Biodegradation of nitroaromatics and other nitrogen-containing xenobiotics

Biodegradation of nitroaromatics and other nitrogen-containing xenobiotics

Jing Ye, Ajay Singh and Owen P. Ward*Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1*Author for correspondence: Tel.: þ1-519-888-4567 ext. 2427, Fax: þ1-519-746-0614,E-mail: [email protected]

Received 25 March 2003; accepted 25 August 2003

Keywords: Glycerol trinitrate, microorganisms, nitrobenzene, nitrobenzoate, nitrophenols, nitrotoluene, nitrateesters, RDX, s-Triazine, TNT

Summary

Nitroaromatic compounds constitute a major class of widely distributed environmental contaminants. Compoundslike nitrobenzene, nitrotoluenes, nitrophenols, nitrobenzoates and nitrate esters are of considerable industrialimportance. They are frequently used as pesticides, explosives, dyes, and in the manufacture of polymers andpharmaceuticals. Many nitroaromatic compounds and their conversion products have been shown to have toxic ormutagenic properties. Most of them are biodegradable in nature by various microorganisms. However, mostcontaminated environments have combinations of nitroaromatic compounds present, which complicates thebioremediation efforts. During the last 10 years, research on the biodegradation of nitroaromatic compounds hasyielded a wealth of information on the microbiological, biochemical and genetic aspects of the process. Newmetabolic pathways have been discovered and genes and enzymes responsible for key transformation reactions havebeen identified and characterized. Knowledge and advances in pathway engineering have helped furtherunderstanding of the nature of nitroaromatic biodegradation and the development of bioremediation solutions.In this paper, an overview of recent developments on the biodegradation of nitrogen-containing xenobiotics ispresented.

Introduction

A wide variety of nitrogen-containing organic com-pounds exist in the environment. They have applicationsas explosives, pesticides, plastics, dyes, pharmaceuticalsand petroleum products, and they take the form ofnitroaromatics, N-heterocyclic compounds and nitrateesters and some other nitrogen-containing structures(Yinon 1990). The industrial manufacture, transportand use of these compounds have generated a seriousdisposal problem. These substances, their byproductsand metabolites can be highly toxic, mutagenic andcarcinogenic, thereby threatening the environment andhuman health (Haghighi-Podeh & Bhattacharya 1996;ASTDA 1999; Gong et al. 2001). Consequently bio-remediation of these compounds is of significant inte-rest.Microorganisms play an important role in transform-

ing these often recalcitrant contaminants and in theassociated recycling of the nitrogen. Recent scientificliterature indicates that microbes have exploited thewide diversity of microbial genes to evolve mechanismsto degrade these synthetic organic structures (Spain1995; Freeman & Sutherland 1998; Hawari 2000;Ralebitso et al. 2002). Genomics research is assisting

microbiologists to understand further the role microor-ganisms play in biodegradation and will assist in thedevelopment of new bioremediation solutions (Wackett& Hershberger 2001a, b; Wackett et al. 2002).In this paper, microbial degradation processes of

some of the key N-containing groups, namely nitroaro-matics, nitrate esters and the N-heterocyclic compounds,atrazine and RDX, which can cause environmentalcontamination, will be reviewed.

Nitroaromatic compounds

Nitroaromatic compounds such as nitrobenzene, nitro-phenol, nitrotoluene and nitrobenzoate are commonstarting materials for the synthesis of complex industrialN-containing compounds and can be major environ-mental contaminants, hazardous metabolic inter-mediates or dead-end products. Understanding themetabolism of these simpler compounds provides per-spectives into the mechanisms for biodegradation ofmore complex nitroaromatics. Although they are ubiq-uitous, biodegradation of these simple compounds hasnot received much attention recently, as compared withmore highly nitrated compounds (Zylstra et al. 2000).

World Journal of Microbiology & Biotechnology 20: 117–135, 2004. 117� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Because of their higher solubility and xenobiotic char-acter, their fate is of serious concern and most are on theUnited States Environmental Protection Agency (EPA)priority pollutant list (EPA 2003a).The specific functional nitro-group in these aromatics

plays a key role in mechanisms for their conversion intocommon intermediary metabolites. Nitroaromatics aredegraded by various pathways using different biochemi-cal mechanisms. The aromatic p electron nucleophilicmechanism with the additional nitro(–NO2) electron-withdrawing property protects nitroaromatics frominitial attack by oxygenases but is favourable forreductive attack (Rieger & Knackmuss 1995). On theother hand, anaerobic reductive attack produces thenitroamine (–NH2), an electron-donating group whichrepresents a barrier to further attack by anaerobes(McCormick et al. 1976). Thus, nitroaromatics ofteneither persist or become amine end products in theenvironment.However, it was found that the nitroaromatic meta-

bolites, 5-aminosalicylate and 4-aminobenzoate, werecompletely mineralized to methane under completelyanaerobic conditions (Razo-Flores et al. 1999). In recentyears, it has been shown that aerobic degradation ofnitroaromatics results in mineralization in most cases.Bacteria appear to have evolved four main strategies

to address the nitro-group under aerobic conditions(Nishino et al. 2000b): (a) dioxygenation of the nitro-aromatic ring, with release of the nitro-group as nitriteand production of dihydroxy intermediates, (b) mon-oxygenation to epoxides, (c) formation of a hydride-Meisenheimer complex, and (d) partial reduction of thenitro-group, formation of hydroxylaminobenzenederivatives and ammonia release, followed by rear-rangement of the hydroxylaminobenzene to the corre-sponding catechol and elimination of another ammoniamolecule.While mono-nitroaromatics and certain di-nitroaro-

matics are more susceptible to initial oxygenation attackunder aerobic conditions (Rieger & Knackmuss 1995)because of the nitroaromatic nucleophilic feature, byusing the above strategies, microbes are able to convertthe nitroaromatic ring into the intermediate with two ormore hydroxyl (–OH) groups necessary for ring cleav-age. Subsequent oxidative ring cleavages are catalysedby a dioxygenase in the presence of NADPH througheither ortho-ring cleavage between two hydroxyl groupsor a meta-ring cleavage at the bond proximal to one oftwo hydroxyl groups (Nishino et al. 2000b).Theoretically, amino-groups, produced from nitroa-

romatic reductive attack by aerobic bacteria serve aselectron-donating constituents and activate the aromaticnucleus for further electrophilic attack by a dioxygenase.But until 2000, there was little evidence showingcomplete reduction of nitroaromatics by a single aerobevia the amine prior to ring cleavage. It appeared that thepathway involving complete reduction of the nitro-group to the amine by aerobic bacteria was not aneffective degradation system (Nishino et al. 2000b).

However, new evidence has shown that Pseudomonas sp.JX165 was able to mineralize nitrobenzene via both 2-aminophenol and aniline, followed by oxidative catab-olism under fully aerobic conditions (Wang et al. 2001).

Nitrobenzene

Nitrobenzene (NB) is the simplest nitroaromatic com-pound. It is produced on a large scale annually and ismainly used for the manufacture of aniline. Also, it isthe starting material for the synthesis of lubricating oils,dyes, drugs, pesticides, and synthetic rubber (ASTDA1999). It has been regulated and monitored in theenvironment because of its airborne toxicity to humansand animals (EPA 2003b). Generally, it is difficult toachieve complete degradation of NB. In the last decadeevidence has been provided that NB is degradable andits complete metabolic breakdown pathway has nowbeen established.Aerobic degradation of NB involves two major

pathways: the widespread partial reductive pathwayand the well-characterized dioxygenase pathway (Nish-ino et al. 2000b) (Figure 1). In the partial reductivepathway, as illustrated in Pseudomonas pseudoalcalig-enes JS45 (Nishino & Spain 1993), NB is reducedthrough nitrosobenzene to hydroxylaminobenzene withno aniline production. The hydroxylaminobenzene ismainly rearranged to aminophenol by a mutase andfinally dioxygenase-mediated meta-ring cleavage occursto produce 2-aminomuconic semialdehyde with subse-quent release of ammonia. The initial reductive enzyme,a NB-inducible and oxygen-insensitive nitroreductasewas characterized as a flavoprotein with a tightly boundFMN cofactor. Unlike most nitroreductases whichconvert nitro compounds to the corresponding amines,the reaction with the purified enzyme stopped athydroxylaminobenzene without producing amine (So-merville et al. 1995). An intramolecular transfer reactionwhich rearranged hydroxylaminobenzene to 2-amin-ophenol mediated by hydroxylaminobenzene mutasewas different from the nonenzymatic transfer whichproduced 4-aminophenol instead of 2-aminophenol (Heet al. 2000). The 2-aminophenol is converted to aminomuconic semialdehyde in a dioxygenase-mediated step.Transformation of 2-aminomuconic semialdehyde to 2-aminomuconate was mediated by 2-aminomuconicsemialdehyde dehydrogenase in the presence of NADand the 2-aminomuconate was further deaminatedstoichiometrically to 4-oxalocrotonic acid by 2-amin-omuconate deaminase (He & Spain 1997).A similar partial reductive metabolism was observed

by Zhao et al. (2001). A 3-nitrophenol-grown strainPseudomonas putida 2NP8 degraded NB through hy-droxylaminobenzene followed by the release of ammo-nia. It was proposed that 3-nitrophenol reductasecatalysed this reductive reaction. Surprisingly, anilinewas observed as a product in the partial aerobicreduction of NB by Pseudomonas sp. JX165, whicheventually led to NB mineralization (Wang et al. 2001).

118 J. Ye et al.

In the early nineties, Pseudomonas putida F1 andPseudomonas sp. strain JS150 were shown to producetoluene dioxygenase and to carry out an initial attack onthe 2,3-position on the NB ring (Haiger & Spain 1991).18O2 uptake evidence was used to propose the dioxy-genation mechanism. Nishino & Spain (1995) observedthat 154/155 strains contained the above mentionedpartial reductive pathway while one Comamonas sp.JS765 appeared to be able to mineralize NB via adioxgenation pathway similar to that described byHaigler & Spain (1991). The dioxygenase attacked the1,2 positions instead of the 2,3 positions to yield anitrohydrodiol. This was spontaneously decomposed tocatechol with the liberation of nitrite (Figure 1).Dioxygenases have a very broad substrate specificity

and NB dioxygenase is inducible (Lessner et al. 2002;Nishino & Spain 1995). Dioxygenation appears to be awidely used strategy for microbial degradation ofnitroaromatics and the mechanism has been extensivelyinvestigated to determine variations in enzymatic prop-erties among different species and strains. The genes ofsome of the enzymes have been sequenced and com-pared. The dioxygenase enzyme system of Comamonassp. strain JS765 has four components including reduc-taseNBZ, feredoxinNBZ, oxygenaseNBZa and oxygen-aseNBZb, with the gene designations nbzAa, nbzAb,nbzAc and nbzAd. Interestingly, this enzyme system

has high level homology with the 2-nitrotoluene dioxy-genase from Pseudomonas sp. strain JS42 (Lessneret al. 2002).Notwithstanding the different characteristics of up-

stream NB metabolism by Pseudomonas pseudoalcalig-enes JS45 (partial reductive pathway), Comamonas sp.JS765 (initial dioxygenase attack) and Pseudomonas sp.AP-3 (alternative metabolism of 2-aminophenol), allthree strains utilized the same meta-ring cleavage in thedownstream pathways. This convergence implies thatthe biodegradation activities of the three strains had acommon evolutionary origin (He & Spain 1999) and thishas been confirmed by genetic evidence of high similar-ity between the 2-aminophenol-1,6-dioxygenase from P.pseudoalcaligenes JS45 and the ring fission dioxygenasefrom Pseudomonas sp. AP-3. These investigations willprovide meaningful clues regarding enzymatic evolutionand should lead to the discovery of new strains andenzymes.To date, there is no evidence to show NB degradation

mediated by a specific NB monoxygenase. Two toluene-induced strains P. mendocina and P. pickettii couldoxidize NB readily in very small amounts to 3-nitro-phenol and 4-nitrophenol, respectively. Because phenolhydroxylase also possibly catalysed this reaction, thismonooxygenase was not considered to be very specific(Haigler & Spain 1991).

