APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p. 949-958 Vol. 31, No.

6
Copyright C 1976 American Society for Microbiology Printed in U.S.A.

Microbial Transformation of 2,4,6-Trinitrotoluene and Other

Nitroaromatic Compounds*
NEIL G. McCORMICK,* FLORENCE E. FEEHERRY, AND HILLEL S. LEVINSON
Food Sciences Laboratory, U.S. Army Natick Development Center, Natick, Massachusetts 01760
Received for publication 17 February 1976

A variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT),

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were reduced by hydrogen in the presence of enzyme preparations from Veillo-
nella alkalescens. Consistent with the proposed reduction pathway, R-NO2
R-NO R-NHOH H, R-NH2, 3 mol of H2 was utilized per mol of nitro group.
The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V.
alkalescens extract were determined. The reactivity of the nitro groups de-
pended on other substituents and on the position of the nitro groups relative to
these substituents. In the case of the nitrotoluenes, the para-nitro group was the
most readily reduced, the 4-nitro position of 2,4-dinitrotoluene being reduced
first. The pattern of reduction of TNT (disappearance of TNT and reduction
products formed) depended on the type of preparation (cell-free extract, resting
cells, or growing culture), on the species, and on the atmosphere (air or H2). The
"nitro-reductase" activity of V. alkalescens extracts was associated with protein
fractions, one having some ferredoxin-like properties and the other possessing
hydrogenase activity. Efforts to eliminate hydrogenase from the reaction have
thus far been unsuccessful. The question of whether ferredoxin acts as a nonspe-
cific reductase for nitroaromatic compounds remains unresolved.

The fate of 2,4,6-trinitrotoluene (TNT) in bio-
logical systems has been a subject of concern for
many years. Toxic effects (including liver dam-
age and anemia) have been reported on workers
engaged in large-scale manufacturing and han-
dling operations (1, 6, 8, 9, 23, 30). TNT, in
concentrations greater than 2 ,.g/ml (ca. 10-;
M), is toxic to some fish (17, 18).
Studies have been undertaken to determine
the fate of TNT in biological systems. In animal
experiments, TNT fed to rabbits, rats, or hu-
man volunteers was excreted in the urine as
the transformed products 4-amino-2,6-dinitro-
toluene (4A), 2,4-diamino-6-nitrotoluene (2,
4DA), or 2,2',6,6'-tetranitro-4,4'-azoxytoluene
(4,4'Az), or as glucuronide conjugates (4, 6, 13).
In vitro experiments with beef heart prepara-
tions (31), slices and homogenates of pig liver
(2), and cell-free extracts of Neurospora (35)
and Escherichia coli (24) suggested that nico-
tinamide adenine dinucleotide and flavopro-
teins were involved in the metabolism of TNT,
leading to the formation of 4A.
Bacteria and fungi that catalyzed the disap-
pearance of TNT during growth in a nutrient
medium in the presence of TNT have been iso-
lated from soil (18). The transformation prod-
ucts were: 4A; 4-hydroxylamino-2,6-dinitrotol-
949
uene (4HA); and 4,4'Az. Several strains of
Pseudomonas, growing in a medium supple-
mented with glucose and yeast extract, also
transformed TNT to these reduction products
(32). In addition, traces of 2-amino-4,6-dinitro-
toluene (2A) and 4,4', 6,6'-tetranitro-2,2'-azoxy-
toluene (2,2'Az) were detected. Cell-free ex-
tracts of the strict anaerobe Veillonella alkales-
cens catalyzed the reduction, by hydrogen gas,
of the nitro groups of a number of nitroaromatic
compounds to the corresponding amino com-
pounds (C. A. Woolfolk, Ph.D. thesis, Univ. of
Washington, Seattle, 1963).
There is no evidence for biological cleavage
and subsequent degradation of the aromatic
nucleus of TNT. The initial steps in the metab-
olism of TNT by a variety of biological systems,
including mammalian, bacterial, and fungal,
appear to involve a stepwise reduction of the
nitro groups, through the nitroso and hydroxyl-
amino, to the amino (27). Although the biologi-
cal reduction of nitroaromatic compounds may
lower or even abolish toxicity, it represents
only a superficial modification of the molecule
and not decomposition (10). The present study
was undertaken to investigate the biochemistry
of bacterial transformation of nitroaromatic
compounds under aerobic as well as anaerobic
950 McCORMICK, FEEHERRY, AND LEVINSON APPL. ENVIRON. MICROBIOL.
conditions and to gain a better understanding methylammonium hydroxide or ethylenediamine
of the apparent recalcitrance of the TNT mole- for nitro-containing derivatives; or by spraying with
cule. a freshly prepared 0.5% aqueous solution of p-ni-
troanilineazo-2,5-dimethoxyaniline-diazotate (fast
MATERIALS AND METHODS black K salt, K and K Laboratories, Inc.) followed
Maintenance and growth of cultures. The growth by 0.1 N NaOH for amino-containing toluenes. In
of V. alkalescens and E. coli was described previ- addition to differences in R,, quite visible differences
in color were noted (Table 1). Due to variability in
ously (15, 16). Clostridium pasteurianum was grown the Rf values, reference compounds were chromato-
in the synthetic medium of Rabinowitz (22). Cul- graphed with the unknown.
tures were incubated at 37 C for 16 to 18 h in 20-liter Chromatography of extracts. Diethylaminoethyl
carboys containing 18 liters of medium. Cells were (DEAE)-cellulose was prepared as described by Ra-
harvested in a Sorvall RC-2 centrifuge equipped binowitz (22), packed in a column (6-cm diameter by

