Biodegradation of 2,4,6-trinitrotoluene (TNT): An enzymatic perspective

Enzymatic degradation of TNT by aerobic bacteria is mediated by oxygen insensitive (Type 1) or by oxygen sensitive nitroreductases (Type II nitroreductases). Transformation by Type I nitroreductases proceeds through two successive electron reductions either by hydride addition to the aromatic ring or by direct nitro group reduction following a ping pong kinetic mechanism. TNT is reduced to the level of hydroxylaminodinitrotoluenes and aminodinitrotoluenes by pure enzyme preparations without achieving mineralization. Interestingly, database gene and amino acid sequence comparisons of nitroreductases reveal a close relationship among all enzymes involved in TNT transformation. They are all flavoproteins which use NADPH/NADH as electron donor and reduce a wide range of electrophilic xenobiotics. TNT degradation by fungi is initiated by mycelia bound nitroreductases which reduce TNT to hydroxylaminodinitrotoluenes and aminodinitrotoluenes. Further degradation of these products and mineralization is achieved through the activity of oxidative enzymes especially lignin degrading enzymes (lignin and manganese peroxidases).     

Two unidentified aerobic bacterial strains that transform 2,4,6-trinitrotoluene

Several strains of aerobic bacteria tolerant to some chemical toxins were isolated from the water of man-made Yerevan Lake and from the soil and air of different regions of the city of Yerevan. Some of these bacteria were capable of degrading 2,4,6-trinitrotoluene (TNT) during shake-flask fermentation in liquid medium. Two bacterial strains with good ability to degrade TNT were isolated. These strains showed visible morphological and physiological differences during growth on numerous elective media. The comparative ability of these strains to transform TNT was investigated.     

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     

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

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

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.     

Transformation of 2,4,6-trinitrotoluene (TNT) by immobilized Phanerochaete chrysosporium under fed-batch and continuous TNT feeding conditions

The cometabolic transformation of 2,4,6-trinitrotoluene (TNT) by an immobilized Phanerochaete chrysosporium culture was investigated under different TNT and/or glycerol feeding conditions in a 5-L reactor. In the fed-batch feeding mode, as a result of four spiking events at an average feeding rate of 20 mg TNT L(-1) d(-1) and 250 mg glycerol L(-1) d(-1), the initial TNT transformation rate and the glycerol uptake rate of the 7-day-old immobilized cell culture were 2.41 mg L(-1) h(-1) and 16.6 mg L(-1) h(-1), respectively. Thereafter, the TNT fed into the reactor depicted a negative effect on the cell physiology of P. chrysosporium, i.e., both rates decreased constantly. At 32 mg TNT L(-1) d(-1) feeding rate, also in the presence of glycerol (200 mg L(-1) d(-1)), this effect on the fungal cell metabolism was even more significant. When TNT was fed alone at 3.7 mg L(-1) d(-1), it showed an initial 0.75 mg L(-1) h(-1) rate of TNT transformation, i.e., one-third the initial level observed in the presence of glycerol. In contrast, in the continuous feeding mode (dilution rate, D = 0.11 d(-1)), at 5.5 mg TNT L(-1) d(-1) and 220 mg glycerol L(-1) d(-1), the immobilized cell culture exhibited a constant TNT transformation rate for cultivation periods of 50 and 61 days, under uncontrolled and controlled pH conditions, respectively. Thereafter, during the latter experiment, 100% TNT biotransformation was achieved at 1,100 mg L(-1) d(-1) glycerol feeding rate. Immobilized cells (115-day-old), sampled from a continuous TNT feeding experiment, mineralized [(14)C]-TNT to a level of 15.3% following a 41-day incubation period in a microcosm.     

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     

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.     

Models of 2,4,6-trinitrotoluene (TNT) initial conversion by yeasts

We devised a screening method to obtain basidiomycetous fungi capable of degrading dioxins. About 200 fungal strains were selected from more than 1500 strains by their ability to decolorize Remazol brilliant blue R dye as an indicator. To attempt to eliminate the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long term or short term to 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), and compared the decreases in the levels of this chemical. In only 11 strains was there a significant difference between the two treatments. We chose Panellus stypticus strain 99-334 as a new, effective dioxin degrader, because it gave a close to 100% decrease in 2,7-DCDD levels (from an initial concentration of 10 microM) after 40 days of exposure. The detection of a metabolic intermediate (1-chloro-3,4-dihydroxybenzene) by gas chromatography-mass spectrometry analysis supported the ability of this strain to degrade 2,7-DCDD.     