Figure 1. Nitrobenzene metabolism.

Biodegradation of N-containing xenobiotics 119

NB-contaminated sites are often treated using biosti-mulation or bioaugmentation. NB-metabolizing bacte-ria could lead to non-specific NB reduction to the moresoluble but less toxic aniline. With sequential aerobictreatment, aniline will be relatively easily mineralized(Dickel et al. 1993). As compared to possible aerobic NBtreatment, the latter approach would reduce NB vola-tilization significantly. By combining a reductive con-sortium with an oxidative Comamonas acidovoransstrain, it was possible to exploit both reductive andoxidative activities to mineralize NB (Peres et al. 1998).In a combined electron beam-biological treatment, NBwas effectively transformed to nitrophenols which werefurther degraded via a nitrophenol-degrading mixedculture (Zhao & Ward 2001).

Nitrobenzoates

Nitrobenzoate (NBA) has received much attentionbecause of its potential use as a substrate for biocatalytictransformation and its involvement as an intermediatein the degradation of nitrotoluene (Yabannavar &Zylstra 1995; James & Williams 1998). While all threeNBA isomers are degradable, the pathways remainunclear. Like most nitroaromatic compounds, NBAscan be transformed to aminoaromatics under anaerobicconditions. However, NBA was one of two nitroaro-matic compounds that were degradable to methane in afully anaerobic system, although the adaptive lag phasewas as long as 225 days (Razo-Flores et al. 1999).2-Nitrobenzoate (2-NBA) reductive degradation un-

der aerobic conditions has been described, with therelease of the nitro-group as ammonia (Durham 1958;Chauhan et al. 2000; Hasegawa et al. 2000). Thisoxygen-insensitive reduction route in Arthrobacterprotophormiae RKJ100 converted 2-NBA to 2-hydro-xylaminaobenzoate and ammonia under aerobicconditions. The presence of anthranilate as a majorand obligate intermediate distinguished this result fromthe similar pathway which was without a detectableamine product such as anthranile (Durham 1958).Pseudomonas fluorescens KU-7 had a similar 2-NBAreductive pathway. The enzyme system appeared to besimilar to the NB partial reductive enzyme system.2-NBA was converted to 2-hydroxylaminobenzoate (2-HABA) by an NADPH-dependent reductase. The2-HABA was rearranged and converted to 3-hydroxy-anthranilate (3-HA) followed by ring cleavage with noanthranilate detected (Figure 2).Degradation of 4-nitrobenzoate (4-NBA) was analo-

gous to that of 2-NBA, again using a predominantreductive pathway (Figure 2). The initial nitroreductase-mediated attack transforms 4-NBA to 4-hydroxyl-aminobenzoate followed by the release of ammoniaand formation of protocatechuate. The NBA reductasewas NAD(P)H-dependent. The formation of protoca-techuate was mediated by a lyase enzyme (hydroxylam-inolyase) rather than a mutase which mediated NBpartial reduction (Zylstra et al. 2000). This metabolic

pattern has been found in 4-NBA degradation byComamonas acidovorans NBA-10 (Groenewegen et al.1992; Groenewegen and De Bont 1992), Pseudomonassp. 4NT (Haigler & Spain 1993), Pseudomonas pickettiiYH105 (Yabannavar & Zylstra 1995) and P. putidaTW3 (Hughes & Williams 2001). In the 4-NBA degra-dation system of P. pickettii YH105, the initial reductivestep was catalysed by 4-NBA reductase. The gene forthis enzyme encoded in a 0.8-kb SalI-ApaI DNAfragment. The gene coding for the hydroxylaminolyasewas located adjacently. Strains 4NT and TW3 wereoriginally characterized as catabolizing 4-nitrotoluenevia 4-NBA as an intermediate. Further genomic researchshowed that TW3 has a 6-kbp fragment of TW3 DNA,which contains five genes including the pnbA (coding for4-NBA reductase) and pnbB (coding for 4-hydrox-ylaminobenzoate lyase).While two 4-aminobenzoate-grown strains, Burk-

holderia cepacia PB4 and Ralstonia paucula SB4, par-tially degraded 4-NBA to protocatechuate and twodead-end products, the inducer 4-aminobenzoate wasnot found as an intermediate in the pathway. Protoc-atechuate was produced, together with two dead-endproducts 3-hydroxy-4-aminobenzoate and 3-hydroxy-4-acetamidobenzoate because of the mutase, which wasnot found in the other cases cited. As stated earlier, thissystem contains strains capable of degrading nitroaro-matic and aminoaromatic mixtures simultaneously withpotential practical applications in bioremediation (Pereset al. 2001).3-Nitrobenzoate (3-NBA) is initially degraded by

dioxygenase attack in Pseudomonas strain JS51 andComamonas strain JS46. With 18O2 being incorporatedinto protocatechuate, the formation of dihydroxynitrointermediates at the 3,4 positions was analogous todioxygenase attack on other nitroaromatics (Nadeau &Spain 1995). 3-NBA degradation by Nocardia wasmediated by two monoxygenation steps producing 3-hydrobenzoate and finally leading to protocatechuate.In both cases, formation of nitrite demonstrated oxida-tive activities (Cartwright & Cain 1958) (Figure 2).

Nitrotoluenes

Nitrotoluenes (NTs), 2- and 4-NT, 2,4- and 2,6-dinitro-toluenes (DNT) are precursors of TNT and are impor-tant by-products of the explosive industry. 2,4- and 2,6-DNT are listed as EPA priority pollutants (EPA 2003a).Under some circumstances, these compounds are subjectto nonspecific nitro reduction to form amino derivativeswhich could be the predominant degradative reactionprior to aromatic ring cleavage. Although these aminoderivatives are resistant to further microbial attack andexhibit significant toxicity, they are often to be neglectedby regulatory bodies and in research studies (McCor-mick et al. 1976; Freeman et al. 1996b).Pseudomonas strain JS42 degrades 2-nitrotoluene (2-

NT) by dioxygenation via 3-methylcatechol with nitriterelease leading to ring cleavage (Parales et al. 1998)

120 J. Ye et al.

(Figure 3). 2-Nitrotoluene-2,3-dioxygenase has broadspecificity towards aromatic compounds and catalysesother dioxygenation and monoxygenation reactions.The system has been cloned and sequenced and appearsto be a three-component dioxygenase. While there was asimilarity with 2,4-DNT-dioxygenase, substrate profileswere different.Pseudomonas strains TW3 and 4-NT degrade 4-

nitrotoluene (4-NT) via 4-NBA followed by the partialreductive pathway. The initial oxidative reaction of themethyl group, which transforms NT into more oxidizednitroaromatics, was possibly mediated by the TOLplasmid. This oxidation reaction differed in the twostrains, one being NADþ-dependent but the other wasnot. Toluene and 4-NT induced a common set of genesin strain TW3 whose products were responsible forconverting the hydrocarbon side chains to carboxylicacids (James & Williams 1998). Mycobacerium sp. strainHL-4NT-1 also degraded 4-NT, but without oxidationof the methyl group but rather by production of 4-hydroxylaminotoluene followed by 6-amino-m-cresolbefore meta-ring cleavage (Figure 3). With evidence of

TOL plasmid-like involvement in the 4-NT upperpathway, a stable transconjugant from 4-NBA-degrad-ing strain Pseudomonas fluorescens 410PR and TOLpWWW0Dpm plasmid was constructed. It was capableof converting 4-NT into 4-NBA. (Michan et al.1997).This transconjugant mineralized 4-nitrotoluene via 4-NBA.Psuedomonas mineralized 2,4-DNT with a nitrite

removal pathway involving dioxygenase and monooxy-genase enzymes which have been characterized andcloned (Suen & Spain 1993; Spanggord et al. 1991). Thisproduces 2-hydroxy-5-methylquinone and leads to2,4,5-trihydroxytoluene. Ring fission of 2,4,5-trihydr-oxytoluene likely occurs at position 5,6 of the aromaticring to yield 3,5-dihydroxy-2-methyl-6-oxo-hexa-2,4-di-enoic acid as ring cleavage product (Haigler et al. 1999)(Figure 4).Nishino et al. (2000a) isolated Burkholderia cepacia

strain JS850 and Hydrogenophaga paleronii strain JS863that were able to mineralize 2,4-DNT in the same waybut degraded 2,6-DNT in a different way. When 2,4-and 2,6-DNT were used as sole source of carbon and

Figure 2. Nitrobenzoate metabolism.


nitrogen together, dioxygenation of the 2,6-DNT to 3-methyl-4-nitrocatechol (3M4NC) was the initial reac-tion, accompanied by the release of nitrite. 3M4NC was

then subjected to meta-ring cleavage. Although 2,4-DNT-degrading strains also could convert 2,6-DNT to3-methyl-4-nitrocatechol, further catabolism was halted

Figure 3. 2-, and 4-Mononitrotoluene metabolism.

Figure 4. Dinitrotoluene metabolism.

122 J. Ye et al.

at that point. The pathway for 2,4-DNT degradationwas different from that for 2,6-DNT degradation. In thelatter case, 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoicacid was the first ring fission product. How 3M4NC isconverted to 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoicacid is unknown. The position 3 methyl group appearsas the determinant recognized by the initial dioxygenaseto produce high specific 3M4NC in the 2,6-DNTpathway that they proposed. The gene encoding thisdioxygenase showed a nucleotide sequence similar to thea subunit among nitroarene dioxygenases.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(Spain 1995; Nishino et al. 2000b). After initial dioxy-genation, the two pathways appear to diverge (Fig-ure 4).How DNT degradation is affected by the presence of

both isomers is important, since 2,4-DNT and 2,6-DNTare produced in a 4:1 ratio (Nishino & Spain 2001), andare therefore often present together in munitions plantwastewater. Lendenmann & Spain (1998) initially failedto observe degradation of 2,4-DNT and 2,6-DNTsimultaneously. Subsequently an aerobic biofilm, ini-tially fed with DNT mixture in low concentrationswhich were then gradually increased, exhibited mineral-ization rates of 98% and 94%, for 2,4- and 2,6-DNT,respectively. The nitrogen was released as nitrate,reflecting oxidative bacterial activity. Isomer concentra-tions needed to be kept below inhibitory levels as highconcentrations of each isomer inhibited degradation ofthe other. Simultaneous degradation of 2,4- and 2,6-DNT may be unpredictable until an adapted populationis established (Nishino & Spain 2001).Although bacteria able to degrade nitrotoluenes are

widely distributed at contaminated sites, the contami-nants still persist for very long periods, leaving unan-swered questions as to how their biodegradation may beproduced by biostimulation (Nishino et al. 1999, 2001).Efficient anaerobic pathways for the degradation eitherof mono- or di-nitrotoluenes are not known and 2,3-DNT does not appear to be degradable (Nishino &Spain 2001).Trinitrotoluene (TNT) is difficult to degrade (Nishino

& Spain 2001). The three nitro-groups with a nucleo-philic aromatic ring structure make TNT vulnerable toreductive attack but resistant to oxygenase attack fromaerobic organisms (Lenke et al. 2000). In most currentreports the reductive mechanism predominates in TNTdegradation. New evidence indicates that TNT could bereduced by carbon monoxide dehydrogenase fromClostridium thermoaceticum (Huang et al. 2000), andby the managanese-dependent peroxidase (MnP) fromthe white-rot fungus Phlebia radiata (Van Aken et al.1999). Based on the discovery of pentaerythritol tetra-nitrate (PETN) reductase from Enterobacter cloacaePB2, French et al. (1998) found that this strain couldgrow slowly on 2,4,6-TNT under aerobic conditions asthe sole nitrogen source without production of dinitro-

toluene as an intermediate and catalysed conversion ofthe TNT via a hydride-Meisenheimer complex with thenitro-group released as nitrite.

