Anaerobic transformation of 2,4,6-trinitrotoluene (TNT)

A sulfate-reducing bacterium using trinitrotoluene (TNT) as the sole nitrogen source was isolated with pyruvate and sulfate as the energy sources. The organism was able to reduce TNT to triaminotoluene (TAT) in growing cultures and cell suspensions and to further transform TAT to still unknown products. Pyruvate, H₂, or carbon monoxide served as the electron donors for the reduction of TNT. The limiting step in TNT conversion to TAT was the reduction of 2,4-diamino-6-nitrotoluene (2,4-DANT) to triaminotoluene. The reduction proceeded via 2,4-diamino-6-hydroxylaminotoluene (DAHAT) as an intermediate. The intermediary formation of DAHAT was only observed in the presence of carbon monoxide or hydroxylamine, respectively. The reduction of DAHAT to triaminotoluene was inhibited by both CO and NH₂OH. The inhibitors as well as DANT and DAHAT significantly inhibited sulfide formation from sulfite. The data were taken as evidence for the involvement of dissimilatory sulfite reductase in the reduction of DANT and/or DAHAT to triaminotoluene. Hydrogenase purified from Clostridium pasteurianum and carbon monoxide dehydrogenase partially purified from Clostridium thermoaceticum also catalyzed the reduction of DANT in the presence of methyl viologen or ferredoxin, however, as the main reduction product DAHAT rather than triaminotoluene was formed. The findings could explain the function of CO as an electron donor for the DANT reduction (to DAHAT) and the concomitant inhibitory effect of CO on triaminotoluene formation (from DAHAT) by the inhibition of sulfite reductase. Triaminotoluene is further anaerobically converted to unknown products by the isolate under sulfate-reducing and by a Pseudomonas strain under denitrifying conditions. Triaminotoluene conversion was also catalyzed in the absence of cells under aerobic conditions by trace elements, especially by Mn²⁺, accompanied by the elimination of ammonia in a stoichiometry of 1 NH₃ released per TAT transformed. The results might be of interest for the bioremediation of wastewater polluted with nitroaromatic compounds.Abbreviations TNT = 2,4,6-Trinitrotoluene DANT - 2,4-DANT = 2,4-Diamino-6-nitrotoluene - 2,6-DANT = 2,6-Diamino-4-nitrotoluene - ADNT = aminodinitrotoluene - 2-ADNT and 4-ADNT amino substituent at positions 2 or 4 - TAT = 2,4,6-Triaminotoluene - DAHAT = 2,4-Diamino-6-hydroxylaminotoluene - MV = Methyl viologen - Fd = Ferredoxin - H₂ase = Hydrogenase - CODH = Carbon monoxide dehydrogenase - Pyr: Fd OR = Pyruvate: ferredoxin oxidoreductase - U = Units = mol of substrate converted per min