TNT biodegradation and production of dihydroxylamino-nitrotoluene by aerobic TNT degrader Pseudomonas sp. strain TM15 in an anoxic environment
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Anaerobic bacteria have been used to produce 2,4-dihydroxylamino-nitrotoluene (2,4DHANT), a reductive metabolite of 2,4,6-trinitrotoluene (TNT). Here, an aerobic TNT biodegrader Pseudomonas sp. strain TM15 produced 2,4DHANT as evidenced by the molecular ion with m/z of 199 identified from LC-TOFMS analyses. TNT biodegradation with a high cell concentration (109 cells/ml) led to a significant accumulation of 2,4DHANT in the culture medium, as well as hydroxylamino-dinitrotoluenes (HADNTs), although these products were not accumulated when a low cell concentration was used; also, the accumulation of diamino-nitrotoluene and of an unidentified metabolite were observed in the culture medium with the high cell concentration (1010 cells/ml). 2,4DHANT overproduction was a function of the aeration speed since cultures with low aeration speeds (30 rpm) had a 19-fold higher DHANT productivity than those aerated with high speeds (180 rpm); this indicates that molecular oxygen was related to the formation of 2,4DHANT. The quantification of dissolved oxygen (DO) in the media demonstrated that the productivity of 2,4DHANT was increased at low DO values. Moreover, supplying oxygen to the culture media produced a remarkable decrease of 2,4DHANT accumulation; these results clearly indicate that high 2,4DHANT production was a consequence of the oxygen deficit in the culture medium. This finding is useful for understanding the TNT biodegradation (bioremediation technology) in an anoxic environment.
KeywordsDihydroxylamino-nitrotoluene 2,4,6-Trinitrotoluene metabolites Accumulation Aerobic bacteria Anoxic environment
We are grateful to Chugoku Kayaku Co. Ltd., Hiroshima, Japan, for their gift of 2,4,6-trinitrotoluene, and to Dr. R. Spanggord, Chemical Sciences and Technology Department, SRI International, Melno Park, CA, for his gift of 2-hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotolulene, and 2,4-diamino-nitrotoluene. We express much gratitude to Dr. Robin Gerlach, Montana State University, Bozeman, MT, for his help in identifying 2,4-dihydroxylamino-6-nitrotoluene, to Dr. Hideaki Uchida and Dr. Yoshizumi Takigawa, Agilent Technologies Japan, Ltd., for their support with LC/TOF–MS, and to Dr. Norikazu Nishino, Department of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, for his support with the LC/MS analyses. We are also grateful for the assistance of Dr. Thomas K. Wood of Texas A & M University, College Station, TX, for his kind helps with preparing this manuscript.
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