Abstract
Extensive use and disposal of 2,4,6-trinitrotoluene (TNT), a primary constituent of explosives, pollutes the environment and causes severe damage to human health. Complete mineralization of TNT via bacterial degradation has recently gained research interest as an effective method for the restoration of contaminated sites. Here, screening for TNT degradation by six selected bacteria revealed that Buttiauxella sp. S19-1, possesses the strongest degrading ability. Moreover, BuP34O (a gene encoding for protocatechuate 3,4-dioxygenase—P34O, a key enzyme in the β-ketoadipate pathway) was upregulated during TNT degradation. A knockout of BuP34O in S19-1 to generate S-M1 mutant strain caused a marked reduction in TNT degradation efficiency compared to S19-1. Additionally, the EM1 mutant strain (Escherichia coli DH5α transfected with BuP34O) showed higher degradation efficiency than DH5α. Gas chromatography mass spectrometry (GC-MS) analysis of TNT degradation by S19-1 revealed 4-amino-2,6-dinitrotolune (ADNT) as the intermediate metabolite of TNT. Furthermore, the recombinant protein P34O (rP34O) expressed the activity of 2.46 µmol/min·mg. Our findings present the first report on the involvement of P34O in bacterial degradation of TNT and its metabolites, suggesting that P34O could catalyze downstream reactions in the TNT degradation pathway. In addition, the TNT-degrading ability of S19-1, a Gram-negative marine-derived bacterium, presents enormous potential for restoration of TNT-contaminated seas.
Highlights
The prolific use of 2,4,6-trinitrotoluene (TNT), as a major component of explosives, during wars dates back to several decades and has caused deleterious effects on the ecosystem
TNT degradation by six selected bacteria (KF-1, PS, S19-1, JP1, P14 and LY1; described in Section 2.1) was determined by High-Performance Liquid Chromatography (HPLC) analysis of the amount of TNT remaining in bacterial cultures after 9 h exposure at 27 ◦C
That the bacteria could hardly grow on mineral medium using TNT as the sole source of carbon and energy, while cometabolism improved the TNT degradation efficiency
Summary
The prolific use of 2,4,6-trinitrotoluene (TNT), as a major component of explosives, during wars dates back to several decades and has caused deleterious effects on the ecosystem. TNT contamination by inappropriate disposal of TNT-containing wastes into the environment was found at levels of about 200 g/kg and 100 mg/L in soil and water, respectively [5,6,7,8,9,10]. The deposition of TNT-containing military wastes at various training and war sites across the globe has contributed remarkably towards environmental pollution [10]. TNT inhibited nitrogenfixation, dehydrogenase, and other microbial activities even at low levels (10 mg/L) of contamination [13,14]. The U.S Environmental Protection Agency recommended a limit of 0.01 mg/L TNT in drinking water for TNT [8]
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