is transformed to
4-
N-AcANT as a dead-end product with no role
in
mineralization. In contrast, with the same bacterium, ethanol
as the C
source, and 2,4-DANT as the sole N source, Naumova et
al.
(
31) reported the formation of phloroglucinol
(1,3,5-trihydroxybenzene)
and pyrogallol (1,2,3-trihydroxybenzene),
both of which require
nitrogen
elimination.
The microbial acylation (acetylation and formylation) of aromatic
amines has been previously described, although the mechanism
for this
remains unclear in most cases (
1,
6,
7,
17).
Also, reviews
of the reactivities of TNT metabolites, particularly
that of HADNT, and
the formation of the corresponding acyl derivatives
have recently been
published (
10,
29). In the case of the
fungus
P. chrysosporium, the formation of 4-
N-FmDNT was suggested
to act as an intermediate in the formation of 2,4-DANT (
29).
The phenolamines 17 and 18, both of which are formed under
nonligninolytic conditions, were observed only in the presence
of
HADNTs, suggesting their coexistence in a reversible manner.
Furthermore, no products directly related to these acid-catalyzed
Bamberger products could be identified. On the other hand the
formation
of 4-HADNT (metabolite 5) together with its Bamberger-rearranged
phenolamine product (metabolite 17) has been reported to occur
under
anaerobic (
C. acetobutylicum) and near-neutral conditions
(
21). Schenzle et al. (
35) have shown that
3-nitrophenol transforms
to 3-hydroxylaminophenol under anaerobic
conditions, which in
turn transforms to 3-aminohydroquinone in a
reaction similar to
that observed for the acid-catalyzed Bamberger
rearrangement.
In the present study, neither
2,4-dihydroxylamino-6-nitrotoluene
nor its Bamberger-rearranged
products were observed; this is possibly
due to the aerobic and acidic
conditions (pH 4.5) used. However,
Fiorella and Spain (
15)
have observed 2,4-dihydroxylamino-6-nitrotoluene
after treatment of TNT
with
Pseudomonas pseudoalcaligenes JS52,
whereas Lewis et
al. (
27) reported the formation of
2,4-dihydroxylamino-6-nitrotoluene
as an intermediate of TNT
biotransformation by the obligate anaerobe
Clostridium
bifermentans. In the present study, the presence of
metabolite 16 (4-
N-AcAHNT) among the detected TNT metabolites
could
support the involvement of 2,4-dihydroxylaminonitrotoluene
as an
intermediate in the biotransformation
process.
The last time course study was constructed to examine the formation and
disappearance profiles of the azoxy intermediates
and their related
reduced products (LC-MS peaks 19 to 24) against
those of the HADNT
prime metabolites (peaks 4 and 5), as shown
in Fig.
6. The azoxy products appeared with the
formation of HADNT
and disappeared with the disappearance of HADNT,
although at a
much lower rate. For instance, the disappearance of HADNT
(10
days) was also marked by the disappearance of the azoxy
intermediates.
However, the disappearance of the two azoxy derivatives
was accompanied
by the appearance of its reduced products, the azo
dimers (TN-2,2'-AzoT
and TN-4,4'-AzoT), followed by the formation of
the hydrazo derivatives
(TN-2,2'-HydrazoT and TN-4,4'-HydrazoT). Figure
6 shows that the
two azoxy derivatives 19 and 20 were clearly
nonligninolytic,
since maximum yields were obtained after only 5 days
of incubation,
long before the onset of the ligninolytic phase of the
fungus
(Fig.
3).
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|
FIG. 6.
Typical time course profile representing the formation
and disappearance of the azoxy products (metabolites 19 and 20) to
produce the corresponding azo derivatives (21 and 22), which finally
are reduced further to the hydrazines 23 and 24. For the TN-AzoT and
the TN-HydrazoT compounds, the peak area counts were used to draw the
curves.
|
|
Figure
6 also shows that another fraction of HADNT transformed
reversibly to give the phenolamines, since the latter could
be observed
only as long as HADNT was present in the system. Other
transformations
for HADNT are shown in Fig.
5, where the prime
products were found to
transform to ADNT and several other acylated
products. After 25 days of
incubation, the only metabolites that
were detected were traces of
4-ADNT and the hydrazo derivatives,
suggesting the partial
decomposition of these hydrazo compounds
back into amines, possibly
through abiotic
means.
As far as azoxy derivatives are concerned, it would be difficult at
present to determine whether these azoxy compounds are
formed via
enzymatic or chemical routes, since both routes have
been reported for
the dimerized coupling of HADNT and NsT to provide
such adducts during
TNT biotransformation (
10,
29). Also,
azoxy compounds have
been reported to biotransform to the corresponding
azo compounds
(---N==N---) during their mineralization with the same
fungus
(
29,
38). Transformation of azoxy dimers to the
corresponding
azo derivatives followed by mineralization has been
reported earlier
(
32,
38).
Neither NO
2 nor NH
4+
ions were detected, indicating that inorganic nitrogen species expected
from the small amount of mineralization
(10%) observed might have
ended up in the biomass. However, cases
of denitration of
polynitroorganics, such as that of 2,4,-dinitrotoluene
with
P. chrysosporium (
41,
47), 2,4-dinitrotoluene with a
Pseudomonas sp. (
39), and TNT with a
Bacillus sp. (
23), have
been reported. When the
mycelia from these cultures were extracted
with acetonitrile (12 h),
only negligible amounts of 4-ADNT and
the hydrazine dimer
TN-4,4'-HydrazoT could be detected. In the
present study, no other
products from the mycelia were included
in the time course study. The
results of the time course study
conducted in the liquid phase are
summarized in the constructed
pathway shown in Fig.
7.
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|
FIG. 7.
Constructed pathway for TNT biotransformation during
treatment with the fungus P. chrysosporium with citric acid
and molasses in agitated cultures at pH 4.5. The dashed arrow indicates
products that were expected but have not been detected.
|
|
Conclusion.
The present LC-MS study demonstrates the
effectiveness of the fungus P. chrysosporium in transforming
TNT into several primary and secondary products, although two
significant changes were introduced in the normal protocol used for
incubation of the fungus: (i) the use of agitated cultures instead of
stationary ones and (ii) addition of TNT with the fungi as opposed to
addition after 6 days. The coexistence of an unusually high number of
intermediates indicates the complexities associated with TNT
biotransformation, exemplified by a unique reactivity and fast
transformation. The formation of several TNT products by one
microorganism (P. chrysosporium) can be taken as proof of
the involvement of several enzymes in the biotransformation process. We
found that the disappearance of TNT and the formation of its prime
metabolites (HADNT and ADNT) together with their Bamberger and azoxy
products occurred prior to the onset of the ligninase activity by the
fungus, while those of the secondary acyl, azo, and hydrazo products
became noticeable during the ligninase state of the fungus, which
started after 6 to 8 days of incubation. A positive practical
conclusion from this work might arise from the formation of the
acylated TNT intermediates, which did not accumulate in the system and
may hold a key to an optimized TNT detoxification process.
| |
ACKNOWLEDGMENTS |
We thank the Department of National Defence, Canada, and the
National Research Council Canada for supporting the work of A. Halasz.
We also thank A. Corriveau and S. Deschamps for their analytical
support and J. Hodgson for the fungal strain.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biotechnology
Research Institute, National Research Council, 6100 Royalmount Ave., Montreal (PQ) H4P 2R2, Canada. Phone: (514) 496-6267. Fax: 514 496-6265. E-mail: Jalal.Hawari{at}NRC.Ca.
National Research Council Canada publication number 41843.
| |
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Abstract
Introduction