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with a continuous-flow attachment. E. coli was cul- 5 cm), and equilibrated with PBSH. Crude extracts
tured aerobically in the same medium as for anaero- of V. alkalescens were treated with streptomycin
bic growth except in 100-ml quantities contained in sulfate (1.5%) to precipitate nucleic acids, the mix-
1-liter Erlenmeyer flasks and incubated on a recip- ture was centrifuged, and the supernatant solution,
rocal shaker (93 3-inch [about 7.5-cm] strokes/min) containing 1.5 g of protein, was passed through the
at 30 C for 16 to 18 h. The pseudomonad FR2 was column followed by PBSH until the buffer came
isolated by F. Rosenberg, Northeastern University, through clear. Column elution was achieved with
from garden soil perfused with Radford (Va.) Army PBSH buffer containing 0.5 M NaCl and was contin-
Ammunition Plant acid waste water containing 50 ued until the dark-brown band was eluted. The ma-
,ug of TNT per ml and 7 ,ug of 2,4-dinitrotoluene terial eluted with 0.5 M NaCl was precipitated with
(2,4DNT) per ml. Cultures of pseudomonad FR2 ammonium sulfate, and the protein precipitating
were grown in the medium of Kitagawa (12) in 100-
ml quantities in 1-liter Erlenmeyer flasks. between 50 and 70% saturated ammonium sulfate
Preparation of cell-free extracts. Approximately was dissolved in PBSH.
1 volume of a buffer consisting of 20 mM potassium Sephadex G-200 was prepared by swelling on a
phosphate, pH 7.0, and 10 mM 2-mercaptoethanol steam bath overnight. After removal of fine parti-
(PBSH) was added to 1 volume of packed cells. The cles by sedimentation and decanting, a column (2-
cell suspensions, cooled in an ice bath, were sub- cm diameter by 50 cm) was prepared, equilibrated
jected to intermittent (1-min intervals) sonic vibra- with PBSH, and loaded with the undialyzed 50 to
tion with a Sonifier cell disruptor (Heat Systems, 70% saturated (NH4)2SO4 protein fraction. The col-
Inc.) until, as determined by microscopic examina- umn was developed with PBSH.
tion, over 95% breakage had occurred. The resulting Polyacrylamide gel electrophoresis was per-
sonic extract was centrifuged at 17,000 x g for 20 formed as described by Davis (7). Protein concentra-
min at 0 C, and the supernatant solution was desig- tion was adjusted so that 100 to 200 ,ug was applied
nated as crude extract. in a volume of 0.05 to 0.10 ml. Gels were stained
Hydrogen uptake studies. Manometry was per- with 0.5% amido black in acetic acid-methanol-wa-
formed as described by Umbreit et al. (28). Double ter (1:5:5) for 60 min and destained electrophoreti-
side-arm Warburg vessels, equipped with vented cally.
outlets in each side arm for flushing with hydrogen Analytical methods. Protein was estimated by
gas, were used. Reaction mixtures consisted of 100
,umol of potassium phosphate buffer, pH 7.5, cell TABLE 1. Colors of complexes from interaction of
suspension or cell-free extract, and 2 ,umol of the sprays with nitro and amino compounds
nitro compound in a total volume of 2 ml for a final
concentration of 50 mM potassium phosphate and 1 Spray Compound Color
mM nitro compound. Hydrogen gas, previously Tetramethylammo- TNT Orange-gold
passed through a Deoxo purifier (Fisher Scientific nium hydroxide 4,4'Az Blue
Co.) to remove traces of oxygen, was flushed (10% aqueous) or 2,2'Az Purple-blue
through the vessels for 10 min before tipping in the ethylenediamine 4A Yellow
enzyme.
Product analysis. Protein was removed by cen- 4HA Gray-tan
trifugation after precipitation by the addition of 0.1 Fast black K salt 2,4DA Blue
