APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, June 1976,
p. 949-958
Copyright C 1976 American Society for Microbiology
Vol. 31, No. 6 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), reduced by hydrogen in the presence of enzyme preparations from Veillonella alkalescens. Consistent with the proposed reduction pathway, R-NO2 R-NHOH H, R-NH2, 3 mol of H2 was utilized per mol of nitro group. R-NO The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V. alkalescens extract were determined. The reactivity of the nitro groups depended 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 were
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 nonspecific reductase for nitroaromatic compounds remains unresolved. The fate of 2,4,6-trinitrotoluene (TNT) in biological systems has been a subject of concern for many years. Toxic effects (including liver damage and anemia) have been reported on workers engaged in large-scale manufacturing and handling 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 human volunteers was excreted in the urine as the transformed products 4-amino-2,6-dinitrotoluene (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 preparations (31), slices and homogenates of pig liver (2), and cell-free extracts of Neurospora (35) and Escherichia coli (24) suggested that nicotinamide adenine dinucleotide and flavoproteins were involved in the metabolism of TNT, leading to the formation of 4A. Bacteria and fungi that catalyzed the disappearance of TNT during growth in a nutrient medium in the presence of TNT have been isolated from soil (18). The transformation products were: 4A; 4-hydroxylamino-2,6-dinitrotol-
uene (4HA); and 4,4'Az. Several strains of Pseudomonas, growing in a medium supplemented with glucose and yeast extract, also transformed TNT to these reduction products (32). In addition, traces of 2-amino-4,6-dinitrotoluene (2A) and 4,4', 6,6'-tetranitro-2,2'-azoxytoluene (2,2'Az) were detected. Cell-free extracts of the strict anaerobe Veillonella alkalescens catalyzed the reduction, by hydrogen gas, of the nitro groups of a number of nitroaromatic compounds to the corresponding amino compounds (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 metabolism 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 hydroxylamino, to the amino (27). Although the biological 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 949
950
APPL. ENVIRON. MICROBIOL.
McCORMICK, FEEHERRY, AND LEVINSON
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 a freshly prepared 0.5% aqueous solution of p-nicule. MATERIALS AND METHODS Maintenance and growth of cultures. The growth of V. alkalescens and E. coli was described previously (15, 16). Clostridium pasteurianum was grown in the synthetic medium of Rabinowitz (22). Cultures were incubated at 37 C for 16 to 18 h in 20-liter carboys containing 18 liters of medium. Cells were harvested in a Sorvall RC-2 centrifuge equipped with a continuous-flow attachment. E. coli was cultured aerobically in the same medium as for anaerobic growth except in 100-ml quantities contained in 1-liter Erlenmeyer flasks and incubated on a reciprocal shaker (93 3-inch [about 7.5-cm] strokes/min) at 30 C for 16 to 18 h. The pseudomonad FR2 was isolated by F. Rosenberg, Northeastern University, from garden soil perfused with Radford (Va.) Army Ammunition 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 FR2 were grown in the medium of Kitagawa (12) in 100ml quantities in 1-liter Erlenmeyer flasks. Preparation of cell-free extracts. Approximately 1 volume of a buffer consisting of 20 mM potassium phosphate, pH 7.0, and 10 mM 2-mercaptoethanol (PBSH) was added to 1 volume of packed cells. The cell suspensions, cooled in an ice bath, were subjected to intermittent (1-min intervals) sonic vibration with a Sonifier cell disruptor (Heat Systems, Inc.) until, as determined by microscopic examination, over 95% breakage had occurred. The resulting sonic extract was centrifuged at 17,000 x g for 20 min at 0 C, and the supernatant solution was designated as crude extract. Hydrogen uptake studies. Manometry was performed as described by Umbreit et al. (28). Double side-arm Warburg vessels, equipped with vented outlets in each side arm for flushing with hydrogen gas, were used. Reaction mixtures consisted of 100 ,umol of potassium phosphate buffer, pH 7.5, cell suspension or cell-free extract, and 2 ,umol of the nitro compound in a total volume of 2 ml for a final concentration of 50 mM potassium phosphate and 1 mM nitro compound. Hydrogen gas, previously passed through a Deoxo purifier (Fisher Scientific Co.) to remove traces of oxygen, was flushed through the vessels for 10 min before tipping in the enzyme.
