Key genomes, transcriptomes, proteins, and metabolic factors involved in the detoxification/tolerance of TNT and its intermediates by bacteria in anaerobic/aerobic environments.

Novel microbial strains capable of efficient degradation of TNT and typical intermediates (2-ADNT and 4-ADNT) in aerobic/anaerobic environment were screened and isolated from ammunition-contaminated sites. The key genomes, transcriptomes, proteins, and metabolic factors for microbial detoxification/tolerance to pollutants in anaerobic and aerobic environments were analyzed for the first time. The bacterial genome, which is rich in metabolism and environmental information-processing functional genes, provides transcriptional and translational-related proteins for detoxifying/tolerating pollutants. At the transcriptional level, bacteria significantly expressed genes related to inositol phosphate metabolism for regulating membrane transport, maintaining the cytoskeleton, and signal transduction. At the protein level, genes involved in antioxidation, fat metabolism, sugar synthesis/degradation, and pyruvate metabolism were significantly expressed. At the metabolic level, riboflavin metabolism, which regulates membrane integrity, protects against oxidative stress, and maintains the sugar-protein-fat balance, showed significant responses. Bacteria simultaneously regulate amino acid metabolism, carbohydrate metabolism, and N/P/S cycles to maintain homeostatic cellular energy supplies. The key pathway for pollutant degradation in bacteria is nitrotoluene degradation. The molecular mechanism of bacterial tolerance to pollutants involves the regulation of oxidative phosphorylation and basic cycle pathways to maintain gene transcription, protein translation, and metabolic cycles.

Overexpressing CYP81D11 enhances 2,4,6-trinitrotoluene tolerance and removal efficiency in Arabidopsis.

Phytoremediation is a promising technology for removing the high-toxic explosive 2,4,6-trinitrotoluene (TNT) pollutant from the environment. Mining dominant genes is the key research direction of this technology. Most previous studies have focused on the detoxification of TNT rather than plants' TNT tolerance. Here, we conducted a transcriptomic analysis of wild type Arabidopsis plants under TNT stress and found that the Arabidopsis cytochrome P450 gene CYP81D11 was significantly induced in TNT-treated plants. Under TNT stress, the root length was approximately 1.4 times longer in CYP81D11-overexpressing transgenic plants than in wild type plants. The half-removal time for TNT was much shorter in CYP81D11-overexpressing transgenic plants (1.1 days) than in wild type plants (t1/2 = 2.2 day). In addition, metabolic analysis showed no difference in metabolites in transgenic plants compared to wild type plants. These results suggest that the high TNT uptake rates of CYP81D11-overexpressing transgenic plants were most likely due to increased tolerance and biomass rather than TNT degradation. However, CYP81D11-overexpressing plants were not more tolerant to osmotic stresses, such as salt or drought. Taken together, our results indicate that CYP81D11 is a promising target for producing bioengineered plants with high TNT removing capability.

Metabolic activation of 2,4,6-trinitrotoluene; a case for ROS-induced cell damage.

The explosive compound 2,4,6-trinitrotoluene (TNT) is well known as a major component of munitions. In addition to its potential carcinogenicity and mutagenicity in humans, recent reports have highlighted TNT toxicities in diverse organisms due to its occurrence in the environment. These toxic effects have been linked to the intracellular metabolism of TNT, which is generally characterised by redox cycling and the generation of noxious reactive molecules. The reactive intermediates formed, such as nitroso and hydroxylamine compounds, also interact with oxygen molecules and cellular components to cause macromolecular damage and oxidative stress. The current review aims to highlight the crucial role of TNT metabolism in mediating TNT toxicity, via increased generation of reactive oxygen species. Cellular proliferation of reactive species results in depletion of cellular antioxidant enzymes, DNA and protein adduct formation, and oxidative stress. While TNT toxicity is well known, its ability to induce oxidative stress, resulting from its reductive activation, suggests that some of its toxic effects may be caused by its reactive metabolites. Hence, further research on TNT metabolism is imperative to elucidate TNT-induced toxicities.

