**2.9. Degradation of Trinitrotoluene (TNT)**

Trinitrotoluene (TNT) is very difficult to degrade [87]. The three nitro groups with a nucleo‐ philic aromatic ring structure make TNT vulnerable to reductive attack but resistant to oxygenase attack from aerobic organisms [92]. In most current reports, the reductive mecha‐ nism predominates in TNT degradation. New evidence indicates that TNT could be reduced by carbon monoxide dehydrogenase from *Clostridium thermoaceticum* [93] and by the manga‐ nese-dependent peroxidase (MnP) from the white-rot fungus *Phlebia radiata* [94]. Based on the discovery of pentaerythritoltetranitrate (PETN) reductase from *Enterobacter cloacae* PB2, French et al. [95] found that this strain could grow slowly on 2,4,6-TNT under aerobic conditions as the sole nitrogen source without the production of dinitrotoluene as an intermediate and catalyzed conversion of the TNT via a hydride–Meisenheimer complex with the nitro group released as nitrite.

Bacteria basically tends to biotransform TNT to aminonitroaromatic compounds through aerobic degradation, which in many cases turn out to be the dead-end products, and this reduced dead-end products sometimes react with themselves and form azotetranitrotoluene [96].

The removal of nitro group from the ring is essential to allow the dioxygenase to act upon it. There are very rare cases where the complete usage of TNT as the sole carbon nitrogen and energy source has also been reported. In general, most bacteria are only capable of transform‐ ing TNT to other simpler and less toxic compounds.

Due to the absence of oxygen in anaerobic processes, the formation of azonitrotoluene does not take place, thereby making the degradation through bacteria more feasible and efficient. Reduction product of TNT is very prominent in case of anaerobic process, which easily forms triaminotoluene, which is far less toxic and more soluble in water than TNT (Figures 12 and 13).

Collie et al. [116] observed the biodegradation of TNT in the liquid phase bioreactor by four different bacterial strain having pure TNT in a liquid medium. The initial concentration of TNT (70mg/L) was periodically extracted from the bioreactor for by-product identification with the help of HPLC. The bacteria used were one strain of *Enterobacter*, one strain of *Pseudomonad*, and two strains of *Alcaligenes.* Two basic intermediates, i.e., 2-amino-4,6-DNT and 4-amino-2,6 DNT, were observed with all the bacteria after 12 hours.

A bench-scale reactor by Cho and coworkers [114] used *P. putida* HK6 (collected from RDXcontaminated soils). This was used for the degradation of several nitroaromatic compounds simultaneously in one go, i.e., TNT–RDX–atrazine–simazine (TRAS). Cells were grown in a liquid media composed of 30–100 mg TNT, 5–15 mg RDX, 20–50 mg atrazine, and 5–15 mg simazine and other basal salts like K2HPO4, NaH2PO4, MgSO4 7H2O, CaCl2 2H2O, FeCl3 6H2O in appropriate quantities, and subsequent experiments on the utilization of nitroaromatic compounds were carried out using a 2.5-L bottom driving type bench-scale reactor with a water condenser at 5°C and operated at 30°C at 150 rpm. A 10% inocula of test culture was

microorganism for first 10 days, and then the whole system was further operated for 140 more days, gradually increasing the concentration of 2,4 DNT. DNT was then transformed to 2-

Trinitrotoluene (TNT) is very difficult to degrade [87]. The three nitro groups with a nucleo‐ philic aromatic ring structure make TNT vulnerable to reductive attack but resistant to oxygenase attack from aerobic organisms [92]. In most current reports, the reductive mecha‐ nism predominates in TNT degradation. New evidence indicates that TNT could be reduced by carbon monoxide dehydrogenase from *Clostridium thermoaceticum* [93] and by the manga‐ nese-dependent peroxidase (MnP) from the white-rot fungus *Phlebia radiata* [94]. Based on the discovery of pentaerythritoltetranitrate (PETN) reductase from *Enterobacter cloacae* PB2, French et al. [95] found that this strain could grow slowly on 2,4,6-TNT under aerobic conditions as the sole nitrogen source without the production of dinitrotoluene as an intermediate and catalyzed conversion of the TNT via a hydride–Meisenheimer complex with the nitro group

Bacteria basically tends to biotransform TNT to aminonitroaromatic compounds through aerobic degradation, which in many cases turn out to be the dead-end products, and this reduced dead-end products sometimes react with themselves and form azotetranitrotoluene [96].

