厌氧条件下Shewanella oneidensis MR-1对2,4-二硝基甲苯的还原转化

【目的】研究Shewanella oneidensis MR-1厌氧生物转化2,4-二硝基甲苯(2,4-DNT)的能力、转化过程和影响因素。【方法】以乳酸钠为电子供体, 2,4-DNT为电子受体, S. oneidensis MR-1为降解菌, 黄素为胞外电子载体, 设立四个不同的对照体系并监测各体系在转化过程中2,4-DNT及其产物的动态变化。同时研究不同2,4-DNT浓度下细胞的生长情况, 以及不同黄素浓度下2,4-DNT的降解情况。【结果】S. oneidensis MR-1菌能够高效还原转化2,4-DNT为4-氨基-2-硝基甲苯(4A2NT)和2-氨基-4-硝基甲苯(2A4NT), 并将其进一步还原为2,4-二氨基甲苯(2,4-DAT), 黄素能加速转化过程。【结论】S. oneidensis MR-1菌具备高效还原转化2,4-DNT的能力, 为实际环境中硝基苯污染的原位修复提供科学依据。     

Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain

Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored.     

Anaerobic transformation of 2,4,6-TNT by bovine ruminal microbes

Degradation of TNT by bovine rumen fluid, a novel source of anaerobic microbes, was investigated. Whole rumen fluid contents were spiked with TNT and incubated for a 24h time period. Supernatant samples taken at 0, 1, 2, 4, and 24h were analyzed by reverse-phase HPLC with diode array detection. Within 1h, TNT was not detectable and reduction products of TNT including 2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were present with smaller amounts of diamino-nitrotoluenes. Within 2h, only the diamino and dihydroxyamino-nitrotoluene products remained. After 4h, 2,4-diamino-6-nitrotoluene and 2,4-dihydroxyamino-6-nitrotoluene were the only known molecular species left. At 24h known UV absorbing metabolites were no longer detected, suggesting further transformation such as complete reduction to triaminotoluene or destruction of the aromatic ring of TNT may have occurred. TNT was not transformed at 24h in autoclaved and buffered controls. This study presents the first direct evidence of biodegradation of TNT by ruminal microbes.     

Rapid separation of reduction products of 2,4,6-trinitrotoluene using TLC

Silica gel TLC methods were developed for the separation of 2,4,6-trinitrotoluene (TNT) in mixtures with possible reduction products. The methods employed repeated elutions with simple binary or ternary solvent systems in either one or two dimensional modes. The resolved analytes include TNT, selected amino derivatives (2-amino-4,6-di-nitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene) and known hydroxylamino derivatives (2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene and 2,4-dihydroxylamino-6-nitrotoluene).     

Identification of biotransformation products from 2,4-dinitrotoluene.

The products of microbial transformation of 2,4-dinitrotoluene by Mucrosporium sp. were identified by thin-layer chromatography and by gas chromatography/mass spectrometry as 2-amino-4-nitrotoluene, 4-amino-2-nitrotoluene, 2,2'-dinitro-4,4'-azoxytoluene, 4,4'-dinitro-2,2'-azoxytoluene, and 4-acetamido-2-nitrotoluene. A third azoxy compound, believed to be a "mixed" type (i.e., 2,4'-azoxy or 4,2'-azoxy), was isolated but not yet identified.     

Transformation of 2,4,6-trinitrotoluene during oxygen and nitrate respiration in Pseudomonas fluorescens

A study of the metabolic pathway and the rate of 2,4,6-trinitrotoluene (TNT) transformation depending on the nature of the electron acceptor in the electron transport chain of Pseudomonas fluorescens B-3468 revealed that the first reaction of nitroreduction of TNT resulting in formation of 2-amino-4,6-dinitrotoluene (2A) and 4-amino-2,6-dinitrotoluene (4A) became more active in case of nitrate respiration as compared to oxygen respiration; a TNT decrease was 100 and 66%, respectively. The same tendency but much more pronounced was observed at the next stage of nitroreduction that lead to 2,4-diamino-6-nitrotoluene (2,4DA). On the contrary, aerobic conditions are more preferable for the subsequent destruction of 2,4DA. Thus monoamino derivatives, 2A and 4A, predominated under anaerobic conditions, whereas 2,4DA under anaerobic ones (85 and 69% of the total nitrogen-containing metabolites), respectively. Phloroglucinol and pyrogallol accumulated in the culture liquid when the bacteria were grown on a medium containing 2,4DA as a sole source of nitrogen. Their role as intermediates was proved by the results obtained by studying oxidative activity of the cells grown in the presence of 2,4DA and phloroglucinol.     

