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 μM) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated (14)CO(2)) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation 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-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene 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 acetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, 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 derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.

本研究考察了在pH值4.5条件下，以糖蜜与柠檬酸为辅助基质，利用黄孢原毛平革菌（Phanerochaete chrysosporium）对2,4,6-三硝基甲苯（2,4,6-trinitrotoluene, TNT）（浓度为175 μM）的生物转化过程。在不到两周的时间内，TNT即可完全消失，但矿化作用（释放¹⁴CO₂）的效率未超过1%。时间进程分析显示，体系中先后出现多种代谢中间体：首先生成两种单羟基氨基二硝基甲苯（hydroxylaminodinitrotoluene, HADNT），即2-HADNT与4-HADNT，随后二者依次转化为包括单氨基二硝基甲苯（aminodinitrotoluene, ADNT）在内的多种其他产物。此外还检测到9种酰化中间体，具体包括2-N-乙酰氨基-4,6-二硝基甲苯及其对位异构体、2-N-甲酰氨基-4,6-二硝基甲苯及其对位异构体（均属于酰化ADNT类）、4-N-乙酰氨基-2-氨基-6-硝基甲苯与4-N-甲酰氨基-2-氨基-6-硝基甲苯（属于乙酰化二氨基硝基甲苯，acetylated DANT）、4-N-乙酰羟基-2,6-二硝基甲苯及4-N-乙酰氧基-2,6-二硝基甲苯（属于乙酰化HADNT），以及最终的4-N-乙酰氨基-2-羟基氨基-6-硝基甲苯。进一步研究发现，部分HADNT可发生班伯格重排（Bamberger rearrangement），转化为对应的酚胺类物质；另有部分HADNT发生二聚反应生成氧化偶氮甲苯，后者可进一步转化为偶氮化合物，最终生成对应的肼基衍生物。培养30天后，除痕量的4-ADNT与肼基衍生物外，所有上述代谢产物均已完全消失；但即使将培养周期延长至120天，矿化率仍未超过10%。TNT的生物转化过程伴随锰过氧化物酶（manganese peroxidase, MnP）与木质素过氧化物酶（lignin-dependent peroxidase, LiP）活性的产生：在TNT完全消失的阶段，即初始代谢产物HADNT与ADNT出现的时期，几乎可即刻检测到MnP活性；而LiP活性则在培养8天后才可观测到，该时间点与酰化衍生物的出现时间相吻合。在接种后的10至15天内，MnP与LiP活性均达到峰值，分别为100 U/L与10 U/L。