APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p. 949-958Copyright C 1976 American Society for Microbiology

Vol. 31, No. 6Printed in U.S.A.

Microbial Transformation of 2,4,6-Trinitrotoluene and OtherNitroaromatic 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),were reduced by hydrogen in the presence of enzyme preparations from Veillo-nella alkalescens. Consistent with the proposed reduction pathway, R-NO2R-NO R-NHOH H, R-NH2, 3 mol ofH2 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 tothese substituents. In the case ofthe nitrotoluenes, the para-nitro group was themost readily reduced, the 4-nitro position of 2,4-dinitrotoluene being reducedfirst. The pattern of reduction of TNT (disappearance of TNT and reductionproducts formed) depended on the type of preparation (cell-free extract, restingcells, or growing culture), on the species, and on the atmosphere (air or H2). The"nitro-reductase" activity of V. alkalescens extracts was associated with proteinfractions, one having some ferredoxin-like properties and the other possessinghydrogenase activity. Efforts to eliminate hydrogenase from the reaction havethus far been unsuccessful. The question ofwhether 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 formany years. Toxic effects (including liver dam-age and anemia) have been reported on workersengaged in large-scale manufacturing and han-dling operations (1, 6, 8, 9, 23, 30). TNT, inconcentrations greater than 2 ,.g/ml (ca. 10-;M), is toxic to some fish (17, 18).

Studies have been undertaken to determinethe fate ofTNT in biological systems. In animalexperiments, TNT fed to rabbits, rats, or hu-man volunteers was excreted in the urine asthe 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 nutrientmedium in the presence of TNT have been iso-lated from soil (18). The transformation prod-ucts were: 4A; 4-hydroxylamino-2,6-dinitrotol-

uene (4HA); and 4,4'Az. Several strains ofPseudomonas, growing in a medium supple-mented with glucose and yeast extract, alsotransformed 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 ofthe strict anaerobe Veillonella alkales-cens catalyzed the reduction, by hydrogen gas,ofthe nitro groups ofa number of nitroaromaticcompounds to the corresponding amino com-pounds (C. A. Woolfolk, Ph.D. thesis, Univ. ofWashington, Seattle, 1963).There is no evidence for biological cleavage

and subsequent degradation of the aromaticnucleus of TNT. The initial steps in the metab-olism ofTNT by a variety of biological systems,including mammalian, bacterial, and fungal,appear to involve a stepwise reduction of thenitro groups, through the nitroso and hydroxyl-amino, to the amino (27). Although the biologi-cal reduction of nitroaromatic compounds maylower or even abolish toxicity, it representsonly a superficial modification of the moleculeand not decomposition (10). The present studywas undertaken to investigate the biochemistryof bacterial transformation of nitroaromaticcompounds under aerobic as well as anaerobic

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950 McCORMICK, FEEHERRY, AND LEVINSON

conditions and to gain a better understandingof the apparent recalcitrance of the TNT mole-cule.

MATERIALS AND METHODSMaintenance and growth of cultures. The growth

of V. alkalescens and E. coli was described previ-ously (15, 16). Clostridium pasteurianum was grownin the synthetic medium of Rabinowitz (22). Cul-tures were incubated at 37 C for 16 to 18 h in 20-litercarboys containing 18 liters of medium. Cells wereharvested in a Sorvall RC-2 centrifuge equippedwith a continuous-flow attachment. E. coli was cul-tured aerobically in the same medium as for anaero-bic growth except in 100-ml quantities contained in1-liter Erlenmeyer flasks and incubated on a recip-rocal shaker (93 3-inch [about 7.5-cm] strokes/min)at 30 C for 16 to 18 h. The pseudomonad FR2 wasisolated by F. Rosenberg, Northeastern University,from garden soil perfused with Radford (Va.) ArmyAmmunition Plant acid waste water containing 50,ug of TNT per ml and 7 ,ug of 2,4-dinitrotoluene(2,4DNT) per ml. Cultures of pseudomonad FR2were grown in the medium of Kitagawa (12) in 100-ml quantities in 1-liter Erlenmeyer flasks.

