Biodegradation of 2,6-dinitrotoluene and plant growth promoting traits by Rhodococcus pyridinivorans NT2: Identification and toxicological analysis of metabolites and proteomic insights

• Laboratory scale aerobic bioremediation of 2,6-DNT at high concentration.
• Degradation products include 2-methyl-3-nitroaniline and 2,6-diaminotoluene.
• Toxicological studies showed less-toxic nature of 2,6-DNT biodegraded metabolites.
• Rhodococcus pyridinivorans NT2 could maintain PGP traits under 2,6-DNT stress.
• One of the few studies showing proteomic analysis of 2,6-DNT biodegradation.

2,6-dinitrotoluene (2,6-DNT), one of the major priority pollutants, is a common isomer produced during 2,4,6-trinitrotoluene (TNT) synthesis and also frequently used in production of herbicides, dyes, and synthetic foams. While 2,6-DNT may be degraded relatively rapidly under batch liquid culture conditions, very limited biodegradation details have been reported particularly at high initial DNT concentrations. In this study, Rhodococcus pyridinivorans NT2 was subjected to 0.5–1.6 mM of 2,6-DNT and shown to grow on 2,6-DNT as a sole carbon and nitrogen source. Although strain NT2 tolerated high concentrations of 2,6-DNT (1.6 mM in 108 h), maximum biodegradation was observed at 0.54 mM of 2,6-DNT (within 48 h) that was described well by both first-order and pseudo-first-order reaction kinetics (R2, 0.927). Biodegradation of 2,6-DNT suggested a reductive metabolic pathway with the formation of 2-methyl-3-nitroaniline and 2,6-diaminotoluene. A nitroreductase catalyzing nitroreduction of 2,6-DNT was detected in the cell lysate of strain NT2. Phytotoxicity (with Triticum aestivum and Vigna radiata), cytogenotoxicity (with Allium cepa root-tip cells), and microbial toxicity (using Escherichia coli DH5α) studies were performed to evaluate the toxicity of metabolites produced after degradation of 2,6-DNT. Besides, strain NT2 possessed important plant-growth promoting traits, both in the presence and absence of 2,6-DNT. Furthermore, proteomic characterization using nano LC-MS/MS identified a total of 516 proteins, of which 75 were up-regulated. These proteins were involved in 2,6-DNT degradation (oxidation-reduction systems related to nitroreductase-like proteins), transport processes, carbon and energetic metabolism, transcription/translational changes and stress tolerance, shedding light on the complexity of 2,6-DNT catabolism by strain NT2.

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