ml of either 100% trichloroacetic acid or 12 N H2S04 (0.5% aqueous) TAT" Blue
into the reaction mixture. Whole cells were removed 2,4DAT0 Blue
by centrifugation of the reaction mixture. The clear 2,6DATe Blue
supernatant solutions were applied to thin-layer sil- 2A4N1N Orange-rust
ica gel glass plates (Analtech, Inc.) containing a
fluorescent background. Developing solvents were: 4A2NTI Orange-rust
chloroform-methanol-acetic acid (80:20:1) for separa- 2,4,6-Triaminotoluene.
tion of aminotoluenes; and benzene-hexane-pen- b 2,4-Diaminotoluene.
tane-acetone (50:40:10:3) for separation of nitrotolu- '2,6-Diaminotoluene.
ene compounds. Visualization was by ultraviolet "2-Amino-4-nitrotoluene.
light at 254 nm; by spraying with either 10% tetra- 4-Amino-2-nitrotoluene.
VOL. 31, 1976 TRANSFORMATION OF NlIROAROMATIC COMPOUNDS
the method of Lowry et al. (14), with bovine serrum
albumin as a standard. Nitrite was determinedI by
the method of Joy and Hageman (11).
RESULTS
The reduction of nitro groups to amino
groups proceeds through the nitroso and hy-
droxylamino compounds (33) according to the
following equations:
R-NO2 H2 :R-NO + H20 (1)
R-NO 2 R-NHOH (2)
R-NHOH -2, R-NH2 + H20 (3)
Thus, 3 mol of hydrogen is required to reduce
each nitro group to the amino group. The stoi-
chiometry of the reduction of TNT and several
mono- and dinitrotoluenes is illustrated in Fig.
1. Hydrogen uptake with the mononitro com-
pounds leveled off at 3 mol of H2 per mol of nitro
compound; that with the dinitrotoluenes lev-
eled off and approached 6 mol; and that with
TNT approached 9 mol of H2. Further studies,
with 40 different nitro compounds, showed that,
with the exception of o-, m-, and p-nitrotolu-
ene, hydrogen uptake with all the compounds
approached the stoichiometric limit of 3 mol of
H2 per mol of nitro group when appropriate
levels of V. alkalescens extract were used. Hy-
drogen uptake with these three compounds was
slow and appeared to level off much below the
value of 3 mol of H2 per mol of nitro group. No
evidence for nonenzymatic reductions was ob-
served in control experiments using heat-inac-
tivated cells or enzyme solutions.
Relative rates of hydrogen uptake with a
number of organic nitro compounds are shown
in Table 2. The most sparingly soluble com-
pounds were dissolved (2 mM) by heating on a
hot plate with vigorous stirring just before ad-
dition to the reaction mixture to give a final
concentration of 1 mM. The rate of H2 consump-
tion by mononitro compounds can be compared,
as can the rates within each of the other two
groups, the dinitro and trinitro compounds.
However, comparing rates based on concen-
tration of nitro groups instead of concentration
of parent molecule may not be valid because
other substituent groups influence the rate of
reduction of nitro groups. An electron-attract-
ing nitro group enhances the reactivity of the
oxygen attached to nitrogen atoms of other ni-
tro groups on the same molecule (19). The reac-
tivity of the nitro groups appears to depend not
only on other substituents but also on the posi-
tion of the nitro groups relative to these substit-
uents. Thus, the para nitro group was more
readily reduced than the ortho group in the
951
aI
E
=
.3
E
6
E
E
'-1
w
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En
u
T I M E (min)
FIG. 1. Stoichiometry of H2 consumption by cell-
free extracts of V. alkalescens. The reaction mixture
was as described in Materials and Methods and con-
tained 20 mg of protein.
case of nitrotoluenes, nitrobenzoic acids, and
dinitrobenzenes, whereas the converse was true