Preparation of cell-free extracts. Approximately1 volume of a buffer consisting of 20 mM potassiumphosphate, pH 7.0, and 10 mM 2-mercaptoethanol(PBSH) was added to 1 volume of packed cells. Thecell suspensions, cooled in an ice bath, were sub-jected to intermittent (1-min intervals) sonic vibra-tion with a Sonifier cell disruptor (Heat Systems,Inc.) until, as determined by microscopic examina-tion, over 95% breakage had occurred. The resultingsonic extract was centrifuged at 17,000 x g for 20min at 0 C, and the supernatant solution was desig-nated as crude extract.Hydrogen uptake studies. Manometry was per-

formed as described by Umbreit et al. (28). Doubleside-arm Warburg vessels, equipped with ventedoutlets in each side arm for flushing with hydrogengas, were used. Reaction mixtures consisted of 100,umol of potassium phosphate buffer, pH 7.5, cellsuspension or cell-free extract, and 2 ,umol of thenitro compound in a total volume of 2 ml for a finalconcentration of 50 mM potassium phosphate and 1mM nitro compound. Hydrogen gas, previouslypassed through a Deoxo purifier (Fisher ScientificCo.) to remove traces of oxygen, was flushedthrough the vessels for 10 min before tipping in theenzyme.

Product analysis. Protein was removed by cen-

trifugation after precipitation by the addition of 0.1ml of either 100% trichloroacetic acid or 12 N H2S04into the reaction mixture. Whole cells were removedby centrifugation of the reaction mixture. The clearsupernatant solutions were applied to thin-layer sil-ica gel glass plates (Analtech, Inc.) containing a

fluorescent background. Developing solvents were:

chloroform-methanol-acetic acid (80:20:1) for separa-tion of aminotoluenes; and benzene-hexane-pen-tane-acetone (50:40:10:3) for separation of nitrotolu-ene compounds. Visualization was by ultravioletlight at 254 nm; by spraying with either 10% tetra-

methylammonium hydroxide or ethylenediaminefor nitro-containing derivatives; or by spraying witha freshly prepared 0.5% aqueous solution of p-ni-troanilineazo-2,5-dimethoxyaniline-diazotate (fastblack K salt, K and K Laboratories, Inc.) followedby 0.1 N NaOH for amino-containing toluenes. Inaddition to differences in R,, quite visible differencesin color were noted (Table 1). Due to variability inthe Rf values, reference compounds were chromato-graphed with the unknown.

Chromatography of extracts. Diethylaminoethyl(DEAE)-cellulose was prepared as described by Ra-binowitz (22), packed in a column (6-cm diameter by5 cm), and equilibrated with PBSH. Crude extractsof V. alkalescens were treated with streptomycinsulfate (1.5%) to precipitate nucleic acids, the mix-ture was centrifuged, and the supernatant solution,containing 1.5 g of protein, was passed through thecolumn followed by PBSH until the buffer camethrough clear. Column elution was achieved withPBSH buffer containing 0.5 M NaCl and was contin-ued until the dark-brown band was eluted. The ma-terial eluted with 0.5 M NaCl was precipitated withammonium sulfate, and the protein precipitatingbetween 50 and 70% saturated ammonium sulfatewas dissolved in PBSH.

Sephadex G-200 was prepared by swelling on asteam bath overnight. After removal of fine parti-cles by sedimentation and decanting, a column (2-cm diameter by 50 cm) was prepared, equilibratedwith PBSH, and loaded with the undialyzed 50 to70% saturated (NH4)2SO4 protein fraction. The col-umn was developed with PBSH.

Polyacrylamide gel electrophoresis was per-formed as described by Davis (7). Protein concentra-tion was adjusted so that 100 to 200 ,ug was appliedin a volume of 0.05 to 0.10 ml. Gels were stainedwith 0.5% amido black in acetic acid-methanol-wa-ter (1:5:5) for 60 min and destained electrophoreti-cally.

Analytical methods. Protein was estimated by

TABLE 1. Colors of complexes from interaction ofsprays with nitro and amino compounds

Spray Compound Color

Tetramethylammo- TNT Orange-goldnium hydroxide 4,4'Az Blue(10% aqueous) or 2,2'Az Purple-blueethylenediamine 4A Yellow

4HA Gray-tan

Fast black K salt 2,4DA Blue(0.5% aqueous) TAT" Blue

2,4DAT0 Blue2,6DATe Blue2A4N1N Orange-rust4A2NTI Orange-rust

2,4,6-Triaminotoluene.b 2,4-Diaminotoluene.'2,6-Diaminotoluene."2-Amino-4-nitrotoluene.4-Amino-2-nitrotoluene.

