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.     

Stability of hydroxylamino- and amino-intermediates from reduction of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene

Hydroxylamines, produced as intermediates in the reductive metabolism of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene between nitroaromatic parent compounds and corresponding amines, were unstable in aqueous solution in the presence of O₂. Reactions of hydroxylamines to compounds other than amines may be the major cause of poor mass balance observations in bioremediation systems where only aminated products are monitored. Results demonstrate the formation of azoxy compounds as products of abiotic aryl-hydroxylamine reactions.     

Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) and the related 2,4-dinitrotoluene (DNT) are toxic environmental pollutants. The biotransformation and detoxication of these persistent compounds in higher organisms are of great significance from a health perspective as well as for the biotechnological challenge of bioremediation of contaminated soil. We demonstrate that different human glutathione transferases (GSTs) and GSTs from the fruit fly Drosophila melanogaster are catalysts of the biotransformation of TNT and DNT. The human GSTs had significant but modest catalytic activities with both DNT and TNT. However, D. melanogaster GSTE6 and GSTE7 displayed outstanding high activities with both substrates.     

Features of the toxic action of 2,4,6-trinitrotoluene on Escherichia coli K12

2,4,6-Trinitrotoluene present in a culture of Escherichia coli K12, at a concentration of 200 mg/l, caused a decrease in the total cell population and the population of colony-forming units, increased permeability of the external lipoprotein envelope, and increased the refractive index of cells. The shape of some cells changed from rod-like to coccoid, and cell size decreased. The specific rate of glucose consumption and the content of NADH (NADPH) in cells decreased. The changes of these morphological and physiological features were reversible, tending to normalize after reduction of 2,4,6-trinitrotoluene concentration in the course of cultivation.     

Sensitivity of various escherichia coli strains to 2,4,6-trinitrotoluene

The sensitivity of Escherichia coli strains K-12 and 055 to 2,4,6-trinitrotoluene (TNT) was found to correlate with the structural and functional properties of the outer lipoprotein membrane. The protective ability of the membrane of strain 055 is much lower than that of K-12. This is the cause of the greater sensitivity of 055 to the toxic action of TNT. High TNT concentrations (100-200 mg/l) suppressed the growth of 055, whereas K-12 grew at all TNT concentrations studied. Both strains adapted to high TNT concentrations by converting it by either nitroreduction or denitritation depending on concentration. The denitritation system of strain 055 started TNT degradation earlier than that of K-12.     

Surface plasmon resonance immunosensor for highly sensitive detection of 2,4,6-trinitrotoluene

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (< or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.     

Promising Strategies for the Mineralisation of 2,4,6-trinitrotoluene

2,4,6-trinitrotoluene (TNT) is known to be one of the most common military explosives. In spite of its established toxicity and mutagenicity for many organisms, soils and groundwater are still being frequently contaminated at manufacturing, disposal and TNT destruction sites. The inability of natural aquatic and soil biota to use TNT as growth substrate has been recognized as the primary limitation in the application of bioremediation processes to contaminated environments. However, promising degradation pathways have been recently discovered which may lead to the mineralisation of TNT. Significant advances have been made in studying the mechanism of TNT denitration, which can be considered as the major reaction and the driving force towards beneficial biodegradation. The possibilities to favour TNT denitration are discussed based on current knowledge of the enzymology and genetics of denitration in nitroaromatic degrading organisms. The literature survey demonstrates that the only enzymes characterized so far for their denitrase activity towards TNT belong to the class I flavin-dependent β/α barrel oxidoreductases, also known as the “Old Yellow Enzyme” family. In addition, this review provides an overview of strategies and future directions towards a rational search for new catabolic activities, including metagenomic library screening, plus new possibilities to improve the activity of known catabolic enzymes acting on TNT, such as DNA shuffling.     

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     

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     

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.     

Engineering Pseudomonas fluorescens for biodegradation of 2,4-dinitrotoluene

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10 degrees C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.     

Derivatization and separation of 2,4,6-trinitrotoluene metabolic products

2,4,6-Triaminotoluene (TAT), an oxygen sensitive metabolic product of 2,4,6-trinitrotoluene (TNT) reduction, was derivatized yielding oxygen stable products using a procedure previously employed for the stabilization of other O2sensitive TNT metabolic products. A HPLC method is presented that allows for the separation of diverse intermediates and products of TNT metabolism following the derivatization procedure. © Rapid Science Ltd. 1998     

Illuminating the detection chain of bacterial bioreporters

Engineering bacteria for measuring chemicals of environmental or toxicological concern (bioreporter bacteria) has grown slowly into a mature research area. Despite many potential advantages, current bioreporters do not perform well enough to comply with environmental detection standards. Basically, the reasons for this are the lack of engineering principles in the detection chain in the bioreporters. Here, we dissect critical steps in the detection chain and illustrate how bioreporter design could be improved by mutagenizing specificity and selectivity of the sensing and regulatory proteins, by newer expression strategies and application of different signalling networks. Furthermore, we describe how redesigning bioreporter assays with respect to pollutant transport into the cells and application of other detection devices can decrease detection limits and increase the speed of detection.     

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.