Nitrophenols

Nitrophenols (NPs) are among the most important andversatile industrial organic compounds with applica-tions as pesticides, pharmaceuticals, pigments, dyes, andrubber chemicals (Haghighi-podeh & Bhattacharya1996). These compounds may accumulate in soil as aresult of hydrolysis of several insectides such as para-thion, methylparathion and other more complex nitro-phenolic herbicides. Under anaerobic conditions, mono-NPs are most likely subject to reductive degradation tothe corresponding aminophenol (Uberoi & Bhattach-arya 1997) although 4-NP was removed by methano-genic bacteria without production of any amino product(Haghighi-Podeh & Bhattacharya 1996). Aminopheno-lics can be further converted to nonaromatic productsand possibly be mineralized completely in differentanaerobic system (Oren et al. 1991; Donlon et al. 1996;Razo-Flores et al. 1999). Compared with other nitro-aromatics, mono-NP degradation pathways are rela-tively diverse among micro-organisms.2-Nitrophenol (2-NP) has the simplest mono-NP

degradation pathway. In Pseudomonas putida B2, 2-NPis attacked by a monoxygenase, forming nitrite andcatechol, with further degradation of the latter by 1,2-dioxygenase-mediated ortho-ring cleavage (Zeyer et al.1985) (Figure 5).P. putida B2 degradation of 3-nitrophenol (3-NP)

involved a different mechanism, mediated by a reductivepathway possibly via 3-hydroxylaminophenol, andeventually leading to 1,2,4-benzenetriol ring cleavage(Meulenberg et al. 1996). A similar pathway wasobserved with Ralstonia eutropha JMP134 which used3-NP as sole nitrogen and carbon source. The intermo-lecular rearrangement was mediated by a mutase ratherthan a lyase (Figure 5). 3-Hydroxylaminophenol mutasewas first purified and characterized as a 62-kDa enzyme,whose amino acid sequence was very similar to gluta-mine synthetase. This may imply the mechanism ofammonia release, although no further evidence hasshown the relationship between this mutase with eitherglutamine or glutamate (Schenzle et al. 1997).4-Nitrophenol (4-NP) can be degraded via three

monooxidative pathways through different intermedi-ates by different species such as Bacillus (Kadiyala et al.1998), Pseudomonas (Chauhan et al. 2000; Qureshi &Purohit 2002), Moraxella (Spain & Gibson 1991),Arthrobacter (Hanne et al. 1993; Bhushan et al. 2000).The major route of degradation of 4-NP in Moraxellasp. is via hydroquinone, possibly partly through 4-nitrocatechol. At that time, there was no evidence thatthe hydroquinone or 4-nitrocatechol could be convertedto 1,2,4-benzenetriol, through a central metabolic path-way (Spain & Gibson 1991; Spain 1995). This pathwayis more common in gram-negative isolates (Kadiyala &


Spain 1998), however, a gram-positive Nocardia mayhave this pathway (Hanne et al. 1993). In Ralstonia sp.SJ98, Arthrobacter protophormiae RKJ100, Burkholderiacepacia RKJ200 earlier proposed intermediates, hydro-quinone and benzoquinone, inhibited degradation of 4-NP (Bhushan et al. 2000).Other degradative pathways involve monoxygenases

with hydroxylation at either the 2- or 3-ring position toform 4-nitroresorcinol or 4-nitrocatechol, with the latterbeing considered the more likely initial oxidative prod-uct (Figure 5). The 1,2,4-benzenetriol undergoes ortho-ring fission via maleylacetate in strain ArthrobacterJS443. A similar metabolic pathway was proposed forBacillus sphaericus JS905 (Kadiyala et al. 1998) with 4-nitrocatechol as intermediate. A two component mono-oxygenase, comprising a flavoprotein reductase andoxygenase, was purified and found to be NADH-dependent. Degradation kinetics demonstrated thatevery mole of 4-NP metabolized consumed 3 mol O2

which indicated that the whole pathway involved twomonooxygenation steps and a subsequent dioxygenationof 1,2,4-trihydroxybenzenetriol (Figure 5).2-NP and 4-NP are hydroxylated at the nitro-group

with release of nitrite to form an ortho- or para-dihydroxybenzene (Nishino & Spain 2001). The sameset of genes likely encodes hydroquinone metabolism inconverting 4-NP through hydroquinone and 4-nitro-catechol via the 1,2,4-benzenetriol and 2-hydroxy-1,4-benzoquinone pathway in Arthrobacter protophormiae

strain RKJ100 (Chauhan & Jain 2000). 2-Hydroxy-1,4-benzoquinone can be dehydroxylated to form hydro-quinone (Chauhan & Jain 2000; Chauhan et al. 2000).Pentachlorophenol-degrading Sphingomonas spp UG30degraded 4-NP via 4-nitrocatechol to 1,2,4-benzenetriol(Leung et al. 1997, 1999), the latter step being catalysedby an aromatic flavoprotein monoxygenase pentachlor-ophenol-4-monoxygenase, which also exhibited someability to convert 4-NP at the para-postion to hydro-quinone (Figure 5).Pseudomonas sp. YTK 17 and Rhodococcus opacus

YTK32 exhibited different 4-NP degradation activities(Shinozaki et al. 2002). Pseudomonas sp. YTK 17required a lower 4-NP concentration for the initialdegradation. Zhao & Ward (1999) described a mixedculture, enriched on 3-mono-nitrophenols, that degra-ded both NP and nitrobenzene. The mixed cultureincluded two isolates identified as Comamonas testoste-roni and Acidovorax delafieldii. 3-NP-induced cells ofPseudomonas putida 2NP8 degraded a wide range ofnitroaromatic substrates, with rapid transformationof most mono- and di-nitroaromatics and with stoichio-metric release of ammonia.2,4,6-Trinitrophenol (TNP, picric acid), an explosive

and major byproduct of large-scale nitration of benzeneis a common anthropogenic compound and envi-ronmental contaminant (Russ et al. 2000). The threenitro-groups make the TNP structure particularly elec-tron-deficient and the aromatic electrophilic property

Figure 5. Mononitrophenol metabolism.

124 J. Ye et al.

protects the nitro-groups from oxygenase-mediatedelectrophilic attack. Under aerobic conditions, the initialoxygenation usually occurs with the mono- and to someextent di-nitrophenol. As the number of nitro-groupsincreases, the reductive reaction becomes the dominantinitial mechanism (Rieger & Knackmuss 1995).Mineralization of TNP by Nocardioides sp. strain

CB22-2 (Behrend & Heesche-Wagner 1999) and Rhodo-coccus erythropolis (Rieger et al. 1999) involved ahydride-Meisenheimer complex of TNP and 2,4-DNP,and also including some 2,6-DNP. The initial hydroge-nation of the TNP nitroaromatic ring happened atposition 3 followed by position 2 protonation to form2,4-DNP (Figure 6). The Meisenheimer complex isrecognized as a key intermediate of denitration inmicrobial degradation of TNP. 2,4-DNP became thesubstrate for the monooxygenase. Whether the Meisen-heimer complex of 2,4-DNP provides it with an addi-tional negative charge facilitating electrophilic attack isnot known. A new product of 3-nitroadipate wasrevealed in a novel strain, Rhodococcus RB1 with thecapability of degrading 2,4-DNP by releasing nitrite.Nitro-group removal occurred in two steps: the 2-nitro-group was removed first to form a new ortho- ringfission product, 3-nitroadipate (Figure 6). Further ni-trite assimilation required an additional carbon sourceas electron donor (Blasco et al. 1999).A two component enzyme system from Nocardioides

simplex FJ2-1A catalysing the aerobic hydrogenation ofthe nitroaromatic ring consisted of an NADPH-depen-dent F420 reductase and a hydride transferase, whichtransferred hydride from reduced coenzyme F420 back

to the reaction system. Although it is not usual to findthis reductase and its coenzyme F420 in aerobic bacte-ria, N-terminal sequences showed high homology withan F420-dependent NADP reductase found in anaerobicArchaea. At the same time, the hydride transferaselacked N-terminal similarity to other hydride transfer-ases and was considered a novel enzyme responsible forhydride transfers involving the nitroaromatic ring (Ebertet al. 1999) (Figure 6).DNOC (2-methyl-4,6-dinitrophenol) and Dinoseb (2-

sec-butyl-4,6-dinitrophenol) are herbicides. Dinoseb hasbeen banned in US because of its high toxicity but haslimited use in Canada. Under well aerated conditions,biodegradation does not occur. Dinoseb can be trans-formed to amino and acetamido forms which retainsignificant toxicity and further form polymeric material(Stevens et al. 1990, 1991; Kaake et al. 1992). DNOChas an electrophilic character which could lead toreductive initial attack, suggesting anaerobic conditionsare probably favoured for an initial attack (Gisi et al.1997).

Organophosphate derivatives: parathion and methyl

parathion

The organophosphates, parathion and methyl parathi-on, have been widely used as pesticides. Hydrolysis ofthese compounds is the major detoxication route and itleads to the formation of dialkylthiophosphates and 4-NP (Munnecke 1979). Researches on biodegradation ofparathion and methyl-parathion have focused not only

Figure 6. Polynitrophenol metabolism.


on the parent compounds but also on the byproduct 4-nitrophenol, because 4-NP is a priority pollutant (EPA2003a). The key enzyme in biodegradation of parathionis organophosphorus hydrolase (OPH). The researchstrategy appears to be to find a strain with good 4-NPdegradation ability, engineering a strain to encode theOPH gene and converting parathion and 4-nitrophenolsimultaneously.Shimazu et al. (2001) investigated a native soil

organism Moraxella sp., which was able to degrade 4-nitrophenol rapidly. A truncated version of ice nucle-ation protein INP–INPNC anchor was used to targetthe organophosphorus hydrolase (OPH) onto the sur-face of Moraxella sp., by constructing a shuttle vectorpPNCO33, coding for INPNCOPH. The functionalitywas demonstrated in both E. coli and Moraxella sp. Thenew microorganism Moraxella sp. with coded INPN-COPH was able to rapidly and simultaneously degrade4-nitrophenol and organophosphates. The gene encod-ing the native OPH (opd) was also cloned into broad-host-range plasmids under the control of tac promoterand resulted in high OPH activity. Originally, P. putidaKT2442 was not able to metabolize the parathion viahydrolysis. The plasmid-harbouring 4-NP operonswhich encoded the enzyme catalysing the conversionof 4-nitrophenol to b-ketoadipate, were transformedinto P. putida KT2442 along with the plasmids har-bouring opd, thereby engineering this strain to utilizeparathion as carbon and energy source and to degradeparathion (Walker & Keasling 2002). Zhongli et al.(2001) cloned another novel OPH gene which wasdesignated as mpd from Plesimonas sp. strain M6. It wasable to degrade methyl-parathion to 4-NP. Meanwhile,sequential cycles of DNA shuffling and screeningstrategies have been developed which will facilitate finetuning and enhancement of OPH activity to create OPHvariants and improve substrate specificities (Cho et al.2002).

