Reduction of the Nitro Group

Anaerobic Reduction

The most common degradation pathway for TNT proceeds along the sequential reduction of the nitro groups. Following sequential steps of two-electron transfers, the corresponding mononitroso, monohydroxylamino and monoamino derivatives

Aerobic

Aerobic

NHCOCH 3

Funketal., 1993

GHcrease and Murphy, 1995

Vanderbergetal., 1995

2-aminodinitrotoluene 4-aminodinitrotoluene (2-ADNT) (4-ADNT)

GHcrease and Murphy, 1995

Vanderbergetal., 1995

Deaminationand formation of nitrogen free metabolites

2-aminodinitrotoluene 4-aminodinitrotoluene (2-ADNT) (4-ADNT)

HO OH

Naumova et a|., 1988

2,4-diaminonitrotoluene (2,4-DANT)

Anaerobic

Bamberger rearrangement and formation ^nhoh of phenolic amine derivate

Hughes et alv 1998

NHCOCH 3

Funketal., 1993

Boopathyand Kulpa, 1992

triaminotoluene

(tah tetraamino azo derivates

Funketal., 1993

methylphloroglucinol p-cresol

Fig. 5.2 Reduction pathway of TNT, and further transformation of the reduced derivatives under aerobic and anaerobic conditions of TNT are sequentially formed (Fig. 5.2). Reduction of the monoamino derivatives can then proceed further to produce a diamino derivative.

The initial reduction toward the formation of diamino derivatives occurs under both aerobic and anaerobic conditions. It is performed by a large variety of microbial strains (Table 5.3) and it can be catalyzed by various enzymes, such as nitroreductase, aldehyde oxidase, dihydrolipic amide dehydrogenase, cytochrome b5 reductase, diaphorases, hydrogenases, xanthine oxidase, and carbon monoxide dehydrogenase (Esteve-Nunez et al. 2001). The reduction of the nitro group has also been shown to occur alternatively via two sequential steps of single-electron

Table 5.3 Microbial isolates of TNT degraders

Isolate

Conditions

Suggested pathway

Reference

Acinetobacter johnsonii

Aerobic

Pathway was not determined

Fuller and Manning (1997)

A. juiiii A8

Aerobic

TNT transforms to 2,6-DN-4-nitrosotoluene, 4-AM-2.6-dinitrotoluene, 4-AM-2,6-dinitrobenzoic acid

Soojhawon et al. (2005)

Agrobacteriurn sp. 2PC

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Alcaligenes eutroplius

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Anabaena sp.

Detection of azoxy-tetranitrotoluene isomers and hydroxylaminodinitrotoluene

Pavlostathis and Jackson (1999)

Arthrobacter globiforrnis

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Arthrobacter sp. RP17

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Bacillus cereus

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Bacillus sp.

Aerobic

(1) Release of nitrite, with final formation of toluene; (2) sequential reduction to the final formation of TAT

Kalafut et al. (1998)

B. subtilis

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Cellulomonas sp. ES6

Anaerobic

Amino and hydroxylamino derivatives

Borch et al. (2005)

Clostridium acetobutylicum

Anaerobic

Degradation via Bamberger rearrangement of 2,4-diHA-6-nitrotoluene to either 2-AM-4-HA-5-hydroxyl-6-nitrotoluene (4-AM-6-HA-3-methyl-2 nitrophenol) or 2-HA-4-AM-5-hydroxyl-6-nitrotoluene (6-AM-4-HA-3-methyl-2-nitrophenol).

Hughes et al. (1998)

C. acetobutylicum

Anaerobic

(1) Partial reduction by Bamberger rearrangement to HA intermediate; (2) TNT-TAT

Khan et al. (1997), Hughes et al. (1998)

C. bifermentans

Anaerobic

Detection of TAT, phenolic products of TAT hydrolysis, and TAT and pyruvic aldehyde condensation products

Lewis et al. (1996)

C. bifermentans

Anaerobic

Reductive TNT transformations to form TAT and phenolic products of TAT hydrolysis

Regan and Crawford (1994), Lewis et al. (1996)

C. bifermentans ATCC 638

Anaerobic

Reductive pathway

Ederer et al. (1997)