Microecological characteristics of water bodies/sediments and microbial remediation strategies after 50 years of pollution exposure in ammunition destruction sites in China.

The effects of long-term ammunition pollution on microecological characteristics were analyzed to formulate microbial remediation strategies. Specifically, the response of enzyme systems, N/O stable isotopes, ion networks, and microbial community structure/function levels were analyzed in long-term (50 years) ammunition-contaminated water/sediments from a contamination site, and a compound bacterial agent capable of efficiently degrading trinitrotoluene (TNT) while tolerating many heavy metals was selected to remediate the ammunition-contaminated soil. The basic physical and chemical properties of the water/sediment (pH (up: 0.57-0.64), nitrate (up: 1.31-4.28 times), nitrite (up: 1.51-5.03 times), and ammonium (up: 7.06-70.93 times)) were changed significantly, and the significant differences in stable isotope ratios of N and O (nitrate nitrogen) confirmed the degradability of TNT by indigenous microorganisms exposed to long-term pollution. Heavy metals, such as Pb, Zn, Cu, Cd, Cs, and Sb, have synergistic toxic effects in ammunition-contaminated sites, and significantly decreased the microbial diversity and richness in the core pollution area. However, long-term exposure in the edge pollution area induced microorganisms to use TNT as a carbon and nitrogen sources for life activities and growth and development. The Bacteroidales microbial group was significantly inhibited by ammunition contamination, whereas microorganisms such as Proteobacteria, Acidobacteriota, and Comamonadaceae gradually adapted to this environmental stress by regulating their development and stress responses. Ammunition pollution significantly affected DNA replication and gene regulation in the microecological genetic networks and increased the risk to human health. Mg and K were significantly involved in the internal mechanism of microbial transport, enrichment, and metabolism of TNT. Nine strains of TNT-utilizing microbes were screened for efficient TNT degradation and tolerance to typical heavy metals (copper, zinc and lead) found in contaminated sites, and a compound bacterial agent prepared for effective repair of ammunition-contaminated soil significantly improved the soil ecological environment.

New insights into xenobiotic tolerance of Antarctic bacteria: transcriptomic analysis of Pseudomonas sp. TNT3 during 2,4,6-trinitrotoluene biotransformation.

The xenobiotic 2,4,6-trinitrotoluene (TNT) is a highly persistent environmental contaminant, whose biotransformation by microorganisms has attracted renewed attention. In previous research, we reported the discovery of Pseudomonas sp. TNT3, the first described Antarctic bacterium with the ability to biotransform TNT. Furthermore, through genomic analysis, we identified distinctive features in this isolate associated with the biotransformation of TNT and other xenobiotics. However, the metabolic pathways and genes active during TNT exposure in this bacterium remained unexplored. In the present transcriptomic study, we used RNA-sequencing to investigate gene expression changes in Pseudomonas sp. TNT3 exposed to 100 mg/L of TNT. The results showed differential expression of 194 genes (54 upregulated and 140 downregulated), mostly encoding hypothetical proteins. The most highly upregulated gene (> 1000-fold) encoded an azoreductase enzyme not previously described. Other significantly upregulated genes were associated with (nitro)aromatics detoxification, oxidative, thiol-specific, and nitrosative stress responses, and (nitro)aromatic xenobiotic tolerance via efflux pumps. Most of the downregulated genes were involved in the electron transport chain, pyrroloquinoline quinone (PQQ)-related alcohol oxidation, and motility. These findings highlight a complex cellular response to TNT exposure, with the azoreductase enzyme likely playing a crucial role in TNT biotransformation. Our study provides new insights into the molecular mechanisms of TNT biotransformation and aids in developing effective TNT bioremediation strategies. To the best of our knowledge, this report is the first transcriptomic response analysis of an Antarctic bacterium during TNT biotransformation.

Biotransformation of 2,4,6-trinitrotoluene by Diaphorobacter sp. strain DS2.