Product analysis. Protein was removed by centrifugation after precipitation by the addition of 0.1 ml of either 100% trichloroacetic acid or 12 N H2S04 into the reaction mixture. Whole cells were removed by centrifugation of the reaction mixture. The clear supernatant solutions were applied to thin-layer silica gel glass plates (Analtech, Inc.) containing a fluorescent background. Developing solvents were: chloroform-methanol-acetic acid (80:20:1) for separation of aminotoluenes; and benzene-hexane-pentane-acetone (50:40:10:3) for separation of nitrotolucompounds. Visualization was by ultraviolet light at 254 nm; by spraying with either 10% tetraene
troanilineazo-2,5-dimethoxyaniline-diazotate (fast black K salt, K and K Laboratories, Inc.) followed by 0.1 N NaOH for amino-containing toluenes. In addition to differences in R,, quite visible differences in color were noted (Table 1). Due to variability in the Rf values, reference compounds were chromatographed with the unknown. Chromatography of extracts. Diethylaminoethyl (DEAE)-cellulose was prepared as described by Rabinowitz (22), packed in a column (6-cm diameter by 5 cm), and equilibrated with PBSH. Crude extracts of V. alkalescens were treated with streptomycin sulfate (1.5%) to precipitate nucleic acids, the mixture was centrifuged, and the supernatant solution, containing 1.5 g of protein, was passed through the column followed by PBSH until the buffer came through clear. Column elution was achieved with PBSH buffer containing 0.5 M NaCl and was continued until the dark-brown band was eluted. The material eluted with 0.5 M NaCl was precipitated with ammonium sulfate, and the protein precipitating between 50 and 70% saturated ammonium sulfate was dissolved in PBSH. Sephadex G-200 was prepared by swelling on a steam bath overnight. After removal of fine particles by sedimentation and decanting, a column (2cm diameter by 50 cm) was prepared, equilibrated with PBSH, and loaded with the undialyzed 50 to 70% saturated (NH4)2SO4 protein fraction. The column was developed with PBSH. Polyacrylamide gel electrophoresis was performed as described by Davis (7). Protein concentration was adjusted so that 100 to 200 ,ug was applied in a volume of 0.05 to 0.10 ml. Gels were stained with 0.5% amido black in acetic acid-methanol-water (1:5:5) for 60 min and destained electrophoretically. Analytical methods. Protein was estimated by TABLE 1. Colors of complexes from interaction of sprays with nitro and amino compounds Spray
Tetramethylammonium hydroxide (10% aqueous) or ethylenediamine Fast black K salt (0.5% aqueous)
Compound
Color
TNT 4,4'Az 2,2'Az 4A 4HA
Orange-gold Blue Purple-blue Yellow
2,4DA TAT"
Blue Blue Blue Blue Orange-rust Orange-rust
2,4DAT0 2,6DATe 2A4N1N 4A2NTI
2,4,6-Triaminotoluene. b 2,4-Diaminotoluene.
'2,6-Diaminotoluene. "2-Amino-4-nitrotoluene. 4-Amino-2-nitrotoluene.