for the nitrophenols and nitroanilines; the nitro
groups of dinitrobenzoic acid and dinitrotoluene
were reduced more rapidly than those of dini-
trophenol and dinitroaniline; and the trinitro
derivatives of benzoic acid were more readily
reduced than the toluene or phenol derivatives.
Each of the biological systems reported in the
literature as acting on TNT catalyzed the re-
duction of at least one nitro group (2, 4, 13, 24,
31, 35). In fact, nitro group reduction appears to
be the only metabolic process noted. Table 3
shows the effects of various anaerobic and aero-
bic bacterial preparations on the transforma-
tion of TNT into reduction products. Cell-free
extracts of the anaerobic organisms, utilizing
molecular H2, reduced the three nitro groups to
the corresponding amino groups. Resting cells
of the strict anaerobes reduced all three nitro
groups, whereas resting cells of anaerobically
grown E. coli reduced two of the nitro groups.
Cultures of V. alkalescens and E. coli, growing
anaerobically in the presence of 100 ,g of TNT
per ml, produced 2,4DA. Apparently, in the
absence of added hydrogen donor, these orga-
nisms could not reduce the remaining nitro
group. E. coli and pseudomonad FR2, actively
growing aerobically in the presence of 100 ,g of
TNT per ml, generated enough reducing poten-
tial to reduce two of the three nitro groups, but
not the third. Resting cells of E. coli, when
supplied with a hydrogen atmosphere, also
formed 2,4DA, whereas pseudomonad FR2 sup-
952 McCORMICK, FEEHERRY, AND LEVINSON
plied with hydrogen did not. Resting cells of
both organisms, in air, formed 4,4'Az. The 4HA
compound was detected only with cell-free ex-
tracts, never with resting cells or growing cul-
tures, with the exception of C. pasteurianum,
with which 4HA was found in the growth me-
dium early in the log phase, but not later in
growth. Traces of 2,2'Az were found in every
instance where 4,4'Az was detected. From
these results, a tentative reaction pathway for
the transformation (reduction) of TNT may be
presented (Fig. 2). The presence of azoxy com-
pounds is believed to be due to nonenzymatic
oxidation of the very reactive intermediate,
4HA (4). With storage, the 4HA content of
freshly analyzed reaction mixtures decreased
with increasing levels of 4,4'Az.
The rate studies (Table 2) suggested that the
reduction of 2,4DNT proceeds with the reduc-
tion of the 4-nitro group first. This was indeed
the case (Table 4). No trace of 2-amino-4-nitro-
toluene was detected, suggesting that essen-
tially all of the 4-nitro group was reduced before
reduction of the 2-nitro group began.
The fact that a large variety of nitroaromatic
compounds are susceptible to nitro group reduc-
tion implies relative nonspecificity of the "ni-
tro-reductase" system. In an attempt to study
this system in more detail, V. alkalescens crude
extract was fractionated by chromatography.
Almost all of the activity was removed by pas-
sage through a DEAE-cellulose column (Table
5). The active material remained at the top of
the column and was eluted with 0.5 M NaCl.
The material absorbed to DEAE-cellulose sepa-
rated into several visible bands upon passage
through Sephadex G-200. The bands were col-
lected separately and tested in all combinations
for the catalysis of the uptake of He with TNT.
Of the eight fractions collected, only three
(fractions 4, 5, and 6) were active. Rechroma-
tography of fractions 4 and 6 on Sephadex G-200
yielded fractions 4' and 6', which exhibited syn-
ergistic reductase properties (Table 6).
The ratio of absorption at 390 nm to 280 nm
(A390/A280) of ferredoxin ranges from approxi-