Biodegradation of 2,4,6-trinitrotoluene (TNT) under sulfate and nitrate reducing conditions

Anaerobic degradation of 2,4,6-trinitrotoluene (TNT) was studied under sulfate- and nitrate-reducing conditions using enrichment cultures developed from a TNT-contaminated soil from the Louisiana Army Ammunition Plant (LAAP) in Minden, Louisiana, USA. The soil samples were enriched using mineral salt media with either nitrate or sulfate as electron acceptors in the presence of TNT under strict anaerobic conditions. The enriched samples were experimented with TNT as either the sole source of carbon or nitrogen and also under co-metabolic conditions with molasses as co-substrate. The results revealed that TNT was removed under both electron acceptor conditions. However, the TNT degradation efficiency was significantly higher under sulfate-reducing conditions than the nitrate-reducing conditions. Under sulfate-reducing conditions, TNT removal was faster when molasses was used as co-substrate. The metabolic analysis showed that TNT was mineralized and the major end product was acetic acid, CO₂, and ammonia. A soil slurry reactor with TNT-contaminated soil showed more than 90% of TNT removal within 60 days of incubation.     

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the white-rot basidiomycete Phlebia radiata

Phlebia radiatatransformed 2,4,6-trinitrotoluene (TNT), as well as its first reduction products, the aminodinitrotoluenes, into 4-hydroxylamino-2,6-dinitrotoluene (4-OHA-2,6-DNT) and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). No extracellular peroxidases were involved in this step. The ligninolytic extracellular fluid, assumed to contain peroxidases, did not reduce TNT. However, ligninolytic peroxidases are implicated in the transformation of the first reduction products of TNT.     

以大肠杆菌为底盘细胞构建XylR-Pu线路检测2,4,6-三硝基甲苯

目的:恶臭假单胞菌中的TOL质粒上的XylR-Pu是经典的甲苯代谢通路,在甲苯类化合物存在时,调控蛋白XylR可以特异性的激活Pu启动子,进而启动相应甲苯代谢通路的表达。基于合成生物学的思想,优化设计此通路并导入遗传背景清楚、操作简单的大肠杆菌中构建全细胞生物传感器,用于检测环境污染物2,4,6-三硝基甲苯(TNT)。方法:以pETDuet-1为载体骨架,构建了XylR-Pu基因线路,并以绿色荧光蛋白GFP为报告分子,GFP的荧光值可以指征结合诱导剂后的XylR蛋白对Pu启动子的诱导强度,并在基因线路中加入四串联终止序列来降低背景值。最后对XylR蛋白的信号识别区进行连续易错PCR,构建随机突变体文库,从中筛选具有更高感应强度、更好灵敏度及特异性的调控蛋白。结果:四串联终止序列可有效降低XylR-Pu通路的背景值,随机突变体文库中筛选出的生物元件eX0-4,对TNT表现出良好的感应强度、灵敏度及特异性。结论:XylR蛋白在大肠杆菌中对硝基甲苯响应不明显,但筛选得到的突变蛋白eX0-4,为后续生物传感器的更深层次地开发提供了优良的元件储备。另外,利用易错PCR构建随机突变体文库从中筛选发挥预定功能的突变蛋白质也可成为挖掘生物元件的一种通用方法。     

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.     

Phytodetoxification of the environmental pollutant and explosive 2,4,6-trinitrotoluene

Our recent study highlights the role of 2 glutathione transferases (GSTs) in the detoxification of the environmental pollutant, 2,4,6-trinitrotoluene (TNT) in Arabidopsis thaliana. TNT is toxic and highly resistant to biodegradation in the environment, raising both health and environmental concerns. Two GSTs, GST-U24 and GST-U25, are upregulated in response to TNT treatment, and expressed predominantly in the root tissues; the site of TNT location following uptake. Plants overexpressing GST-U24 and GST-U25 exhibited significantly enhanced ability to withstand and detoxify TNT, and remove TNT from contaminated soil. Analysis of the catalytic activities of these 2 enzymes revealed that they form 3 TNT-glutathionyl products. Of particular interest is 2-glutathionyl-4,6-dinitrotoluene as this represents a potentially favorable step toward subsequent degradation and mineralization of TNT. We demonstrate how GSTs fit into what is already known about pathways for TNT detoxification, and discuss the short and longer-term fate of TNT conjugates in planta.     