Diaphorobacter strain DS2 degrades 3-nitrotoluene and 2-nitrotoluene via ring oxidation with 3-nitrotoluene dioxygenase (3NTDO). In the current study, we hypothesized that 3NTDO might also be involved in the degradation of 2,4,6-trinitrotoluene (TNT), a major nitroaromatic explosive contaminant in soil and groundwater. Strain DS2 transforms TNT as a sole carbon and nitrogen source when grown on it. Ammonium chloride and succinate in the medium accelerated the TNT degradation rate. A resting cell experiment suggested that TNT does not compete with 3NT degradation (no negative impact of TNT on the reaction velocity for 3NT). Enzyme assay with 3NTDO did not exhibit TNT transformation activity. The above results confirmed that 3NTDO of DS2 is not responsible for TNT degradation. In the resting cell experiment, within 10 h, 4ADNT completely degraded. The degradation of 2ADNT was 97% at the same time. We hypothesized that 3NTDO involve in this reaction. Based on the DS2 genome, we proposed that the N-ethylmaleimide reductases (nemA) were involved in the initial reduction of the nitro group and aromatic ring of TNT. Our findings suggest that strain DS2 could be helpful for the removal of TNT from contaminated sites with or without any additional carbon and nitrogen source and with minimal accumulation of undesirable intermediates.

Oxygen-insensitive nitroreductase bacteria-mediated degradation of TNT and proteomic analysis.

2,4,6-trinitrotoluene (TNT) is a nitroaromatic compound that causes soil and groundwater pollution during manufacture, transportation, and use, posing significant environmental and safety hazards. In this study, a TNT-degrading strain, Bacillus cereus strain T4, was screened and isolated from TNT-contaminated soil to explore its degradation characteristics and proteomic response to TNT. The results showed that after inoculation with the bacteria for 4 h, the TNT degradation rate reached 100% and was transformed into 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT), and 2,6-diamino-4-nitrotoluene (2,6-DANT), accompanied by the accumulation of nitrite and ammonium ions. Through proteomic sequencing, we identified 999 differentially expressed proteins (482 upregulated, 517 downregulated), mainly enriched in the pentose phosphate, glycolysis/gluconeogenesis, and amino acid metabolism pathways. In addition, the significant upregulation of nitroreductase and N-ethylmaleimide reductase was closely related to TNT denitration and confirmed that the strain T4 converted TNT into intermediate metabolites such as 2-ADNT and 4-ADNT. Therefore, Bacillus cereus strain T4 has the potential to degrade TNT and has a high tolerance to intermediate products, which may effectively degrade nitroaromatic pollutants such as TNT in situ remediation in combination with other bacterial communities.

Solid medium for the direct isolation of bacterial colonies growing with polycyclic aromatic hydrocarbons or 2,4,6-trinitrotoluene (TNT).

Isolation of hydrocarbon-degrading bacteria is a key step for the study of microbiological diversity, metabolic pathways, and bioremediation. However current strategies lack simplicity and versatility. We developed an easy method for the screening and isolation of bacterial colonies capable of degrading hydrocarbons, such as diesel or polycyclic aromatic hydrocarbons (PAHs), as well as the pollutant explosive, 2,4,6-trinitrotoluene (TNT). The method uses a two-layer solid medium, with a layer of M9 medium, and a second layer containing the carbon source deposited through the evaporation of ethanol. Using this medium we grew hydrocarbon-degrading strains, as well as TNT-degrading isolates. We were able to isolate PAHs-degrading bacterial colonies directly from diesel-polluted soils. As a proof of concept, we used this method to isolate a phenanthrene-degrading bacteria, identified as Acinetobacter sp. and determined its ability to biodegrade this hydrocarbon.

Enhanced removal of warfare agent tri-nitro-toluene by a Methylophaga-dominated microbiome.