mately 0.6 to 0.85, depending upon the source
(34). The A390/A280 of fraction 6' was 1.08, and
that of fraction 4' was 0.064. Fraction 6' may
contain ferredoxin or a mixture of ferredoxin-
like molecules. Fraction 4' appeared to be more
active than fraction 6' when assayed for hydro-
genase as measured by the evolution of hydro-
gen from hydrosulfite-reduced methyl viologen
in a nitrogen atmosphere (21). Disc gel electro-
phoresis of fractions 4' and 6' indicated that the
major component of fraction 4' was a protein
(amido black staining) that fluoresced blue-
white when the gel was exposed to ultraviolet
APPL. ENVIRON. MICROBIOL.
light at 360 nm; fraction 6', more heteroge-
neous, contained no predominant band. These
preliminary data suggested that the active
components of the V. alkalescens "nitro-reduc-
tase" were hydrogenase and a ferredoxin-like
molecule. Efforts to eliminate hydrogenase
from the reaction by providing hydrogen donors
other than H. gas have been unsuccessful.
DISCUSSION
Biological degradation of nitroaromatic com-
pounds involves the initial conversion of nitro
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groups to hydroxyl groups. Two modes ofattack
on nitroaromatic compounds appear to be pres-
ent in bacteria, one involving the reduction of a
nitro group to an amino group followed by oxi-
dative deamination to a phenol with release of
ammonia, and the other involving the release
of a nitro group as nitrite with the concomitant
formation of a phenol. Nitrite was produced
from nitrophenols (26) and nitrobenzoic acids
(3), and, depending upon the strain of bacte-
rium used, ammonia was also produced from
nitrobenzoic acids (3). An Arthrobacter sp. de-
graded the dinitro pesticide 4,6-dinitro-o-cresol,
in addition to 2,4-dinitrophenol and the trinitro
compound 2,4,6-trinitrophenol (picric acid),
with the release of nitrite (10); a Pseudomonas
degraded 4,6-dinitro-o-cresol with the produc-
tion of ammonia (M. S. Tewfik and W. C. Ev-
ans, Biochem J. 99: 31P, 1966). The two path-
ways converged to give the same intermediate,
2,3,5-trihydroxytoluene, followed by meta
cleavage of the ring (between the 1 and 2 car-
bon atoms) and eventual degradation of the
molecule (Tewfik and Evans, Biochem J. 99:
31P, 1966). The report that picric acid was me-
tabolized with the formation of nitrite indicates
that trinitro compounds are not immune to deg-
radation. Picric acid already possesses one hy-
droxyl group and only requires the conversion
of one nitro group to a phenolic hydroxyl group
to yield a necessary intermediate for ring cleav-
age; i.e., the aromatic nucleus must carry at
least two hydroxyl groups, ortho orpara to each
other, in order for ring cleavage to occur (5). On
the other hand, TNT, in order to be converted
to a catechol, requires not only the conversion
of one nitro group to a hydroxyl group but also
the hydroxylation of an adjacent unsubstituted
ring position.
We have shown, as have others, that nitro
groups on the TNT molecule are reduced by
both aerobic and anaerobic systems. Depending
upon the reducing potential of the system, one,
two, or three of the nitro groups may be reduced
to amino groups. The pathway involving the
release of nitro groups as nitrite does not ap-
pear to be a major pathway (at least in pseudo-
VOL. 31, 1976 TRANSFORMATION OF NITROAROMATIC COMPOUNDS 953
TABLE 2. Rate of hYdrogen c-onsumlption by (ell-free extracts of Veillonella alkalescens on carious nitro-
aromatic compounds
Compound Sp act" Compound Sp act'
No,) Nitrobenzene" 15 NHs m-Nitroanilineb 42
(CD, ""VIVL