C. bifermentans KMR-1

Anaerobic

Reductive pathway

Ederer et al. (1997)

(continued )

Isolate

Conditions

Suggested pathway

Reference

C. pasteurianum DSM 525

Anaerobic

Reductive TNT transformations to form TAT and further transformation of TAT to undefined products

Preuss et al. (1993)

C. sordellii

Anaerobic

Reductive pathway

Ederer et al. (1997)

C. sporogenes

Anaerobic

Reductive pathway

Ederer et al. (1997)

C. thennoaceticurn

Anaerobic

Huang et al. (2000)

Corynebacterium glutarnicurn

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Corynebacterium sp. Nap2

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Cytopliaga pectinovora

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Desulfovibrio gigas

Anaerobic

Boopathy and Manning (1996)

D. indolicum

Anaerobic

Boopathy et al. (1997)

Desulfovibrio sp.

Anaerobic

Detection of TNT, DANT and TAT isomers

Drzyzga et al. (1999)

Desulfovibrio sp. strain B

Anaerobic

In the absence of external N sources, reductive deamination of amino derivatives to the final formation of toluene

Boopathy et al. (1993)

D. vulgaris

Anaerobic

Boopathy and Kulpa (1994)

Enterobacter cloacae PB2

Aerobic

Reduction to hydride-Meisenheimer complex with release of nitrite

French et al. (1998)

Escherichia coli

Aerobic

Pathway was not determined

Fuller and Manning (1997)

E. coli

Anaerobic

Reductive pathway

Ederer et al. (1997)

Flavobacteriurn odoraturne

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Klebsiella sp. IPC

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Klebsiella sp. CI

Anaerobic

Reduced to hydroxylamino dinitrotoluenes, aminodinitrotoluenes and to nitrite via denitration

Kim et al. (2002)

Lactobacillus acidophilus

Anaerobic

Reductive pathway

Ederer et al. (1997)

L. casei

Anaerobic

Reductive pathway

Ederer et al. (1997)

L. lactis

Anaerobic

Reductive pathway

Ederer et al. (1997)

Methanococcus deltae

Boopathy (1994)

Methanococcus sp. strain B

Anaerobic

Boopathy (1994)

Isolate

Conditions

Suggested pathway

Reference

M. thennolithotrophicus

Boopathy (1994)

Methylobacterium sp. BJ001

Anaerobic

Reduced to ADNT

Van Aken et al. (2004)

Micrococcus Intens

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Mycobacterium sp. strain HL 4-NT-1

Aerobic

Formation of a hydride-Meisenheimer complex

Vorbeck et al. (1994)

M. vaccae strain JOB-5

Aerobic

(1 ) Detection of products as 4-AM-2,6-dinitrobenzoic acid and

Vanderberg et al. (1995)

2,4-diAM-6-nitrobenzyl methyl ether; (2) detection of

azoxy compounds; (3) evidence of ring cleavage

Myxococcus xantlius

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Nocardiodes CB22-2

Aerobic

Dinitration leads to formation of Meisenheimer complex

Behrend and Heesche-Wagner

(1999)

Pseudomonas

Aerobic

Production of hydride-Meisenheimer complex and

Duque et al. (1993)

transformation to DNT, NT and toluene

P. aeruginosa

Aerobic

(1) Release of nitrite, with final formation of toluene; (2)

Kalafut et al. (1998)

sequential reduction to the final formation of TAT

P. aeruginosa

Aerobic

Detection of HA and AM derivatives

Oh et al. (2001)

P. aeruginosa

Aerobic

Pathway was not determined

Fuller and Manning (1997)

P. aeruginosa strain MA01

Aerobic

Oxidation of ADNT isomers to N(4)-acetyl-2,4-diAM-6-

Alvarez et al. (1995)

nitrotoluene and tetranitroazoxytoluene isomers

P. cepacia

Aerobic

Pathway was not determined

Fuller and Manning (1997)

P. fluorescein

Aerobic

Reduction by addition of hydride to form dihydride

Pak et al. (2000)

Meisenheimer complex to catalyze reduction of nitro group

P. fluorescein

Aerobic

Pathway was not determined

Fuller and Manning (1997)