Gray-tan
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 hydroxylamino compounds (33) according to the following equations: R-NO2 H2 :R-NO + H20 (1) (2) R-NO 2 R-NHOH R-NHOH -2, R-NH2 + H20 (3) reduce to Thus, 3 mol of hydrogen is required each nitro group to the amino group. The stoichiometry of the reduction of TNT and several mono- and dinitrotoluenes is illustrated in Fig. 1. Hydrogen uptake with the mononitro compounds leveled off at 3 mol of H2 per mol of nitro compound; that with the dinitrotoluenes leveled 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-nitrotoluene, 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. Hydrogen 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 observed in control experiments using heat-inactivated 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 compounds were dissolved (2 mM) by heating on a hot plate with vigorous stirring just before addition to the reaction mixture to give a final concentration of 1 mM. The rate of H2 consumption 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 concentration 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-attracting nitro group enhances the reactivity of the oxygen attached to nitrogen atoms of other nitro groups on the same molecule (19). The reactivity of the nitro groups appears to depend not only on other substituents but also on the position of the nitro groups relative to these substituents. Thus, the para nitro group was more readily reduced than the ortho group in the
951
aI E
= .3
E
E
6
E '-1 w
En
u
T I M E (min) FIG. 1. Stoichiometry of H2 consumption by cellfree extracts of V. alkalescens. The reaction mixture was as described in Materials and Methods and contained 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 dinitrophenol 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 reduction 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 aerobic bacterial preparations on the transformation 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 organisms 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 potential 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 extracts, never with resting cells or growing cultures, with the exception of C. pasteurianum, with which 4HA was found in the growth medium 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 compounds 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 reduction of the 4-nitro group first. This was indeed the case (Table 4). No trace of 2-amino-4-nitrotoluene was detected, suggesting that essentially 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 reduction implies relative nonspecificity of the "nitro-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 passage 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 separated into several visible bands upon passage through Sephadex G-200. The bands were collected 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. Rechromatography of fractions 4 and 6 on Sephadex G-200 yielded fractions 4' and 6', which exhibited synergistic reductase properties (Table 6). The ratio of absorption at 390 nm to 280 nm (A390/A280) of ferredoxin ranges from approximately 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 ferredoxinlike molecules. Fraction 4' appeared to be more active than fraction 6' when assayed for hydrogenase as measured by the evolution of hydrogen from hydrosulfite-reduced methyl viologen in a nitrogen atmosphere (21). Disc gel electrophoresis of fractions 4' and 6' indicated that the major component of fraction 4' was a protein (amido black staining) that fluoresced bluewhite when the gel was exposed to ultraviolet
APPL. ENVIRON. MICROBIOL.
light at 360 nm; fraction 6', more heterogeneous, contained no predominant band. These preliminary data suggested that the active components of the V. alkalescens "nitro-reductase" 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 compounds involves the initial conversion of nitro groups to hydroxyl groups. Two modes ofattack on nitroaromatic compounds appear to be present in bacteria, one involving the reduction of a nitro group to an amino group followed by oxidative 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 bacterium used, ammonia was also produced from nitrobenzoic acids (3). An Arthrobacter sp. degraded 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 production of ammonia (M. S. Tewfik and W. C. Evans, Biochem J. 99: 31P, 1966). The two pathways converged to give the same intermediate, 2,3,5-trihydroxytoluene, followed by meta cleavage of the ring (between the 1 and 2 carbon atoms) and eventual degradation of the molecule (Tewfik and Evans, Biochem J. 99: 31P, 1966). The report that picric acid was metabolized with the formation of nitrite indicates that trinitro compounds are not immune to degradation. Picric acid already possesses one hydroxyl group and only requires the conversion of one nitro group to a phenolic hydroxyl group to yield a necessary intermediate for ring cleavage; 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 appear 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 nitroaromatic compounds Compound Sp act" Compound Sp act'
