2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp. , and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO2 from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO2 from the nitroaromatic ring.

The microbial reduction of the aromatic ring of 2,4,6-trinitrotoluene (TNT) can lead to its complete destruction. The acid-tolerant yeast Yarrowia lipolytica AN-L15 transformed TNT through hydride ion-mediated reduction of the aromatic ring (as the main pathway), resulting in the accumulation of nitrite and nitrate ions, as well as through nitro group reduction (as minor pathway), resulting in hydroxylamino- and aminoaromatics. TNT transformation depended on the yeasts' ability to acidify the culture medium through the production of organic acids. Aeration and a low medium buffer capacity favored yeast growth and resulted in rapid acidification of the medium, which influenced the rate and extent of TNT transformation. This is the first time that nitrate has been detected as a major product of microbial TNT degradation, and this work demonstrates the importance of pH on TNT biotransformation. The ability of Y. lipolytica AN-L15 to reduce the TNT aromatic ring to form TNT-hydride complexes, followed by their denitration, makes this strain a potential candidate for bioremediation of sites contaminated with explosives.

2,4,6-Trinitrotoluene (TNT) transformation by the yeast strain Yarrowia lipolytica AN-L15 was shown to occur via two different pathways. Direct aromatic ring reduction was the predominant mechanism of TNT transformation, while nitro group reduction was observed to be a minor pathway. Although growth of Y. lipolytica AN-L15 was inhibited initially in the presence of TNT, TNT transformation was observed, indicating that the enzymes necessary for TNT reduction were present initially. Aromatic ring reduction resulted in the transient accumulation of eight different TNT-hydride complexes, which were characterized using highperformance liquid chromatography, UV-visible diode array detection, and negative-mode atmospheric pressure chemical ionization mass spectrometry (APCI-MS). APCI-MS analysis revealed three different groups of TNT-hydride complexes with molecular ions at m/z 227, 228, and 230, which correspond to TNT-mono-and dihydride complexes and protonated dihydride isomers, respectively. One of the three protonated dihydride complex isomers detected appears to release nitrite in the presence of strain AN-L15. This release of nitrite is of particular interest since it can provide a pathway towards complete degradation and detoxification of TNT.

: The addition of exogenous to poultry manure and synthetic NH 4Cl medium was used to study the e †ect of ammonia-nitrogen on the activity and composition of a methanogenic consortium. Results indicated that the production of biogas and methane was not a †ected by the variation in con-NH 4 Cl centration within the range 2È10 g dm~3 (0É5È2É6 g dm~3). At higher N-NH 4 values of ammonium (10È30 g dm~3 or 2È8 g dm~3) a signiÐcant N-NH 4 decline in both parameters (by 50È60% for biogas and 80È90% for methane) was observed. A signiÐcant decrease in the numbers of bacteria of all physiological groups (especially proteolytic and methanogenic) was observed when more than 30 g dm~3 (7É8 g dm~3) was added to the fermentation medium. NH 4 Cl N-NH 4 The addition of 10% (w/v) of powdered phosphorite ore enhanced the production of biogas and methane at concentrations up to 30 g dm~3, and NH 4 Cl also changed the composition of the methanogenic consortium. A partial recovery in the numbers of proteolytic and methanogenic bacteria coupled with the decrease in the density of sulphate-reducers was observed. High concentrations (more than 50 g dm~3) of seemed to cause irreversible inhibition of NH 4 Cl methanogenesis which could not be eliminated by the addition of phosphorites.

The yeast strain Geotrichum candidum AN Z4 isolated from an anthropogenically polluted site was able to transform 2,4,6 trinitrotoluene (TNT) via the formation of unstable intermediate hydride Meisenheimer complexes with their subsequent destruction and accumulation of nitrite and nitrate ions as the end mineral forms of nitrogen. Aeration of the medium promoted more profound destruction of this xenobiotic by the strain G. candidum AN Z4 than static conditions. The yeast strain was shown to produce citrate, succinate, and isocitrate, which sharply acidified the medium and influenced the TNT destruction. Two possible pathways of TNT biodegradation were confirmed experimentally: (1) via the destruction of the TNT monohydride complex (3 H-TNT) and (2) via the destruction of one protonated TNT dihydride complex (3,5 2H-TNT • H +). The strain G. candidum AN Z4, due to its ability for TNT degradation, may be promising for bioremediation of TNT contaminated soil and water.