P. pseudoalcaligenes JS52

Aerobic and

Detection of mono- and di-HADNTs, monoAM monoHA NT,

Fiorella and Spain (1997)

anaerobic

and monoAM DNT

P. putida

Aerobic

Pathway was not determined

Fuller and Manning (1997)

P. putida HK-6

Aerobic

Reductive pathway

Cho et al. (2008)

(continued )

Isolate

Conditions

Suggested pathway

Reference

P. piitida KP-T20 1

Aerobic

Amino derivatives of TNT, and denitrated products (DNT)

Park et al. (2003)

Pseudomonas sp. clone A

Aerobic

Formation of hydride-Meisenheimer complex, followed by denitration to form 2,4-DNT and an unidentified compound. AM and HA products were also detected, and condensed to azoxytoluenes

Haidour and Ramos (1996)

Pseudomonas sp. DFC49

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Pseudomonas sp. JLR11

Anaerobic

Mineralization, via the formation of products such as 2,4,6-trinitrobenzaldehyde, 2-nitro-4-hydroxybenzoic acid, and 4-hydroxybenzaldehyde, and 4-hydroxybenzoic acid

Esteve-Nunez and Ramos (1998)

Pseudomonas sp. JS150

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Pseudomonas sp. TollA

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Ralinella aquitilis BFB

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Raoultella terrigena HB

Aerobic

AM derivatives and azoxy dimers

Claus et al. (2007)

Rliodococcus erythropolis

Aerobic

Dinitration leads to formation of Meisenheimer complex by F420 reductase and hydride transferase

Rieger et al. (1999)

R. etythropolis

Aerobic

Formation of hydride and dihydride TNT-Meisenheimer complexes

Vorbeck et al. (1998)

R. etythropolis

Aerobic

Pathway was not determined

Fuller and Manning (1997)

R. globerulus

Aerobic

Pathway was not determined

Fuller and Manning (1997)

R. rhodocrous

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Rliodococcus sp. TF2

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Salmonella typhimutium

Anaerobic

Reductive pathway

Ederer et al. (1997)

Serratia marcescens

Aerobic

Not defined. ADNT isomers were detected

Montpas et al. (1997)

SPlb (coryneform)

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Sphingomonas capsulata

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Staphylococcus sp.

Aerobic

(1) Release of nitrite, with final formation of toluene; (2) sequential reduction to final formation of TAT

Kalafut et al. (1998)

Isolate

Conditions

Suggested pathway

Reference

Streptomyces albus

Aerobic

Pathway was not determined

Fuller and Manning (1997)

S. chrornofuscus All

Aerobic

Pasti-Grigsby et al. (1996)

S. griseus

Aerobic

Pathway was not determined

Fuller and Manning (1997)

Sulphate-reducing bacterium

Anaerobic

TNT reduction to TAT and further transformation to unknown

Preuss et al. (1993)

products

Veillonella alkalescens

McCormick et al. (1976)

Yarrowia lipolytica AN-L15

Aerobic

Detection of eight different hydride complexes and nitrite

Ziganshin et al. (2007)

release from the ring

DN dinitro, AM amino, HA hydroxylamino, DANT diaminonitrotoluene, ADNT aminodinitrotoluene, TAT triaminotoluene

DN dinitro, AM amino, HA hydroxylamino, DANT diaminonitrotoluene, ADNT aminodinitrotoluene, TAT triaminotoluene transfers, forming first a nitroanion radical and then the corresponding nitroso derivative. This is catalyzed by the oxygen-sensitive nitroreductase enzymes, which are found in bacteria such as Clostridium (Angermaier and Simon 1983) and Escherichia coli (Peterson et al. 1979).

Because of the high electron deficiency on the nitro groups of TNT, its microbial degradation is often initiated by reductive rather than oxidative reactions, even under aerobic conditions (Vorbeck et al. 1998). Nevertheless, further transformation of the mono and diamino derivatives toward the formation of the most reduced product—triaminotoluene (TAT)—proceeds only under strictly anaerobic conditions in which redox potential values are essentially below -200 mV (Hawari et al. 2000a). Thus, under oxic conditions, diamino derivatives tend to accumulate, while the presence of TAT is indicative of strictly reduced conditions.