CH3 o-Nitrotolueneb 5 kNO2

NO2 NH2 p-Nitroanilineb 6

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CH3 m -Nitrotoluene"b 12

NO2
~N02 CH3 5-Nitro-o-toluidine' 59
CH3 p-Nitrotoluene" 20 NH2

NO2
NO2 CH3 3-Nitro-p-toluidine" 26
COOH o-Nitrobenzoic acidb 14 +,N02

32- NH2
COOH m-Nitrobenzoic acid"

CH3 2-Nitro-p-toluidinec 24
kN02
0NO2
COOH p-Nitrobenzoic acid" 41 NH2

CH3 4-Nitro<-otoluidinec 15
NO2
OH o-Nitrophenol" 32 02N."
3-Nitro<-otoluidinec 28
NO2 CH3
02N NH2
m-Nitrophenolb 27
OH
CH3 2-Methyl-5-nitrophenol" 39
4OH
NO2
OH p-Nitrophenol' 6 N02
CH3 4-Methyl-3-nitrophenolc 31

NO2 2N32
NH2 o-Nitroaniline" 23 O
kN02
 TABLE 2-Continued
Compound Sp act' Compound Sp actV
2,4-Dinitroanilineb 18
OH 2-Nitroresorcinol" 59
NH
iN02
4-Nitrotoluene-2-sul-
fonic acid"
47 NO2
kSO3H CH3 2,6-Dinitrotolueneb 39
C 3

02 N02
NO2
3,5-Dinitrobenzoic acidb 139
50 COOH

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m-Nitrobenzene-sul-
1 rS03H fonic acid'

N02 02NANO2
COOH 5-Nitro-m-phthalic acid" 40 CH3 2,6-Dinitro-p-toluidinel' 8

02N COOH 02N N02

o-Dinitrobenzene' 79 NH2
aN02 COOH 3,5-Dinitro-salicylic
acidb
64

m-Dinitrobenzene"
j<OH
NO2
02N N02
kNO2 CH3 4,6-Dinitro-o-cresol" 51

NO2 p-Dinitrobenzeneb 210 O2

02N NO2
1,3,5-Trinitrobenzeneb 288
NO2
02N"r N02
2,4-Dinitrotolueneb 58 NO2
2,4,6-Trinitrotolueneh 147
CH3
NO2
k NO2
COH 2,4-Dinitrobenzoic acid' 87 NO2
NO2
0
OH 2,4,6-Trinitrophenoli 81

NO2 02N N02

OH 2,4-Dinitrophenolb 33 N2
N2
eN02
COOH 2,4,6-Trinitrobenzoic
acidb
210
N2 02N N02
NO
954
VOL. 31, 1976 TRANSFORMATION OF NITROAROMATIC COMPOUNDS 955
TABLE 2-Continued
Expressed as nanomoles of H, consumed per minute per milligram of protein.
"From Eastman Kodak Co.
From Aldrich Chemical Co.
"From Pfaltz and Bauer.
From K and K Laboratories.
f From ICN Pharmaceuticals, Inc.
From Alfred Bader Chemical Co.
"From Radford Army Ammunition Plant.
'From Merck and Co., Inc.

monad FR2); the maximum amount of nitrite buffer. Rabbits fed TNT oxidized the methyl

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found was less than 6% of the nitrogen avail- group to an alcohol and reduced the 4-nitro
able from the nitro groups of the TNT that had group to an amino group (4). The resulting 4-
disappeared from the medium. amino-2,6-dinitrobenzyl alcohol in turn formed
According to present theory, formation of a a glucuronide that was eliminated in the urine.
diphenol is a necessary prerequisite to degrada- Conversion of TNT to a catechol, through oxida-
tion of an aromatic nucleus. Thus far, no phe- tion and decarboxylation at the methyl group
nolic intermediate or other evidence for di- and hydroxylation of an adjacent nitro group,
phenol formation has been found in the case of might be more energy demanding than con-
TNT. During the degradation of p-nitrobenzoic version of a nitro group and hydroxylation of
acid by Nocardia (3), p-hydroxybenzoate accu- an adjacent carbon atom. The inability of bio-
mulated in the presence of tris(hydroxymeth- logical systems to perform this operation may
yl)aminomethane but not in phosphate buffer. be due to inhibitory effects of pendant nitro or
All of our experiments were done in phosphate amino groups on hydroxylation enzymes.
TABLE 3. Effect of variouts enzyme sources on TNT disappearance and on product formation"
Product formedb
Organism Prepn Atmosphere
4HA 4,4'Az 4A 2,4DA TA¶P
C. pasteurianum Cell-free extract H2 +d +d _ - +
Resting cells H2 - - - +
Growing culture Deep, standing +e - _ _ _
V. alkalescens Cell-free extract H2 +f +f +f - +
Resting cells H2 - - - +
Growing culture Deep, standing - - - +
E. coli (anaerobic) Cell-free extract H2 + - - + +
Resting cells H2 - + - + -
Resting cells Air 0 0 0 0 0
Growing culture Deep, standing - - - + -
E. coli (aerobic) Cell-free extract H2 + + - - -
Cell-free extract Air 0 0 0 0 0
Resting cells H2 - + + + -
Resting cells Air - +
Growing culture Shallow, shaking - + - + -
Pseudomonad FR2 Cell-free extract H2 0 0 0 0 0
Cell-free extract Air - - - - -
Resting cells H2 - +
Resting cells Air - +
Growing culture Shallow, shaking - + + +
aReaction mixtures were as described in Materials and Methods. Crude extracts used were equivalent to
20 mg of protein. Cells for resting-cell experiments were grown overnight, harvested by centrifugation,
washed twice with ice-cold distilled water, and resuspended at a concentration of about 1011 cells/ml, and 0.2
ml of the suspension was added to the reaction mixture. Growing cultures were inoculated with a 10%
inoculum of an actively growing culture.
b
Products were detected as described in Materials and Methods. Symbols: +, TNT disappeared, com-
pound observed; -, TNT disappeared, compound not observed; 0, TNT did not disappear.
'2,4,6-Triaminotoluene.
d Extracts were prepared from aged frozen cells.
e Was detected early in log phase of growth and then disappeared completely from medium.
f Extracts were treated with DEAE-cellulose before
testing.
956 McCORMICK, FEEHERRY, AND LEVINSON APPL. ENVIRON. MICROBIOL.

