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2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound and is highly resistant to degradation. Most aerobic microorganisms reduce TNT to amino derivatives via formation of nitroso- and hydroxylamine intermediates. Although pathways of TNT degradation are well studied, proteomic analysis of TNT-degrading bacteria was done only for some individual Gram-negative strains. Here, we isolated a Gram-positive strain from TNT-contaminated soil, identified it as Bacillus pumilus using 16S rRNA sequencing, analyzed its growth, the level of TNT transformation, ROS production, and revealed for the first time the bacillary proteome changes at toxic concentration of TNT. The transformation of TNT at all studied concentrations (20-200 mg/L) followed the path of nitro groups reduction with the formation of 4-amino-2,6-dinitrotoluene. Hydrogen peroxide production was detected during TNT transformation. Comparative proteomic analysis of B. pumilus showed that TNT (200 mg/L) inhibited expression of 46 and induced expression of 24 proteins. Among TNT upregulated proteins are those which are responsible for the reductive pathway of xenobiotic transformation, removal of oxidative stress, DNA repair, degradation of RNA and cellular proteins. The production of ribosomal proteins, some important metabolic proteins and proteins involved in cell division are downregulated by this xenobiotic., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

This study investigated the impact of ferrihydrite on the pathway and rate of 2,4,6-trinitrotoluene (TNT) transformation by Yarrowia lipolytica AN-L15. The presence of ferrihydrite in the culture medium decreased the rate of TNT biotransformation but resulted in the accumulation of the same TNT metabolites as in the absence of ferrihydrite, albeit at slightly different concentrations. Transformation products observed included aromatic ring reduction products, such as hydride-Meisenheimer complexes, and nitro group reduction products, such as hydroxylamino- and amino-dinitrotoluenes. Independently of the presence of ferrihydrite the subsequent degradation of the hydride complex(es) resulted in the release of nitrite followed by its conversion to nitrate and nitric oxide at the low pH values observed during yeast cultivation. Nitric oxide generation was ascertained by electron spin resonance spectroscopy. In addition, increased Fe(3+)-reduction was observed in the presence of TNT and Y. lipolytica. This study demonstrates that in the presence of yeast cells, TNT-hydride complexes were formed at approximately the same level as in the presence of ferrihydrite, opening up the possibility of aromatic ring cleavage, instead of promoting the production of potentially toxic nitro group reduction products in the presence of iron minerals.

2,4,6-Trinitrotoluene (TNT) is a toxic and persistent explosive compound occurring as a contaminant at numerous sites worldwide. Knowledge of the microbial dynamics driving TNT biodegradation is limited, particularly in native aquifer sediments where it poses a threat to water resources. The purpose of this study was to quantify the effect of organic amendments on anaerobic TNT biodegradation rate and pathway in an enrichment culture obtained from historically contaminated aquifer sediment and to compare the bacterial community dynamics. TNT readily biodegraded in all microcosms, with the highest biodegradation rate obtained under the lactate amended condition followed by ethanol amended and naturally occurring organic matter (extracted from site sediment) amended conditions. Although a reductive pathway of TNT degradation was observed across all conditions, denaturing gradient gel electrophoresis (DGGE) analysis revealed distinct bacterial community compositions. In all microcosms, Gram-negative γ- or β-Proteobacteria and Gram-positive Negativicutes or Clostridia were observed. A Pseudomonas sp. in particular was observed to be stimulated under all conditions. According to non-metric multidimensional scaling analysis of DGGE profiles, the microcosm communities were most similar to heavily TNT-contaminated field site sediment, relative to moderately and uncontaminated sediments, suggesting that TNT contamination itself is a major driver of microbial community structure. Overall these results provide a new line of evidence of the key bacteria driving TNT degradation in aquifer sediments and their dynamics in response to organic carbon amendment, supporting this approach as a promising technology for stimulating in situ TNT bioremediation in the subsurface.

Evaluation of multiple-stressor effects stemming from habitat degradation, climate change, and exposure to chemical contaminants is crucial for addressing challenges to ecological and environmental health. To assess the effects of multiple stressors in an understudied taxon, the western fence lizard (Sceloporus occidentalis) was used to characterize the individual and combined effects of food limitation, exposure to the munitions constituent 2,4,6-trinitrotoluene (TNT), and Plasmodium mexicanum (lizard malaria) infection. Three experimental assays were conducted including: Experiment I--TNT × Food Limitation, Experiment II--Food Limitation × Malaria Infection, and Experiment III--TNT × Malaria Infection. All experiments had a 30 day duration, the malaria treatment included infected and non infected control lizards, food limitation treatments included an ad libitum control and at least one reduced food ration and TNT exposures consisting of daily oral doses of corn oil control or a corn oil-TNT suspension at 5, 10, 20, 40 mg/kg/day. The individual stressors caused a variety of effects including: reduced feeding, reduced testes mass, anemia, increased white blood cell (WBC) concentrations and increased mass of liver, kidney and spleen in TNT exposures; reduced cholesterol, WBC concentrations and whole body, testes and inguinal fat weights given food limitation; and increased WBC concentrations and spleen weights as well as decreased cholesterol and testes mass in malaria infected lizards. Additive and interactive effects were found among certain stressor combinations including elimination of TNT-induced hormesis for growth under food limitation. Ultimately, our study indicates the potential for effects modulation when environmental stressors are combined.

