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Bioelimination of trinitroaromatic compounds: immobilization versus mineralization Gesche Heiss* and Hans-Joachim Knackmuss Electron deficiency of trinitroaromatic compounds favors gratuitous reduction of nitro groups or unique ring hydrogenation. From nitro-group reduction of 2,4,6-trinitrotoluene (TNT), some highly reactive products are generated that are subject to further transformation or interaction with diverse electrophiles. Up to now, only initial ring hydrogenation of picric acid (2,4,6-trinitrophenol) opens perspectives of complete degradation. This review focuses on recent findings that may be relevant for bioremediation or complete degradation of TNT or picric acid. Addresses Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany *Author for correspondence: Gesche Heiss; e-mail: [email protected] Current Opinion in Microbiology 2002, 5:282–287 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 7 May 2002 Abbreviations 2H––PA 2,4-DNP H––2,4-DNP H––PA TAT TNT

dihydride σ-complex of picric acid 2,4-dinitrophenol hydride σ-complex of 2,4-dinitrophenol hydride σ-complex of picric acid 2,4,6-triaminotoluene 2,4,6-trinitrotoluene

Introduction Production and use for military operations have made polynitroaromatic compounds such as 2,4,6-triaminotoluene (TNT) and picrate-relevant organic contaminants of soil. They are of major concern, owing to their toxicity and relatively high solubility in water. Numerous nitroaromatic-compound-degrading microorganisms have been described, the most notable of which are the few bacterial strains that harbor complete catabolic sequences for mineralization of these xenobiotics. This work has been compiled in recent reviews [1•,2•,3,4]. Nitroaromatic compounds are rare amongst natural compounds. Two or more nitro groups on the aromatic ring generate a highly xenobiotic character (which is partly due to the strong electron-withdrawing properties of the nitro substituents) that confers a high electron deficiency on the entire π-electron system [5,6•]. Consequently, these xenobiotic compounds are subject to initial reductive, rather than the usual oxidative, transformation by oxygenases of aerobic microorganisms. Nitro-group reduction can readily proceed via one or two electron transfers [5,7]. A great number of reductive transformations of polynitroaromatic compounds were described as taking place in vivo [5,8],

in vitro [9•,10–12] or chemically [7]. The extent of nitro-group reduction and the kind of products formed depend on the type of organisms involved and the chemical composition of the medium. Although most nitro-group reductions have to be considered to be gratuitous reactions, in Pseudomonas sp. strain JLR11, TNT reduction was shown to be coupled to proton translocation and ATP synthesis [13]. In contrast, biological hydride transfer to the aromatic ring system seems to be a rare initial reaction. It is observed in aerobic, mostly Gram-positive, bacteria that generate hydride σ-complexes, particularly in picric-acid-utilizing strains (see later). This review focuses on recent findings that may be relevant for bioremediation or complete degradation of TNT or picrate [5].

Reduction of nitro groups — treatment of soil The resistance to complete mineralization and utilization of TNT appears to be due to the ease of gratuitous reduction of nitro groups and subsequent chemical misrouting of reactive intermediates. Hydroxylaminodinitro-, aminodinitro- and diaminonitrotoluenes, as well as bi- and polynuclear condensation products such as nitro- and/or aminosubstituted azoxytoluenes [1•,5] and even biphenyls [9•], have been identified (Figure 1). All these partially reduced derivatives of TNT are dead-end products under aerobic conditions and, consequently, constitute proven or potential co-contaminants at sites of aged TNT contamination. Co-reduction of TNT can be enhanced enormously by readily metabolizable substrates such as carbohydrates [14], so that, under strict anaerobic conditions (≤–200 mV), TNT and its partially reduced secondary products finally converge into 2,4,6-triaminotoluene (TAT) [5,8]. For immobilization-based remediation (see later), it is important that the abundance of products generated under aerobic or semiaerobic conditions decreases substantially under highly reducing conditions (see Figure 10 in [15]). TAT is highly reactive towards electrophiles and oxidizing agents. In the presence of oxygen and, in particular, heavy metal ions as redox mediators, TAT is subject to rapid autoxidation, polymerization and irreversible binding to electrophilic matrices [15]. Despite its high chemical reactivity, which prevents productive mineralization, TAT holds the position of an attractive key metabolite under anaerobic or anoxic conditions. Firstly, TNT and all its partially reduced derivatives can readily and quantitatively be funneled to TAT under highly reducing conditions [5,15]. Secondly, it disappears on prolonged incubation through spontaneous hydrolytic deamination. Therefore, TAT should either serve as a nitrogen source or in the presence of an electron