Degradation of 2,4,6-trinitrotoluene (TNT) by immobilized microorganism-biological filter

The combined process of immobilized microorganism-biological filter was used to degrade TNT in an aqueous solution. The results showed that the process could effectively degrade TNT, which was not detected in the effluent of the system. GC/MS analysis identified 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT), 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT), 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2,4-diamino-6-nitrotoluene (2,6-DA-4-NT) as the main anaerobic degradation products. In addition, the Haldane model successfully described the anaerobic degradation of TNT with high correlation coefficients (R² = 0.9803). As the electron donor, ethanol played a major role in the TNT biodegradation. More than twice the theoretical requirement of ethanol was necessary to achieve a high TNT degradation rate (above 97.5%). Moreover, Environment Scan Electron Microscope (ESEM) analysis revealed that a large number of globular microorganisms were successfully immobilized on the surface of the carrier. Further analysis by Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DGGE) demonstrated that the special bacterial for TNT degradation may have generated during the domestication with TNT for 150 days. The dominant species for TNT degradation were identified by comparing gene sequences with Genebank.     

Analysis of nitroaromatic compounds in urine by gas chromatography–mass spectrometry for the biological monitoring of explosives

Organic nitrocompounds are the most frequently used constituents of explosives and some of them have been evaluated to be highly toxic or even carcinogenic. Human contact with explosives may originate from a variety of sources, including occupational exposure during the production of ammunition as well as environmental exposure due to the contamination of soil and ground water reservoirs on former military production sites and training areas. This paper describes two gas chromatography–mass spectrometry–selected ion monitoring methods for the determination of twelve nitroaromatic compounds in urine (nitrobenzene, 1,2-dinitrobenzene, 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2-nitrotoluene, 3-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2,4,6-trinitrotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene). The analytes are detectable in the lowest μg/l range, with imprecisions of 3–22% within series and 5–29% between series, depending on the compound of interest. Both procedures are rapid and relatively easy to perform and, therefore, are advantageous for the screening of occupationally or environmentally exposed persons. We analysed urine samples obtained from nine workers from an ammunition dismantling workshop and from twelve control persons. 2,4,6-Trinitrotoluene was detected in six samples at concentrations between 4 and 43 μg/l. The main metabolites of 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene, were found in a concentration range from 143 to 16 832 μg/l and from 24 to 5787 μg/l, respectively. Nonconjugated aminodinitrotoluenes were present as varying percentages of the total amount. 2,4-Dinitrotoluene and 2,6-dinitrotoluene were found in two samples (2–9 μg/l). Nitroaromatics were not detectable in urine specimens from control persons.     

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

Biotransformation of 2,4,6-trinitrotoluene with Phanerochaete chrysosporium in agitated cultures at pH 4.5.

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 microM) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated 14CO2) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-N-acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4, 6-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-N-acetylhydroxy-2,6-dinitrotoluene and 4-N-acetoxy-2, 6-dinitrotoluene (as acetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.     

Biotransformation of 2,4,6-Trinitrotoluene with Phanerochaete chrysosporium in Agitated Cultures at pH 4.5

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 μM) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated ¹⁴CO₂) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-N-acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4,6-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-N-acetylhydroxy-2,6-dinitrotoluene and 4-N-acetoxy-2,6-dinitrotoluene (as acetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.     

Biotransformation of 2,4,6-trinitrotoluene (TNT) by the fungus Fusarium oxysporum

The fungus Fusarium oxysporum was isolated and identified from the aquatic plant M. aquaticum. The capability of this fungus to transform 2,4,6-trinitrotoluene (TNT) in liquid cultures was investigated TNT was added to shake flask cultures and transformed into 2-amino-4,6-dinitrotoluene (2-A-DNT), 4-amino-2,6-dinitrotoluene (4-A-DNT), and 2,4-diamino-6-nitrotoluene (2,4-DAT) via 2- and 4-hydroxylamino-dinitrotoluene derivatives, which could be detected as intermediate metabolites. Transformation of TNT, 2-A-DNT, and 4-A-DNT was observed by whole cultures and with isolated mycelium. Cell-free protein extracts from the extracellular, soluble, and membrane-bound fractions were prepared from this fungus and tested for TNT-reducing activity. The concentrated extracellular culture medium was unable to transform TNT; however, low levels of TNT transformation were observed by the membrane fraction in the presence of nicotinamide adenine dinucleotide phosphate in an argon atmosphere. A concentrated extract of soluble enzymes also transformed TNT, but to a lesser extent. When TNT toxicity was studied with this fungus, a 50% decrease in the growth of F. oxysporum mycelium was observed when exposed to 20 mg/L TNT.     