Historical exposure of the marine environment to 2,4,6-trinitrotoluene (TNT) happened due to the dumping of left-over munitions. Despite significant research on TNT decontamination, the potential of marine microbiome for TNT degradation remains only little explored. In this study, TNT degradation experiments were conducted with sediment located near the World War I munition dumpsite - Paardenmarkt in the Belgian part of North Sea. A slow removal was observed using TNT as sole source of C and N, which could be enhanced by adding methanol. Degradation was reflected in nitro-reduced metabolites and microbial growth. 16S Illumina sequencing analysis revealed several enriched genera that used TNT as a sole source of C and N - Colwellia, Thalossospira, and Methylophaga. Addition of methanol resulted in increased abundance of Methylophaga, which corresponded to the rapid removal of TNT. Methanol enhanced the degradation by providing additional energy and establishing syntrophic association between methanol-utilizing and TNT-utilizing bacteria.

Critical Role of Monooxygenase in Biodegradation of 2,4,6-Trinitrotoluene by Buttiauxella sp. S19-1.

2,4,6-Trinitrotoluene (TNT) is an aromatic pollutant that is difficult to be degraded in the natural environment. The screening of efficient degrading bacteria for bioremediation of TNT has received much attention from scholars. In this paper, transcriptome analysis of the efficient degrading bacterium Buttiauxella sp. S19-1 revealed that the monooxygenase gene (BuMO) was significantly up-regulated during TNT degradation. S-ΔMO (absence of BuMO gene in S19-1 mutant) degraded TNT 1.66-fold less efficiently than strain S19-1 (from 71.2% to 42.9%), and E-MO mutant (Escherichia coli BuMO-expressing strain) increased the efficiency of TNT degradation 1.33-fold (from 52.1% to 69.5%) for 9 h at 180 rpm at 27 °C in LB medium with 1.4 µg·mL-1 TNT. We predicted the structure of BuMO and purified recombinant BuMO (rBuMO). Its specific activity was 1.81 µmol·min-1·mg-1 protein at pH 7.5 and 35 °C. The results of gas chromatography mass spectrometry (GC-MS) analysis indicated that 4-amino-2,6-dinitrotoluene (ADNT) is a metabolite of TNT biodegradation. We speculate that MO is involved in catalysis in the bacterial degradation pathway of TNT in TNT-polluted environment.

Comparative Genomic Analysis of Antarctic Pseudomonas Isolates with 2,4,6-Trinitrotoluene Transformation Capabilities Reveals Their Unique Features for Xenobiotics Degradation.

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Since physicochemical methods for remediation are poorly effective, the use of microorganisms has gained interest as an alternative to restore TNT-contaminated sites. We previously demonstrated the high TNT-transforming capability of three novel Pseudomonas spp. isolated from Deception Island, Antarctica, which exceeded that of the well-characterized TNT-degrading bacterium Pseudomonas putida KT2440. In this study, a comparative genomic analysis was performed to search for the metabolic functions encoded in the genomes of these isolates that might explain their TNT-transforming phenotype, and also to look for differences with 21 other selected pseudomonads, including xenobiotics-degrading species. Comparative analysis of xenobiotic degradation pathways revealed that our isolates have the highest abundance of key enzymes related to the degradation of fluorobenzoate, TNT, and bisphenol A. Further comparisons considering only TNT-transforming pseudomonads revealed the presence of unique genes in these isolates that would likely participate directly in TNT-transformation, and others involved in the ß-ketoadipate pathway for aromatic compound degradation. Lastly, the phylogenomic analysis suggested that these Antarctic isolates likely represent novel species of the genus Pseudomonas, which emphasizes their relevance as potential agents for the bioremediation of TNT and other xenobiotics.

Bacillus pumilus proteome changes in response to 2,4,6-trinitrotoluene-induced stress.

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.

The plant streptolysin S (SLS)-associated gene B confers nitroaromatic tolerance and detoxification.