Bacillus cereus strain PU, isolated from soil contaminated with explosive waste, tolerated up to 1.3 mM 2,4,6 trinitrophenol (TNP) and utilize it aerobically as sole nitrogen and carbon source. Degradation of TNP was accompanied by stoichiometric release of 2.1 ± 0.15 mol nitrite/mol TNP at 539 μmol/h g dry cell wt. Metabolism of TNP was accompanied by transient accumulation of an orange-red metabolite, hydride meisenheimer complex (H-TNP), indicating a metabolic pathway involving complete reductive removal of the nitro group as nitrite., (© Springer Science+Business Media B.V. 2011)

Anaerobic bacteria have been used to produce 2,4-dihydroxylamino-nitrotoluene (2,4DHANT), a reductive metabolite of 2,4,6-trinitrotoluene (TNT). Here, an aerobic TNT biodegrader Pseudomonas sp. strain TM15 produced 2,4DHANT as evidenced by the molecular ion with m/z of 199 identified from LC-TOFMS analyses. TNT biodegradation with a high cell concentration (10(9) cells/ml) led to a significant accumulation of 2,4DHANT in the culture medium, as well as hydroxylamino-dinitrotoluenes (HADNTs), although these products were not accumulated when a low cell concentration was used; also, the accumulation of diamino-nitrotoluene and of an unidentified metabolite were observed in the culture medium with the high cell concentration (10(10) cells/ml). 2,4DHANT overproduction was a function of the aeration speed since cultures with low aeration speeds (30 rpm) had a 19-fold higher DHANT productivity than those aerated with high speeds (180 rpm); this indicates that molecular oxygen was related to the formation of 2,4DHANT. The quantification of dissolved oxygen (DO) in the media demonstrated that the productivity of 2,4DHANT was increased at low DO values. Moreover, supplying oxygen to the culture media produced a remarkable decrease of 2,4DHANT accumulation; these results clearly indicate that high 2,4DHANT production was a consequence of the oxygen deficit in the culture medium. This finding is useful for understanding the TNT biodegradation (bioremediation technology) in an anoxic environment.

The contamination of the environment by explosives is a worldwide problem resulting in part from 2,4,6-trinitrotoluene (TNT) production. In situ phytoremediation is an appropriate, alternative, cost-effective technology to detoxify extended contamination of surface soil. The ability of rice (Oriza sativa) to both tolerate and assimilate 14C-labeled TNT was investigated over a 40-day exposure period. The germination rate decreased at 500 mg/kg TNT whereas root and shoot length increased significantly at high TNT concentrations, from 150 to 500 mg/kg. Rice took up TNT residues from soil and accumulated most in roots. Less than 25% of radioactivity taken up was translocated to aerial parts. Above 200 mg/kg TNT, the concentration of TNT residues in roots reached a maximum of approximately 0.7 mg/g. No TNT was found in plant extracts, good evidence for rapid metabolism of TNT. More than 60% of (14)C activity was found as unextractable residues in roots. It was concluded that TNT metabolized and subsequently sequestered by roots could not be translocated to aerial parts.

Energetic compounds have been used in a variety of industrial and military applications worldwide leading to widespread environmental contamination. Many of these compounds are toxic and resist degradation by oxidative enzymes resulting in a need for alternative remediation methods. It has been shown that trinitrotoluene (TNT)-contaminated soil subjected to treatment in strictly anaerobic bioreactors results in tight binding of TNT transformation products to soil organic matter. The research presented here examined the fate of TNT and its metabolites in bioreactors under three different aeration regimes. In all treatment regimes, the typical metabolites of aminodinitrotoluenes and diaminonitrotoluenes were observed prior to irreversible binding into the soil fraction of the slurry. Significant transformation of TNT into organic acids or simple diols, as others report in prior work, was not observed in any of the treatments and is an unlikely fate of TNT in anaerobic soil slurries. These results indicate that aeration does not dramatically affect transformation or fate of TNT in reactor systems that receive a rich carbon source but does affect the rate at which metabolites become tightly bound to the soil. The most rapid transformations and lowest redox potentials were observed in reactors in which an aerobic headspace was maintained suggesting that aerobes play a role in establishing conditions that are most conducive to TNT reduction.

Contamination of ground and surface water with 2,4,6-trinitrotoluene (TNT) and its biological and chemical transformation products are a persisting problem at former TNT production sites. We have investigated the photochemical degradation of TNT and its aminodinitro-(ADNT) and diaminonitrotoluene (DANT) metabolites using OH-radical generating systems like Fenton and hydrogen peroxide irradiated with UV, in order to compare the degradation and mineralization rate of ADNT- and DANT-isomers with TNT itself. As a result, we find that the aminoderivatives were mineralized much faster than TNT. Consequently, as ADNTs and DANTs are the known dead-end products of biological TNT degradations, we have combined our photochemical procedure with a preceding biological treatment of TNT by a mixed culture from sludge of a sewage plant. This consecutive degradation procedure, however, shows a reduced mineralization rate of the ADNTa and DANTs in the biologically derived supernatant as compared to the pure substances, suggesting that during the biological TNT treatment by sludge competing substrates are released into the solution, and that a more defined biological procedure would be necessary in order to achieve an effective, ecologically and economically acceptable mineralization of TNT from aqueous systems.

