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.     

Observations on the use of guaiacol and 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as peroxidase substrates

Aging of aqueous guaiacol (o-methoxyphenol) solutions over a period of several months led to the spontaneous formation of peroxidatic compound(s) and other unidentified oxidation products of guaiacol. This accelerated the oxidation of guaiacol catalyzed by lactoperoxidase (LPO) severalfold depending on the pH of the reaction mixture. The peroxide(s) acted like H₂O₂ while the aromatic oxidation products may be more reactive than guaiacol. Five- to 12-month-old 20 mm stock solutions contained even 0.05-0.3% of H₂O₂ equivalents. The formation of the peroxidatic compound(s) was found to be a photochemical process which progressed in a few hours at 254 nm and slowly (detectable in 2-week-old solutions) in regular glass bottles kept under normal laboratory illumination. The kinetics and pH dependence of the oxidation of aged guaiacol solutions by LPO were distinctly different from those found with fresh substrate. The spontaneously formed peroxidatic compound is possibly a better oxygen donor in LPO assays than H₂O₂. The spontaneously formed aromatic oxidation products of guaiacol may include compounds that contain diphenoquinone groups. The complexity of the oxidation of guaiacol and the multitude of reaction products formed require special consideration in kinetic studies of LPO. The use of 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) as a LPO substrate was studied. The published method utilizing this substrate was modified into a more sensitive procedure by readjusting some of the reaction conditions.     

Biotransformation and partial mineralization of the explosive 2,4,6-trinitrotoluene (TNT) by rhizobia

Three strains, T10, B5, and M8, each belonging to a different species of the family Rhizobiaceae and isolated from atrazine-contaminated soils, were tested for their ability to transform 2,4,6-trinitrotoluene (TNT) (50 microg x mL(-1)) in liquid cultures using glucose as the C-source. All three strains were able to transform TNT to hydroxylaminodinitrotoluenes (2-HADNT, 4-HADNT), aminodinitrotoluenes (2-ADNT, 4-ADNT), and diaminonitrotoluene (2,4-DANT). The transformation was significantly faster in the presence of glutamate. Furthermore, the major metabolites that accumulated in cultures were 2-ADNT with glucose, and 4-ADNT with glutamate plus glucose. Rhizobium trifolii T10 was also tested for its ability to transform high levels of TNT (approximately 350 microg x mL(-1)) in a soil slurry. Almost 60% of the TNT was transformed within 2 days in bioaugmented soil slurries, and up to 90% when cultures were supplemented with glucose and glutamate. However, mineralization was minimal in all cases, less than 2% in 78 days. This is the first report on the degradation of TNT by rhizobial strains, and our findings suggest that rhizobia have the potential to play an important role in the safe decontamination of soils and sites contaminated with TNT if bioaugmentation with rhizobia is shown to have no ecotoxicological consequence.     

Anaerobic transformation of 2,4,6-trinitrotoluene (TNT)

