Nitrate Esters and Methylene Nitramine Cyclic Esters

Nitrate esters, including glycerol trinitrate (nitroglycerin) and pentaerythritol tetra nitrate (PETN) (Figure 1b) are pharmacologically active at low concentrations and at high concentrations they are acutely toxic (Gorontzy et al. 1994). Other nitrate esters, like cellulose nitrate (Figure 1c), are nontoxic and relatively stable. Naturally occurring, biologically generated, organic nitrate esters appear very rarely. There is a single report of an insect sex pheromone that contains a nitrate ester (Hall et al. 1992).

Another group of energetic compounds that are significant environmental contaminants are cyclic trimers of methylene nitramine (Gorontzy et al. 1994) (Figure 1d and e). This group includes the most powerful military explosives in use today, RDX (cyclotrimethlenetrinitramine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine). RDX is often mixed with TNT and is therefore a common environmental co-contaminant. It also has a limited aqueous solubility of 42.3 mg/l at 20°C (Gorontzy et al. 1994). HMX is used in shaped charges or as a rocket propellant and is a by-product of

In contrast to the newly evolved pathways we see in the bacteria, fungi have the capability to biodegrade explosives using existing, initially co-metabolic, pathways that lead to mineralization. Parrish (1977) published the results of a screen of the explosive degrading capability of 190 fungi. This study showed that fungi were capable of TNT degradation, but the sensitivity of the fungi to levels exceeding 20 ppm was such that Parrish discounted their use in bioremediation. Bennett (1994) comments that the interpretation of results from this work appears to have delayed more serious consideration of fungi as explosive degraders for some years. However, more recent screens have shown that the ability to degrade TNT to some degree was distributed across many genera within the Zygomycota, Ascomycota, and Basidiomycota (Scheibner et al. 1997b; Weber et al. 2002). Recent data suggests that under the right conditions fungi are capable of achieving mineralization of TNT at rates far higher than bacteria (Hawari et al. 2000a).

The best studied fungal TNT decomposer is the lignin decomposing Basidiomycete Phanaerochaeta chryso-sporium. This fungus was first noted for its lignolytic capabilities when it was found to cause the overheating of woodchip piles (Burdsall 1981) but to date its natural niche is unknown. It has been demonstrated in numerous studies to be capable of TNT degradation (Esteve-Nunez et al. 2001; Fernando et al. 1990). The initial reduction of TNT is independent of the ligninase enzymes associated with its mineralization, and is mediated by nonspecific nitro-reductases that catalyze the conversion of highly oxidized nitro-functional groups to mono- and di-amino toluenes (ADNT and DANT), and nitroso- and hydroxylamine containing intermediates (Esteve-Nunez et al. 2001). Stahl and Aust (1993) demonstrated that this reaction is dependent on the presence of a living mycelium and appears to be associated with a membrane bound redox system. This work was recently confirmed by Van Aken et al. (1999). However, Michels and Gottschalk (1995) have reported intracellular NADPH+ H+ dependent TNT reductase activity. A further mechanism was reported by Eiler et al. (1999). Using Bjerkandera adusta, they observed TNT breakdown was associated with a microsomal cytochrome P 450.

The initial co-metabolic reactions increase the electron density of the aromatic ring and facilitate electrophilic attack by lignin-degrading enzymes (Field et al. 1993). Lignin and TNT degradation occurs by a series of co-metabolic, synergistic reactions that involve three enzymes, manganese peroxide (MnP), lignin peroxidase (LiP), and laccase. The peroxidase enzymes are haem-containing glycoproteins that require hydrogen peroxide to function and they catalyze single electron oxidations that generate free radicals. Hydrogen peroxide is generated by oxidase enzymes including glyoxyl- and aryl-oxidase. Laccase is a copper containing phenol oxidase, which uses molecular oxygen as its terminal electron acceptor (Fritsche et al. 2000). Laccase can catalyze both polymerization and depolymerization reactions via oxidation (Harvey and Thurston 2001). Subsequent mineralization steps of TNT reduction products, including nitroso-toluene (NST), ortho-hydroxyl amino 2,4-dinitrotoluene (HADNT), ADNT, and DANT are illustrated in a putative pathway constructed by Hawari et al. (2000a) (Figure 2) where the initial products are azo, azoxy, phenolic, and acylated derivatives. These compounds are already known to be mineralized by white rot fungi (Esteve-Nunez et al. 2001). However in P. chrysosporum, HADNT is known to inhibit veratryl alcohol oxidation by LiP (Bumpus and Tartako 1994). The veratryl alcohol to veratryl aldehyde conversion is essential for the production of free radicals that are involved in the oxidation of primary substrates of LiP. Thus biodegradation of TNT will be inhibited if levels of HADNT are allowed to accumulate. Bumpus and Tatarko (1994) reported that levels as low as 30 mM will inhibit enzyme activity. This sensitivity is regarded as a key limitation to the use of this fungus in biodegradation in the field (Michels and Gottschalk 1995).