The removal of nitro group from the ring is essential to allow the dioxygenase to act upon it. There are very rare cases where the complete usage of TNT as the sole carbon nitrogen and energy source has also been reported. In general, most bacteria are only capable of transform‐

Due to the absence of oxygen in anaerobic processes, the formation of azonitrotoluene does not take place, thereby making the degradation through bacteria more feasible and efficient. Reduction product of TNT is very prominent in case of anaerobic process, which easily forms triaminotoluene, which is far less toxic and more soluble in water than TNT (Figures 12 and 13). Collie et al. [116] observed the biodegradation of TNT in the liquid phase bioreactor by four different bacterial strain having pure TNT in a liquid medium. The initial concentration of TNT (70mg/L) was periodically extracted from the bioreactor for by-product identification with the help of HPLC. The bacteria used were one strain of *Enterobacter*, one strain of *Pseudomonad*, and two strains of *Alcaligenes.* Two basic intermediates, i.e., 2-amino-4,6-DNT and 4-amino-2,6

A bench-scale reactor by Cho and coworkers [114] used *P. putida* HK6 (collected from RDXcontaminated soils). This was used for the degradation of several nitroaromatic compounds simultaneously in one go, i.e., TNT–RDX–atrazine–simazine (TRAS). Cells were grown in a liquid media composed of 30–100 mg TNT, 5–15 mg RDX, 20–50 mg atrazine, and 5–15 mg simazine and other basal salts like K2HPO4, NaH2PO4, MgSO4 7H2O, CaCl2 2H2O, FeCl3 6H2O in appropriate quantities, and subsequent experiments on the utilization of nitroaromatic compounds were carried out using a 2.5-L bottom driving type bench-scale reactor with a water condenser at 5°C and operated at 30°C at 150 rpm. A 10% inocula of test culture was

amino-4-nitrotoluene and 4-amino-2 nitrotoluene and 2,4-diaminotoluene.

**2.9. Degradation of Trinitrotoluene (TNT)**

66 Wastewater Treatment Engineering

ing TNT to other simpler and less toxic compounds.

DNT, were observed with all the bacteria after 12 hours.

released as nitrite.

**Figure 12.** Different reductive pathways in different bacteria: (*A*) *Clostridium acetobutylicum, Escherichia coli*, *Lactobacillus* sp. [97]; (*B*) *Clostridium bifermentans* CYS-1 [98]; (*C*) *Clostridium bifermentans* LJP-1 [99]; (*D*) *Disulphovibriosp* strain B [100]; (*E*) *Disulphovibrio* sp. [101]; (*F*) *Disulphovibrio* sp. [102]; (*G*) *Methanococcus* sp. strain B [103]; (*H*) *Veillonella alkales‐ cens* [57].

grown with nitroaromatic compound, and the turbidity observed showed that the bacteria *P. putida* HK6 was able to degrade 100 mg/l TNT, 15 mg/l RDX, 50 mg/l atrazine, and simazine in 4, 24, 2, and 4 days after incubation, respectively. In this experiment, it is noteworthy that the presence of Tween-80 in the culture led to the complete degradation of TRAS compounds, whose otherwise partial degradation was TNT (80%), RDX (35%), and simazine (78%) during the incubation period [114].

**Figure 13.** Complete representation of the aerobic pathway of TNT conversion to its products performed by the group of bacteria shown in the figure: (*A*) *Bacillus* sp., *Pseudomonas aeruginosa, Staphylococcus* sp. [104]; (*B*) *Enterobacter cloacae* PB2 [105]; (*C*) *Enterobacter* sp. [89]; (*D*) *Pseudomonas aeruginosa* MA101 [106]; (*E*) *Pseudomonas florescence* [107]; (*F*) *Pseu‐ domonas florescence* B3468 [108]; (*G*) *Pseudomonas florescence* [109]; (*H*) *Pseudomonas pseudoalcaligenes* [110]; (*I*) *Rhodococcus erythropolis* [111]; (*J*) *Serratia marcensens* [112].

Similarly, the degradation of some other nitroaromatic compounds was reported on the basis of field trials. A highly active microbial consortium was chosen by Oh et al. [115] for field trials to degrade the wastewater samples having 4,6-dinitro-*ortho*-cresol (DNOC) [116]. Fixed bed column reactors were employed to increase the volume density of active biomass and to degrade DNOC in wastewater stream. In this case, glass bead matrix was used to immobilize the bacterial colonies. The mixed bacterial culture CDNOC1-3 metabolizes DNOC with concomitant liberation of nitrate. The efficiency of the bioreactor was 86%. This was substan‐ tially higher over a batch culture mode (60–65%). The following schematic diagram (Figure 14) shows the fixed bed column reactor system [116].