A sulfate-reducing bacterium using trinitrotoluene (TNT) as the sole nitrogen source was isolated with pyruvate and sulfate as the energy sources. The organism was able to reduce TNT to triaminotoluene (TAT) in growing cultures and cell suspensions and to further transform TAT to still unknown products. Pyruvate, H₂, or carbon monoxide served as the electron donors for the reduction of TNT. The limiting step in TNT conversion to TAT was the reduction of 2,4-diamino-6-nitrotoluene (2,4-DANT) to triaminotoluene. The reduction proceeded via 2,4-diamino-6-hydroxylaminotoluene (DAHAT) as an intermediate. The intermediary formation of DAHAT was only observed in the presence of carbon monoxide or hydroxylamine, respectively. The reduction of DAHAT to triaminotoluene was inhibited by both CO and NH₂OH. The inhibitors as well as DANT and DAHAT significantly inhibited sulfide formation from sulfite. The data were taken as evidence for the involvement of dissimilatory sulfite reductase in the reduction of DANT and/or DAHAT to triaminotoluene. Hydrogenase purified from Clostridium pasteurianum and carbon monoxide dehydrogenase partially purified from Clostridium thermoaceticum also catalyzed the reduction of DANT in the presence of methyl viologen or ferredoxin, however, as the main reduction product DAHAT rather than triaminotoluene was formed. The findings could explain the function of CO as an electron donor for the DANT reduction (to DAHAT) and the concomitant inhibitory effect of CO on triaminotoluene formation (from DAHAT) by the inhibition of sulfite reductase. Triaminotoluene is further anaerobically converted to unknown products by the isolate under sulfate-reducing and by a Pseudomonas strain under denitrifying conditions. Triaminotoluene conversion was also catalyzed in the absence of cells under aerobic conditions by trace elements, especially by Mn²⁺, accompanied by the elimination of ammonia in a stoichiometry of 1 NH₃ released per TAT transformed. The results might be of interest for the bioremediation of wastewater polluted with nitroaromatic compounds.Abbreviations TNT = 2,4,6-Trinitrotoluene DANT - 2,4-DANT = 2,4-Diamino-6-nitrotoluene - 2,6-DANT = 2,6-Diamino-4-nitrotoluene - ADNT = aminodinitrotoluene - 2-ADNT and 4-ADNT amino substituent at positions 2 or 4 - TAT = 2,4,6-Triaminotoluene - DAHAT = 2,4-Diamino-6-hydroxylaminotoluene - MV = Methyl viologen - Fd = Ferredoxin - H₂ase = Hydrogenase - CODH = Carbon monoxide dehydrogenase - Pyr: Fd OR = Pyruvate: ferredoxin oxidoreductase - U = Units = mol of substrate converted per min     

Transformation of 2,4,6-trinitrotoluene (TNT) by <Emphasis Type="Italic">Raoultella</Emphasis> <Emphasis Type="Italic">terrigena</Emphasis>

Manufacture of nitroorganic explosives generates toxic wastes leading to contamination of soils and waters, especially groundwater. For that reason bacteria living in environments highly contaminated with 2,4,6-trinitrotoluene (TNT) and other nitroorganic compounds were investigated for their capacity for TNT degradation. One isolate, Raoultella terrigena strain HB, removed TNT at concentrations between 10 and 100 mg l⁻¹ completely from culture supernatants under optimum aerobic conditions within several hours. Only low concentrations of nutrient supplements were needed for the cometabolic transformation process. Radioactivity measurements with ring-labelled ¹⁴C–TNT detected about 10–20% of the initial radioactivity in the culture supernatant and the residual 80–90% as water-insoluble organic compounds in the cellular pellet. HPLC analysis identified aminodinitrotoluenes (2-ADNT, 4-ADNT) and diaminonitrotoluenes (2,4-DANT) as the metabolites which remained soluble in the culture medium and azoxy-dimers as the main products in the cell extracts. Hence, the new isolate could be useful for the removal of TNT from contaminated waters.     

Lime Treatment of Explosives-Contaminated Soil from Munitions Plants and Firing Ranges

Microcosms were prepared using soils from munitions plants and active firing ranges and treated with hydrated lime. The presence of particulate explosives and co-contaminants, and the concentration of soil total organic carbon (TOC) on the alkaline hydrolysis reaction were studied. Trinitrobenzene (TNB) and dinitrobenzene (DNB) were sensitive to alkaline hydrolysis under these experimental conditions. The TNT metabolites, 2A- and 4A-DNT, were also removed, although more slowly than the parent compound, and the reaction required a higher pH (>12). RDX retention in the soil was proportional to the TOC content. The degradation intermediates of the alkaline hydrolysis reaction partitioned in the soil matrix in a manner similar to the parent. Solid particles of explosives are also degraded by alkaline hydrolysis. RDX and HMX exhibited 74 and 57% removal, respectively, in 21 days. TNT, as whole and broken grains, showed 83 and 99.9% removal in 21 days, respectively. The propellants, 2,4- and 2,6-DNT, were insensitive to alkaline hydrolysis. Alkaline hydrolysis is an inexpensive and effective means of reducing the varied explosives contamination.     