A few studies have aimed to identify further transformation steps of TAT under anaerobic conditions. Hawari et al. (1998) identified its further transformation to tetraaminoazo derivatives in an anaerobic sludge, which, in turn, disappeared from the solution and was suspected of polymerization (Hawari et al. 1998). Boopathy and Kulpa (1992) detected toluene as a product in TNT degradation by Desulfovibrio sp. strain B. They proposed that the toluene was formed by reductive elimination of the amino groups from TAT, but this pathway was never verified. Funk et al. (1993) identified the formation of para-hydroxytoluene (p-cresol) and methylphloroglucinol (2,4,6-trihydroxytoluene) in anaerobic mixed cultures. They suggested that these products were formed from TAT.

The reductive pathway of TNT towards the formation of amino derivatives may be desirable in contaminated environments, since the toxicity and mutagenic characteristics of the amino derivatives are found to be morally lower than those of TNT (Drzyzga et al. 1995; Lachance et al. 1999; Neuwoehner et al. 2007). In addition, the amino derivatives, and most significantly TAT, present higher sorption characteristics than TNT itself, with TAT even presenting irreversible sorption to soil particles (Daun et al. 1998; Achtnich et al. 1999). This strong affinity to soil material decreases the pollutant concentration in water. Nevertheless, it remains in the environment.

Some studies have shown that the anaerobic reductive pathway does not necessarily proceed to TAT, but to the alternative transformation of the partly reduced monohydroxylamine derivatives to dihydroxylamine derivatives which may be further transformed to phenolic amine products (Hughes et al. 1998). This was shown to occur anaerobically via Bamberger rearrangement of 2,4-dihydroxyla-mino-6-nitrotoluene by anaerobic Clostridium acetobutylicum cell extracts, as well as in whole-cell systems. The products of this mechanism were identified as either

2-amino-4-hydroxylamino-5-hydroxyl-6-nitrotoluene (4-amino-6-hydroxylamino-

3-methyl-2-nitrophenol) or 2-hydroxylamino-4-amino-5-hydroxyl-6-nitrotoluene (6-amino-4-hydroxylamino-3-methyl-2-nitrophenol). Similar products were identified during incubation of TNT with the enzyme CO dehydrogenase purified from Clostridium thermoaceticum (Huang et al. 2000).

Aerobic Reduction

Reduced TNT derivatives are also commonly detected under aerobic conditions (Table 5.3). Nevertheless, under these conditions, the mono and diamino derivatives often accumulate in the medium without further metabolism (Esteve-Nunez et al. 2001) and the formation of TAT is halted. In the presence of oxygen, however, it has been shown that both the nitroso and the monohydroxylamino metabolites can follow an alternative abiotic transformation pathway to form tetranitroazoxytoluene dimmers, also referred as azoxytetranitrotoluene as shown in Fig. 5.2 (McCormick et al. 1976; Hai'dour and Ramos 1996). These azoxy products were shown to cause a higher rate of mutations than TNT (George et al. 2001) and were thought to suppress the degradation of RDX and HMX which may coexist with TNT in the environment (Sagi-Ben Moshe et al. 2009). The accumulation of azoxy derivatives in the environment is hence clearly undesirable, but laboratory experiments have shown that these derivatives themselves may further degrade in microcosm experiments with the fungal strain Phanerochaete chry-sosporium (Hawari et al. 1999), or in soil slurries with a mixed culture (Sagi-Ben Moshe et al. 2009), as well as in other experimental systems. In these two examples, complete disappearance of tetranitroazoxytoluene was achieved within a few weeks, indicating the non-recalcitrant nature of the compound. Its removal was followed by its further transformation to azo derivatives (tetranitroazotolu-ene), which, in turn, formed hydrazo derivatives (tetranitrohydrazotoluene), that also disappeared from the solution. Sorption of the azoxy metabolite to soil material in slurries was shown to occur in trace amounts (Sagi-Ben Moshe et al. 2009).

Under aerobic conditions, the amino derivatives may alternatively follow deaminization (Naumova et al. 1988), and be transformed to benzoic acid (Vanderberg et al. 1995) or N-acetylamino derivatives (Gilcrease and Murphy 1995). Thus, although the amino derivatives are often accumulated under aerobic conditions, they should not be treated as dead-end products.

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