Nitroaromatic compounds, as the important chemical feedstock, have caused widespread environmental contaminations, and exhibited high toxicity and mutagenic activity to nearly all living organisms. The clean-up of nitroaromatic-contaminated soil and water has long been a major international concern. Here, we uncovered the role of a novel nitroreductase family gene, streptolysin S (SLS)-associated gene B (SagB), in enhancing nitroaromatic tolerance and detoxification of plants, and its potential application in phytoremediation of nitroaromatic contaminations. The expression of both the Arabidopsis and rice SagB genes is significantly induced by multiple hazardous nitroaromatic substances, including explosive pollutant 2,4,6-trinitrotoluene (TNT), natural compound 1-nitropyrene (1-NP) and herbicide pendimethalin (Pen). In vitro and in vivo evidences revealed that plant SagBs possess activities in degradation of these nitroaromatic substances. Arabidopsis and rice transgenic assays suggested that plant SagB genes increase tolerance and detoxification of nitroaromatic through facilitating its transformation to the amino derivative. More importantly, overexpression of plant SagBs increase their ability in TNT uptake, and remove more TNT from the growth culture. Our findings shed novel insights into a plant endogenous nitroreductase-mediated nitroaromatic tolerance and detoxification, and provide a new gene target for phytoremediation of nitroaromatic-contaminated environments.

Transgenerational reproductive toxicity of 2,4,6-trinitrotoluene (TNT) and its metabolite 4-ADNT in Caenorhabditis elegans.

2,4,6-trinitrotoluene (TNT) as an energetic compound widely used in military applications has aroused great concerns in recent years due to its large-scale contamination in soil and water; however, its toxicity is still largely unknown. In this study, we investigated the reproductive toxicity and the transgenerational effects of TNT on Caenorhabditis elegans (C. elegans). Our data showed that exposure to TNT at concentrations ranging from 10 to 100 ng/mL resulted in decreasing the lifespan, brood size, number of oocytes and eggs in uterus, while increasing the number of germ cell apoptosis in C. elegans. The apoptotic effects of TNT were blocked in mutants of cep-1 (w40), egl-1 (n487), and hus-1 (op241), indicating conserved genotoxic response genes was involved in mediating TNT-induced germ cell apoptosis. Parental exposure to TNT significantly increased the germ cell apoptosis from P0 to F2 generation, but the toxicity faded away in F3 and F4 generations. Furthermore, TNT was rapidly metabolized in P0, and the accumulation of 4-aminodinitrotoluene (4-ADNT), the main metabolite of TNT in C. elegans, showed a significant decrease from P0 to F1 and a slow decrease in the subsequent generations. Our results demonstrated that ingested TNT can cause severe transgenerational reproductive toxicity and be rapidly converted to 4-ADNT in the nematodes. These data provided basis for future studies on the effects of energetic compounds across generations.

Toxicity analysis of TNT to alfalfa's mineral nutrition and secondary metabolism.

KEY MESSAGE: Alfalfa has the ability to degrade TNT. TNT exposure caused root disruption of mineral nutrient metabolism. The exposure of TNT imbalanced basal cell energy metabolism. The mechanism of 2,4,6-trinitrotoluene (TNT) toxicity effects was analyzed in alfalfa (Medicago sativa L.) seedlings by examining the mineral nutrition and secondary metabolism of the plant roots. Exposure to 25-100 mg·L-1 TNT in a hydroponic solution for 72 h resulted in a TNT absorption rate of 26.8-63.0%. The contents of S, K, and B in root mineral nutrition metabolism increased significantly by 1.70-5.46 times, 1.38-4.01 times, and 1.40-4.03 times, respectively, after TNT exposure. Non-targeted metabolomics analysis of the roots identified 189 significantly upregulated metabolites and 420 significantly downregulated metabolites. The altered metabolites were primarily lipids and lipid-like molecules, and the most significant enrichment pathways were alanine, aspartate, and glutamate metabolism and glycerophospholipid metabolism. TNT itself was transformed in the root system into several intermediate products, including 4-hydroxylamino-2,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2-hydroxylamino-4,6-dinitrotoluene, 2,4',6,6'-tetranitro-2',4-azoxytoluene, 4,4',6,6'-tetranitro-2,2'-azoxytoluene, and 2,4-dinitrotoluene. Overall, TNT exposure disturbed the mineral metabolism balance, and significantly interfered with basic plant metabolism.