Nitrate esters

Nitrate esters are abundantly used in the ammunitionand pharmaceutical industries. Their metabolites aresoluble at high concentrations where they also exhibithigher toxicity. Important nitrate esters include glycer-ol trinitrate (GTN), pentaerythritol tetranitrate(PETN), nitrocellulose (NC), isosorbide dinitrate(ISDN), ethylene glycol dinitrate (EGDN) and thenitrate ester chemical treatment products, epoxidesglycidol and glycidyl nitrate (White & Snape 1993). Innitrate esters, abundant carbon atoms, which arederived from the polyhydric alcohols can be assimilatedby bacteria and the abundant nitrogen thus maysupport microbial growth. Biodegradation appears tobe potentially the most effective remediation method,as chemical treatment methods require follow-on sec-ondary treatments of reaction products (Wendt et al.1978).

Glycerol trinitrate

Glycerol trinitrate (GTN) is toxic and inhibitory tobacterial growth and until recently information on itsbacterial degradation was minimal and insufficient forthe design of effective treatment systems (White & Snape1993). Wendt et al. (1978) monitored changes in thenitrate ester composition and observed the final effluentto be free from tri-di- and mono-nitrate esters within 8–15 h. A sequential conversion of GTN and GDN toGMN was proposed, but the relationship among thetwo di-nitrate esters, mono-nitrate ester intermediatesand final products was unknown.Researches revealed that nitrogen removal from GTN

to GDN and from GDN to GMN is regioselective.While random attack of the three ester bonds of GTNwould be expected to yield a ratio for 1,3-GDN:1,2-GDN of 1:2, the ratio actually was found of 2:1 in theGeotrichum candidum transformation (Ducrocq et al.1989) and even higher ratios than 2:1 occurred inPseudomonas sp. R1-NG1(White et al. 1996a). 1,3-GDNwas the major isomer in both cases which was theevidence that the GTN conversion to GDN was C2regioselective. In contrast to the lack of significantregioselectivity in the formation of GMN observed inthe G. candidum transformation (Ducrocq et al. 1989),Pseudomonas R1-NG1 showed regiospecific preferencefor C1 but not C2 in the further conversion from GDNto GMN (White et al. 1996a).Although most researches showed sequential denitra-

tions of GTN through GDN, GMN and glycerol, theenzymatic mechanism of nitrogen removal from nitrateesters has not yet been clarified (Figure 7). White et al.(1996a) found that the nitro-group was released asnitrite (NO2) but not nitrate (NO3

)) from GTN inPseudomonas R1-NG1. Although Pseudomonas speciesare well known producers of extracellular lipases andesterases, it is an esterase-mediated mechanism in thiscase. This finding is consistent with the findings formammalian and fungal cells. Servent et al. (1991) foundthe filamentous fungus Phanerochaete chrysosporium toconvert GTN and its dinitrate and mono-nitrate deriv-atives concurrently with the formation of nitric oxide(NO) and nitrite (NO2

)). These two pieces of evidenceshowed that esterase-type activity was not involved.EPR spectra related the enzymatic activity to catabolismof GTN in mammalian cells. A glutathione transferase-like system seemed responsible for nitrite and nitricoxide formation. Biodegradation of GTN and PETN byAgrobacterium radiobacter appeared to be catalysed by anitroreductase, with NADH as a cofactor and nitrogenwas released as nitrite (White et al. 1996b).Ester hydrolytic cleavage mechanism may also exist

in GTN degradation. In cell extracts of Bacillusthuringiensis/cereus and Enterobacter agglomerans, ni-trogen was removed from GTN as nitrate and possiblyfollowed by reduction of nitrate to nitrite. No cofactoris required in this transformation. The problem remainsunsolved because stoichiometric evidence is not suffi-

126 J. Ye et al.

cient and it may be a multi-enzyme system (Meng et al.1995).Sequential denitrations were also confirmed by other

mixed culture studies, which were carried out underboth anaerobic and aerobic conditions. The degradationrates between each successive denitration were found tobe decreased significantly (Christodoulatos et al. 1997;Bhaumik et al. 1997; Accashian et al. 1998).Marshall & White (2001) isolated four species: P.

putida, Arthrobacter sp., Klebsiella sp. and Rhodococcussp. from GTN-contaminant soil. All isolates were ableto degrade GTN sequentially, but only Klebsiella sp.showed regioselectivity to C2 from GTN. Moreover,Rhodococcus sp. was able to convert GMN to glycerol,thus it became the first reported single strain with thecapability of mineralizing GTN.

Nitrocellulose

While nitrocellulose is not toxic to humans, its explosiveproperty and air-polluting effect following combustionmake it hazardous (White & Snape 1993; Freeman et al.1996b). It is very recalcitrant to microbial degradationand can be degraded under anaerobic conditions (Duranet al. 1995; Freeman et al. 1996a) and aerobically bycombination of cellulolytic and denitrifying fungi (Shar-ma et al. 1995).

Pentaerythritol tetranitrate

Pentaerythritol tetranitrate (PETN) is a powerful ex-plosive and commonly used coronary vasodilator. It isrecalcitrant to microbial attack. Recent research hasfocused on PETN reductase. Binks et al. (1996) foundPETN reductase in the PETN-grown microorganismEnterobacter cloacae PB2, which was isolated fromexplosive-contaminated soil. The strain PB2 used two ofthe four nitrogen atoms from PETN for growth, thus

degrading PETN aerobically. Nitro-groups were re-moved as nitrites by the PETEN reductase-mediatedreductive reaction. PETN reductase was detected incrude cell extracts of PB2 and characterized as anNADPH-dependent flavoprotein with noncovalentbinding of FMN. It was cloned and overexpressed inE. coli and its properties were elucidated (French et al.1996).Several similar nitrate ester-degrading enzymes, being

capable of catalysing the NADPH-dependent reductivecleavage of nitrate esters to give alcohol and nitrate andwith noncovalently linked FMN-dependent reductaseactivity, have been cloned and sequenced (Williams &Bruce 2000). Blehert et al. (1997, 1999) purified andcharacterized xenA and xenB genes encoding nitroesterreductases from Pseudomonas putida IIB and Pseudo-monas fluorescens I-C. These enzymes share biochemicalproperties and sequence similarities with old yellowenzyme (OYE), a dimeric FMN-containing flavoproteinfrom several yeasts. There are seven homologous bac-terial flavoproteins in this enzyme family, which canreduce a variety of electrophilic substrates such asnitroaromatics, including TNT and picric acid (Frenchet al. 1998). This type of enzyme has general xenobioticreductase activity and is referred as xenobiotic reduc-tase, although many xenobiotics are not substrates forall seven reductases (French et al. 1996; Blehert et al.1999). On the other hand, Williams & Bruce (2002)reviewed the OYE family in the context of these newbiodegradative applications. Khan et al. (2002) estab-lished the reductive half-reaction of a PETN reductasemodel with mechanistic similarities with OYE andprovided kinetic and structural data and demonstratedrelationship and clear difference in reactivity betweenOYE and PETN reductase. The characterization ofmetabolism of nitrate esters by bacteria and the poten-tial to adapt the bacteria and to direct evolution of thedegrading enzymes provides an opportunity for futurebioremediation processes.

Figure 7. Enzymatic removal of nitrate esters.


Nitrogen heterocyclic ring structure contaminants

Numerous industrial compounds contain the N-hetero-cyclic ring structure. The s-triazines constitute a groupof heterocyclic compounds, characterized by a symmet-rical hexameric nitrogen-containing ring. Most s-tria-zines are recalcitrant (Wackett & Hershberger 2001b).These compounds are xenobiotics and exhibit toxicity,mutagenicity and carcinogenicity. Biodegradation ofatrazine and RDX are reviewed. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is one of the most widely usedexplosive contaminants on the world because of itsrelative stability and great explosive power.

Atrazine

Biodegradation of atrazine (2-chloro-4(ethylamino)-6-(isopropylamino)-s-triazine, the most widely used s-triazine herbicide, occurs predominantly by a series ofbiological processes, including N-dealkylation, dechlo-rination and ring cleavage. Several bacterial strainsutilized s-triazine as nitrogen source, in most casesthrough oxidative N-dealkylation of the side chainrather than through ring cleavage. While side chainsappear accessible to microbial attack and less heavilysubstituted and nonchlorinated s-triazines are morebiodegradable (Cook et al. 1985), the mineralization ofs-triazine is very slow (Yanze-Kontchou & Gschwind1994). Dead-end metabolites with the s-triazine ringstructure still remain after transformation by Rhodococ-cus, (Behki et al. 1993; Mulbry 1994; Nagy et al.1995),by Phanerochaete chrysosporium (Mougin et al. 1994),and by Nocardioides (Topp et al. 2000).Very little is known regarding the mechanism of s-

triazine ring degradation (Karns 1999). Cyanuric acid,having the simplest triazine structure, was formedduring degradation of atrazine by most strains such as

Pseudomonas (Jutzi et al. 1982), Arthrobacter aurescensTC1 (Strong et al. 2002).Pseudomonas sp. strains A and D and Klebsiella

pneumoniae were proved to be able to utilize atrazine asnitrogen source. Successive deamination to cyanuricacid with further ring cleavage appears to be hydrolytic(Jutzi et al. 1982; Cook et al. 1985; Karns & Eaton 1997;Karns 1999). Karns & Eaton (1997) reported that the s-triazine degradation plasmid pPDL12 from Klebsiellapneumoniae 99 is a member of the IncIa incompatibilitygroup. pPDL 12 may be an agent for the disseminationof the s-triazine degradation gene(s) between bacteria.Further, Karns (1999) reported that the gene sequence(trzD) encoded the s-triazine ring cleavage enzyme,cyanuric acid amidohydrolase, from Pseudomonas sp.strain NRRLB-12227.An atrazine-degrading Pseudomonas sp. ADP was

isolated from an enriched mixed culture which wasfound to be able to degrade and release 14CO2 from theatrazine ring at a high rate (Mandelbaum et al. 1993,1995). This strain has become a reference strain (Rale-bitso et al. 2002) and the plasmid pADP-1 on which arelocalized genes atzABC from this strain has beencompletely sequenced (Wackett et al. 2002) to elucidatethe catabolic enzymes involved in atrazine degradationunder different conditions. Genes atzABC, which areresponsible for s-triazine degradation prior to theformation of cyanuric acid, have been cloned fromdifferent genera and described (Shao & Behki 1995; DeSouza et al. 1998a, b; Sadowsky & Wackett 2000;Martinez et al. 2001; Clausen et al. 2002; Rousseauxet al. 2002; Shapir, et al. 2002). These evidencesdemonstrate that atzABC genes are widespread in otheratrazine-degrading bacteria. A very similar structurewas found between atzA from Pseudomonas sp. ADPand TriA from NRRLB-12227, but their functions aredifferent, for while TriA is responsible for deamination,atzA is responsible for dehalogenation (Figure 8) (Sef-

Figure 8. s-Triazine metabolism.

128 J. Ye et al.

fernick et al. 2001). The s-triazine hydrolyase in Nocar-dioides sp. proved to be different from the deaminohy-drolase in the ADP strain (Topp et al. 2000). Therefore,atrazine catabolic pathways appear to be the result ofrecruitment of different genes in broad microbial com-munities rather than a genetic element encoding theentire pathway (Rousseaux et al. 2002).