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TRANSFORMATION OF NlIROAROMATIC COMPOUNDS 951

the method of Lowry et al. (14), with bovine seralbumin as a standard. Nitrite was determinedthe method of Joy and Hageman (11).

rumI by

RESULTSThe reduction of nitro groups to amino

groups proceeds through the nitroso and hy-droxylamino compounds (33) according to thefollowing equations:

R-NO2 H2 :R-NO + H20

R-NO 2 R-NHOH

R-NHOH -2, R-NH2 + H20

(1)(2)(3)

Thus, 3 mol of hydrogen is required to reduceeach nitro group to the amino group. The stoi-chiometry of the reduction of TNT and severalmono- and dinitrotoluenes is illustrated in Fig.1. Hydrogen uptake with the mononitro com-pounds leveled off at 3 mol ofH2 per mol of nitrocompound; that with the dinitrotoluenes lev-eled off and approached 6 mol; and that withTNT 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 compoundsapproached the stoichiometric limit of 3 mol ofH2 per mol of nitro group when appropriatelevels of V. alkalescens extract were used. Hy-drogen uptake with these three compounds wasslow and appeared to level off much below thevalue of 3 mol of H2 per mol of nitro group. Noevidence for nonenzymatic reductions was ob-served in control experiments using heat-inac-tivated cells or enzyme solutions.

Relative rates of hydrogen uptake with anumber of organic nitro compounds are shownin Table 2. The most sparingly soluble com-pounds were dissolved (2 mM) by heating on ahot plate with vigorous stirring just before ad-dition to the reaction mixture to give a finalconcentration of 1 mM. The rate ofH2 consump-tion by mononitro compounds can be compared,as can the rates within each of the other twogroups, the dinitro and trinitro compounds.However, comparing rates based on concen-tration of nitro groups instead of concentrationof parent molecule may not be valid becauseother substituent groups influence the rate ofreduction of nitro groups. An electron-attract-ing nitro group enhances the reactivity of theoxygen attached to nitrogen atoms of other ni-tro groups on the same molecule (19). The reac-tivity of the nitro groups appears to depend notonly on other substituents but also on the posi-tion ofthe nitro groups relative to these substit-uents. Thus, the para nitro group was morereadily reduced than the ortho group in the

E

E

E

=.3

6

E

'-1w

En

aI

u

T I M E (min)FIG. 1. Stoichiometry of H2 consumption by cell-

free extracts of V. alkalescens. The reaction mixturewas as described in Materials and Methods and con-tained 20 mg ofprotein.

case of nitrotoluenes, nitrobenzoic acids, anddinitrobenzenes, whereas the converse was truefor the nitrophenols and nitroanilines; the nitrogroups of dinitrobenzoic acid and dinitrotoluenewere reduced more rapidly than those of dini-trophenol and dinitroaniline; and the trinitroderivatives of benzoic acid were more readilyreduced than the toluene or phenol derivatives.Each ofthe 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 tobe the only metabolic process noted. Table 3shows the effects of various anaerobic and aero-bic bacterial preparations on the transforma-tion of TNT into reduction products. Cell-freeextracts of the anaerobic organisms, utilizingmolecular H2, reduced the three nitro groups tothe corresponding amino groups. Resting cellsof the strict anaerobes reduced all three nitrogroups, whereas resting cells of anaerobicallygrown E. coli reduced two of the nitro groups.Cultures of V. alkalescens and E. coli, growinganaerobically in the presence of 100 ,g of TNTper ml, produced 2,4DA. Apparently, in theabsence of added hydrogen donor, these orga-nisms could not reduce the remaining nitrogroup. E. coli and pseudomonad FR2, activelygrowing aerobically in the presence of 100 ,g ofTNT per ml, generated enough reducing poten-tial to reduce two of the three nitro groups, butnot the third. Resting cells of E. coli, whensupplied with a hydrogen atmosphere, alsoformed 2,4DA, whereas pseudomonad FR2 sup-

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952 McCORMICK, FEEHERRY, AND LEVINSON