02N N 02 C02N H3 N2
NCCH3 CH -N
D.-NN-0

rK>N
23
02NK)NO202 N
CH3 3jNHOH
NO2 CH3
0 2NjjN02 2t1N 1N02
CR3
Vill N02 V IINHOH i

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002N2
NH2 02N jJN02
N02 CR3 NH2
02N1j1NH2

NH2

CR3
H2N NH 2
IX
NH2
Ix

FIG. 2. Proposed pathway for transformation of TNT by the reduction of the nitro groups. Compounds
illustrated are: I, TNT; II, 4HA; III, 4A; IV, 2,4DA; V, 2HA; VI, 2A; VII, 4,4'Az; VIII, 2,2'Az; IX, TAT.

TABLE 4. Formation of 4-amino-2-nitrotoluene TABLE 5. Activity of DEAE-cellulose-treated extracts

(4A2NT) from the reduction of 2,4-dinitrotoluene of Veillonella alkalescens"
(2,4DNT) by V. alkalescens" Enzyme source Sp act
Reaction time (min) 1. Crude extract 15.2
Compound 2. DEAE-cellulose-treated 0.7
0 10 60
crude extract
2,4DNT + + - 3. 0.5 M NaCl eluate 15.1
2A4NT - - - 4. (2) + (3) 14.2
4A2NT - + -
2,4DAT - + + "DEAE-cellulose chromatography was performed
as described in Materials and Methods. Total pro-
Reaction mixture was as described in Materials tein per reaction mixture was 13 mg.
and Methods, using V. alkalescens crude extract. h Expressed as in Table 2.
The mixture was deproteinized with H2SO4 and cen-
trifuged, the solution was made alkaline with TABLE 6. Hydrogen uptake by TNT in the presence
NaOH and extracted twice with benzene, and the of Sephadex G-200 fractions
benzene solution was evaporated to a small volume
and applied to a thin-layer plate. Separation of Fraction no. Activity"
authentic 2-amino-4-nitrotoluene (2A4NT) from 4' 36.7
4A2NT was achieved by five successive develop- 6' 7.5
ments in benzene. Detection was as described in 4' + 6' 89.2
Materials and Methods. 2,4DAT, 2,4-Diamino-
toluene. a
Expressed as nanomoles of H2 consumed per
minute.
Our results agree with those expected from
the accepted pathway of reduction of nitro com- Although the extent of reduction depended on
pounds. The proposed intermediate nitroso the redox potential of the system, our observa-
compound (equation 1) was not detected, but tions agree with those of Channon et al. (4) that
the second intermediate, the hydroxylamino in an aerobic environment the hydroxylamino
compound (equation 2), was identified. All the compound may be nonenzymatically oxidized to
major products arising from TNT metabolism the azoxy compound. Thus, the finding of
originate from the hydroxylamino compound. 2,2'Az presupposes the formation of 2-hydrox-
VOL. 31, 1976 TRANSFORMATION OF NITROAROMATIC COMPOUNDS
ylamino-4,6-dinitrotoluene, which in turn sug-
gests that 2A, and even hybrids such as 2,4'-
azoxy compounds, might be formed. Won et al.
(32) reported the presence of 2A in their sys-
tem. Thus it becomes apparent that complex
molecules may arise by condensation reactions
of such possible intermediates as 2,4-dihydrox-
ylamino-6-nitrotoluene.
The order of reduction rate of nitro com-
pounds is consistent both with that reported by
Woolfolk (Ph.D. thesis) and with the "electro-
negativity rule" (25), which states that the rate
of reduction of nitro compounds increases with
increasing electron withdrawing power of the
groups at the para position: -NH2 < -OH <
-H < CH: < -COOH < -NO2. In the tri-
nitro series of compounds (Table 2), the benzoic
acid derivative should be more readily reduced
than the aniline derivatives. The rate of reduc-
tion of trinitrobenzoic acid is indeed high, but
picramide (the trinitroaniline derivative), which
should have the lowest rate, was not available