Hexahydro-1,3,5-trinitro-1,3,5-triazine

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has beenused in detonators, primers, mines, rocket boosters, andplastic explosive both in military and civilian applica-tions (Yinon 1990). It does not adsorb strongly to soil(Price et al. 1998; Sheremata et al. 2001; Pennington &Brannon 2002), thus it can move into the ground waterand migrate offsite. Little is known regarding thebiodegradation of RDX. It is less toxic to indigenousmicrobes than TNT (Gong et al. 2001). Its three weak –N–N– bonds make it vulnerable to enzymatic attack atthe nitro-groups, potentially leading to destabilizationof the inner C–N bonds causing rapid ring cleavage(Hawari et al. 2000a, b; Bhushan et al. 2002b). However,the nitro-groups are often not actively metabolized andhence RDX can persist over long periods of time in theenvironment (Gong et al. 2001). Specially, it resistsaerobic degradation (McCormick et al. 1976).Based on early anaerobic research showing disap-

pearance of RDX and formation of formaldehyde, areductive pathway was proposed resulting in the forma-tion of a series of nitroso compounds followed byhydroxylamine during the anaerobic attack (McCor-mick et al. 1976, 1981). These unstable structureseventually lead to ring cleavage. This degradationpathway remains controversial (Hawari et al. 2001).Different anaerobic conditions have been examined. A

sulphate-reducing consortium of Desulfovibrio usedRDX as sole nitrogen source and eliminated it within12 days with concurrent release of NH3 (Boopathy et al.1998). The biotransformation of RDX was inhibitedunder nitrate-reducing conditions. Only after the nitratewas depleted by excess electron donors, such as byrepeatedly adding ethanol, would RDX be rapidlyreduced to nitroso metabolites which were subject tofurther degradation (Freeman & Sutherland 1998).Research also suggested that Fe0-filing system-amendedmunicipal anaerobic sludge was able to produce H2 aselectron donor to enhance the reduction of RDX. Thus,the mineralization of RDX would be more efficient andcomplete (Oh et al. 2001).Halasz et al. (2002) first demonstrated the formation

of methylenedinitramine as a key RDX anaerobic ringcleavage metabolite. At least two degradation routeswere suggested and a novel way that the RDX C–Nbond could be hydroxylated to methylenedinitramineand bis(hydroxymethyl) as two ring cleavage intermedi-ates was proposed (Figure 9). A facultative anaerobicbacterium Klebsiella pneumoniae strain SCZ-1 wasisolated which degraded RDX completely (Zhao et al.

2002). RDX and hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) were transformed at very similar rates,indicating a possible common initial attack mechanism.They also detected methylenedinitramine as well as traceamounts of hexahydro-1,3,-dinitroso-5-nitro-1,3,5-tri-azine (DNX) and MNX.An oxygen-insensitive nitroreductase (type I nitrore-

ductase), responsible for nitroreduction of RDX bothunder aerobic and anaerobic condition, appeared tofacilitate reduction of the nitro-group of RDX or TNTby the enteric bacterium Morganella morganii strain B2and another enteric Enterobacter cloacae strain 96-3(ATCC 43560). Strain B2 also carried out an RDX-cyclic nitramine reductive reaction under anaerobicconditions (Kitts et al. 1994, 2000). Nitroreductases donot reduce inorganic nitrate or nitrite like nitrate/nitritereductases and they do not provide a source of reducednitrogen for cell growth. Obviously, the contradictionneeds to be resolved. A nitrate reductase from Asper-gillus niger was found to be able to use NADPH aselectron donor to reduce the RDX nitro-group, andproduced MNX and methylenedinitramine. Stoichio-metric evidence (Bhushan et al. 2002a) showed that itwas a two-electron reduction (Figure 9).A flavoenzyme, diaphorase from an anaerobic bacte-

rium Clostridium kluyveri used NADH as electron donorto remove RDX (Bhushan et al. 2002a). The initialenzymatic attack was oxygen sensitive and formedRDX.) whose spontaneous denitration would generatenitrite and the free radical RDX.. The existence ofRDX.) is a possible reason for the transformation ofRDX being inhibited by O2. With conversion of O2 toO:�

2 , RDX.) reverted to original RDX, thus furtherdegradation was blocked. FMN played a key role in thisnet two redox equivalents transferring system. Mono-denitration was responsible and sufficient for the ringcleavage and secondary decomposition. Methylenedi-nitramine, NO�

2 , HCHO, NHþ4 , and N2O were observed

but no nitroso intermediates were detected (Figure 9).Far less aerobic degradation studies have been re-

ported for RDX. Stenotrophomonas maltophilia PB1 wasable to use RDX as sole nitrogen for growth underaerobic conditions. With three out of the six nitrogensfrom RDX, two intermediates, MW167, MW 171 wereobserved (Binks et al. 1995). Hawari (2000) related theMW 171 intermediate to the proved anaerobic interme-diate methylenedinitramine, because chloride methylen-edinitramine has the same molecular mass MW171.Coleman et al. (1998) isolated a Rhodococcus sp. strainDN22, which was the first gram-positive aerobic bacte-rium known to degrade RDX, used it as sole nitrogensource and depleted RDX from medium within 24 h.Further research showed that nitrogen was removed asnitrite in the ratio of 2 mol NO�

2 for every mole of RDX(Fournier et al. 2002). An RDX degradation pathwayinvolving an initial enzymatic denitration step wasproposed (Figure 9). Removal of the first NO2

) pro-duced a cyclohexenyl product and the second denitra-tion produced cyclohexadienyl intermediates. Following


the denitration, ring cleavage occurred with formationof CO2 and HCHO, possibly through a hydrolyticreaction. A dead end product C2H5N3O3. was alsodetected and not all the RDX was mineralized (Fournieret al. 2002). The DN22 has a plasmid-encoded cyto-chrome P-450 enzyme, which could be potentiallytransferred between bacteria (Coleman et al. 2002).The white rot fungus Phanerochaete chrysosporium

used RDX as nitrogen source and mineralized 52.9% ofit in 60 days. Only MNX (hexahydro-1-nitroso-2,5,-dinitro-1,3,5-triazine) was detected as intermediate butno DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-tri-azine) was detected. The final product N2O was foundwith one nitrogen originating from the RDX ring andthe other from a ring substituent, which suggested ringcleavage (Sheremata et al. 2001; Sheremata & Hawari2000).Rhodococcus rhodochrous 11Y, isolated from explo-

sive-contaminated land, used RDX as sole nitrogensource under aerobic conditions and mineralized RDXcompletely. Denitration preceded RDX ring break-down. The gene which conferred RDX degradationability was constitutively expressed as cytochrome P-450-like gene xplA. It was found with a flavodoxindomain N-terminus. Notwithstanding the role of theflavodoxin domain in XplA as a redox agent and the

possible use of XplB as a reductase, the evidence that thecytochrome P-450 inhibitor metyrapone reduced theRDX removal rate strongly supported the function ofcytochrome P-450 in the aerobic degradation of RDX.The mechanism of action of XplA is thought to involveinitial denitration followed by spontaneous ring cleav-age and mineralization (Seth-Smith et al. 2002).While degradation of RDX has been investigated

under both anaerobic and aerobic conditions and itsenzymatic pathway is far from being understood,common correlations have not been found betweenbiodegradation of RDX and that of s-triazine andatrazine, which is well understood. This is probablybecause the nitro-group in RDX presents a barrier. Alsoevidence suggests that RDX ring cleavage could occurwithout complete denitration. S. maltophila PB1 whichused RDX as nitrogen source was unable to usecyanuric acid as nitrogen source but it could grow onother s-triazines as the limiting nitrogen source (Binkset al. 1995). There is no report showing RDX ringcleavage via the cyanuric acid pathway. It appears thatring cleavages occur differently, with dealkylation ofatrazine preceding triazine ring cleavage. Atrazine was amoderate inducer of the P-450 enzyme in DN22 strain,which induces RDX biodegradation ability (Colemanet al. 2002).

Figure 9. RDX metabolism.

130 J. Ye et al.

Conclusions

The current research on biodegradation of nitroaromat-ics is focussed on development of strategies to allowmore recalcitrant compounds to serve as growthsubstrates for microorganisms. Since many of thecontaminated environments have combinations of ni-troaromatic compounds present, this further compli-cates the bioremediation efforts. Research in explorationof new metabolic pathways has considerably improvedour understanding of the genes and enzymes responsiblefor key nitroaromatic biotransformation reactions. Al-though the field of metabolic engineering is still young, ithas helped further understanding the nature of nitro-aromatic biodegradation systems, which in turn isbeginning to have an impact on the development ofbioremediation solutions. A potential area to explore isthe possibility of producing value-added products fromnitroaromatic wastes. Development of biocatalytic pro-cesses that utilize relatively inexpensive nitroaromaticwaste feedstocks for commercially useful productswould potentially reduce waste at source and reducecomplications in bioremediation efforts. A combinationof increasing commercial interest and advances in ourunderstanding of the genetic and biochemical basis ofbiodegradation is expected to produce a more rationalapproach to bioremediation technology.

References

Accashian, J.V., Vinopal, R.T., Kim, B.-J. & Smets, B.F. 1998 Aerobic

growth on nitroglycerin as the sole carbon nitrogen, and energy

source by a mixed bacterial culture. Applied and Environmental

Microbiology 64, 3300–3304.

ASTDA. 1999 Toxicological profile for nitrobenzene. Web site:

www.atsdr.cdc.gov/tfacts140.html. Agency for Toxic Substances

and Disease Registry, Department of Health and Human Services,

Public Health Service. Atlanta, GA, USA.

Behki, R., Topp, E., Dick, W. & Germon, P. 1993 Metabolism of the

herbicide atrazine by Rhodococcus strain. Applied and Environ-

mental Microbiology 59, 1955–1959.

Behrend, C. & Heesche-Wagner, K. 1999 Formation of Hydride-

Meisenheimer complexes of picric acid (2,4,6-trinitrophenol) and

2,4-dinitrophenol during mineralization of picric acid by Nocar-

dioides sp. Strain CB 22-2. Applied and Environmental Microbiology

65, 1372–1377.

Bhaumik, S., Christodoulatos, C., Korfiatis, G.P. & Brodman, B.W.

1997 Aerobic and anaerobic biodegradation of nitroglycerin in

batch and packed bed bioreactors. Water Science and Technology

36, 139–146.

Bhushan, B., Chauhan, A., Samanta, S.K. & Jian, R.K. 2000 Kinetics

of biodegradation of p-nitrophenol by different bacteria. Biochem-

ical and Biophysical Research Communications 274, 626–630.

Bhushan, B., Halasz, A., Spain, J. & Hawari, J. 2002a Diaphorase

catalysed biotransformation of RDX via N-denitration mecha-

nism. Biochemical and Biophysical Research Communications 296,

779–784.

Bhushan, B., Halasz, A., Spain, J., Thiboutot, S., Ampleman, G. &

Hawari, J. 2002b Biotransformation of hexahydro-1,3,5-trinitro-

1,3,5-triazine catalysed by a NAD(P)H: nitrate oxidoreductase

from Aspergillus niger. Environmental Science and Technology 36,

3104–3108.

Binks, P.R., French, C.E., Nicklin, S. & Bruce, N.C. 1996 Degradation

of pentaerythritol tetranitrate by Enterobacter cloacae PB2.

Applied and Environmental Microbiology 62, 1214–1219.

Binks, P.R., Nicklin, S. & Bruce, N.C. 1995 Degradation of hexahy-

dro-1,3,5-trinitro-1,3,5-triazine (RDX) by Stenotrophomonas

maltophilia PB1. Applied and Environmental Microbiology 61,

1318–1322.

Blasco, R., Moore, E., Wray, V., Pieper, D., Timmis, K. & Castillo, F.

1999 3-Nitroadipate, a metabolic intermediate for mineralization

of 2,4-dinitrophenol by a new strain of a Rhodococcus species.

Journal of Bacteriology 181, 149–152.

Blehert, D.S., Fox, B.G. & Chambliss, G.H. 1999 Cloning and

sequence analysis of two Pseudomonas flavoprotein xenobiotic

reductases. Journal of Bacteriology 181, 6254–6263.

Blehert, D.S., Knoke, K.L., Fox, B.G. & Chambliss, G.H. 1997

Regioselectivity of nitroglycerin denitration by flavoprotein nitro-

esters reductase purified from two Pseudomonas species. Journal of

Bacteriology 179, 6254–6263.