No,)
(CD,
""VIVL
CH3
Nitrobenzene"
15
o-Nitrotolueneb
5
NO2 CH3
m-Nitroanilineb
42
p-Nitroanilineb
6
kNO2 NH2
m -Nitrotoluene"b
12
NO2
~N02 CH3
NHs
p-Nitrotoluene"
20
CH3
5-Nitro-o-toluidine'
59
3-Nitro-p-toluidine"
26
2-Nitro-p-toluidinec
24
CH3
4-Nitro
AND
ENVIRONMENTAL MICROBIOLOGY, June 1976,
p. 949-958
Copyright C 1976 American Society for Microbiology
Vol. 31, No. 6 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), reduced by hydrogen in the presence of enzyme preparations from Veillonella alkalescens. Consistent with the proposed reduction pathway, R-NO2 R-NHOH H, R-NH2, 3 mol of H2 was utilized per mol of nitro group. R-NO The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V. alkalescens extract were determined. The reactivity of the nitro groups depended 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 were
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 nonspecific reductase for nitroaromatic compounds remains unresolved. The fate of 2,4,6-trinitrotoluene (TNT) in biological systems has been a subject of concern for many years. Toxic effects (including liver damage and anemia) have been reported on workers engaged in large-scale manufacturing and handling 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 human volunteers was excreted in the urine as the transformed products 4-amino-2,6-dinitrotoluene (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 preparations (31), slices and homogenates of pig liver (2), and cell-free extracts of Neurospora (35) and Escherichia coli (24) suggested that nicotinamide adenine dinucleotide and flavoproteins were involved in the metabolism of TNT, leading to the formation of 4A. Bacteria and fungi that catalyzed the disappearance of TNT during growth in a nutrient medium in the presence of TNT have been isolated from soil (18). The transformation products were: 4A; 4-hydroxylamino-2,6-dinitrotol-
uene (4HA); and 4,4'Az. Several strains of Pseudomonas, growing in a medium supplemented with glucose and yeast extract, also transformed TNT to these reduction products (32). In addition, traces of 2-amino-4,6-dinitrotoluene (2A) and 4,4', 6,6'-tetranitro-2,2'-azoxytoluene (2,2'Az) were detected. Cell-free extracts of the strict anaerobe Veillonella alkalescens catalyzed the reduction, by hydrogen gas, of the nitro groups of a number of nitroaromatic compounds to the corresponding amino compounds (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 metabolism 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 hydroxylamino, to the amino (27). Although the biological 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 949
950
APPL. ENVIRON. MICROBIOL.
McCORMICK, FEEHERRY, AND LEVINSON
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 a freshly prepared 0.5% aqueous solution of p-nicule. MATERIALS AND METHODS Maintenance and growth of cultures. The growth of V. alkalescens and E. coli was described previously (15, 16). Clostridium pasteurianum was grown in the synthetic medium of Rabinowitz (22). Cultures were incubated at 37 C for 16 to 18 h in 20-liter carboys containing 18 liters of medium. Cells were harvested in a Sorvall RC-2 centrifuge equipped with a continuous-flow attachment. E. coli was cultured aerobically in the same medium as for anaerobic growth except in 100-ml quantities contained in 1-liter Erlenmeyer flasks and incubated on a reciprocal shaker (93 3-inch [about 7.5-cm] strokes/min) at 30 C for 16 to 18 h. The pseudomonad FR2 was isolated by F. Rosenberg, Northeastern University, from garden soil perfused with Radford (Va.) Army Ammunition 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 FR2 were grown in the medium of Kitagawa (12) in 100ml quantities in 1-liter Erlenmeyer flasks. Preparation of cell-free extracts. Approximately 1 volume of a buffer consisting of 20 mM potassium phosphate, pH 7.0, and 10 mM 2-mercaptoethanol (PBSH) was added to 1 volume of packed cells. The cell suspensions, cooled in an ice bath, were subjected to intermittent (1-min intervals) sonic vibration with a Sonifier cell disruptor (Heat Systems, Inc.) until, as determined by microscopic examination, over 95% breakage had occurred. The resulting sonic extract was centrifuged at 17,000 x g for 20 min at 0 C, and the supernatant solution was designated as crude extract. Hydrogen uptake studies. Manometry was performed as described by Umbreit et al. (28). Double side-arm Warburg vessels, equipped with vented outlets in each side arm for flushing with hydrogen gas, were used. Reaction mixtures consisted of 100 ,umol of potassium phosphate buffer, pH 7.5, cell suspension or cell-free extract, and 2 ,umol of the nitro compound in a total volume of 2 ml for a final concentration of 50 mM potassium phosphate and 1 mM nitro compound. Hydrogen gas, previously passed through a Deoxo purifier (Fisher Scientific Co.) to remove traces of oxygen, was flushed through the vessels for 10 min before tipping in the enzyme.
