Anaerobic incorporation of the radiolabeled explosive TNT and metabolites into the organic soil matrix of contaminated soil after different treatment procedures.

Four bioreactor designs were performed to evaluate the level of incorporation of 14C-labeled 2,4,6-trinitrotoluene (TNT) and metabolites into the organic soil matrix of different anaerobically treated contaminated soils. The contaminated soils were amended with molasses slivers (80:20% per weight) as auxiliary substrate to enhance microbial activity. After 5 weeks (bioreactors 1 and 2), 8 weeks (bioreactor 3) and 12 weeks (bioreactor 4) of anaerobic incubation, we determined 41%, 58%, 72%, and 54%, respectively, of the initially applied radioactivity immobilized in various soil fractions. After alkaline hydrolyses of the solvent-extracted soils, low quantities of radiolabel were found in the humic and fulvic acid fractions, whereas the bulk of 14C activity was found to be strongly bound to the humin fraction (solid soil residues). The amounts of solvent extractable radioactivity were 53%, 40%, 16%, and 29% for bioreactors 1, 2, 3, and 4, respectively. The level of TNT transformation at the end of the experiments was within 90-94%. Regarding the results presented in this study, we can assume that there is the possibility of high incorporation levels of TNT metabolites into the soil organic matrix mediated by microbial cometabolism under strictly anoxic conditions.

Mass balance studies with 14C-labeled 2,4,6-trinitrotoluene (TNT) mediated by an Anaerobic desulfovibrio species and an Aerobic serratia species.

Investigations were carried out to evaluate the level of incorporation of radiolabeled 2,4,6-trinitrotoluene (TNT) and metabolites into the bacterial biomass of two different bacterial species after cometabolically mediated TNT transformation. Biotransformation experiments with 14C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with the cells. It was found that more than 42% of the initially applied radiolabel was associated with the cell biomass after cometabolic 14C-TNT transformation with the strictly anerobic Desulfovibrio species strain SHV, whereas with the strictly aerobic Serratia plymuthica species strain B7, 32% of cell-associated 14C activity was measured. The remainder of the radiolabel was present in the supernatants of the liquid cultures in the form of different TNT metabolites. Under anoxic conditions with the Desulfovibrio species, TNT was ultimately transformed to 2,4,6-triaminotoluene (TAT) and both diaminonitrotoluene isomers, whereas under oxic conditions with the Serratia species, TNT was converted to hydroxylaminodinitrotoluenes and aminodinitrotoluenes, with 4-amino-2,6-dinitrotoluene (4ADNT) being the major end product. In both culture supernatants, small amounts of very polar, radiolabeled, but unidentified metabolites were detected. At the end of the experiments approximately 92% and 96% of the originally applied radioactivity was recovered in the studies with the Serratia and Desulfovibrio species, respectively.

Anaerobic degradation and transformation of p-toluidine by the sulfate-reducing bacterium Desulfobacula toluolica.

The ability of the strictly anaerobic sulfate-reducing bacterium Desulfobacula toluolica (strain Tol2) to cometabolically degrade p-toluidine (p-methylaniline) while using toluene as the primary source of carbon and energy has been studied. This organism has been shown to modify and degrade toluidine in dense cell suspensions when no other source of carbon and energy is added. The metabolism led to the formation of a variety of metabolites. From these metabolites a biphenyl-like compound as well as phenylacetic acid have been identified by means of HPLC/MS techniques. The probable conversion of p-toluidine to p-aminophenylacetic acid and phenylacetic acid as dead end products suggested that this organism initiates p-toluidine degradation by the carboxylation of the methyl group. If this could be validated in further experiments, it would be the first time that a toluidine was carboxylated at the methyl moiety by an anaerobic, sulfate-reducing bacterium.

Microbial transformation of 2,4,6-trinitrotoluene in aerobic soil columns.

2,4,6-Trinitrotoluene (TNT)-contaminated soil material of a former TNT production plant was percolated aerobically in soil columns. Nineteen days of percolation with a potassium phosphate buffer supplemented with glucose or glucose plus ammonium sulfate caused an over 90% decline in the amount of extractable nitroaromatics in soils containing 70 to 2,100 mg of TNT per kg (dry weight). In the percolation solution, a complete elimination of TNT was achieved. Mutagenicity and soil toxicity were significantly reduced by the percolation process. 4-N-Acetylamino-2-amino-6-nitrotoluene was generated in soil and percolation fluid as a labile TNT metabolite.

Microbial degradation of explosives and related compounds.

The pollution of soil and water with explosives and related compounds caused by military activities has been known for a long time, but progress in understanding the environmental fate of such substances has only been made in the last few years. Microbial processes could be used for the remediation of explosives-contaminated soils and waste waters because it has been shown that a variety of different microorganisms are able to metabolize these chemical compounds. In some cases even a complete mineralization has been found, whereas in others only biotransformation reactions took place, producing more or less toxic and/or recalcitrant metabolites. Studies with pure cultures of bacteria and fungi have given detailed insights into the biodegradation pathways of at least some nitroorganic compounds. Additionally, some of the key enzymes have been isolated and purified or studied in crude extracts. This review summarizes information on the biodegradation and biotransformation pathways of several important explosives. This may be useful in developing microbiological methods for a safe and economic clean-up of soil and water contaminated with such compounds. It also shows the necessity of further investigations concerning the microbial metabolism of these substances.

Microbial transformation of nitroaromatic compounds under anaerobic conditions.

The transformation of several mono- and dinitroaromatic compounds (tested at 50-200 microM) by methanogenic bacteria, sulphate-reducing bacteria and clostridia was studied. Some of the nitroaromatics tested were transformed chemically by 1.5 mM quantities of culture media reducing agents, like cysteine or sulphide. This abiotic reduction occurred at the o-nitro-groups preferentially. Nitrophenols, p-nitroaniline and p-nitrobenzoic acid were completely transformed biologically into the corresponding amino derivatives. The nitroaromatics were transformed by all of the bacterial strains tested. While growing cells of sulphate-reducing bacteria and Clostridium spp. carried out nitroreduction, methanogen cells lysed in the presence of nitroaromatics. Nevertheless these culture suspensions converted nitroaromatics to the corresponding amino derivatives. This was also confirmed by crude cell extracts of methanogenic bacteria. The rate of nitroreduction by sulphate-reducing bacteria depended on the electron donors supplied and the cell density, with molecular hydrogen being the most effective donor of reducing equivalents. The toxicity of p-nitrophenol to some of the organisms tested depended on the concentration of the nitroaromatic compound and the type of organism.