Biotransformation of 2,4,6-trinitrotoluene by Diaphorobacter sp. strain DS2.

Diaphorobacter strain DS2 degrades 3-nitrotoluene and 2-nitrotoluene via ring oxidation with 3-nitrotoluene dioxygenase (3NTDO). In the current study, we hypothesized that 3NTDO might also be involved in the degradation of 2,4,6-trinitrotoluene (TNT), a major nitroaromatic explosive contaminant in soil and groundwater. Strain DS2 transforms TNT as a sole carbon and nitrogen source when grown on it. Ammonium chloride and succinate in the medium accelerated the TNT degradation rate. A resting cell experiment suggested that TNT does not compete with 3NT degradation (no negative impact of TNT on the reaction velocity for 3NT). Enzyme assay with 3NTDO did not exhibit TNT transformation activity. The above results confirmed that 3NTDO of DS2 is not responsible for TNT degradation. In the resting cell experiment, within 10 h, 4ADNT completely degraded. The degradation of 2ADNT was 97% at the same time. We hypothesized that 3NTDO involve in this reaction. Based on the DS2 genome, we proposed that the N-ethylmaleimide reductases (nemA) were involved in the initial reduction of the nitro group and aromatic ring of TNT. Our findings suggest that strain DS2 could be helpful for the removal of TNT from contaminated sites with or without any additional carbon and nitrogen source and with minimal accumulation of undesirable intermediates.

Transformation of 2, 4, 6-trinitrotoluene by Stenotrophomonas strain SG1 under aerobic and anaerobic conditions.

TNT, or 2,4,6-trinitrotoluene, is a common explosive that can contaminate soil and groundwater in production sites, military training areas, and disposal locations. The compound is highly toxic; therefore, there is an urgent need to rehabilitate the impacted environments. Harnessing the microbial ability to biodegrade TNT into environmentally harmless compound(s) is one approach to remediating contaminated sites. In our study, we report on the genomic and metabolic ability of Stenotrophomonas strain SG1 to degrade TNT under aerobic and anaerobic conditions. The bacterial strain SG1 was first isolated as a contaminant from a culture of Diaphorobacter sp. strain DS2 over minimal media supplemented with TNT. The draft genome assembly of strain SG1 is ∼4.7 Mb and is distributed among 358 contigs. The homology search against the custom database of enzymes responsible for TNT biodegradation revealed the presence of three N-ethylmaleimide reductases (NemA) with a defined KEGG ortholog and KEGG pathway of TNT degradation. The presence of respiratory nitrate reductases has also been mapped, which supports denitrification under anaerobic conditions. Experimentally, the TNT transformation rate accelerated when carbon sources, such as sodium acetate, sodium citrate, sodium succinate, sucrose, and glucose (final concentration of 5 mM), were added. Citrate promoted the highest growth and TNT transformation ratio (88.35%) in 120 h. With the addition of 5 mM ammonium chloride, TNT completely disappeared in the citrate and sucrose-containing treatments in 120 h. However, higher biomass was obtained in the sucrose and glucose-containing treatments in 120 h. During incubation, the formation of amino dinitrotoluene isomers, dinitrotoluene isomers, trinitrobenzene, azoxy isomers, diaryl hydroxylamines, and corresponding secondary amines was confirmed by GC/MS and UPLC/MS. 2-Amino-4-nitrotoluene, 4-amino-2-nitrotoluene, and 2-amino-6-nitrotoluene were also identified in the culture supernatant by GC/MS. Under anaerobic conditions, TNT completely disappeared in the citrate and citrate plus nitrate treatments. Since the strain shows the ability to remove TNT, this research should be useful in basic research and practical applications for removing TNT from wastewater.