plied with hydrogen did not. Resting cells ofboth organisms, in air, formed 4,4'Az. The 4HAcompound 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 ingrowth. Traces of 2,2'Az were found in everyinstance where 4,4'Az was detected. Fromthese results, a tentative reaction pathway forthe transformation (reduction) of TNT may bepresented (Fig. 2). The presence of azoxy com-pounds is believed to be due to nonenzymaticoxidation of the very reactive intermediate,4HA (4). With storage, the 4HA content offreshly analyzed reaction mixtures decreasedwith 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 indeedthe case (Table 4). No trace of 2-amino-4-nitro-toluene was detected, suggesting that essen-tially all ofthe 4-nitro group was reduced beforereduction 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 studythis system in more detail, V. alkalescens crudeextract was fractionated by chromatography.Almost all of the activity was removed by pas-sage through a DEAE-cellulose column (Table5). The active material remained at the top ofthe column and was eluted with 0.5 M NaCl.The material absorbed to DEAE-cellulose sepa-rated into several visible bands upon passagethrough Sephadex G-200. The bands were col-lected separately and tested in all combinationsfor 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-200yielded 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, andthat of fraction 4' was 0.064. Fraction 6' maycontain ferredoxin or a mixture of ferredoxin-like molecules. Fraction 4' appeared to be moreactive than fraction 6' when assayed for hydro-genase as measured by the evolution of hydro-gen from hydrosulfite-reduced methyl viologenin a nitrogen atmosphere (21). Disc gel electro-phoresis of fractions 4' and 6' indicated that themajor component of fraction 4' was a protein(amido black staining) that fluoresced blue-white when the gel was exposed to ultraviolet

light at 360 nm; fraction 6', more heteroge-neous, contained no predominant band. Thesepreliminary data suggested that the activecomponents of the V. alkalescens "nitro-reduc-tase" were hydrogenase and a ferredoxin-likemolecule. Efforts to eliminate hydrogenasefrom the reaction by providing hydrogen donorsother than H. gas have been unsuccessful.

DISCUSSIONBiological degradation of nitroaromatic com-

pounds involves the initial conversion of nitrogroups to hydroxyl groups. Two modes ofattackon nitroaromatic compounds appear to be pres-ent in bacteria, one involving the reduction ofanitro group to an amino group followed by oxi-dative deamination to a phenol with release ofammonia, and the other involving the releaseof a nitro group as nitrite with the concomitantformation of a phenol. Nitrite was producedfrom nitrophenols (26) and nitrobenzoic acids(3), and, depending upon the strain of bacte-rium used, ammonia was also produced fromnitrobenzoic acids (3). An Arthrobacter sp. de-graded the dinitro pesticide 4,6-dinitro-o-cresol,in addition to 2,4-dinitrophenol and the trinitrocompound 2,4,6-trinitrophenol (picric acid),with the release of nitrite (10); a Pseudomonasdegraded 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 metacleavage of the ring (between the 1 and 2 car-bon atoms) and eventual degradation of themolecule (Tewfik and Evans, Biochem J. 99:31P, 1966). The report that picric acid was me-tabolized with the formation of nitrite indicatesthat trinitro compounds are not immune to deg-radation. Picric acid already possesses one hy-droxyl group and only requires the conversionof one nitro group to a phenolic hydroxyl groupto yield a necessary intermediate for ring cleav-age; i.e., the aromatic nucleus must carry atleast two hydroxyl groups, ortho orpara to eachother, in order for ring cleavage to occur (5). Onthe other hand, TNT, in order to be convertedto a catechol, requires not only the conversionof one nitro group to a hydroxyl group but alsothe hydroxylation of an adjacent unsubstitutedring position.We have shown, as have others, that nitro

groups on the TNT molecule are reduced byboth aerobic and anaerobic systems. Dependingupon the reducing potential of the system, one,two, or three of the nitro groups may be reducedto amino groups. The pathway involving therelease of nitro groups as nitrite does not ap-pear to be a major pathway (at least in pseudo-

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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

o-Nitrotolueneb

m -Nitrotoluene"b

p-Nitrotoluene"

o-Nitrobenzoic acidb

m-Nitrobenzoic acid"

p-Nitrobenzoic acid"

o-Nitrophenol"

m-Nitrophenolb

p-Nitrophenol'

o-Nitroaniline"

5 kNO2NH2

12

NO2

CH320 NH2

NO2

CH314 +,N02

32- NH2

CH3

0NO241 NH2

CH3

02N."32

CH302N NH2

27

CH34OH

6 N02

CH32N32

23 O

p-Nitroanilineb

5-Nitro-o-toluidine'

3-Nitro-p-toluidine"

2-Nitro-p-toluidinec

4-Nitro<-otoluidinec

3-Nitro<-otoluidinec

2-Methyl-5-nitrophenol"

4-Methyl-3-nitrophenolc

6

59

26

24

15

28

39

31

CH3NO2

CH3

~N02CH3

NO2COOH

COOH

kN02COOH

NO2

OHNO2

OH

NO2OH

NO2NH2kN02

(CD,""VIVL

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OH

kSO3H

NO2

1 rS03HN02

COOH

02N COOH

aN02NO2

kNO2

NO2

NO2

k NO2NO2COH

0NO2

NO2

OHeN02

N 2

Compound

2-Nitroresorcinol"

