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.

Cometabolic transformation and cleavage of nitrodiphenylamines by three newly isolated sulfate-reducing bacterial strains.

Three sulfate-reducing bacterial strains (Desulfovibrio sp. strain SHV, Desulfococcus sp. strain WHC, and Desulfomicrobium sp. strain WHB) with the capacity to cometabolize 2-nitrodiphenylamine, 4-nitrodiphenylamine, and 2,4-dinitrodiphenylamine were newly isolated. Before breaking down the diphenylamine structure, these strains cometabolically reduce the nitrodiphenylamines to the corresponding aminodiphenylamines during anaerobic oxidation of the growth substrate lactate (Desulfovibrio strain SHV and Desulfomicrobium strain WHC) or benzoate (Desulfococcus strain WHB), leading to the formation of aniline and a smaller quantity of methylaniline. These compounds were not further metabolized by the sulfate reducers. The anaerobic metabolism of aminodiphenylamines also led to the formation of heterocyclic condensation products such as phenazine and acridine derivatives, provided that they contained an amino group in the ortho position of the diphenylamine (e.g., 2-aminodiphenylamine or 2,4-diaminodiphenylamine). In addition, low levels of indole and benzothiazole derivatives were identified, but these also were not further metabolized by the three sulfate-reducing strains.

Reduction of Nitrated Diphenylamine Derivatives under Anaerobic Conditions.

2-Nitrodiphenylamine, 4-nitrodiphenylamine, and 2,4-dinitrodiphenylamine were anaerobically metabolized in sediment-water batch enrichments inoculated with mud from the German North Sea coast. The first intermediate in 2,4-dinitrodiphenylamine degradation was 2-amino-4-nitrodiphenylamine, which appeared in large (nearly stoichiometric) amounts before being completely reduced to 2,4-diaminodiphenylamine. Of the second theoretically expected metabolite, 4-amino-2-nitrodiphenylamine, only traces were detected by gas chromatographic-mass spectrometric analysis in highly concentrated extracts. In addition, low levels of 4-nitrodiphenylamine, which may be the product of ortho deamination of intermediately produced 2-amino-4-nitrodiphenylamine, were observed. 2-Nitrodiphenylamine and 4-nitrodiphenylamine were primarily reduced to 2-aminodiphenylamine and 4-aminodiphenylamine, respectively. Diphenylamine was never detected in any experiment as a theoretically possible intermediate. Results from studies with dense cell suspensions of anaerobic, aromatic-compound-mineralizing bacteria confirmed the transformation reactions, which were carried out by microorganisms indigenous to the anaerobic coastal water sediment.

Toxicity of diphenylamine and some of its nitrated and aminated derivatives to the luminescent bacterium Vibrio fischeri.

Aqueous samples containing various nitrated and aminated diphenylamine derivatives were subjected to the luminescent bacterium Vibrio fischeri NRRL-B-11177 to determine their ecotoxicological potential. As the most important toxicological parameter, EC50, the concentration needed to reduce bacterial luminescence by 50%, was calculated. All compounds tested must be classified to the category "very toxic to aquatic organisms" using the widely accepted classification scheme of D. Strupp, H.P. Lühr, H. T. Grunder, J. Gerdesmann, and J. Ahlers (1990, UWSF--Z. Umweltchem. Okotox. 2, 151-156). Only 2, 4-diaminodiphenylamine can be classified as "less toxic to aquatic organisms". EC50 values after 30, 60, and 90 min of incubation of the test compounds are presented. For many of the compounds tested in this study there are no toxicological data in the literature.

Mineralization of monofluorobenzoate by a diculture under sulfate-reducing conditions.

A mesophilic, dehalogenating, sulfate-reducing diculture was isolated from an anaerobic lake sediment. One strain of the diculture is proposed to be an endospore-forming Desulfotomaculum species, the second strain was a vibrioid, motile and non-sporeforming species which is tentatively assigned to the genus Desulfovibrio. The diculture was able to mineralize 4- and 2-fluorobenzoate both isomers being incompletely oxidized with the release of acetate, which was subsequently used by both sulfate-reducing strains. Other electron donors used for growth included benzoate, 3- and 4-hydroxybenzoate, protocatechuate, catechol, phenol, 2,5-dimethoxyphenol, fatty acids up to C8, malate and pyruvate. The culture obtained from a freshwater habitat grew optimally at NaCl concentrations of 0.3-0.5 g l-1, 33-37 degrees C, and pH 7.4. Our experiments showed that certain fluorinated aromatic hydrocarbons could serve as sole sources of carbon and energy for sulfate-reducing bacteria.

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.

Isolation and characterization of a new spore-forming sulfate-reducing bacterium growing by complete oxidation of catechol.

A new mesophilic sulfate-reducing bacterium, strain Groll, was isolated from a benzoate enrichment culture inoculated with black mud from a freshwater ditch. The isolate was a spore-forming, rod-shaped, motile, gram-positive bacterium. This isolate was able of complete oxidation of several aromatic compounds including phenol, catechol, benzoate, p- and m-cresol, benzyl alcohol and vanillate. With hydrogen and carbon dioxide, formate or O-methylated aromatic compounds, autotrophic growth during sulfate reduction or homoacetogenesis was demonstrated. Lactate was not used as a substrate. SO4(2-), SO3(2-), and S2O3(2-) were utilized as electron acceptors. Although strain Groll originated from a freshwater habitat, salt concentrations of up to 30 g.l-1 were tolerated. The optimum temperature for growth was 35-37 degrees C. The G + C content of DNA was 42.1 mol%. This isolate is described as a new species of the genus Desulfotomaculum.

Complete oxidation of benzoate and 4-hydroxybenzoate by a new sulfate-reducing bacterium resembling Desulfoarculus.

A new sulfate-reducer "strain SAX" was isolated from an anaerobic marine sediment [Saxild, Denmark]. The isolate was a gram-negative, motile and non-spore-forming rod which sometimes appeared as a curved rod. Strain SAX differed from all described Desulfovibrio-, Desulfobotulus- and Desulfoarculus-species by the ability to degrade aromatic compounds such as benzoate, 4-hydroxybenzoate and phenol completely to CO2. Electron donors used included lactate, pyruvate, malate, fumarate, crotonate and butyrate, while pyruvate was fermented in the absence of an external electron acceptor. Sulfate, thiosulfate or sulfite served as electron acceptors with benzoate as the donor, while nitrate and nitrite did not. The sulfate-reducing bacterium required vitamins and NaCl-concentrations of about 20 g/l. The optimum temperature for growth of strain SAX was 30 degrees C and the optimum pH value was 7.3. The DNA base composition was 62.4 mol% G+C. The strain possessed cytochrome c3, but no desulfoviridin. On the basis of these characteristics and because strain SAX could not be ascribed to any of the existing species therefore assignment as a new species to the genus Desulfoarculus was suggested.