Microbial transformation of nitroaromatic compounds in sewage effluent

The transformation of mono- and dinitroaromatic compounds was measured in sewage effluent maintained under aerobic or anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much of the 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence of oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disappeared slowly, but no loss was evident in 28 days in aerated sewage. Aromatic amines did not accumulate during the aerobic decomposition of the mononitro compounds. They did appear in nonsterile, but not in sterile, sewage incubated aerobically with the dinitro compounds and anaerobically with all the chemicals. Analysis by gas chromatography and combined gas chromatography-mass spectrometry showed that aniline was formed from nitrobenzene, toluidine was formed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3- and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formed from 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotoluene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid under both conditions. The isomeric forms of the metabolites were not established. Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared from aerated nonsterile, but not from sterile, sewage or sewage in the absence of oxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid added to sewage persisted for at least 60 days in aerobic or anaerobic conditions. Gas chromatographic and gas chromatographic-mass spectrometric analyses demonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimidazoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, and that 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-nitroaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence of oxygen. It is suggested that the transformations of widely used nitroaromatic compounds should be further studied because of the persistence and possible toxicity of products of their metabolism.     

Microbial Transformation of Nitroaromatic Compounds in Sewage Effluent

The transformation of mono- and dinitroaromatic compounds was measured in sewage effluent maintained under aerobic or anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much of the 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence of oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disappeared slowly, but no loss was evident in 28 days in aerated sewage. Aromatic amines did not accumulate during the aerobic decomposition of the mononitro compounds. They did appear in nonsterile, but not in sterile, sewage incubated aerobically with the dinitro compounds and anaerobically with all the chemicals. Analysis by gas chromatography and combined gas chromatography-mass spectrometry showed that aniline was formed from nitrobenzene, toluidine was formed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3- and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formed from 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotoluene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid under both conditions. The isomeric forms of the metabolites were not established. Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared from aerated nonsterile, but not from sterile, sewage or sewage in the absence of oxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid added to sewage persisted for at least 60 days in aerobic or anaerobic conditions. Gas chromatographic and gas chromatographic-mass spectrometric analyses demonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimidazoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, and that 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-nitroaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence of oxygen. It is suggested that the transformations of widely used nitroaromatic compounds should be further studied because of the persistence and possible toxicity of products of their metabolism.     

TNT Biotransformation and Detoxification by a Pseudomonas Aeruginosa Strain

Successful microbial-mediated remediation requires transformationpathways that maximize metabolism and minimize the accumulation of toxic products. Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, degraded 100 mg TNT L^-1 in culture medium within 10 h under aerobic conditions. The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2'-azoxytoluene (2,2'AZT, primarily in the cell fraction), which accumulated as major products via the intermediate2-hydroxylamino-4,6-dinitrotoluene (2HADNT). The 2HADNT and2,2'AZT were relatively less toxic to the strain than TNT and 2ADNT. Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium. While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH. The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P. aeruginosa strain MX.     

Implication of manganese (III), oxalate, and oxygen in the degradation of nitroaromatic compounds by manganese peroxidase (MnP)