Structural Factors That Determine the Activity of the Xenobiotic Reductase B Enzyme from Pseudomonas putida on Nitroaromatic Compounds.

Xenobiotic reductase B (XenB) catalyzes the reduction of the aromatic ring or nitro groups of nitroaromatic compounds with methyl, amino or hydroxyl radicals. This reaction is of biotechnological interest for bioremediation, the reuse of industrial waste or the activation of prodrugs. However, the structural factors that explain the binding of XenB to different substrates are unknown. Molecular dynamics simulations and quantum mechanical calculations were performed to identify the residues involved in the formation and stabilization of the enzyme/substrate complex and to explain the use of different substrates by this enzyme. Our results show that Tyr65 and Tyr335 residues stabilize the ligands through hydrophobic interactions mediated by the aromatic rings of these aminoacids. The higher XenB activity determined with the substrates 1,3,5-trinitrobenzene and 2,4,6-trinitrotoluene is consistent with the lower energy of the highest occupied molecular orbital (LUMO) orbitals and a lower energy of the homo orbital (LUMO), which favors electrophile and nucleophilic activity, respectively. The electrostatic potential maps of these compounds suggest that the bonding requires a large hydrophobic region in the aromatic ring, which is promoted by substituents in ortho and para positions. These results are consistent with experimental data and could be used to propose point mutations that allow this enzyme to process new molecules of biotechnological interest.

A novel method development and validation for determination of 2,4,6-Trinitrotoluene and its metabolites on LC-MS/MS.

LC-MS/MS has recently emerged as the best practice for simultaneous analysis of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites. We have developed and validated an LC-MS/MS method for simultaneous quantification of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites 4-ADNT, 2-ADNT, 2,4-DNT, and 2,6-DNT in urine samples. These four metabolites were acid hydrolyzed using 1 mL of urine followed by extraction using n-Hexane and ethyl acetate as an extracting solvent. Separation was achieved by centrifugation, and the supernatant was dried under nitrogen, reconstituted with water and acetonitrile, and then filtered. Chromatographic separation was achieved on Agilent Poroshel 120 EC-C18 column (2.1 mm × 75 mm × 2.7 µm) utilizing two mobile phases 0.1% formic acid in water and 0.1% formic acid in acetonitrile in gradient flow. The validated AMR of TNT and its metabolites was 7.8-1000 ng/mL. The method showed an excellent correlation (>0.99) for TNT and its metabolites. Accuracy and within/between day precision of TNT and its metabolites were within ±15%. The integrity of diluted samples was maintained for each dilution factor. The method was found stable after storage and freeze-thaw cycle. The presented method can be used for TNT screening in occupationally exposed ordnance factory workers.

Enhanced phytoremediation of TNT and cobalt co-contaminated soil by AfSSB transformed plant.

2,4,6-trinitrotoluene (TNT) and cobalt (Co) contaminants have posed a severe environmental problem in many countries. Phytoremediation is an environmentally friendly technology for the remediation of these contaminants. However, the toxicity of TNT and cobalt limit the efficacy of phytoremediation application. The present research showed that expressing the Acidithiobacillus ferrooxidans single-strand DNA-binding protein gene (AfSSB) can improve the tolerance of Arabidopsis and tall fescue to TNT and cobalt. Compared to control plants, the AfSSB transformed Arabidopsis and tall fescue exhibited enhanced phytoremediation of TNT and cobalt separately contaminated soil and co-contaminated soil. The comet analysis revealed that the AfSSB transformed Arabidopsis suffer reduced DNA damage than control plants under TNT or cobalt exposure. In addition, the proteomic analysis revealed that AfSSB improves TNT and cobalt tolerance by strengthening the reactive superoxide (ROS) scavenging system and the detoxification system. Results presented here serve as strong theoretical support for the phytoremediation potential of organic and metal pollutants mediated by single-strand DNA-binding protein genes. SUMMARIZES: This is the first report that AfSSB enhances phytoremediation of 2,4,6-trinitrotoluene and cobalt separately contaminated and co-contaminated soil.