Interaction of reactive and inert chemicals in the presence of oxidoreductases: Reaction of the herbicide bentazon and its metabolites with humic monomers

The herbicide bentazon (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3 H)-one-2,2-dioxide), a relatively inert chemical, and some of its metabolites were incubated with a laccase or a peroxidase in the presence or absence of humic monomers to evaluate the incorporation of the herbicide and its metabolites into humic material by oxidative enzymes. Guaiacol and ferulic acid were used as representative electron donor co-substrates in most of the oxidative coupling reactions. Bentazon and its metabolites, with the exception of hydroxy metabolites, underwent little or no transformation by the two enzymes in the absence of guaiacol and ferulic acid,but in the presence of these co-substrates transformation occurred. The reaction of bentazon with guaiacol in the presence of the laccase or a peroxidase was almost complete in30 min. 6-Hydroxy- and 8-hydroxy-bentazon were completely transformed by each enzyme both with and with out co-substrates. At pH 3.0 and in the presence of laccase and guaiacol, the concentrations of bentazon and its metabolites2-amino-N-isopropyl-benzamide (AIBA), des-isopropyl-bentazon and 8-chloro-bentazon decreased by 27, 57, 20 and 4%,respectively. The corresponding levels of transformation with peroxidase at pH 3.0 were 9, 70, 30 and 5%, respectively. The extent of transformation decreased with increasing pH. At low pH, the hydroxy-bentazons were completely transformed,followed by (in order of percentage transformation) AIBA,des-isopropyl-bentazon, bentazon and 8-chloro-bentazon. Transformation of bentazon by the laccase increased with increasing guaiacol concentration. In the presence of the peroxidase, the most effective co-substrates for transformation of bentazon were (in decreasing order) catechol, vanillicacid, protocatechuic acid, syring aldehyde and caffeic acid,while in the presence of the laccase, catechol was most effective, followed by caffeic acid, protocatechuic acid and syringaldehyde. This revised version was published online in August 2006 with corrections to the Cover Date.     

Polymerization of guaiacol and a phenolic beta-O-4-substructure by Trametes hirsuta laccase in the presence of ABTS

The reaction of the monomeric lignin model compound guaiacol and the beta-O-4-type dimer erol (1-(4-hydroxy-3-methoxyphenyl)-2(2-methoxyphenoxy)-propane-1,3-diol with laccase from Trametes hirsuta was studied in the presence of the mediator ABTS (2,2'-azino-di[3ethylbenzothiazoline-6-sulfonic acid]). The product mixtures were analyzed by means of aqueous-phase size exclusion chromatography (SEC) with 50 mM NaOH as eluent. Interestingly, in the laccase-catalyzed reaction with both substrates, the mediator not only functioned as an electron carrier but underwent coupling reactions with the substrate to give polymeric coupling products. The molecular weight of these copolymeric products was significantly higher than the molecular weight of products obtained without ABTS. After ultrafiltration, 33% and 21% of the initially applied ABTS could be found in the polymeric product fraction for the substrates guaiacol and erol, respectively, on the basis of nitrogen analysis. When ABTS was added to substrates after full laccase-catalyzed polymerization, the reaction proceeded toward higher molecular weights.     

Inertness of horseradish peroxidase to 2,4-dichlorophenoxyacetic acid

The possibility of mutual effects of 2,4-D and horseradish (Armoracia lapathifolia L.) peroxidase on each other has been explored by four procedures. (i) Compounds I, II, and III of horseradish peroxidase (HRP) and H₂O₂ were exposed to 2,4-D. (ii) Extracts from batchwise operations of HRP + H₂O₂ and 2,4-D were analyzed for oxidation products by means of thin layer chromatography. (iii) The velocity of the IAA oxidase reaction with HRP as catalyst, and (iv) K_m and V_s of the overall peroxidation of guaiacol by HRP + H₂O₂, were determined in the absence and presence of 2,4-D. The results failed to show any effect of 2,4-D; only at very high concentrations did 2,4-D slightly inhibit the oxidation of IAA by one isoperoxidase. It is concluded that 2,4-D does not promote growth in plants by hampering a peroxidase-catalyzed IAA oxidation. It seems probable that 2,4-D perturbs the isoperoxidase pattern by acting at some step prior to the release of the enzyme from its site of synthesis.     