The fungal ligninolytic enzyme manganese peroxidase (MnP) is known to function by oxidizing Mn(II) to Mn(III), a powerful oxidant. In this work, an abiotic system consisting of Mn(III) in oxalate buffer under aerobic conditions (Mn(III)/oxalate/O2 system) was shown to be capable of extensively transforming 2-amino-4,6-dinitrotoluene (2A46DNT)--one of the main reduction products of 2,4,6-trinitrotoluene (TNT). No significant transformation occurred in the presence of other organic acids or under anaerobic conditions. The Mn(III)/oxalate/O2 system was also able to transform other nitroaromatic compounds such as 2-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene, TNT - the latter to a lesser extent -, and their reduction derivatives. The Mn(III)/oxalate/O2 system mineralized 14C-U-ring labeled 2A46DNT slightly, while no significant mineralization of 14C-U-ring labeled TNT was observed. Unidentified 14C-transformation products were highly polar. Electron spin resonance experiments performed on the Mn(III)/oxalate/O2 system revealed the generation of formyl free radicals (*COO-). The oxygen requirement for the transformation of nitroaromatic compounds suggests the involvement of superoxide free radicals (O2-*). produced through autoxidation of *COO- by molecular oxygen. The implication of such a Mn(III)/oxalate/O2 system in the MnP-catalyzed degradation of nitroaromatic pollutants by white-rot fungi is further discussed.     

Urinary metabolites of workers exposed to nitrotoluenes.

Nitrotoluenes are important intermediates in the chemical industry. 2,6-Dinitrotoluene (26DNT), 2,4-dinitrotoluene (24DNT) and 2-nitrotoluene (2NT) are carcinogenic in animals and possibly carcinogenic in humans. Thus, it is important to develop methods to biomonitor workers exposed to such chemicals. The authors have monitored the air and urine metabolite levels for a group of workers in China exposed to 24DNT, 26DNT, 2NT and 4-nitrotoluene (4NT). The metabolites 2,4-dinitrobenzylalcohol (24DNBAlc), 2-amino-4-nitrobenzoic acid (2A4NBA), 4-amino-2-nitrobenzoic acid (4A2NBA) and 2,4-dinitrobenzoic acid (24DNBA) resulting from exposure to 24DNT were found in 89, 88, 91 and 78% of the exposed workers, respectively. The metabolites 2,6-dinitrobenzylalcohol (26DNBAlc) and 2,6-dinitrobenzoic acid resulting from 26DNT exposure were found in 99 and 86% of the exposed workers, respectively. Quantitatively, 2A4NBA, 4A2NBA and 26DNBAlc were the major metabolites. The nitrobenzoic acids were the major metabolites resulting from exposure to 2NT and 4NT and were present in 96 and 73% of the exposed workers, respectively. Air concentrations of DNT and 2NT did not correlate with the levels of metabolites in the urine. In conclusion, the dinitrobenzyl alcohols and aminonitrobenzoic acids determined in the urine provided a good marker for recently absorbed dose and were intrinsically related to the bioactivation and detoxification pathways of DNT. Air measurements were not a good measure to predict internal exposure.     

Bioconversion of 2,4-diamino-6-nitrotoluene to a novel metabolite under anoxic and aerobic conditions.

Under nitrate-reducing, nongrowth conditions, a Pseudomonas fluorescens species reduced 2,4,6-trinitrotoluene to aminodinitrotoluenes, which were then further reduced to diaminonitrotoluenes. 2,4-Diamino-6-nitrotoluene (2,4-DANT) was further transformed to a novel metabolite, 4-N-acetylamino-2-amino-6-nitrotoluene (4-N-AcANT), while 2,6-diamino-4-nitrotoluene (2,6-DANT) was persistent. Efforts to further degrade 2,4-DANT and 2,6-DANT under aerobic, nitrogen-limited conditions were unsuccessful; 2,6-DANT remained persistent, and 2,4-DANT was again transformed to the 4-N-AcANT compound.     

Anaerobic transformation of 2,4,6-TNT and related nitroaromatic compounds by Clostridium acetobutylicum

The transformation of TNT and related aminated nitrotoluenes by Clostridium acetobutylicum was investigated. 2,4,6-trinitrotoluene (TNT) was rapidly reduced (537 nM min⁻¹ mg protein⁻¹) to undetermined end products via monohydroxylamino derivatives. TNT reduction was more rapid than that of 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene. The metabolic phase of clostridial cultures affected rates and extents of transformation of TNT and its intermediates. Acidogenic cultures showed rapid transformation rates and the ability to transform TNT and its primary reduction products to below detection limits; solventogenic cultures did not transform TNT completely, and showed accumulation of its hydroxylamino derivatives. Carbon monoxide-induced solventogenesis was capable of slowing the transformation of TNT and intermediates. Studies employing [ring-U-¹⁴C]-TNT demonstrated that no significant mineralization occurred and that products of transformation were water-soluble. Received 06 November 1995/ Accepted in revised form 15 August 1996