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Figure 1

(a) Reduction of NO2– groups (R=CH3) Polynuclear azo- and azoxy-derivatives

O2 N

Anoxic

Coupling reactions

CH3

n[H]

NO2

O2 N 2[H]

CH3 H2 N

NO2

NH2

O2

2[H]

2[H] NO

NO2

12[H]

CH3

HONH

NH2

Sequestration and humification

NH2

≥ –200 mV

Successive reduction of nitro groups Increasing basicity and reactivity with acidic and electrophilic structures

(b) Ring hydrogenation (R=OH, CH3) R

R O 2N

NO2

[ H–]

O2 N

NO2 H H

NO2

NO2–

–O N 2 [ H–]

H H

[ H–]

R = OH

R NO2 H H

NO2–

NO2– R = CH3 NO2–

Total and productive degradation 2NO2–

Biphenyl derivatives as dead-end products

Coupling reactions Current Opinion in Microbiology

Initial reductive reactions for, and fate of, trinitroaromatic compounds. [H–], hydride transfer reactions.

acceptor like NO3– as a carbon and energy source. The latter possibility holds much promise, more so given that NO3– does not react with TAT under physiological conditions (O Dickel, H-J Knackmuss, unpublished data). In spite of the efforts from different laboratories to isolate bacteria that utilize TNT or TAT under anaerobic or anoxic conditions, strict evidence for productive breakdown of these xenobiotics is still lacking. Work in this direction has been initiated by Esteve-Nuñez et al. [13,16], who demonstrated a kind of TNT respiration in a facultative Pseudomonas strain. Progressive reduction of TNT also proceeds in soil amended with appropriate auxiliary substrates as electron donors. Characteristically, aminodinitro- and diaminonitrotoluenes are observed as intermediates, whereas the highly reactive hydroxylaminodinitrotoluenes and TAT were transiently detectable only at very high loads of TNT (4g per kg) [17–19]. Biologically induced immobilization and humification of TNT and congeneric compounds is the basis of simple and cost-effective anaerobic or aerobic bioslurry or windrow-composting processes [2•]. Transformation by bioslurrying is often faster and makes nitro-group reduction more complete, particularly if readily available electron donors such as glucose or saccharose are present in excess and mass transfer is enhanced by mechanical agitation [5]. Composting in windrow piles involves mixing with organic wastes and bulking material, allowing intermittent

aeration through regular turning. During aeration, the temperature may rise to ≤75°C, which enhances disappearance of the contaminants [20]. As shown by 14C–TNT experiments and NMR studies with 15N–TNT, extensive anaerobic and subsequent aerobic treatment in a bioslurry does not result in mineralization, but largely covalent and irreversible incorporation of labeled material into fulvic acid (7%), humic acid (8%) and humin (85%). Less than 0.2% of the total radioactivity was extractable and associated with soluble soil organic matter [20]. 15N-NMR studies with silylated whole soil revealed that primary amines produced during extensive reduction disappeared during subsequent aerobic treatment. The signals indicating amide and tertiary amine structures increased simultaneously [21,22]. Characteristically, the capability of fungi, which, by generating highly efficient oxidants, can attack a broad spectrum of xenobiotic compounds, mineralize TNT in soil to a limited extent (≤20%). Again, irreversible chemisorption is due to the intermediates that react with humic substances [23]. Comparative studies using highly contaminated soil from a former production plant (Werk Tanne, near ClausthalZellerfeld, Germany) show that bioslurrying or composting can eliminate TNT from contaminated soil by chemisorption. As demonstrated by remobilization studies, the bioslurry process allows an extended nitro group reduction and, thus, multivalent chemisorption. In contrast, the

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Figure 2

(a)

O–

NO2

NO2

O

First ring (b) hydrogenation O N 2

NO2–

NpdI

F420H2

H

F420

H –

NO2

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NpdG

Proposed upper picric-acid-degradation pathway in R. (opacus) erythropolis HL PM-1: (a) picrate; (b) H––PA; (c) 2H––PA; (d) and (e) tautomeric forms of the protonated 2H––PA; (f) H––2,4-DNP. NpdC, hydride transferase I; NpdG, NADPH-dependent F420 reductase; NpdH, converts 2H––PA to a tautomer of the protonated 2H––PA; NpdI, hydride transferase II.

