*2.8.1. 2,4- and 2,6-dinitrotoluene*

*Burkholderia* sp. strain DNT and strain R34 mineralized 2,4-DNT with a nitrite removal pathway involving dioxygenase and mono-oxygenase enzyme. Here both nitro groups are not removed simultaneously but in a stepwise fashion. First, dinitrotoluene dioxygenase (DNTDO) attacks on the benzene ring converting DNT into 4-methyl-5-nitrocatachol (4M5NC) with the simultaneous removal of a nitrite group. Further, MNC mono-oxygenase removes another nitrite and converts the substrate into 2-hydroxy-5-methylquinone. These dioxyge‐ nase and mono-oxygenase enzymes have been cloned and characterized [68, 85]. This 2 hydroxy-5-methylquinone eventually leads to the formation of 2,4,5-trihydroxytoluene, a reaction that is catalyzed by HMQ reductase. Ring fission of 2,4,5-trihydroxytoluene likely occurs at position 5,6 of the aromatic ring to yield 2,4-dihydroxy-5-methyl-6-oxo-hexa-2,4 dienoic acid as ring cleavage product (Figure 10).

Nishino et al. [61, 86] isolated *Burkholderia cepacia* strain JS850 and *Hydrogenophaga paleronii* strain JS863 that were able to mineralize 2,4-DNT in the same way but degraded 2,6-DNT in a different way. When 2,4- and 2,6-DNT were used as the sole source of carbon and nitrogen together, the dioxygenation of 2,6-DNT to 3-methyl-4-nitrocatachol (3M4NC) was the initial reaction, accompanied by the release of nitrite. 3M4NC was then subjected to *meta*-ring cleavage (Figure 11) without releasing the second nitro group prior to the ring cleavage. Although 2,4-DNT-degrading strains also could convert 2,6-DNT to 3M4NC, further catabo‐ lism was halted at the point. The pathway for 2,4-DNT degradation was different from that for 2,6-DNT degradation. In the latter case, 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid was the first ring fission product. How 3M4NC is converted to 2-hydroxy-5-nitro-6-oxohepta-2,4 dienoic acid is unknown. In this pathway, the second nitrite group is released at the latter stages of the pathway. Position 3 methyl group appears as the determinant recognized by the initial dioxygenase to produce highly specific 3M4NC in the 2,6-DNT pathway proposed. The gene encoding this dioxygenase showed a nucleotide sequence similar to the α subunit among nitroarene dioxygenase.

The genes for the initial dioxygenases involved in 2,4-DNT and 2,6-DNT degradation are all closely related, but the enzymes are produced at low constitutive levels [27, 86]. After initial dioxygenation, the two pathways appear to diverge (Figures 10 and 11).

How does DNT degradation get affected by the presence of both isomers is important since 2,4-DNT and 2,6-DNT are produced in a 4:1 ratio [87]. These are often present together in munitions plant wastewater. Lendenmann and Spain [88] initially failed to observe the degradation of 2,4-DNT and 2,6-DNT simultaneously. Subsequently, an aerobic biofilm, initially fed with low concentrations of DNT mixture, was tested. These concentrations were then gradually increased and exhibited mineralization rates of 98% and 94% for 2,4- and 2,6- DNT, respectively. The nitrogen was released as nitrite, reflecting oxidative bacterial activity. Isomer concentration needed to be kept below inhibitory levels as high concentrations of each isomer inhibited the degradation of the other. The simultaneous degradation of 2,4- and 2,6- DNT may be unpredictable until an adapted population is established [87].

**Figure 10.** 2,4-DNT metabolism pathway in *Burkholderia* sp. strain DNT and strain R34.

**2.8. Degradation of dinitrotoluene**

dienoic acid as ring cleavage product (Figure 10).

*Burkholderia* sp. strain DNT and strain R34 mineralized 2,4-DNT with a nitrite removal pathway involving dioxygenase and mono-oxygenase enzyme. Here both nitro groups are not removed simultaneously but in a stepwise fashion. First, dinitrotoluene dioxygenase (DNTDO) attacks on the benzene ring converting DNT into 4-methyl-5-nitrocatachol (4M5NC) with the simultaneous removal of a nitrite group. Further, MNC mono-oxygenase removes another nitrite and converts the substrate into 2-hydroxy-5-methylquinone. These dioxyge‐ nase and mono-oxygenase enzymes have been cloned and characterized [68, 85]. This 2 hydroxy-5-methylquinone eventually leads to the formation of 2,4,5-trihydroxytoluene, a reaction that is catalyzed by HMQ reductase. Ring fission of 2,4,5-trihydroxytoluene likely occurs at position 5,6 of the aromatic ring to yield 2,4-dihydroxy-5-methyl-6-oxo-hexa-2,4-