Catalytic properties of tryptophanless recombinant horseradish peroxidase

Heme-containing plant peroxidases (EC 1.11.1.7) contain a highly conserved single tryptophan residue. Its replacement with Phe in recombinant horseradish peroxidase (rHRP) increased the stability of the mutant enzyme in acid media. The kinetic properties of native, wild-type, and W117F mutant recombinant horseradish peroxidase in the reactions of ammonium 2, 2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), guaiacol, and o-phenylenediamine oxidation are very similar. However, significant changes in the reaction rate constant characteristic for the monomolecular rate-limiting step ascribed either to product dissociation from its complex with the enzyme or electron transfer from the substrate to the active site within the Michaelis complex were observed. The data indirectly indicate the participation of the single Trp residue in oxidation of ABTS and guaiacol and possible differences in kinetic mechanisms for oxidation of ABTS, guaiacol, and o-phenylenediamine.     

Transformation of 2,4,6-Trinitrotoluene (TNT) Reduction Products by Lignin Peroxidase (H8) from the White-Rot Basidiomycete Phanerochaete chrysosporium

White-rot fungi are known to degrade a wide range of xenobiotic environmental pollutants, including the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). TNT is first reduced by the fungal mycelium to aminodinitrotoluenes and diaminonitrotoluenes. In a second phase, reduced TNT metabolites are oxidatively transformed and mineralized. The extracellular oxidative enzyme of the ligninolytic system of these fungi includes the lignin peroxidases (LiP) and the manganese-dependent peroxidases (MnP). In the present study, we have shown that a cell-free enzymatic system containing fast protein liquid chromatography (FPLC)-purified LiP (H8) from the white-rot fungus Phanerochaete chrysosporium was able to completely transform 50 mg/L of 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) in 1 and 48 h, respectively. Veratryl alcohol (VA), often described as a mediator in the LiP-catalyzed oxidative depolymerization of lignin, was not required for the enzymatic transformation of 2,4-DA-6-NT or 2-A-4,6-DNT. 2,4-DA-6-NT was also shown to be a competitive inhibitor of the LiP activity measured through the oxidation of VA. Experiments using ¹⁴C-U-ring labeled compounds showed that 2-A-4,6-DNT was converted to 2,2'-azoxy-4,4' ,6,6'-tetranitrotoluene. No significant mineralization, measured by the release of ¹⁴CO2, was observed over 5 d.     

Molecular modeling approach to elucidate gas phase hydrodeoxygenation of guaiacol over a Pd(111) catalyst within DFT framework

Excessive amounts of oxy-functional groups in unprocessed bio-oil vitiate its quality as fuel; therefore, it has to be channelized to upgrading processes, and catalytic hydrodeoxygenation is one of the most suitable routes for the upgrading of crude bio-oil. In this computational work, catalytic hydrodeoxygenation (HDO) of guaiacol, which is an important phenolic compound of crude bio-oil, has been carried out using density functional theory (DFT) over a Pd(111) catalyst. The Pd(111) catalyst surface does not endorse direct eliminations of functional groups of guaiacol; however, it is found to perform excellently in stepwise dehydrogenation reactions of oxy-functionals of guaiacol according to present DFT results. The catechol product, formed through dehydrogenation of the methoxy group, followed by elimination of CH₂ and association of the hydrogen atom, has been identified as one of the major products. The overall reaction rate is controlled by scission of CH₂ from 2-methylene-oxy-phenol with an activation energy demand of 23.06 kcal mol^–1. Further, the kinetic analysis of each reaction step involved in HDO of guaiacol over the Pd(111) catalyst surface has also been carried out at atmospheric pressure and at a wide range of temperatures from 473 to 673 K, with temperature intervals of 50 K. In the kinetic analysis part, various kinetic parameters, such as forward and reverse reaction rate constants, Arrhenius constants, and equilibrium rate constants, are reported. The kinetic modeling of the dominating reaction steps has revealed that even a lower temperature of 473 K provides a favorable reaction environment; and the temperature increment further improves the reaction favorability.     