For several years, research concentrated on P. chryso-sporium as a prime candidate for explosive degradation. However, other fungi have been screened and Table 1 summarizes published work to date. In some of the other fungal species screened, tolerance to TNT and its breakdown products can be much higher than that of P. chrysosporium. For example, Rhizopus nigricans was reported as being able to remove TNT from a medium containing 100mg/ml TNT (Klausmeier et al. 1974) and Irpex lacteus tolerated up to 50 mg/ml of TNT, and was able to degrade TNT by more than one route, forming transient hydride-Meisen-heimer complexes (Kim and Song 2000).

Figure 2 One of the putative pathways of TNT degradation by fungi.
Table 1 Species of ñingi reported as capable of TNT degradation







G. separium

Brown rot



P. chrysosporium

White rot

P. sórdida

White rot


P. radiata

White rot

P. brevispora

White rot

P. ochracheofidva

White rot


T. versicolor

White rot

T. suevolens

White rot

L. sulphureas

White rot

F. fomentarius

White rot

S. commune

White rot

C. versicolour

White rot

P. coccineus

White rot

L. lacteus

White rot


T. fibrillosa


B. adusta

White rot



H. annosum

White rot



H. fasciculare

White rot

K. mutablis

White rot

N. frowardii

White rot

S. ruguloso-annulata


C. dusenii

White rot


C. stercorus

White rot


C. (Lepista) nebularis

C. odora


A. muscaria

H. fasciculare

White rot


A. aestivalis

A. bisporus

A. praecox



S. granulatus


S. variegates


P. ti neto rus



P. frequentens


Pénicillium sp.

P. chrysogenum

A. terreus


Wood Hess et al. 1998

Wood ATCC 24725

Wood HHB 8922, Donelly et al. (1997)

Wood ATCC 64658, Van Aken et al. (1999)

Wood HHB7030, Donelly et al. (1997)

Wood Scheibner et al. (1997a, b)

Wood DSM 11269, Scheibner et al. (1997a, b)

Wood Scheibner et al. (1997a, b)

Wood Kim and Song (2000)

Wood KR11W, KR 65W, Kim and Song (2000)

Wood Kim and Song (2000)

Wood Kim and Song (2000)

Ectomycorrhizal Meharg et al. 1997

Wood Field et al. 1993

Wood TM5P2. Scheibner et al. (1997a, b)

Wood ATCC 201144, DSM 1239, Scheibner et al. (1997a, b)

Litter DSM 11373, TME, Scheibner et al. (1997a, b)

Wood DSM 11238, TMb 12, Scheibner et al. (1997a, b)

Wood 36910 Donelly et al. (1997)

Litter TM, Scheibner et al. (1997a, b)

Litter TM3, Scheibner et al. (1997a, b)

Ectomycorrhizal Scheibner et al. (1997a, b)

Wood TM5.2

Litter TMAEST1, Scheibner et al. (1997a, b)

Litter MWA 80-7, Scheibner et al. (1997a, b)

Litter TM 70 84, TM70.3.1 Scheibner et al. (1997a, b)

Ectomycorrhizal Scheibner et al. (1997a, b)

Ectomycorrihazal Meharg et al. 1997

Ectomycorrihizal Meharg et al. 1997

Ubiquitous ATCC 96048, Scheibner et al. (1997a, b)

Ubiquitous DSM 11168, Scheibner et al. (1997a, b)

Ubiquitous IFO 31249, Kim and Song 2000

Ubiquitous MW 458, Scheibner et al. (1997a, b)

Ov Ov

13 2




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