Nishino et al. [61, 86] isolated *Burkholderia cepacia* strain JS850 and *Hydrogenophaga paleronii* strain JS863 that were able to mineralize 2,4-DNT in the same way but degraded 2,6-DNT in a different way. When 2,4- and 2,6-DNT were used as the sole source of carbon and nitrogen together, the dioxygenation of 2,6-DNT to 3-methyl-4-nitrocatachol (3M4NC) was the initial reaction, accompanied by the release of nitrite. 3M4NC was then subjected to *meta*-ring cleavage (Figure 11) without releasing the second nitro group prior to the ring cleavage. Although 2,4-DNT-degrading strains also could convert 2,6-DNT to 3M4NC, further catabo‐ lism was halted at the point. The pathway for 2,4-DNT degradation was different from that for 2,6-DNT degradation. In the latter case, 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid was the first ring fission product. How 3M4NC is converted to 2-hydroxy-5-nitro-6-oxohepta-2,4 dienoic acid is unknown. In this pathway, the second nitrite group is released at the latter stages of the pathway. Position 3 methyl group appears as the determinant recognized by the initial dioxygenase to produce highly specific 3M4NC in the 2,6-DNT pathway proposed. The gene encoding this dioxygenase showed a nucleotide sequence similar to the α subunit among

The genes for the initial dioxygenases involved in 2,4-DNT and 2,6-DNT degradation are all closely related, but the enzymes are produced at low constitutive levels [27, 86]. After initial

How does DNT degradation get affected by the presence of both isomers is important since 2,4-DNT and 2,6-DNT are produced in a 4:1 ratio [87]. These are often present together in munitions plant wastewater. Lendenmann and Spain [88] initially failed to observe the degradation of 2,4-DNT and 2,6-DNT simultaneously. Subsequently, an aerobic biofilm, initially fed with low concentrations of DNT mixture, was tested. These concentrations were then gradually increased and exhibited mineralization rates of 98% and 94% for 2,4- and 2,6- DNT, respectively. The nitrogen was released as nitrite, reflecting oxidative bacterial activity. Isomer concentration needed to be kept below inhibitory levels as high concentrations of each isomer inhibited the degradation of the other. The simultaneous degradation of 2,4- and 2,6-

dioxygenation, the two pathways appear to diverge (Figures 10 and 11).

DNT may be unpredictable until an adapted population is established [87].

*2.8.1. 2,4- and 2,6-dinitrotoluene*

64 Wastewater Treatment Engineering

nitroarene dioxygenase.

Although nitrotoluene degraders are widely distributed at contaminated sites, the contami‐ nants still persist for very long periods, leaving unanswered questions as to why biodegrada‐ tion is ineffective to remove them. Efficient anaerobic pathways for the degradation either of mono- or dinitrotoluenes are not known, and 2,3-DNT currently does not appear to be degradable [87].

**Figure 11.** 2,6-DNT metabolism pathway in *Burkholderiacepacia* strain JS850 and *Hydrogenophaga paleroni* strain JS863.

Bae et al. [89] found that in an anaerobic fluidized-bed granular carbon bioreactor, 2,4 DNT can be converted completely to 2,4-diamino toluene, which subsequently mineralized in batch activated sludge reactor. Paca et al. [90] took a mixture of microbes found in the mononitro‐ toluene, 2,4-dintrotoluene, and 2,6-dinitrotoluene contaminated soil. These microbes were extracted and immobilized on the packing material of the packed bed reactor (PBR). Varying concentrations of 2,4-DNT and 2,6-DNT were used. In this case, two types of packing material were used out of which the reactor packed with Poraver removed 97% DNT in 11 days and the one packed with fine clay achieved the efficiency of 78%. After 20 days, the metabolites detected were 2-amino-4-nitrotoluene and 2,4-diamino toluene.

Wang et al. [91] reported that in wastewater enriched with contaminated DNT taken from Qingyang chemical industry with a DNT concentration of 3.55–95.65 mg/L, ethanol was mixed in the wastewater to act as an electron donor. The reactor in this case was made of polymethyl metacrylate containing polyurethane foams for microorganism immobilization. The microor‐ ganism used was B925. Initially, the reactors were domesticated and immobilized with 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 amino-4-nitrotoluene and 4-amino-2 nitrotoluene and 2,4-diaminotoluene.