Product analysis. Protein was removed by centrifugation after precipitation by the addition of 0.1 ml of either 100% trichloroacetic acid or 12 N H2S04 into the reaction mixture. Whole cells were removed by centrifugation of the reaction mixture. The clear supernatant solutions were applied to thin-layer silica gel glass plates (Analtech, Inc.) containing a fluorescent background. Developing solvents were: chloroform-methanol-acetic acid (80:20:1) for separation of aminotoluenes; and benzene-hexane-pentane-acetone (50:40:10:3) for separation of nitrotolucompounds. Visualization was by ultraviolet light at 254 nm; by spraying with either 10% tetraene
troanilineazo-2,5-dimethoxyaniline-diazotate (fast black K salt, K and K Laboratories, Inc.) followed by 0.1 N NaOH for amino-containing toluenes. In addition to differences in R,, quite visible differences in color were noted (Table 1). Due to variability in the Rf values, reference compounds were chromatographed with the unknown. Chromatography of extracts. Diethylaminoethyl (DEAE)-cellulose was prepared as described by Rabinowitz (22), packed in a column (6-cm diameter by 5 cm), and equilibrated with PBSH. Crude extracts of V. alkalescens were treated with streptomycin sulfate (1.5%) to precipitate nucleic acids, the mixture was centrifuged, and the supernatant solution, containing 1.5 g of protein, was passed through the column followed by PBSH until the buffer came through clear. Column elution was achieved with PBSH buffer containing 0.5 M NaCl and was continued until the dark-brown band was eluted. The material eluted with 0.5 M NaCl was precipitated with ammonium sulfate, and the protein precipitating between 50 and 70% saturated ammonium sulfate was dissolved in PBSH. Sephadex G-200 was prepared by swelling on a steam bath overnight. After removal of fine particles by sedimentation and decanting, a column (2cm diameter by 50 cm) was prepared, equilibrated with PBSH, and loaded with the undialyzed 50 to 70% saturated (NH4)2SO4 protein fraction. The column was developed with PBSH. Polyacrylamide gel electrophoresis was performed as described by Davis (7). Protein concentration was adjusted so that 100 to 200 ,ug was applied in a volume of 0.05 to 0.10 ml. Gels were stained with 0.5% amido black in acetic acid-methanol-water (1:5:5) for 60 min and destained electrophoretically. Analytical methods. Protein was estimated by TABLE 1. Colors of complexes from interaction of sprays with nitro and amino compounds Spray
Tetramethylammonium hydroxide (10% aqueous) or ethylenediamine Fast black K salt (0.5% aqueous)
Compound
Color
TNT 4,4'Az 2,2'Az 4A 4HA
Orange-gold Blue Purple-blue Yellow
2,4DA TAT"
Blue Blue Blue Blue Orange-rust Orange-rust
2,4DAT0 2,6DATe 2A4N1N 4A2NTI
2,4,6-Triaminotoluene. b 2,4-Diaminotoluene.
'2,6-Diaminotoluene. "2-Amino-4-nitrotoluene. 4-Amino-2-nitrotoluene.