Boopathy, R., Gurgas, M., Ullian, J. & Manning, J.F. 1998 Meta-

bolism of explosive compounds by sulfate-reducing bacteria.

Current Microbiology 37, 127–131.

Cartwright, N.J. & Cain, R.B. 1958 Bacterial degradation of the

nitrobenzoic acids. Biochemical Journal 71, 248–261.

Chauhan, A. & Jain, R.K. 2000 Degradation of o-nitrobenzoate via

anthranilic acid (o-aminobenzoate) by Arthrobacter protophormiae:

a plasmid-encoded new pathway. Biochemical and Biophysical

Research Communications 267, 236–244.

Chauhan, A., Chakraborti, A.K., & Jain, R.K. 2000 Plasmid-encoded

degradation of p-nitrophenol and 4-nitrocatechol by Arthrobacter

protophormiae. Biochemical and Biophysical Research Communica-

tions 270, 733–740.

Cho, C.M., Mulchandani, A. & Chen, W. 2002 Bacterial cell surface

display of organophosphorus hydrolase for selective screening of

improved hydrolysis of organophosphate nerve agents. Applied and

Environmental Microbiology 68, 2026–2030.

Christodoulatos, C., Bhaumik, S. & Brodman, B.W. 1997 Anaerobic

biodegradation of nitroglycerin. Water Research 31, 1462–

1470.

Clausen, G.B., Larsen, L., Johnsen, K., De Lipthay, J.R. & Aamand,

J. 2002 Quantification of the atrazine-degrading Pseudomonas sp.

strain ADP in aquifer sediment by quantitative competitive

polymerase chain reaction. FEMS Microbiology Ecology 41, 221–

229.

Coleman V.N., Nelson, D.R. & Duxbury, T. 1998 Aerobic biodegra-

dation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as a ni-

trogen source by a Rhodococcus sp. strain DN22. Soil Biology and

Biochemistry 30, 1159–1167.

Coleman, V.N., Spain, J.C. & Duxbury, T. 2002 Evidence that RDX

biodegradation by Rhodococcus sp. Strain DN22 is plasmid-borne

and involves a cytochrome P-450. Journal of Applied Microbiology

93, 463–472.

Cook, A.M., Beilstein, P., Grossenbacher, H. & Hutter, R. 1985 Ring

cleavage and degradative pathway of cyanuric acid in bacteria.

Biochemical Journal 231, 25–30.

De Souza M.L., Newcombe, D., Alvey, S., Crowley, D., Hay, A.,

Sadowaky, M.J. & Wackett, L.P. 1998a Molecular basis of a

bacterial consortium: interspecies catabolism of atrazine. Applied

and Environmental Microbiology 64, 178–184.

De Souza M.L., Wackett L.P. & Sadowaky, M.J. 1998b The atzABC

genes encoding atrazine catabolism are located on a self-transmis-

sible plasmid in Pseudomonas sp. strain ADP. Applied and


Dickel, O., Huang, W. & Knackmuss, H.J. 1993 Biodegradation of

nitrobenzene by a sequential anaerobic–aerobic process. Biodegra-

dation 4, 187–194.

Donlon, BA., Razo-Flores, E., Lettinga, G. & Field, J.A. 1996

Continuous detoxification, transformation, and degradation of

nitrophenols in upflow anaerobic sludge blanket (UASB) reactors.

Biotechnology and Bioengineering 51, 439–449.


Ducrocq, C., Servy, C. & Lenfant, M. 1989 Bioconversion of glyceryl

trinitrate into mononitrates by Geotrichum candidum. FEMS

Microbiology Letters 65, 219–222.

Duran, M., Kim, B.J. & Speece, R.E. 1995 Anaerobic biotransforma-

tion of nitrocellulose. Waste Management 14, 481–487.

Durham, N.N. 1958 Studies on the metabolism of p-nitrobenzoic acid.

Canadian Journal of Microbiology 4, 141–148.

Ebert, S., Rieger, P.G. & Knackmuss, H.J. 1999 Function of coenzyme

F420 in aerobic catabolism of 2,4,6-TNP and 2,4-dinitrophenol by

Nocardioides simplex FJ2-1A. Journal of Bacteriology 181, 2669–

2674.

EPA 2003a. Numeric criteria. http://oaspub.epa.gov/wqsdatabase/

wqsi_epa_criteria.rep_parameter, last updated on February 15,

2003.

EPA 2003b Technology transfer network. Air toxicity website, http://

www.epa.gov/ttn/atw/hlthef/nitroben.html#ref1, last updated on

February 15, 2003.

Fournier, D., Halasz, A., Spain, J., Fiurasek, P. & Hawari, J. 2002

Determination of key metabolites during biodegradation of

hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp.,

Strain DN22. Applied and Environmental Microbiology 68, 66–172.

Freeman, D.L. & Sutherland, K.W. 1998 Biodegradation of hexahy-

dro-1,3,5-trinito-1,3,5-triazine (RDX) under nitrate-reducing con-

dition. Water Science and Technology 38, 33–40.

Freeman, D.L., Caenepeel, B.M. & Kim, R.J. 1996a. Biotransforma-

tion of nitrocellulose under methanogenic conditions. Water

Science and Technology 34, 327–334.

Freeman, D.L., Shanley, R.S. & Scholze, R.J. 1996b. Aerobic

biodegradation of 2,4-dinitrotoluene, aminonitrotoluene isomers

and 2,4-diaminotoluene. Journal of Hazardous Materials 49, 1–14.

French, C.E., Nicklin, S. & Bruce, N.C. 1996 Sequence and properties

of pentaerythritol tetranitrate reductase from Enterobacter cloacae

PB2. Journal of Bacteriology 178, 6623–6627.

French, C.E., Nicklin, S. & Bruce, N.C. 1998 Aerobic degradation of

2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by penta-

erythritol tetranitrate reductase. Applied and Environmental Mi-

crobiology 64, 2864–2868.

Gisi, D., Stucki, G. & Hanselmann, K.W. 1997 Biodegradation of the

pesticide 4,6-dinitro-ortho-cresol by microorganisms in batch

cultures and in fixed-bed column reactors. Applied Microbiology

and Biotechnology 48, 441–448.

Gong, P., Hawari, J., Thiboutot, S., Ampleman, G. & Sunahara, G.I.

2001 Ecotoxicological effects of hexahydro-1,3,5-trinitro-1,3,5-

triazine on soil microbial activities. Environmental Toxicology

and Chemistry 20, 947–951.

Groenewegen, P.E.J. & De Bont, J.A.M. 1992 Degradation of 4-

nitrobenzoate via 4-hydroxylaminobenzoate and 3,4-hydroxy-

benzoate in Comamonas acidovorans NBA-10. Archives of Micro-

biology 158, 381–386.

Groenewegen, P.E.J, Breeuwer, P., Van Helvoort, J.M.L.M., Langen-

hoff, A.A.M., De Vries, F.P. & De Bont, J.A.M. 1992 Novel

degradative pathway of 4-nitrobenzoate in Comamonas acidovo-

rans NBA-10. Journal of General Microbiology 138, 1599–1605.

Haghighi-Podeh, M.R. & Bhattacharya, S.K. 1996 Fate and toxic

effects of nitrophenols on anaerobic treatment systems Water

Science and Technology 34, 345–350.

Haigler, B.E. & Spain, J.C. 1991 Biotransformation of nitrobenzene by

bacteria containing toluene degradation pathway. Applied and


Haigler, B.E. & Spain, J.C. 1993 Biodegradation of 4-nitrotoluene

by Pseudomonas sp. strain 4NT. Applied and Environmental


Haigler, B.E., Johnson, G.R., Suen, W.-C. & Spain, J.C. 1999

Biochemical and genetic evidence for meta-ring cleavage of 2,4,5-

trihydroxytoluene in Burkholderia sp. strain DNT. Journal of

Bacteriology 181, 965–972.

Halasz, A., Spain, J., Paquet, L., Beaulieu, C. & Hawari, J. 2002

Insights into the formation and degradation mechanisms of

methylenedinitramine during the incubation of RDX with anaer-

obic sludge. Envrionmental Science and Technology 36, 633–638.

Hanne, L.F., Kirk, L.L., Apple, S.M., Marayan, A.D. & Bains, K.K.

1993 Degradation and induction specificity in actinomycetes that

degrade p-nitrophenol. Applied and Environmental Microbiology

59, 3505–3508.

Hasegawa, Y., Muraki, T., Tokuyama, T., Iwaki, H., Tatsuno, M. &

Lau, P.C.K. 2000 A novel degradative pathway of 2-nitrobenzoate

via 3-hydroxyanthranilate in Pseudomonas fluorescens strain KU-7.

FEMS Microbiology Letters 190, 185–190.

Hawari, J. 2000 Biodegradation of RDX and HMX: From basic

research to field application. In Biodegradation of Nitroaromatic

Compounds and explosives, eds. Spain, J.C., Highes, J.B. &

Knackmuss, H.-J., pp. 277–300. Boca Raton, FL: Lewis Publish-

ers. ISBN 1-56670-522-3.

Hawari, J., Beaudet, S., Halasz, A., Thiboutot, S. & Ampleman, G.

2000a Microbial degradation of explosives: biotransformation

versus mineralization. Applied Microbiology and Biotechnology 54,

605–618.

Hawari, J., Halasz, A., Sheremata, T., Beaudet, S., Groom, C., Paquet,

L., Rhofir, C., Ampleman, G. & Thiboutot, S. 2000b Character-

ization of metabolites during biodegradation of hexahydro-1,3,5-

trinitro-1,3,5-triazine (RDX) with municipal anaerobic sludge.


Hawari, J., Halasz, A., Beaudet, S., Paquet, L., Ampleman, G. &

Thiboutot, S. 2001 Biotransformation routes of octahydro-1,3,5,7-

tetraznitro-1,3,5,7-tetrazocine by municipal anaerobic sludge. En-

vironmental Science and Technology 35, 70–75.

He, Z. & Spain, J.C. 1997 Studies of the catabolic pathway of

degradation of nitrobenzene by Pseudomonas pseudoalcaligenes

JS45: removal of the amino group from 2-aminomuconic semial-

dehyde. Applied and Environmental Microbiology 63, 4839–4843.

He, Z. & Spain, J.C. 1999 Comparison of the downstream pathways

for degradation of nitrobenzene by Pseudomonas pseudoalcaligenes

JS45 (2-aminophenol pathway) and by Comamonas sp. JS765

(catechol pathway). Archives of Microbiology 171, 309–316.

He, Z., Nadeau, L.J. & Spain, J.C. 2000 Characterization of

hydroxylaminobenzene mutase from pNBZ139 cloned from

Pseudomonas pseudoalcaligenes JS45 A highly associated SDS-

stable enzyme catalyzing an intramolecular transfer of hydroxy

groups. European Journal of Biochemistry 267, 1110–1116.

Huang, S., Lindahl, P.A., Wang, C., Bennett, G.N., Rudolph, F.B.

Hugnes, J.B. 2000 2,4,6-Trinitrotoluene reduction by carbon

monoxide dehydrogenase from Clostridium thermoaceticum. Ap-

plied and Environmental Microbiology 66, 1474–1478.

Hughes, M.C. & Williams, P.A. 2001 Cloning and characterization of

the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in

Pseudomonas putida TW3. Journal of Bacteriology 183, 1225–1232.

James, K.D. & Williams, P.A. 1998 ntn Genes determining the early

steps in the divergent catabolism of 4-nitrotoluene and toluene in

Pseudomonas sp. strain TW3. Journal of Bacteriology 180, 2043–

2049.