Titration study of guaiacol oxidation by horseradish peroxidase.

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.     

Aromatic ring cleavage of 4,6-di(tert-butyl)guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor

It was found that 2,4-di(tert-butyl)-4-(methoxycarbonylmethyl)-2-buten-4-ol ide (II) was formed as an aromatic ring cleavage product of a phenolic lignin model compound, 4,6-di(tert-butyl)guaiacol (I), by laccase of Coriolus versicolor. Based on isotopic experiments with 18O2 and H2 18O, the mechanism of formation of II from I is discussed.     

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.     

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.     

Differences in the biotransformation of 2,4,6-trinitrotoluene (TNT) between wild and axenically grown isolates of Myriophyllum aquaticum

The aim of this study was to demonstrate the potential for aquatic plants and their associated microbes to bioremediate wetland sites contaminated with 2,4,6-trinitrotoluene (TNT). The transformation of TNT was studied using both wild and axenically grown isolates of Myriophyllum aquaticum (parrot feather). Differences in TNT transformation rates and nitroaromatic metabolites were observed between different plants. The wild isolates, containing a consortium of associated microorganisms, transformed TNT into 2-amino-4,6-dinitrotoluene (2-A-DNT) and 4-amino-2,6-dinitrotoluene (4-A-DNT) via 2- and 4-hydroxylamino-dinitrotoluene, which were detected as intermediates. The wild M. aquaticum also converted the metabolites, 2-A-DNT and 4-A-DNT, into low levels of 2,4-diaminotoluene (2,4-DAT). The axenically grown plants, containing no cultureable microorganisms, also transformed TNT into 2-A-DNT and 4-A-DNT, but at a much lower rate than that observed for the wild isolates. Unlike the wild plants, axenically grown M. aquaticum could not transform either 2-A-DNT or 4-A-DNT into 2,4-DAT over the incubation period. The differences in the performance between these plants could indicate that plant-associated microorganisms assisted in the overall transformation of TNT. For each plant, unidentifiable metabolites were observed and the soluble monoamino-derivatives present in the wild and axenic medium accounted for 14 and 7% of the initial TNT concentration, respectively. Thus, the majority of nitroaromatic derivatives remained associated with the plant tissues. Furthermore, only 7 and 3% of the initial TNT concentration were extracted as monoamino-derivatives from the tissues of the wild and axenically grown plants, respectively.     

2,4,6-trinitrotoluene reduction by carbon monoxide dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2, 4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K(m) and k(cat) values of TNT reduction were 165 +/- 43 microM for TNT and 400 +/- 94 s(-1), respectively. Cyanide, an inhibitor for the CO/CO(2) oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

2,4,6-Trinitrotoluene Reduction by Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K_m and k_cat values of TNT reduction were 165 ± 43 μM for TNT and 400 ± 94 s⁻¹, respectively. Cyanide, an inhibitor for the CO/CO₂ oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

Rapid and unbiased colorimetric quantification of nitrite and ammonium ions released from 2,4,6-trinitrotoluene during biodegradation studies: Eliminating interferences

Worldwide contamination of soils and groundwater by 2,4,6-trinitrotoluene (TNT) has drawn considerable attention to bioremediation solutions. To evaluate the environmental relevance of a biodegradation process or to discover new TNT degrader microorganisms, effective and reliable monitoring strategies are essential, including the sensitive detection of inorganic nitrogen species released from TNT. In this study we describe improved colorimetric methods for a rapid and unbiased monitoring of nitrite and/or ammonium ions produced in TNT-biodegradation experiments. Considerable interferences in the colorimetric detection of nitrite by the Griess reaction were observed in the presence of various chemicals used in biodegradation assays including reduced pyridine nucleotides (NAD(P)H) and commonly used biological buffers and buffer constituents. In the colorimetric quantification of ammonium through the Berthelot reaction, significant interferences were generated by TNT itself and some reduced TNT metabolites. We highlight these pitfalls and give solutions to overcome these problems, including buffer selection, appropriate dilutions, oxidation of NAD(P)H and selective removal of TNT and some of its metabolites.