Gray-tan
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 hydroxylamino compounds (33) according to the following equations: R-NO2 H2 :R-NO + H20 (1) (2) R-NO 2 R-NHOH R-NHOH -2, R-NH2 + H20 (3) reduce to Thus, 3 mol of hydrogen is required each nitro group to the amino group. The stoichiometry of the reduction of TNT and several mono- and dinitrotoluenes is illustrated in Fig. 1. Hydrogen uptake with the mononitro compounds leveled off at 3 mol of H2 per mol of nitro compound; that with the dinitrotoluenes leveled 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-nitrotoluene, 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. Hydrogen 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 observed in control experiments using heat-inactivated 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 compounds were dissolved (2 mM) by heating on a hot plate with vigorous stirring just before addition to the reaction mixture to give a final concentration of 1 mM. The rate of H2 consumption 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 concentration 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-attracting nitro group enhances the reactivity of the oxygen attached to nitrogen atoms of other nitro groups on the same molecule (19). The reactivity of the nitro groups appears to depend not only on other substituents but also on the position of the nitro groups relative to these substituents. Thus, the para nitro group was more readily reduced than the ortho group in the
951
aI E
= .3
E
E
6
E '-1 w
En
u
T I M E (min) FIG. 1. Stoichiometry of H2 consumption by cellfree extracts of V. alkalescens. The reaction mixture was as described in Materials and Methods and contained 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 dinitrophenol 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 reduction 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 aerobic bacterial preparations on the transformation 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 organisms 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 potential 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 extracts, never with resting cells or growing cultures, with the exception of C. pasteurianum, with which 4HA was found in the growth medium 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 compounds 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 reduction of the 4-nitro group first. This was indeed the case (Table 4). No trace of 2-amino-4-nitrotoluene was detected, suggesting that essentially 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 reduction implies relative nonspecificity of the "nitro-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 passage 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 separated into several visible bands upon passage through Sephadex G-200. The bands were collected 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. Rechromatography of fractions 4 and 6 on Sephadex G-200 yielded fractions 4' and 6', which exhibited synergistic reductase properties (Table 6). The ratio of absorption at 390 nm to 280 nm (A390/A280) of ferredoxin ranges from approximately 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 ferredoxinlike molecules. Fraction 4' appeared to be more active than fraction 6' when assayed for hydrogenase as measured by the evolution of hydrogen from hydrosulfite-reduced methyl viologen in a nitrogen atmosphere (21). Disc gel electrophoresis of fractions 4' and 6' indicated that the major component of fraction 4' was a protein (amido black staining) that fluoresced bluewhite when the gel was exposed to ultraviolet
APPL. ENVIRON. MICROBIOL.
light at 360 nm; fraction 6', more heterogeneous, contained no predominant band. These preliminary data suggested that the active components of the V. alkalescens "nitro-reductase" 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 compounds involves the initial conversion of nitro groups to hydroxyl groups. Two modes ofattack on nitroaromatic compounds appear to be present in bacteria, one involving the reduction of a nitro group to an amino group followed by oxidative 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 bacterium used, ammonia was also produced from nitrobenzoic acids (3). An Arthrobacter sp. degraded 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 production of ammonia (M. S. Tewfik and W. C. Evans, Biochem J. 99: 31P, 1966). The two pathways converged to give the same intermediate, 2,3,5-trihydroxytoluene, followed by meta cleavage of the ring (between the 1 and 2 carbon atoms) and eventual degradation of the molecule (Tewfik and Evans, Biochem J. 99: 31P, 1966). The report that picric acid was metabolized with the formation of nitrite indicates that trinitro compounds are not immune to degradation. Picric acid already possesses one hydroxyl group and only requires the conversion of one nitro group to a phenolic hydroxyl group to yield a necessary intermediate for ring cleavage; 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 appear 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 nitroaromatic compounds Compound Sp act" Compound Sp act'
No,)
(CD,
""VIVL
CH3
Nitrobenzene"
15
o-Nitrotolueneb
5
NO2 CH3
m-Nitroanilineb
42
p-Nitroanilineb
6
kNO2 NH2
m -Nitrotoluene"b
12
NO2
~N02 CH3
NHs
p-Nitrotoluene"
20
CH3
5-Nitro-o-toluidine'
59
3-Nitro-p-toluidine"
26
2-Nitro-p-toluidinec
24
CH3
4-Nitro
