*2.7.1. Bacterial degradation of nitrobenzene*

The aerobic degradation of nitrobenzene involves two major pathways: a most commonly found partial reductive pathway and a dioxygenase catalyzed pathway (Figure 6). In the oxidative degradation of NB, degradation starts with the action of nitrobenzene-1,2-dioxyge‐ nase, which converts nitrobenzene into catechol. This catechol is further cleaved by the action of catechol 2,3-dioxygenase and degraded by the *meta* cleavage pathway. This type of pathway is reported in *Comamonas* JS 765, *Acidovorax* sp. JS42, and *Micrococcus* sp. strain SMN1.

**Figure 6.** Degradation pathways for nitrobenzene. Aerobic dioxygenation route is reported in *Comamonas* JS765 [67]. The aerobic partial reductive pathway is from *Pseudomonas pseudoalkaligenes* JS45 [61].

## *2.7.2. Bacterial degradation of mononitrotoluenes*

for the microbe. During the past few decades, several reports came up with isolation of microbes mineralizing different nitroaromatic compound and their degradation pathway. Few of them are extensively studied and characterized. There are different strategies in the aerobic

Mono-oxygenases are known to add single oxygen atom at a time and cause the release of nitro group. Simpson and Evans [62] reported the role of mono-oxygenase in a *Pseudomonas* sp., where 4-nitrophenol was converted to hydroquinone with the concomitant release of nitrite. Subsequently, Spain and Gibson (1991) reported accumulation of hydroquinone and release of nitrite from 4-nitrophenol by partially purified mono-oxygenase from a *Moraxella* sp.

Dioxygenases are known to add two -OH groups simultaneously on the benzene ring of nitroaromatic compounds with the release of nitro group as nitrite. This type of mechanism is reported for 2,6-dinitrotoluene biotransformation by Alcaligenes eutrophus [63]. Other examples include the degradation of 2-NT [64, 65], 3-NT [66], nitrobenzene [67], and 2,4-DNT

The addition of a hydride ion to the aromatic ring of nitroaromatic compound leads to the formation of a Meisenheimer complex [27]. The complex rearomatizes after the release of nitrite

In this mechanism, the nitro group is partially reduced to the corresponding hydroxylamine, which upon hydrolysis yields ammonia. This mechanism was reported in Comamonas

**2.7. Different bacterial pathways reported for the degradation of mononitrotoluenes and**

The aerobic degradation of nitrobenzene involves two major pathways: a most commonly found partial reductive pathway and a dioxygenase catalyzed pathway (Figure 6). In the oxidative degradation of NB, degradation starts with the action of nitrobenzene-1,2-dioxyge‐ nase, which converts nitrobenzene into catechol. This catechol is further cleaved by the action of catechol 2,3-dioxygenase and degraded by the *meta* cleavage pathway. This type of pathway is reported in *Comamonas* JS 765, *Acidovorax* sp. JS42, and *Micrococcus* sp. strain SMN1.

acidovorans, where 4-nitrobenzoate is converted to 4-hydroxyl-aminobenzoate [69].

degradation of nitroaromatics [61], which is used in nature as shown in Figure 5.

**2.3. Reactions catalyzed by mono-oxygenases**

58 Wastewater Treatment Engineering

**2.4. Dioxygenase catalyzed reactions**

**2.5. Meisenheimer complex formation**

**2.6. Partial reduction of nitro groups**

*2.7.1. Bacterial degradation of nitrobenzene*

[68].

anion [69].

**nitrobenzene**

Different bacterial strains have been isolated from various sources, which can utilize nitroto‐ luenes as carbon source or both carbon and nitrogen source. There are several reports on different degradation pathways for mononitrotoluenes as described here.

Nitrotoluenes may be subjected to reductive pathways (formation of aminotoluenes) [70] or partial reductive pathway, wherein a nitro group is reduced to hydroxyl amino group and finally releases ammonia [71–72]. For example, during the degradation of 4-NT by *Pseudomo‐ nas* sp., initially 4-NT is converted into 4-nitrobezoic acid via the formation of 4-nitrobenzyl alcohol and 4-nitrobenzaldehyde. Then the nitro group is partially reduced to hydroxylamino derivative (rather than amino derivative), which is further converted to protocatechuate without the utilization of oxygen and release of ammonia [71, 72]. This type of mechanism was first reported for the degradation of 4-nitrobenzoate by *C. acidovorans* NBA-10 [69]. In yet another mechanism of 4-NT metabolism by *Micobacterium* sp., 4-NT was first converted to 4 hydoxyl aminotoluene followed by 6-amino-*m*-cresol. Here ammonia is released only after the ring cleavage [72].

An oxidative pathway is reported for 2-NT degradation by *Acidovorax* JS42 (Figure 7), wherein the initial oxidation of the aromatic ring takes place to form methylcatechols by simultaneous incorporation of both atoms of molecular oxygen and subsequent removal of the nitro group as nitrite by the action of a dioxygenase enzyme [64, 65, 73].

The role of mono-oxygenases and dioxygenases in the removal of nitro group from *p*-nitro‐ phenol has also been reported from a *Pseudomonas* sp. [74–76].

**Figure 7.** Degradation of 2-NT by the formation of 3-methylcatechol in *Acidovorax* sp. JS 42.

The toluene mono-oxygenase encoded by TOL plasmid oxidizes only the methyl group of 3- NT and 4-NT but not of the 2-NT [77]. Toluene dioxygenase from *Pseudomonas putida* F1 and *Pseudomonas* sp. strain JS-150 oxidatively attacks on the methyl group of 2- and 3-NT to form corresponding nitrobenzylalcohols. The enzyme, however, attacks on the aromatic ring of 4- NT to produce 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol [81]. In both cases (either with toluene mono-oxygenase or dioxygenase as described above), the nitro group was not removed from the benzene ring and mononitrotoluene isomers did not serve as growth substrate. Degradations of monosubstituted 2-, 3-, and 4-nitrotoluenes were also reported from an adapted activated sludge system [79].

The two strains of C*omamonas* JS47 and JS46 capable of degrading 4-nitrobenzoate and 3 nitrobenzoate respectively were immobilized on alginate beads jointly and separately, and these beads were loaded in the reactor and fed to different regimes of alternating nitrobenzoate isomer or mixed nitrobenzoate isomer. Through this experiment, it was deduced that same beads containing both strains were able to recover faster from change in input composition than different beads containing different strains [80].
