Microbial transformation of 2,4,6-trinitrotoluene

The manufacture and decommissioning of explosives has generated, and continues to generate, large quantities of waste material whose primary toxic and mutagenic component is 2,4,6-trinitrotoluene (TNT). The magnitude of this problem has motivated a great deal of research into treatment processes and environmental fate studies, including characterization of microbial transformations of TNT. This work has encompassed studies with mixed cultures and pure cultures of microorganisms derived from either TNT-exposed or unexposed sources, and studies using microorganisms chosen for their known capacities to degrade other pollutants. Several of these studies are discussed with regard to whether they identified a process that may lead to the complete detoxification or mineralization of TNT. Since oxygen can have a significant influence on the types of biochemical reactions that can occur and on the oxidation of intermediates of TNT transformation processes, studies in which oxygen was not excluded are discussed separately from studies conducted under anaerobic conditions. Received 31 October 1995/ Accepted in revised form 29 March 1996     

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     

Alternative pathways of the initial transformation of 2,4,6-trinitrotoluene by yeasts

A new model for the initial transformation of 2,4,6-trinitrotoluene (TNT) by facultatively anaerobic and aerobic yeasts is presented. The model is based on the data that Saccharomyces sp. ZS-A1 was able to reduce the nitrogroups of TNT with the formation of 2- and 4-hydroxyaminodinitrotoluenes (2-HADNT and 4-HADNT) as the major early TNT metabolites (the molar HADNT/TNT ratio reached 0.81), whereas aminodinitrotoluenes (ADNTs) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Candida sp. AN-L13 almost completely transformed TNT into H-TNT through the reduction of the aromatic ring. Candida sp. AN-L14 transformed TNT through a combination of the two mechanisms described. Aeration stimulated the production of HADNT from TNT, whereas yeast incubation under stationary conditions promoted the formation of HADNT. The transformation of TNT into HADNT led to a tenfold increase in the acute toxicity of the TNT preparation with respect to Paramecium caudatum, whereas the increase in the toxicity was about twofold in the case of the alternative attack at the aromatic ring.     

Fungal transformation of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene.

Screening of 190 fungi representing 98 genera showed that the ability to transform 2,4,6-trinitrotoluene was common, whereas transformation of 2,4-dinitrotoluene was rare.     

Participation of oxygen in the bacterial transformation of 2,4,6-trinitrotoluene

The exposure of Bacillus cereus ZS18 cell suspensions to 2,4,6-trinitrotoluene (TNT) in the absence of other oxidizable substrates increases oxygen uptake, exceeding the basal level of respiration of the bacterium 1.5- and 2-fold with 50 and 100 mg/liter of TNT, respectively. The interaction of both living and to less extent dead bacterial cells with TNT results in the accumulation of superoxide anion (O2*-) in the extracellular medium, which was revealed by the EPR spectroscopy. The accumulation of O2*- decreased by 50-70% in the presence of Cu,Zn-superoxide dismutase of animal origin. In the presence of living bacterial cells, the level of TNT decreased progressively, yielding hydroxylaminodinitrotoluenes together with O2*-. In the presence of heat-killed cells, a moderate decrease in TNT was observed, and the appearance of O2*- was not accompanied by the production of any detectable TNT metabolites. Chelating agents inhibited the transformation of TNT and decreased the formation of O2*-. The demonstrated generation of O2*- during the interaction of TNT with K4[Fe(CN)6] together with the observed effects of chelating agents suggest the participation of iron in the one-electron reduction of TNT and the functioning of an extracellular redox cycle with the involvement of molecular oxygen.     

Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds.

A variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT), were reduced by hydrogen in the presence of enzyme preparations from Veillonella alkalescens. Consistent with the proposed reduction pathway, R-NO2 H2 leads to R-NO H2 leads to R-NHOH H2 leads to R-NH2, 3 mol of H2 was utilized per mol of nitro group. The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V. alkalescens extract were determined. The reactivity of the nitro groups 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-dinitrotulene being reduced first. The pattern of reduction of TNT (disappearance of TNT and reduction products formed) depended on the type of preparation (cell-free extract, resting cells, or growing culture), on the species, and on the atmosphere (air or H2). The "nitro-reductase" activity of V. alkalescens extracts was associated with protein fractions, one having some ferredoxin-like properties and the other possessing hydrogenase activity. Efforts to eliminate hydrogenase from the reaction have thus far been unsuccessful. The question of whether ferredoxin acts as a nonspecific reductase for nitroaromatic compounds remains unresolved.     