Jutzi, K., Cook, A.M. & Hutter, R. 1982 The degradative pathway of

s-triazine and melamine: the steps to ring cleavage. Biochemical

Journal 208, 679–684.

Kaake, R.H., Roberts, D.J., Stevens, T.D., Crawford, R.L. &

Crawford, D.L. 1992 Bioremediation of soils contaminated with

the herbicide 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Applied and


Kadiyala, V. & Spain, J.C. 1998 A two-component monoxygenase

catalyzes both the hydroxylation of p-nitrophenol & the oxidative

release of nitrite from 4-nitrocatechol in Bacillus sphaericus JS905.


Kadiyala, V., Smets, B.F., Chandran, K. & Spain, J.C. 1998 High

affinity p-nitrophenol oxidation by Bacillus sphaericus JS905.

FEMS Microbiology Letters 166, 115–120.

Karns J.S. 1999 Gene sequence and properties of an s-triazine ring-

cleavage enzyme from Pseudomonas sp. strain NRRLB-12227.


Karns J.S. & Eaton, R.W. 1997 Genes encoding s-triazine degradation

are plasmid-born in Klebsislla pneumoniae strain 99. Journal of

Agricultural and Food Chemistry 45, 1017–1022.

132 J. Ye et al.

Khan, H., Harris, R.J., Barna, T., Craig, D.H., Bruce, N.C., Munro,

A.W., Moody, P.C.E. & Scrutton, N.S. 2002 Kinetic and structural

basis of reactivity of pentaerythriol tetranitrate reductase with

NADPH, 2-cyclohexenone, nitroesters and nitroaromatic explo-

sives. Journal of Biological Chemistry 227, 21906–21912.

Kitts, C.L., Cunningham, D.P. & Unkefer, P.J. 1994 Isolation of three

hexahydro-1,3,5-trinitro-1,3,5-triazine degrading species of family

Enterobacteriacaea from nitramine explosive contaminated soil.


Kitts, C.L., Geen, C.E., Otley, R.A., Alvarez, M.A. & Unkefer, P.J.

2000 Type I nitroreductases in soil enterobacteria reduce TNT

(2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-

triazine). Canadian Journal of Microbiology 46, 278–282.

Lendenmann, U. & Spain, J.C. 1998 Simultaneous biodegradation of

2,4-dinitrotoluene and 2,6-Dinitrotoluene in an aerobic fluidized-

bed biofilm reactor. Environmental Science and Technology 32, 82–

87.

Lenke, H., Achtnich, C. & Knackmuss, H.-J. 2000 Perceptives of

bioelimination of polynitroaromatic compounds. In Biodegrada-

tion of Nitroaromatic Compounds and Explosives, eds. Spain, J.C.,

Highes, J.B. & Knackmuss, H.-J. pp. 91–126. Boca Raton, FL:

Lewis Publishers. ISBN 1-56670-522-3.

Lessner, D.J., Johnson, G.R., Parales, R.E., Spain, J.C. & Gibson,

D.T. 2002 Molecular characterization and substrate specificity of

nitrobenzene dioxygenase from Comamonas sp. JS765. Applied and


Leung, K.T., Campbell, S., Gan, Y., White, D.C., Lee, H. & Trevors,

J.T. 1999 The role of the Sphingomonas sp. UG30 pentachloro-

phenol-4-monooxygenase in p-nitrophenol degradation. FEMS

Microbiology Letters 173, 247–253.

Leung, K.T., Tresse, O., Errampalli, D., Lee, H. & Trevors, J.T. 1997

Mineralization of p-nitrophenol by pentachlorophenol-degrading

Sphingomonas spp. FEMS. Microbiology Letters 155, 107–114.

Mandelbaum, R.T., Allan, D. & Wackett, L.P. 1995 Isolation and

characterization of a Pseudomonas sp. that mineralizes the s-

triazine herbicide atrazine. Applied and Environmental Microbiol-

ogy 61, 1451–1457.

Mandelbaum, R.T., Wackett, L. P. & Allan, D. 1993 Mineralization of

the s-triazine ring of atrazine by stable bacterial mixed cultures.


Marshall, S.J. & White, G.F. 2001 Complete denitration of nitrogly-

cerin by bacteria isolated from a washwater soakway Applied


Martinez, B., Tomkins, J., Wackett, L., Wing, R. & Sadowsky, M.J.

2001 Complete nucleotide sequence and organization of the

atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain

ADP. Journal of Bacteriology 183, 5684–5697.

McCormick, N.G., Cornell, K.H. & Kaplan, A.M. 1981 Biodegrada-

tion of hexahydro-1,3,5-trinitro-1,3,5-triazine. Applied and Envi-

ronmental Microbiology 42, 817–823.

McCormick, N.G., Feeherry, F.F. & Levinson, H.S. 1976 Microbial

transformation of 2,4,6-trinitrotoluene and other nitroaromatic

compounds. Applied and Environmental Microbiology 31, 949–

958.

Meng, M., Sun, W-Q., Geelhaar, L.A., Kumar, G., Patel, A.R., Payne,

G.F., Speedie, M.K. & Stacy, J.R. 1995 Denitration of glycerol

trinitrate by resting cells and cell extracts of Bacillus thuringiensis/

cereus and Enterobacter agglomerans. Applied and Environmental


Meulenberg, R., Pepi, M. & De Bont, J.A.M. 1996 Degradation of 3-

nitrophenol by Pseudomonas putida B2 occurs via 1,2,4-benzene-

triol. Biodegradation 7, 303–311.

Michan, C., Delgado, A., Haidour, A., Lucchesi, G. & Ramos, J.L.

1997 In vivo construction of a hybrid pathway for metabolism of

4-nitrotoluene in Pseudomonas fluorescens. Journal of Bacteriology

179, 3036–3038.

Mougin, C., Laugero, C., Asther, M., Dubroca, J., Frasse, P. &

Asther, M. 1994 Biotransformation of the herbicide atrazine by the

white rot fungus Phanerochaete chrysosporium. Applied and Envi-

ronmental Microbiology 60, 705–708.

Mulbry, W.W. 1994 Purification & characterization of an inducible s-

triazine hydrolase from Rhodococcus corallinus NRRL B-15444R.


Munnecke, D.M. 1979 Hydrolysis of organophosphate insecticides by

an immobilized-enzyme system. Biotechnology and Bioengineering

21, 2247–2261.

Nadeau, L.J. & Spain, J.C. 1995 Bacterial degradation of m-nitroben-

zoic acid. Applied and Environmental Microbiology 61, 840–843.

Nagy, I., Compernoll, Ghys, K., Vanderleyden, J. & De Mot, R. 1995

A single cytochrome P-450 system is involved in degradation of the

herbicide EPTC (s-ethyl dipropylthiocarbamate) and atrazine by

Rhodococcus sp. strain NI86/21. Applied and Environmental Mi-

crobiology 61, 2056–2060.

Nishino, S.F. & Spain, J.C. 1993 Degradation of nitrobenzene by a

Pseudomonas pseudocalcaligens. Applied and Environmental Micro-

biology 59, 2520–2525.

Nishino, S.F. & Spain, J.C. 1995 Oxidative pathway for the biode-

gradation of nitrobenzene by Comamonas sp. strain JS765. Applied


Nishino, S.F. & Spain, J.C. 2001 Technology status review: Bioreme-

diation of dinitrotoluene (DNT). http://www.estcp.org/documents/

techdocs/DNT_Report.pdf, last access: February 15 2003.

Nishino, S.F., Paoli, G.C. & Spain, J.C. 2000a. Aerobic degradation of

dinitrotoluenes and pathway for bacterial degradation of 2,6-

dinitrotoluene. Applied and Environmental Microbiology 66, 2139–

2147.

Nishino, S.F., Spain, J.C. & He, Z. 2000b Strategies for aerobic

degradation of nitroaromatic compounds by bacteria s process

discovery to field application, In Biodegradation of Nitroaromatic

Compounds and Explosives, eds. Spain, J.C., Highes, J.B. &

Knackmuss, H.-J. pp. 9–61: Boca Raton, FL: Lewis Publishers.

ISBN 1-56670-522–3.

Nishino, S.F., Spain, J.C., Lenke, H. & Knackmuss, H.-J. 1999

Mineralization of 2,4-and 2,6-dinitrotoluene in soil slurries.

Environmental Science and Technology 33, 1060–1064.

Oh, B.-T., Just, C.L. & Alvarez, P.J.J. 2001 Hexahydro-1,3,5-trinitro-

1,3,5-triazine mineralization by zerovalent iron and mixed anaer-

obic culture. Environmental Science and Technology 35, 4341–4346.

Oren, A., Gurevich, P. & Henis, Y. 1991 Reduction of nitrosubstituted

aromatic compounds by the halophilic anaerobic eubacteria

Haloanaerobium praevalens and Sporohalobacter marismortui. Ap-

plied and Environmental Microbiology 57, 3367–3370.

Parales, J.V., Parales, R.E., Resnick, S.M. & Gibson, D.T. 1998

Enzyme specificity of 2-nitrotoluene 2,3-dioxygenase from Pseudo-

monas sp. strain JS42 is determined by the C-terminal region of the

alpha subunit of the oxygenase component. Journal of Bacteriology

180, 1194–1199.

Pennington, J.C. & Brannon, J.M. 2002 Environmental fate of

explosives. Thermochimica Acta 384, 163–172.

Peres, C.M., Haveau, H. & Agathos, S.N. 1998 Biodegradation of

nitrobenzene by its simultaneous reduction into aniline and

mineralization of the aniline formed. Applied Microbiology and

Biotechnology 49, 343–349.

Peres, C.M., Russ, R., Lenke, H. & Agathos, S.N. 2001 Biodegrada-

tion of 4-nitrobenzoate, 4-aminobenzoate and their mixtures: new

strains, unusual metabolites and insight into pathway regulation.

FEMS Microbiology Ecology 37, 151–159.

Price, C.B., Brannon, J.M. & Yost, S. 1998 Technical report IRRP-98-2

US Army Engineer Waterways Experiment Station, Viksburg, MS.

Qureshi, A.A. & Purohit, H.J. 2002 Isolation of bacterial consortia

for degradation of p-nitrophenol from agricultural soil. Annals of

Applied Biology 140, 159–162.

Ralebitso, T.K., Senior, E. & Van Verseveld, H.W. 2002 Microbial

aspects of strazine degradation in natural environments. Biode-

gradation 13, 11–19.

Razo-Flores, E., Lettinga, G. & Field, J.A. 1999 Biotransformation

and biodegradtion of selected nitroaromatics under anaerobic

conditions. Biotechnology Progress 15, 358–365.

Rieger, P.G. & Knackmuss, H.-J. 1995 Basic knowledge and perspec-

tives on biodegradation of 2,4,6-trinitrotoluene and related


nitroaromatic compounds in contaminated soil. In Biodegradation

of Nitroaromatic Compounds. Environmental Science Research, vol.

49. ed. Spain, J.C. pp. 1–18. New York, NY: Plenum Press, ISBN

0-306-45014-3.

Rieger, P.G., Sinnwell, V., Preub, A., Francke, W. & Knackmuss, H-J.

1999 Hydride-Meisenheimer complex formation and protonation

as key reactions of 2,4,6-trinitrophenol biodegradation by Rhodo-

coccus erythropolis. Journal of Bacteriology 181, 1189–1195.

Rousseaux, S., Soulas, G. & Hartmann, A. 2002 Plasmid localization

of atrazine-degrading genes in newly described Chelatobacter and

Arthrobacter strains. FEMS Microbiology Ecology 41, 69–75.