**Figure 14.** Schematic representation of the reactor used for the degradation of 4-nitrobenzoate and 4-aminobenzoate by *B. cepacia* PB4 in the abovementioned study [59].

A similar kind of experiment was performed in year 1999 in which *B. cepacia* strain PB4 was isolated from 4-aminobenzoate. This also possessed the capability to degrade 4-nitrobenzoate. Thus, considering both classes of the contamination to be toxic and mutagenic, an efficient strategy of decontamination was applied because *B. cepacia* was able to use both as the sole source of carbon, nitrogen, and energy. In order to prevent toxic effects, these compounds were supplied in lower concentration, i.e., 10–100 ppm and also in order to increase the efficiency of the procedure at such low concentration the degradative bacteria was immobilized on porous diatomaceous celite. This degradation was carried out in a packed bed reactor (PBR). The bioreactor consisted of a glass tube (296 × 41 mm) filled with 150 mm packed bed of celitegrade R-633 or R-635, which was placed at 30°C. It was shown that the nitroaromatic and aromatic amino compounds, which are otherwise unlikely to degrade together if present in any affected area, had been degraded simultaneously by single microorganism supply.

The eventual objective of all the biochemical and molecular characterization of the bacterial degradation of pollutants is to develop strains, which could be used in the bio remediation process. In this respect, another good field trial experiment was described by Labana et al. [117, 118] with bacterial strain *Arthrobacter protophormiae* RKJ100. The result clearly showed that the disappearance of the nitroaromatic pollutants.

Similarly, the degradation of some other nitroaromatic compounds was reported on the basis of field trials. A highly active microbial consortium was chosen by Oh et al. [115] for field trials to degrade the wastewater samples having 4,6-dinitro-*ortho*-cresol (DNOC) [116]. Fixed bed column reactors were employed to increase the volume density of active biomass and to degrade DNOC in wastewater stream. In this case, glass bead matrix was used to immobilize the bacterial colonies. The mixed bacterial culture CDNOC1-3 metabolizes DNOC with concomitant liberation of nitrate. The efficiency of the bioreactor was 86%. This was substan‐ tially higher over a batch culture mode (60–65%). The following schematic diagram (Figure

CH3 NO2

(unreactive polymer)

Ar-N+=N-Ar' O Azoxynitrotoluene

4-ADNT 2-ADNT

CH3 NH2

O2N

*F*

OH

HO OH

NH2

*F*

NH4 +

> CH3 NHOH

NO2

*H,I*

CH3 O2N NHOH

NO2

SH CH3

NHOH

Yellow product

O2N NH2 CH3

*A,D,F*

O2N

OH HO OH

2,4-DANT

NO2

O2N

<sup>H</sup> Meisenheimer

NO2

\_ \_

*J*

CH3 O2N NO2

CH3 NO2

O2N

COOH NO2

O2N

68 Wastewater Treatment Engineering

O2N

NH2

CH3 NO2

*H*

NHCOCH3

NH2

CH3 O2N NO2

> NO2 H H

H

*erythropolis* [111]; (*J*) *Serratia marcensens* [112].

NHOH

NO2

TNT

*B,E*

CH3 O2N NO2

> NO2 H H

complex

CH3 NO2

NO2

**Figure 13.** Complete representation of the aerobic pathway of TNT conversion to its products performed by the group of bacteria shown in the figure: (*A*) *Bacillus* sp., *Pseudomonas aeruginosa, Staphylococcus* sp. [104]; (*B*) *Enterobacter cloacae* PB2 [105]; (*C*) *Enterobacter* sp. [89]; (*D*) *Pseudomonas aeruginosa* MA101 [106]; (*E*) *Pseudomonas florescence* [107]; (*F*) *Pseu‐ domonas florescence* B3468 [108]; (*G*) *Pseudomonas florescence* [109]; (*H*) *Pseudomonas pseudoalcaligenes* [110]; (*I*) *Rhodococcus*

14) shows the fixed bed column reactor system [116].

Similarly, *Pseudomonas* sp. ST53 was also used as a microbe to degrade TNT and other explosives, but it is best suited on land and water only when the contamination is low [119]. Qureshi et al. [120] reported a bacterial strain *Arthrobacter* sp. HPC1223, which was capable of degrading 2,4,6-trinitrophenol prominently. This also poses the capability to degrade dinitro‐ phenol and mononitrophenol showing broad substrate specificity.