4-Nitrotoluene-2-sul-fonic acid"

m-Nitrobenzene-sul-fonic acid'

5-Nitro-m-phthalic acid"

o-Dinitrobenzene'

m-Dinitrobenzene"

p-Dinitrobenzeneb

2,4-Dinitrotolueneb

2,4-Dinitrobenzoic acid'

2,4-Dinitrophenolb

TABLE 2-ContinuedSp act'

59 NHiN02

47 NO2CH3

02C 3

N02

50 COOH

02NANO240 CH3

02N N0279 NH2

COOHj<OH

02N N02

CH3210 O2

02N NO2

02N"rN02

58 NO2

CH3

87 NO2

OH02N N02

33 N2N2

COOH02N N02

NO

Compound2,4-Dinitroanilineb

2,6-Dinitrotolueneb

3,5-Dinitrobenzoic acidb

2,6-Dinitro-p-toluidinel'

3,5-Dinitro-salicylicacidb

4,6-Dinitro-o-cresol"

1,3,5-Trinitrobenzeneb

2,4,6-Trinitrotolueneh

2,4,6-Trinitrophenoli

2,4,6-Trinitrobenzoicacidb

954

Sp actV18

39

139

8

64

51

288

147

81

210

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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 nitritefound was less than 6% of the nitrogen avail-able from the nitro groups of the TNT that haddisappeared from the medium.According to present theory, formation of a

diphenol is a necessary prerequisite to degrada-tion of an aromatic nucleus. Thus far, no phe-nolic intermediate or other evidence for di-phenol formation has been found in the case ofTNT. During the degradation ofp-nitrobenzoicacid by Nocardia (3), p-hydroxybenzoate accu-mulated in the presence of tris(hydroxymeth-yl)aminomethane but not in phosphate buffer.All of our experiments were done in phosphate

buffer. Rabbits fed TNT oxidized the methylgroup to an alcohol and reduced the 4-nitrogroup to an amino group (4). The resulting 4-amino-2,6-dinitrobenzyl alcohol in turn formeda glucuronide that was eliminated in the urine.Conversion ofTNT to a catechol, through oxida-tion and decarboxylation at the methyl groupand hydroxylation of an adjacent nitro group,might be more energy demanding than con-version of a nitro group and hydroxylation ofan adjacent carbon atom. The inability of bio-logical systems to perform this operation maybe due to inhibitory effects of pendant nitro oramino groups on hydroxylation enzymes.

TABLE 3. Effect of variouts enzyme sources on TNT disappearance and on product formation"

Product formedbOrganism Prepn Atmosphere

4HA 4,4'Az 4A 2,4DA TA¶PC. 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 0Growing culture Deep, standing - - - + -

E. coli (aerobic) Cell-free extract H2 + + - - -

Cell-free extract Air 0 0 0 0 0Resting cells H2 - + + + -

Resting cells Air - +Growing culture Shallow, shaking - + - + -

Pseudomonad FR2 Cell-free extract H2 0 0 0 0 0Cell-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 to20 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 0.2ml 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.

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956 McCORMICK, FEEHERRY, AND LEVINSON

02N N 02 C02N H3N2

NCCH3 CH-N

D.-NN-0

rK>N23CH3 NO2 CH302NK)NO2 02N 3jNHOH 0 2NjjN02 2t1N 1N02CR3

Vill N02 V IINHOH i

0202N NH2 02N jJN02

N02 CR3 NH2

02N1j1NH2

NH2

IX

CR3H2N NH2

NH2

Ix

FIG. 2. Proposed pathway for transformation of TNT by the reduction of the nitro groups. Compoundsillustrated 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(4A2NT) from the reduction of 2,4-dinitrotoluene

(2,4DNT) by V. alkalescens"

Reaction time (min)Compound

0 10 60

2,4DNT + + -2A4NT - - -4A2NT - + -2,4DAT - + +

Reaction mixture was as described in Materialsand Methods, using V. alkalescens crude extract.The mixture was deproteinized with H2SO4 and cen-trifuged, the solution was made alkaline withNaOH and extracted twice with benzene, and thebenzene solution was evaporated to a small volumeand applied to a thin-layer plate. Separation ofauthentic 2-amino-4-nitrotoluene (2A4NT) from4A2NT was achieved by five successive develop-ments in benzene. Detection was as described inMaterials and Methods. 2,4DAT, 2,4-Diamino-toluene.