NADP+

F420H2

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NpdG NADPH/H+

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(d) –O N 2

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H H

H NO2

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–H+

H

(c) O

+H+ –

NO2–

O2N

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H

O –

–

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H H

–H+ +H+

H NO2–

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H

H

H N HO

OH

Elimination of nitrite NO2–

(f)

O NO2– H H NO2– Current Opinion in Microbiology

composted material, as shown by solid state 15N-NMR analysis, still contains intact nitro groups and considerable amounts of water-soluble humic substances that carry bound TNT derivatives [20]. In the composted soil, ecotoxicity is high (including background toxicity), whereas the soil from the bioslurry process exhibits no residual toxicity. The tests were carried out in soil eluates using aquatic test organisms or by analyzing the habitat function of treated soil [5,18]. As demonstrated by neuroblastoma cell lines, the most prevalent initial transformation products of TNT, the hydroxylaminodinitrotoluenes, cause the most toxic effects [24]. This supports the above suggestion that

extensive reduction of the nitro groups of TNT during anaerobic treatment in bioslurrying is necessary for multivalent and irreversible binding to the soil matrix. Using an immunoassay able to detect TNT residues covalently linked to humic acids, it became clear that the initially present signal for bound trinitrophenylmethyl residues is reduced at a considerably slower rate than that of free TNT or its partially reduced products [5].

Ring hydrogenations — total degradation Picric acid is more amenable to complete degradation than is TNT [5,25,26]. The initial degradative step is a unique F420H2-dependent, reductive attack on the aromatic ring, creating the hydride σ-complex of picric acid

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Figure 3

npdH orfA

orfB

npdC

orfD

orfE

orfF

npdR

npdG

npdI

orfJ

orfK

Picric acid degradation gene cluster of R. (opacus) erythropolis HL PM-1 (GenBank accession number AF323606 [28•]). The open reading frames and direction of transcription are indicated by the open arrows. The binding sites of NpdR are shown by open circles. NpdC, gene for hydride transferase I; npdG, gene for

NADPH-dependent F420 reductase; npdH, gene for protein converting 2H––PA to a tautomer of the protonated 2H––PA; npdI, gene for hydride transferase II; npdR, gene for the transcriptional regulator of the IclR type. Reproduced, with permission, from [29].

(H––PA; Figure 2). Both the responsible NADPH-dependent F420 reductase and the hydride transferase have been identified in the picric-acid-degrading strains Rhodococcus (opacus) erythropolis HL PM-1 and Nocardioides simplex FJ2-1A [27,28•]. The genes for NpdG (NADPHdependent F420 reductase) and NpdI (hydride transferase II) in R. (opacus) erythropolis HL PM-1 are contained in an npd gene cluster (Figure 3) [29]. NpdC (hydride transferase I) performs a second reductive step, creating the dihydride σ-complex of picric acid (2H––PA). As opposed to R. (opacus) erythropolis HL PM-1, it appears that N. simplex FJ2-1A contains a hydride transferase with a broader substrate specificity, catalyzing both ring hydrogenations [30].

DNA sequence similarities of 80–100% to the npd genes of R. (opacus) erythropolis HL PM-1 [33]. Hence, the ability to hydrogenate the aromatic ring may be fairly widespread. Preliminary experiments further indicate that some of these npd genes may be located on plasmids, suggesting that these npd genes may be mobilizable (GS Heiss, N Trachtmann, H-J Knackmuss, unpublished data).