for testing. The high rate obtained with 2,4,6-
trinitrobenzoic acid may be the consequence of
the position of the amino group (meta relative
to the remaining nitro groups) resulting from
the reduction of the first nitro group. Amino
groups in the ortho or para position would be
more inhibitory to further reduction than those
in the meta position. In the case ofp-dinitroben-
zene, which has a high rate of reduction, both
nitro groups probably undergo reduction simul-
taneously; otherwise a strongly inhibitory ef-
fect of the first-formed amino group on the re-
duction of the second nitro group (as with p-
nitroaniline) would be expected.
In the case of 2,4DNT, the 4-nitro group is
reduced first (Table 4). Evidence in the litera-
ture and from our own work suggests that with
TNT the 4-nitro group is reduced first. In the
case of 2,4-dinitrophenol (20; Woolfolk, Ph.D.
thesis), the 2-nitro group is reduced first. It
might be predicted that the reduction of picric
acid should proceed via reduction of the 2-nitro
group first. This is borne out by our observation
of spectral changes characteristic of picramic
acid (2-amino-4,6-dinitrophenol) formation dur-
ing the reduction of picric acid.
V. alkalescens "nitro-reductase" appears to
consist essentially of hydrogenase and a ferre-
doxin-like material that can be separated from
each other on a Sephadex G-200 column. Even
after rechromatography of fraction 4 (the hy-
drogenase fraction) on Sephadex, this fraction
may still be contaminated with traces of a fer-
redoxin-like material. The high A3,,/A2-1 of
fraction 6', the absorption as a brown band to
the top of a DEAE-cellulose column, and the in-
957
volvement in reactions of low reducing poten-
tial are all properties suggesting the presence
of ferredoxin, but the question of whether frac-
tion 6' contains other "reductases" or whether
ferredoxin acts as a nonspecific reductase for
nitroaromatic compounds and for intermediate
nitroso and hydroxylamino compounds must
await further experiments. Villanueva (29)
purified a "nitro-reductase" from Nocardia and
concluded that it was one enzyme or a group of
enzymes with similar properties.
The aerobic systems pseudomonad FR2 and
Downloaded from http://aem.asm.org/ on May 19, 2020 by guest
aerobically grown E. coli have the capability of
reducing two of the three nitro groups of TNT
in the absence of the enzyme hydrogenase, sug-
gesting that "nitro-reductase" activity in aero-
bic systems may involve reduced nicotinamide
adenine dinucleotide and flavoproteins as pre-
viously reported (2, 24, 35).
Growing cultures of C. pasteurianum formed
large amounts of 4HA in early log phase, which
disappeared in late log and stationary phase,
when no products of any kind could be detected.
None of the usual amino or azoxy compounds,
nor any phenolic compounds, were detected,
although resting cells or cell-free extracts pro-
duced 2,4,6-triaminotoluene from TNT, nor was
there any residual TNT. Experiments using
TNT (ring-L-[U-'4C]) with growing cultures of
C. pasteurianum are in progress in order to
search for definite evidence, thus far lacking,
for metabolism or degradation of TNT.
ACKNOWLEDGMENTS
We thank G. R. Mandels and D. F. Carpenter for their
critical reviews of the manuscript. Compounds 4HA; 2A;
4A; 2,4DA; 2,2'Az; and 4,4'Az were supplied as reference
compounds through the generosity of J. Hoffsommer, U.S.
Naval Surface Weapons Center, Silver Spring, Md.
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