Our results agree with those expected fromthe accepted pathway of reduction of nitro com-pounds. The proposed intermediate nitrosocompound (equation 1) was not detected, butthe second intermediate, the hydroxylaminocompound (equation 2), was identified. All themajor products arising from TNT metabolismoriginate from the hydroxylamino compound.

TABLE 5. Activity ofDEAE-cellulose-treated extractsof Veillonella alkalescens"

Enzyme source Sp act1. Crude extract 15.22. DEAE-cellulose-treated 0.7

crude extract3. 0.5 M NaCl eluate 15.14. (2) + (3) 14.2

"DEAE-cellulose chromatography was performedas described in Materials and Methods. Total pro-tein per reaction mixture was 13 mg.

h Expressed as in Table 2.

TABLE 6. Hydrogen uptake by TNT in the presenceof Sephadex G-200 fractions

Fraction no. Activity"

4' 36.76' 7.5

4' + 6' 89.2

a Expressed as nanomoles of H2 consumed perminute.

Although the extent of reduction depended onthe redox potential of the system, our observa-tions agree with those ofChannon et al. (4) thatin an aerobic environment the hydroxylaminocompound may be nonenzymatically oxidized tothe azoxy compound. Thus, the finding of2,2'Az presupposes the formation of 2-hydrox-

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TRANSFORMATION OF NITROAROMATIC COMPOUNDS 957

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 complexmolecules may arise by condensation reactionsof 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 byWoolfolk (Ph.D. thesis) and with the "electro-negativity rule" (25), which states that the rateof reduction of nitro compounds increases withincreasing electron withdrawing power of thegroups at the para position: -NH2 < -OH <-H < CH: < -COOH < -NO2. In the tri-nitro series of compounds (Table 2), the benzoicacid derivative should be more readily reducedthan the aniline derivatives. The rate of reduc-tion of trinitrobenzoic acid is indeed high, butpicramide (the trinitroaniline derivative), whichshould have the lowest rate, was not availablefor testing. The high rate obtained with 2,4,6-trinitrobenzoic acid may be the consequence ofthe position of the amino group (meta relativeto the remaining nitro groups) resulting fromthe reduction of the first nitro group. Aminogroups in the ortho or para position would bemore inhibitory to further reduction than thosein the meta position. In the case ofp-dinitroben-zene, which has a high rate of reduction, bothnitro 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 isreduced first (Table 4). Evidence in the litera-ture and from our own work suggests that withTNT the 4-nitro group is reduced first. In thecase of 2,4-dinitrophenol (20; Woolfolk, Ph.D.thesis), the 2-nitro group is reduced first. Itmight be predicted that the reduction of picricacid should proceed via reduction of the 2-nitrogroup first. This is borne out by our observationof spectral changes characteristic of picramicacid (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 fromeach other on a Sephadex G-200 column. Evenafter rechromatography of fraction 4 (the hy-drogenase fraction) on Sephadex, this fractionmay still be contaminated with traces of a fer-redoxin-like material. The high A3,,/A2-1 offraction 6', the absorption as a brown band tothe top of a DEAE-cellulose column, and the in-

volvement in reactions of low reducing poten-tial are all properties suggesting the presenceof ferredoxin, but the question of whether frac-tion 6' contains other "reductases" or whetherferredoxin acts as a nonspecific reductase fornitroaromatic compounds and for intermediatenitroso and hydroxylamino compounds mustawait further experiments. Villanueva (29)purified a "nitro-reductase" from Nocardia andconcluded that it was one enzyme or a group ofenzymes with similar properties.The aerobic systems pseudomonad FR2 and

aerobically grown E. coli have the capability ofreducing two of the three nitro groups of TNTin the absence of the enzyme hydrogenase, sug-gesting that "nitro-reductase" activity in aero-bic systems may involve reduced nicotinamideadenine dinucleotide and flavoproteins as pre-viously reported (2, 24, 35).Growing cultures ofC. pasteurianum formed

large amounts of4HA in early log phase, whichdisappeared in late log and stationary phase,when no products ofany 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 wasthere any residual TNT. Experiments usingTNT (ring-L-[U-'4C]) with growing cultures ofC. pasteurianum are in progress in order tosearch for definite evidence, thus far lacking,for metabolism or degradation of TNT.

ACKNOWLEDGMENTSWe 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 referencecompounds through the generosity of J. Hoffsommer, U.S.Naval Surface Weapons Center, Silver Spring, Md.

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