Biotransformation of 2,4,6-trinitrotoluene with Phanerochaete chrysosporium in agitated cultures at pH 4.5

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 mu M) by Phaneroc haete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberat ed (CO2)-C-14) did not exceed 1%. A time study revealed the presence of sev eral intermediates, marked by the initial formation of two monohydroxylamin odinitrotoluenes (2- and 4-HADNT) followed by their successive transformati on to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-N- acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4,6-dinitrot oluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitro toluene and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4 N-acetylhydroxy-2,6-dinitrotoluene and 4-N-acetoxy-2,6-dinitrotoluene (as a cetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluen e. Furthermore, a fraction of HADNTs were found to rearrange to their corre sponding phenolamines (Bamberger rearrangement), while another group dimeri zed to azoxytoluenes which in turn transformed to azo compounds and eventua lly to the corresponding hydrate derivatives. After 30 days, all of these m etabolites, except traces of 4-ADNT and the hydrate derivatives, disappeare d, but mineralization did not exceed 10% even after the incubation period w as increased to 120 days. The biotransformation of TNT was accompanied by t he appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT d isappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the Lip activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivative s. Both MnP and LiP activities reached their maximum levels (100 and 10 U/l iter, respectively) within 10 to 15 days after inoculation.