Microbial transformation of 14C-labeled 2,4,6-trinitrotoluene in an activated-sludge system

The fate of 14C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14CO2 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The major part of the 14C present in the lipid and protein fractions was found in precipitates that formed in both fractions. The solubility properties and infrared spectra of these precipitates suggested that they are macromolecular structures of the polyamide type formed by the reaction of TNT biotransformation products with lipids, fatty acids, and protein constituents of the microbial flora. This hypothesis is further supported by the correspondence of the infrared spectrum of the lipid precipitate with that of a model compound synthesized from TNT transformation products and lipid precursors. The resistance of these macromolecules to further biodegradation was paralleled by the reported resistance to microbial attack of polyamides containing similar linkages.     

Final stages of the preliminary metabolism of 2,4,6-trinitrotoluene in Pseudomonas fluorescens

The work was aimed at studying the transformation of 2,4-diamino-6-nitrotoluene (2,4-DA), an intermediate product in 2,4,6-trinitrotoluene catabolism by microorganisms. The results allow one to propose the following scheme for the terminal steps of TNT preparatory metabolism: 2,4-DA----[phloroglucinol carboxylic acid]----phloroglucinol----pyrogallol----ring cleavage.     

Degradation of 2,4,6-trinitrotoluene by Serratia marcescens

A strain of Serratia marcescens, isolated from the soil of a contaminated site, degraded 2,4,6-trinitrotoluene (TNT) as the sole source of carbon and energy. At an initial concentration of 50mg , TNT was totally degraded in 48h under aerobic conditions in a minimal salt medium. Reduction intermediates (4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene) were observed. The presence of a surfactant (Tween 80) is essential to facilitate rapid degradation.     

Microbial transformation of 14C-labeled 2,4,6-trinitrotoluene in an activated-sludge system.

The fate of 14C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14CO2 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The major part of the 14C present in the lipid and protein fractions was found in precipitates that formed in both fractions. The solubility properties and infrared spectra of these precipitates suggested that they are macromolecular structures of the polyamide type formed by the reaction of TNT biotransformation products with lipids, fatty acids, and protein constituents of the microbial flora. This hypothesis is further supported by the correspondence of the infrared spectrum of the lipid precipitate with that of a model compound synthesized from TNT transformation products and lipid precursors. The resistance of these macromolecules to further biodegradation was paralleled by the reported resistance to microbial attack of polyamides containing similar linkages.     

Studies on inhibition of transformation of 2,4,6-trinitrotoluene catalyzed by Fe-only hydrogenase from Clostridium acetobutylicum

The major enzyme in Clostridium acetobutylicum ATCC 824 leading to transformation of TNT has been reported to be the Fe-only hydrogenase. In this study, we examine the effect of inhibitors of hydrogenase on TNT reduction by Clostridial extracts. These experiments further demonstrate the major role of hydrogenase in TNT transformation. The C. acetobutylicum hydrogenase is closely related to that of C. pasteurianum; and can be fitted to the X-ray crystal structure with a root mean square deviation of 1.18 Å for the Cα atoms of the generated 3D simulation model. The Hyd1, Hyd2, and Hyd3 antibodies generated against hydrogenase reacted with both the hydrogenase in cell extracts and with C. acetobutylicum hydrogenase expressed in Escherichia coli. Inhibition studies using antibodies against Fe-only hydrogenase from C. acetobutylicum indicated that the transformation of TNT by crude cell extracts was completely inhibited by Hyd2 antibody (to amino acid 415–428) whereas antibodies Hyd1 (to residues 1–16) and Hyd3 (to amino acid 424–448) inhibited less effectively. The TNT transforming activity of the cell extract was retained when Hyd2 antibody pretreated with purified but enzymatically inactive recombinant hydrogenase was added to the extract. Addition of the transition metal Cu (2+) to extracts completely inhibited the transformation of TNT suggesting the destruction of [4Fe–4S] centers which are essential for transfer of electrons from the H2-activating site to TNT. Growth of C. acetobutylicum was also inhibited by 0.5 mM Cu(2+) and Hg(2+) ions. The triazine dye, procion red and the nitroimidazole drug, metronidazole inhibit TNT reduction. The inhibition studies using antibodies, procion red, metronidazole, and transition metals suggest that different portions of hydrogenase are required for effective TNT reduction.     

