*2.7.2.2. Degradation of mononitrotoluenes by Diaphorobacter sp. strain DS2*

There are very few reports available on complete mineralization of mono-nitroaromatics by single bacterial strains. The isolation and characterization of three *Diaphorobacter* sp. strains DS1, DS2, and DS3, which are capable of mineralizing and utilizing 3-NT as the sole source of carbon, nitrogen, and energy, was reported by our group [66]. The mineralization of 3-NT by *Diaphorobacter* sp. strain DS2 was found by the initial reaction catalyzed by a dioxygenase with the formation of mixtures of 3- and 4-methylcatechols. These methylcatechols were further degraded by the *meta* cleavage pathway. This strain was able to degrade other compounds like 2-NT, 4-NT, nitrobenzene (NB), 2CNB, and 3CNB through an oxidative degradation route. Cloning and sequencing of first enzyme of the pathways showed the presence of a multicom‐ ponent dioxygenase, i.e., 3NTDO. This 3NTDO gene was found to be present on the genomic DNA of the strain on a 5-kb DNA stretch. Its subunits were identified as a reductase, a ferredoxin, an oxygenase large and small subunit, and a regulatory gene product [83]. Subunits of 3NTDO were individually expressed in *E. coli* and purified by various purification techni‐ ques. To get active enzyme, all the subunits were mixed together in a certain proportion with added NADH. Its active recombinant-reconstituted enzyme showed the conversion of nitrotoluenes and nitrobenzenes to methylcatechols and catechols as measure products with the release of nitrite [84]. Some other minor products are also formed as shown in Table 1. Products other than (methyl)catechols are dead-end products and observed in the culture broth during the degradation of its corresponding substrates with the *Diaphorobacter* sp. strain DS2. This strain did not grow on any dinitrotoluenes (2,4 or 2,6), but recombinant-reconstituted 3NTDO released nitrite from 2,6-dinitrotoluene.

toluenes and hydroxyl nitrotoluenes (pathways A and B, Figure 9) in *P. putida* OU83. Both oxidative as well as reductive attack is reported during metabolism of 3-NT [71]. Here 70% of the 3-NT was reduced to aminotoluene, whereas 30% was converted to 3-nitrophenol via the formation of 3-nitrobenzylalcohol, 3-nitrobenzaldehyde, and 3-nitrobenzoic acid. 3-Nitrophe‐

> CH3 NO2

CH3 NH2

OH

NO2

NO2

2-nitrotoluene 2-aminotoluene

CH3 HO NO2

6-hydroxy-2- nitrotoluene

COOH

3-nitrobenzaldehyde 3-nitrobenzoic 3-nitrophenol

NO2

nol was further metabolized with the release of nitrite (pathway C, Figure 9).

CH3

NH2

4-Nitrotoluene 4-aminotoluene

CH2OH

NO2

CHO

NO2

**Figure 9.** Degradation of mononitrotoluenes by *Pseudomonas putida* strain OU83 [82]. The strain converts 2- and 4-nitro‐ toluenes to corresponding aminotoluenes and hydroxyl nitrotoluenes (pathways A and B), whereas 70% of 3-NT was

There are very few reports available on complete mineralization of mono-nitroaromatics by single bacterial strains. The isolation and characterization of three *Diaphorobacter* sp. strains DS1, DS2, and DS3, which are capable of mineralizing and utilizing 3-NT as the sole source of carbon, nitrogen, and energy, was reported by our group [66]. The mineralization of 3-NT by *Diaphorobacter* sp. strain DS2 was found by the initial reaction catalyzed by a dioxygenase with the formation of mixtures of 3- and 4-methylcatechols. These methylcatechols were further degraded by the *meta* cleavage pathway. This strain was able to degrade other compounds like 2-NT, 4-NT, nitrobenzene (NB), 2CNB, and 3CNB through an oxidative degradation route. Cloning and sequencing of first enzyme of the pathways showed the presence of a multicom‐ ponent dioxygenase, i.e., 3NTDO. This 3NTDO gene was found to be present on the genomic DNA of the strain on a 5-kb DNA stretch. Its subunits were identified as a reductase, a ferredoxin, an oxygenase large and small subunit, and a regulatory gene product [83]. Subunits

converted to 3-aminotoluene and 30% was degraded via the formation of the 3-nitrophenol (pathway C).

*2.7.2.2. Degradation of mononitrotoluenes by Diaphorobacter sp. strain DS2*

A B

CH3

NO2

CH3 OH

NO2

NO2

3-nitrotoluene 3-nitrobenzyl alcohol

NH2

3-aminotoluene

CH3

C

62 Wastewater Treatment Engineering

CH3

4-hydroxy-4-nitrotoluene

**Table 1.** Products from the various substrates by recombinant 3NTDO.