Transformation of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109 and exploration of the associated enzymes.

The nitrated compounds 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) are toxic xenobiotics widely used in various industries. They often coexist as environmental contaminants. The aims of this study were to evaluate the transformation of 100 mg L-1 of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109c and identify enzymes that may participate in the transformation. These strains were selected from 34 TNT transforming bacteria. Cupriavidus metallidurans DNT was used as a reference strain for comparison purposes. Strains DNT, M30b and M109c transformed 2,4-DNT (100%), TNT (100, 94.7 and 63.6%, respectively), and PETN (72.7, 69.3 and 90.7%, respectively). However, the presence of TNT negatively affects 2,4-DNT and PETN transformation (inhibition > 40%) in strains DNT and M109c and fully inhibited (100% inhibition) 2,4-DNT transformation in R. planticola M30b.Genomes of R. planticola M30b and R. radiobacter M109c were sequenced to identify genes related with 2,4-DNT, TNT or PETN transformation. None of the tested strains presented DNT oxygenase, which has been previously reported in the transformation of 2,4-DNT. Thus, unidentified novel enzymes in these strains are involved in 2,4-DNT transformation. Genes encoding enzymes homologous to the previously reported TNT and PETN-transforming enzymes were identified in both genomes. R. planticola M30b have homologous genes of PETN reductase and xenobiotic reductase B, while R. radiobacter M109c have homologous genes to GTN reductase and PnrA nitroreductase. The ability of these strains to transform explosive mixtures has a potentially biotechnological application in the bioremediation of contaminated environments.

13C and 15N NMR identification of product compound classes from aqueous and solid phase photodegradation of 2,4,6-trinitrotoluene.

Photolysis is one of the main transformation pathways for 2,4,6-trinitrotoluene (TNT) released into the environment. Upon exposure to sunlight, TNT is known to undergo both oxidation and reduction reactions with release of nitrite, nitrate, and ammonium ions, followed by condensation reactions of the oxidation and reduction products. In this study, compound classes of transformation products from the aqueous and solid phase photodegradation of 2,4,6-trinitrotoluene (TNT) have been identified by liquid and solid state 13C and 15N NMR. Aqueous phase experiments were performed on saturated solutions of T15NT in deionized water, natural pond water (pH = 8.3, DOC = 3.0 mg/L), pH 8.0 buffer solution, and in the presence of Suwannee River Natural Organic Matter (SRNOM; pH = 3.7), using a Pyrex-filtered medium pressure mercury lamp. Natural sunlight irradiations were performed on TNT in the solid phase and dissolved in the pond water. In deionized water, carboxylic acid, aldehyde, aromatic amine, primary amide, azoxy, nitrosophenol, and azo compounds were formed. 15N NMR spectra exhibited major peaks centered at 128 to 138 ppm, which are in the range of phenylhydroxylamine and secondary amide nitrogens. The secondary amides are proposed to represent benzanilides, which would arise from photochemical rearrangement of nitrones formed from the condensation of benzaldehyde and phenylhydroxylamine derivatives of TNT. The same compound classes were formed from sunlight irradiation of TNT in the solid phase. Whereas carboxylic acids, aldehydes, aromatic amines, phenylhydroxylamines, and amides were also formed from irradiation of TNT in pond water and in pH 8 buffer solution, azoxy and azo compound formation was inhibited. Solid state 15N NMR spectra of photolysates from the lamp irradiation of unlabeled 2,6-dinitrotoluene in deionized water also demonstrated the formation of aromatic amine, phenylhydroxylamine/ 2° amide, azoxy, and azo nitrogens.