Catabolism of 2,4,6-trinitrotoluene byMycobacterium vaccae

Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of propane as a carbon and energy source. Two novel oxidized metabolites, as well as several known reduced products, were generated during catabolism of TNT byM. vaccae. During the cometabolic process, there was transient production of a brown chromophore. This compound was identified as 4-amino-2,6-dinitrobenzoic acid. WhenM. vaccae was incubated with [^14CTNT and propane, 50% of the added radiolabel was incorporated into the cellular lipid fraction. These results suggest that ring cleavage occurred prior to the incorporation of radiolabelled carbon into phosphatidyl-l-serine, phosphatidylethanolamine, cardiolipin, and other polar lipids.     

Biological degradation of 2,4,6-trinitrotoluene.

Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives. These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms. 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound. Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes. Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates. Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes. Anaerobic microorganisms can also degrade TNT through different pathways. One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known. Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized. Another pathway has been described in Pseudomonas sp. strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells. It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis. In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils. These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material. These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.     

Biodegradation of TNT (2,4,6-trinitrotoluene) by Phanerochaete chrysosporium

Extensive biodegradation of TNT (2,4,6-trinitrotoluene) by the white rot fungus Phanerochaete chrysosporium was observed. At an initial concentration of 1.3 mg/liter, 35.4 +/- 3.6% of the [14C]TNT was degraded to 14CO2 in 18 days. The addition of glucose 12 days after the addition of TNT did not stimulate mineralization, and, after 18 days of incubation with TNT only, about 3.3% of the initial TNT could be recovered. Mineralization of [14C]TNT adsorbed on soil was also examined. Ground corncobs served as the nutrient for slow but sustained degradation of [14C]TNT to 14CO2 such that 6.3 +/- 0.6% of the [14C]TNT initially present was converted to 14CO2 during the 30-day incubation period. Mass balance analysis of liquid cultures and of soil-corncob cultures revealed that polar [14C]TNT metabolites are formed in both systems, and high-performance liquid chromatography analyses revealed that less than 5% of the radioactivity remained as undegraded [14C]TNT following incubation with the fungus in soil or liquid cultures. When the concentration of TNT in cultures (both liquid and soil) was adjusted to contamination levels that might be found in the environment, i.e., 10,000 mg/kg in soil and 100 mg/liter in water, mineralization studies showed that 18.4 +/- 2.9% and 19.6 +/- 3.5% of the initial TNT was converted to 14CO2 in 90 days in soil and liquid cultures, respectively. In both cases (90 days in water at 100 mg/liter and in soil at 10,000 mg/kg) approximately 85% of the TNT was degraded. These results suggest that this fungus may be useful for the decontamination of sites in the environment contaminated with TNT.     

Metabolism of 2,4,6-trinitrotoluene by aPseudomonas consortium under aerobic conditions

An aerobic bacterial consortium was shown to degrade 2,4,6-trinitrotoluene (TNT). At an initial concentration of 100 ppm, 100% of the TNT was transformed to intermediates in 108 h. Radiolabeling studies indicated that 8% of [¹⁴C]TNT was used as biomass and 3.1% of [¹⁴C]TNT was mineralized. The first intermediates observed were 4-amino-2,6-dinitrotoluene and its isomer 2-amino-4,6-dinitrotoluene. Prolonged incubation revealed signs of ring cleavage. Succinate or another substrate—e.g., malic acid, acetate, citrate, molasses, sucrose, or glucose—must be added to the culture medium for the degradation of TNT. The bacterial consortium was composed of variousPseudomonas spp. The results suggest that the degradation of TNT is accomplished by co-metabolism and that succinate serves as the carbon and energy source for the growth of the consortium. The results also suggest that this soil bacterial consortium may be useful for the decontamination of environmental sites contaminated with TNT.     

Transformation of 2,4,6-trinitrotoluene by white rot fungus Irpex lacteus

Up to 200 mg 2,4,6-trinitrotoluene (TNT) l^–1 was removed within 12 h after adding it to a 5-day old culture of Irpex lacteus. The initial formation of hydroxylamino-dinitrotoluenes (2- and 4-OHAmDNT) from TNT was detected, followed by their successive transformation to aminodinitrotoluenes (2- and 4-AmDNT). Transformation of TNT to AmDNT via OHAmDNT was fast, but the next step was slow and seemed to be a rate-limiting step in TNT degradation. OHAmDNT isomers were also rapidly transformed by an in vitro enzymatic system. Both the mycelium and extracellular enzymes of I. lacteus were required for the TNT degradation.     

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

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 an Fe-only hydrogenase in Clostridium acetobutylicum

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K(m) of 152 micro M. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R(2) = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.