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

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

Biological removal of explosive 2,4,6-trinitrotoluene byStenotrophomonas sp. OK-5 in bench-scale bioreactors

The biological removal of 2,4,6-trinitrotoluene (TNT) was studied in a bench-scale bioreactor using a bacterial culture of strain OK-5 originally isolated from soil samples contaminated with TNT. The TNT was completely removed within 4 days of incubation in a 2.5 L benchscale bioreactor containing a newly developed medium. The TNT was catabolized in the presence of different supplemented carbons. Only minimal growth was observed in the killed controls and cultures that only received TNT during the incubation period. This catabolism was affected by the concentration ratio of the substrate to the biomass. The addition of various nitrogen sources produced a delayed effect for the TNT degradation. Tween 80 enhanced the degradation of TNT under these conditions. Two metabolic intermediates were detected and identified as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene based on HPLC and GC-MS analyses, respectively. Strain OK-5 was characterized using the BIOLOG system and fatty acid profile produced by a microbial identification system equipped with a Hewlett packard HP 5890 II gas chromatograph. As such, the bacterium was identified as aStenotrophomonas species and designated asStenotrophomonas sp. OK-5.     

Metabolism of 2,4,6-trinitrotoluene (TNT) by Desulfovibrio sp. (B strain)

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain) isolated from an anaerobic reactor treating furfural-containing waste-water was studied for its ability to metabolize trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm TNT within 7 to 10 days of incubation at 37°C, when grown with 30 mm pyruvate as the primary carbon source and 20 mm sulfate as electron acceptor. Under these conditions, the main intermediate produced was 2,4-diamino-6-nitrotoluene. Under culture conditions where TNT served as the sole source of nitrogen for growth with pyruvate as electron donor and sulfate as electron acceptor, TNT was first converted to 2,4-diamino-6-nitrotoluene within 10 days of incubation. This intermediate was further converted to toluene by a reductive deamination process via triaminotoluene. Apart from pyruvate, various other carbon sources such as ethanol, lactate, formate and H₂ + CO₂ were also studied as potential electron donors for TNT metabolism. The rate of TNT biotransformation by Desulfovibrio sp. (B strain) was compared with other sulfate-reducing bacteria and the results were evaluated. This new strain may be useful in decontaminating TNT-contaminated soil and water under anaerobic conditions in conjunction with toluene-degrading denitrifiers (Pseudomonas spp.) or toluene-degrading sulfate reducers in a mixed culture system. Correspondence to: R. Boopathy     

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.     

Comparative analysis of 2,4,6-trinitrotoluene (TNT)-induced cellular responses and proteomes in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 in two types of media

TNT-induced cellular responses and proteomes in Pseudomonas sp. HK-6 were comparatively analyzed in two different media: basal salts (BS) and Luria broth (LB). HK-6 cells could not degrade more than 0.5 mM TNT with BS medium, while in LB medium, they exhibited the enhanced capability to degrade as much as 3.0 mM TNT. Analysis of total cellular fatty acids in HK-6 cells suggested that the relative abundance of several saturated or unsaturated fatty acids is altered under TNT-mediated stress conditions. Scanning electron microscopy showed the presence of perforations, irregular rod formations, and wrinkled extracellular surfaces in cells under TNT stress. Proteomic analysis of soluble protein fractions from HK-6 cultures grown with TNT as a substrate revealed 11 protein spots induced by TNT. Among these, seven proteins (including Alg8, AlgB, NirB, and the AhpC/Tsa family) were detected only in LB medium containing TNT. The proteins AspS, Tsf, and assimilatory nitrate reductase were increasingly expressed only in BS medium containing TNT. The protein dGTPase was found to be induced and expressed when cells were grown in either type of TNT-containing media. These results provide a better understanding of the cytotoxicity and survival mechanism used by Pseudomonas sp. HK-6 when placed under TNT stress conditions.     

Non-Ligninolytic TNT Mineralization in Contaminated Soil by Phanerochaete chrysosporium

The explosive 2,4,6-trinitrotoluene (TNT) is widely used and results in widespread soil contamination. The white-rot fungus Phanerochaete chrysosporium has been shown to degrade TNT, using the peroxidase enzyme. In this study, we report peroxidase-independent degradation of TNT by non-ligninolytic P. chrysosporium. Significant disappearance of TNT from highly contaminated soil using P. chrysosporium has been observed. Soil highly contaminated with TNT (2270 ppm [10 mM]) was diluted to 100 ppm (0.44 mM) with malt extract medium. Pregrown (48 hours) mycelial pellets of P. chrysosporium were added in 100 mL malt extract medium and incubated in Gledhill flasks. Analysis by high-performance liquid chromatography (HPLC) was conducted on soil extracts at specific time points to estimate the disappearance of TNT from contaminated soil incubated with P. chrysosporium. When the pregrown mycelial pellets were added, TNT disappeared within 48 hours. The dissolved concentration of 2-amino-4,6-dinitrotoluene (2Am-DNT) increased up to the third day, then declined before its final disappearance by day 10. Results show that the pregrown mycelial pellets of P. chrysosporium mineralized up to 17.3±6.3% [¹⁴C]-TNT within 30 days.