High concentrations of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds are commonly found in soil and groundwater at former explosive plants. The bacterium, Raoultella terrigena strain HB, isolated from a contaminated site, converts TNT into the corresponding amino products. Radio-HPLC analysis with [(14)C]TNT identified aminodinitrotoluene, diaminonitrotoluene and azoxy-dimers as the main metabolites. Transformation rate and the type of metabolites that predominated in the culture medium and within the cells were significantly influenced by the culture conditions. The NAD(P)H-dependent enzymatic reduction of nitro-substituted compounds by cell-free extracts of R. terrigena was evaluated in vitro.

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 (14)C-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.

The explosive 2,4,6-trinitrotoluene (TNT), one of the most abundant and persistent contaminants at former armament factories and military sites, was cometabolically reduced by sludge (mixed culture) from a sewage plant in order to facilitate mineralization in a subsequent photochemical treatment. Under aerobic conditions, the main reduction products were aminodinitrotoluenes (ADNTs). A greater amount of the nitroaromatics (ca. 30%) was adsorbed by the sludge as was shown by a complete balance of the process using 14C-TNT. Under anaerobic conditions, TNT was further converted into ADNTs and diaminonitrotoluenes (DANTs) while only negligible adsorption to the sludge occured.

Extensive manufacturing of explosives in the last century has resulted in widespread contamination of soils and waters. Decommissioning and cleanup of these materials has also led to concerns about the explosive hazards associated with residual energetics still present on the surfaces of ordnance and explosives scrap. Typically, open burning or detonation is used to decontaminate ordinance and explosive scrap. Here the use of an anaerobic microbiological system applied as a bioslurry to decontaminate energetics from the surfaces of metal scrap is described. Decontamination of model metal scrap artificially contaminated with 2,4,6-trinitrotoluene and of decommissioned mortar rounds still containing explosives residue was examined. A portable ion mobility spectrometer was employed for the detection of residual explosives residues on the surfaces of the scrap. The mixed microbial populations of the bioslurries effectively decontaminated both the scrap and the mortar rounds. Use of the ion mobility spectrometer was an extremely sensitive field screening method for assessing decontamination and is a method by which minimally trained personnel can declare scrap clean with a high level of certainty.

Successful microbial-mediated remediation requires transformation pathways 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 intermediate 2-hydroxylamino-4,6-dinitrotoluene (2HADNT). The 2HADNT and 2,2'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.

The degradation of the nitroaromatic pollutant 2,4,6-trinitrotoluene (TNT) by the manganese-dependent peroxidase (MnP) of the white-rot fungus Phlebia radiata and the main reduction products formed were investigated. In the presence of small amounts of reduced glutathione (10 mM), a concentrated cell-free preparation of MnP from P. radiata exhibiting an activity of 36 nkat/ml (36 nmol Mn(II) oxidized per sec and per ml) transformed 10 mg/l of TNT within three days. The same preparation was capable of completely transforming the reduced derivatives of TNT. When present at 10 mg/l, the aminodinitrotoluenes were transformed in less than two days and the diaminonitrotoluenes in less than three hours. Experiments with 14C-U-ring labeled TNT and 2-amino-4,6-dinitrotoluene showed that these compounds were mineralized by 22% and 76%, respectively, within 5 days. Higher concentrations of reduced glutathione (50 mM) led to a severe inhibition of the degradation process. It is concluded that Phlebia radiata is a good candidate for the biodegradation of TNT as well as its reduction metabolites.

Microcosm tests simulating bioslurry reactors with 40% soil content, containing high concentrations of TNT and/or RDX, and spiked with either [14C]-TNT or [14C]-RDX were conducted to investigate the fate of explosives and their metabolites in bioslurry treatment processes. RDX is recalcitrant to indigenous microorganisms in soil and activated sludge under aerobic conditions. However, soil indigenous microorganisms alone were able to mineralize 15% of RDX to CO2 under anaerobic condition, and supplementation of municipal anaerobic sludge as an exogenous source of microorganisms significantly enhanced the RDX mineralization to 60%. RDX mineralizing activity of microorganisms in soil and sludge was significantly inhibited by the presence of TNT. TNT mineralization was poor (< 2%) and was not markedly improved by the supplement of aerobic or anaerobic sludge. Partitioning studies of [14C]-TNT in the microcosms revealed that the removal of TNT during the bioslurry process was due mainly to the transformation of TNT and irreversible binding of TNT metabolites onto soil matrix. In the case of RDX under anaerobic conditions, a significant portion (35%) of original radioactivity was also incorporated into the biomass and bound to the soil matrix.