Enhanced humification as an in-situ bioremediation technique for 2,4,6-trinitrotoluene (TNT) contaminated soils

The microbial remediation of 2,4,6-trinitrotoluene (TNT) contaminated soils is difficult because it is not possible to achieve mineralization of this substance. TNT can only be transformed via the sequence TNT --> 4-aminodinitrotoluene (4-ADNT) --> 2,4-diaminonitrotoluene (2,4-DANT) --> 2,4,6-triaminotoluene (TAT). TAT is formed when redox potentials are below - 250 mV, otherwise 2,4-DANT is accumulated. If under in-situ conditions TNT or its metabolites are irreversibly incorporated into the humic matrix (humification) instead of being sorbed, the humification might be used as a remediation technology. Equilibrium dialysis showed that TNT, 4-ADNT and 2,4-DANT did not adsorb on humic substances. However, in TNT-degradation studies under nitrogen limitation, the more humic substances were added the less metabolites were recovered proposing an efficient microorganism-mediated incorporation (humification), probably based on stress-induced radical enzyme reactions. This assumption was supported by the observation that 2,4-DANT but not TNT or 4-ADNT is attacked by the radicalic working enzyme peroxidase. Molar mass chromatography showed that 2,4-DANT is incorporated into the humic matrix by the peroxidase. The microbiologically formed ''bound residues'' could not be released by drastic pH shifts to pH 2 or 12. Only methanolic saponification resulted in the release of about 10% of the bound residues. An in-situ remediation technology may include at first a stepwise microbial reduction of TNT to 2,4-DANT. Although under in-situ conditions TAT cannot be formed in a further step rue to the redox potential higher than required, this is not necessary because the metabolite 2,4-DANT can be efficiently incorporated into humic substances.