Russ, R., Walters, D.M. & Knackmuss, H-J. 2000 Identification of

genes involved in picric acid and 2,4-dinitrophenol degradation by

mRNA differential display. In Biodegradation of Nitroaromatic

Compounds and Explosives, eds. Spain, J.C., Highes, J.B. &

Knackmuss, H.-J. pp. 127–143. Boca Raton, FL: Lewis Publishers.

ISBN 1-56670-522-3.

Sadowskym, M.J. & Wackett, L.P. 2000 Genetics of atrazine and s-

triazine degradation by Pseudomonas sp. strain ADP and other

bacteria, In Pesticide Biotransformation in Plants and Microorgan-

isms: Similarities and Divergences. ACS Symposium Series. eds.

Hall, J.C, Hoagland, R.E. & Zablotowicz, R.M. pp. 268–281.

Washington, DC: American Chemical Society.

Schenzle, A., Lenke, H., Fischer, P., Williams, P.A. & Knackmuss,

H.-J. 1997 Catabolism of 3-nitrophenol by Ralstonia eutropha

JMP 134. Applied and Environmental Microbiology 63, 1421–1427.

Seffernick, J.L., De Souza, M., Sadowsky, M.J. & Wackett, L. 2001

Melamine deaminase and atrazine chlorohydrolase: 98 percent

identical but functionally different. Journal of Bacteriology 183,

2405–2410.

Servent, D., Ducrocq, C., Henry, Y., Guissani, A. & Lenfant, M. 1991

Nitroglycerin metabolism by Phanerochaete chrysosporium: evi-

dence for nitric oxide and nitrite formation. Biochimica et

Biophysica Acta 1074, 320–325.

Seth-Smith, H.M.B., Rosser, S., Basran, A., Travis, E.R., Dabbs,

E.R., Nicklin, S. & Bruce, N.C. 2002 Cloning sequencing, and

characterization of the hexahydro-1,3,5-trinitro-1,3,5-triazine de-

gradation gene cluster from Rhodococcus rhodochrous. Applied and


Shao, Z.Q. & Behki, R. 1995 Cloning of the genes for degradation of

the herbicides EPTC (s-ethyl dipropylthiocarbamate) and atrazine

from Rhodococcus sp. strain TE1. Applied and Environmental


Shapir, N., Osborne, J.P., Johnsin, G., Sadowsky, M.J. & Wackett,

L.P. 2002 Purification substrate range, and metal center of AtzC:

N-isopropylammelide aminohydrolase involved in bacterial at-

razine metabolism. Journal of Bacteriology 184, 5376–5384.

Sharma, A., Sundaram, S.T., Zhang, Y.-Z. & Brodman, B.W. 1995

Nitrocellulose degradation by a coculture of Sclertium rolfsii and

Fusarium solani. Journal of Industrial Microbiology 15, 1–4.

Sheremata, T.W. & Hawari, J. 2000 Mineralization of RDX by the

white rot fungus Phanerochaete chrysosporium to carbon dioxide

and nitrous oxide. Environmental Science and Technology 34, 3384–

3388.

Sheremata, T.W., Halasz, A., Paquet, L., Thiboutot, S., Ampleman,

G. & Hawari, J. 2001 The fate of the cyclic nitramine explosive

RDX in natural soil. Environmental Science and Technology 35,

1037–1040.

Shimazu, M., Mulchandani, A. & Chen, W. 2001 Simultaneous

degradation of organophosphorus pesticides and p-nitrophenol

by a genetically engineered Moraxella sp. with surface-expressed

organophosphorus hydrolase. Biotechnology and Bioengineering

76, 318–324.

Shinozaki, Y., Nobutada, K. & Nakahara, T. 2002 Difference in

degradation p-Nitrophenol between indigenous bacteria in reactor.

Journal of Bioscience and Bioengineering 93, 512–514.

Somerville, C.C., Nishino, S.F. & Spain, J.C. 1995 Purification

and characterization of nitrobenzene nitroreductase from Pseudo-

monas pseudoalcaligenes JS45. Journal of Bacteriology 177, 3837–

3482.

Spain, J.C. 1995 Bacterial degradation of nitroaromatic compound

under aerobic conditions, In Biodegradation of Nitroaromatic

Compounds. Environmental Science Research, vol. 49, eds Spain,

J.C. pp. 19–35, New York: Plenum Press, N.Y. ISBN 0-306-45014-

3.

Spain, J.C. & Gibson, D.T. 1991 Pathway for biodegradation of p-

nitrophenol in a Moraxella sp. Applied and Environmental Micro-

biology 57, 812–819.

Spanggord, R.J., Spain, J.C., Nishino, S.F. & Mortelmans, K.E. 1991

Biodegradation of 2,4,-dinitrotoluene by Pseudomonas sp. Applied


Stevens, T.O., Crawford, R.L. & Crawford, D.L. 1990 Biodegradation

of dinoseb (2-sec-butyl-4,6-dinitrophenol) in several Idaho soils

with various dinoseb exposure histories. Applied and Environmental


Stevens, T.O., Crawford, R.L. & Crawford, D.L. 1991 Selection and

isolation of bacteria capable of degrading dinoseb 2-sec-butyl-4,6-

dinitrophenol. Biodegradation 2, 1–13.

Strong L.C., Rosendahl, C., Johnson, G., Sadowsky, M.J. & Wackett,

L.P. 2002 Arthrobacter aurescens TC1 metabolizes diverse s-

triazine ring compound. Applied and Environmental Microbiology

68, 5973–5980.

Suen, W.C. & Spain, J.C. 1993 Cloning and characterization of

Pseudomonas sp. strain dinitrotoluene genes for 2,4-dinitrotoluene

degradation. Journal of Bacteriology 175, 1831–1837.

Topp. E., Mulbry, W.M., Zhu, H., Nour, S.M. & Cuppels, D. 2000

Characterization of s-triazine herbicide metabolism by a Nocar-

dioides sp. isolated from agricultural soils. Applied and Environ-

mental Microbiology 66, 3134–3141.

Uberoi, V. & Bhattacharya, S.K. 1997 Toxicity and degradability of

nitrophenol in anaerobic system. Water Environment Research 69,

146–156.

Van Aken, B., Hofrichter, M., Scheibner, K., Hatakka, A.I., Naveau,

H. & Agathos, S.N. 1999 Transformation and mineralization of

2,4,6-trinitrotoluene (TNT) by manganese peroxidase from the

white-rot Basidiomycete phlebiaradiata Biodegradation 10, 83–91.

Wackett, L.P. & Hershberger, C.D. 2001a Evolution of catabolic

enzymes and pathways, In Biocatalysis and Biodegradation: Mi-

crobial Transformation of Organic Compounds, eds. Wackett, L.P.

& Hershberger C.D. pp.115–134. Washington, DC: ASM Press,

ISBN 1555811795.

Wackett, L.P. & Hershberger, C.D. 2001b The impact of genomics on

microbial catalysis, In Biocatalysis and Biodegradation Microbial

Transformation of Organic Compounds, eds. Wackett, L.P. &

Hershberger, C.D. pp. 191–204: Washington, DC: ASM Press.

Wackett, L.P., Sadowsky, M.J. & Martinez, N.S. 2002 Biodegradation

of atrazine and related s-triazine compounds: from enzymes to

field studies. Applied Microbiology and Biotechnology 58, 39–45.

Walker, A.W. & Keasling, J.D. 2002 Metabolic engineering of

Pseudomonas putida for the utilization of parathion as a carbon

and energy source. Biotechnology and Bioengineering 78, 715–721.

Wang, J., Zhou, J.-T., Zhang, J.-S., Zhang, A.-L. & Hong, L.U. 2001

Aerobic degradation of nitrobenzene by Pseudomonas sp. JX165

and its intact cells. China Environmental Science 21, 144–147.

Wendt, T.N., Cornell, J.H. & Kaplan, A.M. 1978 Microbial degra-

dation of glycerol nitrates. Applied and Environmental Microbiol-

ogy 36, 693–699.

White, G.F. & Snape, J.R. 1993 Microbial cleavage of nitrate esters:

defusing the environment. Journal of General Microbiology 139,

1947–1957.

White, G.F., Snape, J.R. & Nicklin, S. 1996a Bacterial biodegradation

of glycerol trinitrate. International Biodeterioration and Biodegra-

dation 19, 77–82.

White, G.F., Snape, J.R. & Nicklin, S. 1996b Biodegradation of

glycerol, trinitrate and pentaerythritol tetranitrate by Agrobacte-

rium radiobacter. Applied and Environmental Microbiology 62, 637–

642.

Williams, R.E. & Bruce, N.C. 2000 Strategies for aerobic degradation

of nitroaromatic compounds by bacteria s process discovery to

field application, In Biodegradation of Nitroaromatic Compound

134 J. Ye et al.

and Explosives, eds. Spain, J.C., Highes, J.B. & Knackmuss, H.-J.

pp. 161–184. Boca Raton, FL: Lewis Publishers. ISBN 1-56670-

522-3.

Williams, R.E. & Bruce, N.C. 2002 New uses for an old enzyme-the

old yellow enzyme family of flavoenzymes. Microbiology 148,

1607–1614.

Yabannavar, A.V. & Zylstra, G. 1995 Cloning and characterization of

the genes for p-nitrobenzoate degradation from Pseudomonas

pickettii YH105. Applied and Environmental Microbiology 61,

4284–4290.

Yanze-Kontchou, C. & Gschwind, N. 1994 Mineralization of the

herbicide atrazine as a carbon source by a Pseudomonas stain.


Yinon, J. 1990 Toxicity and Metabolism of Explosive. CRC Press, Ann

Arbor, Michigan. ISBN 0-8493-5128-6.

Zeyer, J., Wasserfallen, A. & Timmis, K.N. 1985 Microbial minerali-

zation of ring-substituted anilines through an ortho-cleavage

pathway. Applied and Environmental Microbiology 50, 447–

453.

Zhao, J.-S. & Ward, O.P. 1999 Microbial degradation of nitrobenzene

and mono-nitrophenol by bacteria enriched from municipal

activated sludge. Canadian Journal of Microbiology 45, 427–

432.

Zhao, J.-S. & Ward, O.P. 2001 Substrate selectivity of a 3-nitrophenol-

induced metabolic system in Pseudomonas putida 2NP8 transform-

ing nitroaromatic compounds into ammonia under aerobic con-

ditions. Applied and Environmental Microbiology 67, 1388–1391.

Zhao, J.-S., Ward, O.P, Lubicki, P., Cross, J.D. & Huck, P. 2001

Process for degradation of nitrobenzene: combining electron beam

irradiation with biotransformation. Biotechnology and Bioengi-

neering 73, 306–312.

Zhao, J.-S., Halasz, A., Paquet, L., Beaulieu, C. & Hawari, J. 2002

Biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine and its

mononitroso derivative hexahydro-1-nitroso-3,5-dinitro-1,3,5-tri-

azine by Klebsiella pneumoniae strain SCZ-1 isolated from an

anaerobic sludge. Applied and Environmental Microbiology 68,

5336–5341.

Zhongli, C., Shunpeng, L., & Guoping, F. 2001 Isolation of methyl

parathion-degrading strain M6 and cloning of the methyl parathi-

on hydrolase gene. Applied and Environmental Microbiology 67,

4922–4925.

Zylstra, G.J., Bang, S-W., Newman, L.M. & Perry, L.L. 2000

Microbial degradation of mononitrophenols and mononitrobenzo-

ates, In Biodegradation of Nitroaromatic Compound and Explosives,

eds. Spain, J.C., Highes, J.B. & Knackmuss, H.-J. pp. 145–160.

Boca Raton. FL: Lewis Publishers. ISBN 1-56670-522–3.


Biodegradation of nitroaromatics and other nitrogen-containing xenobiotics

Documents

Transcript of Biodegradation of nitroaromatics and other nitrogen-containing xenobiotics