When R. (opacus) erythropolis HL PM-1 is exposed to TNT, the hydride σ-complex of TNT (H––TNT) and the dihydride σ-complex of TNT (2H––TNT) are detected [31]. These are analogous to the hydride complexes of picric acid. However, no further productive breakdown takes place, whereas 2H––PA can be further degraded. A possible scenario could be that the initial hydrogenation steps of TNT are catalyzed by NpdG, NpdC and NpdI, albeit less efficiently than for picric acid and its hydride complexes. Hence, the amount of 2H––TNT generated may be too low for subsequently efficient nitrite release. NpdH (putative ‘tautomerase’) of strain HL PM-1 converts 2H––PA to a product that has been tentatively identified as a tautomeric form of the protonated 2H––PA [29]. A similar observation has been made with 2H––TNT during TNT degradation by the same strain [31]. Other groups have also detected several protonated forms of 2H––TNT [9•,32]. (We would like to point out that Pak et al. [9•] referred to the hydride complexes as H–TNT and 2H–TNT, which suggests that the complexes may have been derived from the addition of a hydrogen atom [H], rather than a hydride anion [H–]. Hence, we think the hydride complexes should be abbreviated as we have done above, to show that it is the hydride ion that is being transferred.) npdC, npdG, npdH and npdI have been amplified from several 2,4-dinitrophenol (2,4-DNP) degraders, exhibiting

The hydride σ-complex of 2,4-dinitrophenol (H––2,4-DNP) was originally identified as an intermediate of picric acid degradation in Nocardioides sp. strain CB 22-2 [25]. Later, it was suggested that H––2,4-DNP is a metabolite of picric acid degradation and not 2,4-DNP, as previously thought [30]. This is feasible by assuming that nitrite is released either from 2H––PA or a tautomer of the protonated 2H––PA. Following nitrite release and hydrolytic ring cleavage, two metabolites have been proposed, 4,6-dinitrohexanoate [34] and 3-nitroadipate [35]. Several of the npd genes of R. (opacus) erythropolis HL PM-1, showing sequence similarities to enzymes responsible for the β-oxidation of fatty acids [28•], might be involved in the oxidation of 4,6-dinitrohexanoate. The picric acid degradation capacity of R. (opacus) erythropolis HL PM-1 is inducible by 2,4-DNP [28•,36]. Gel mobility shift assays showed that binding of NpdR to the DNA is affected by the presence of 2,4-DNP, picric acid, 2-chloro-4,6-dinitrophenol and 4,6-dinitro-2-methylphenol, but not TNT (DP Nga, H-J Knackmuss, GS Heiss, unpublished data). This indicates that the -OH group of the effector may be necessary for altering the conformation of NpdR for DNA binding, whereas the -CH3 group on TNT may not have an effect. Gel mobility shift assays also showed that NpdR binds to two different intervening regions in the operon, indicating that the npd gene cluster consists of at least two operons (Figure 3). As discussed by Blehert, Fox and Chambliss [37], the detoxification role of the xenobiotic reductases, such as the old yellow enzyme, is an interesting proposition, considering their broad substrate range. Although npdC, npdG and npdI probably do not serve as detoxification enzymes, they may have an additional function in the cell not specific to picric acid degradation. Firstly, as already pointed out in [29], enzymes similar to the NADPH-dependent F420 reductase (NpdG) have been found in methanogenic Archaea and Streptomyces. Secondly,

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npdC, npdG and npdI are interspersed in the operon amongst genes involved in the β-oxidation of fatty acids (Figure 3). Lastly, as mentioned above, H––TNT and 2H––TNT have been found in culture supernatants of R. (opacus) erythropolis incubated with TNT [31]. These hydride complexes may be due to the hydrogenation activities of NpdI and NpdC. These findings suggest that the genes, originally evolved for a different role, may have been recruited for utilization of picric acid as a sole nitrogen, carbon and energy source.

Conclusions Owing to its pronounced electron deficiency, TNT is preferentially subject to reductive transformation of its nitro groups. Extensive metabolic and chemical misrouting prevents highly reactive intermediates from being mineralized. Chemisorption, particularly of TAT to humic material, is a way out of the dilemma and the basis for remediation technologies. For mineralization of TNT, some possibilities have not been utilized. For example, by a two-stage process, it could be reduced chemoselectively and, thus, generate hydroxylaminotoluenes [4]. These could be transformed by mutases or hydroxylaminolyases generating amino-phenols or catechols as potential key metabolites of oxidative ring cleavage pathways. Also, TAT holds a potential key function for productive mineralization. The ease of its hydrolytic deamination could generate 1,3,5-trihydroxytoluenes that open the possibility of total degradation under fermentative or denitrifying conditions. Finally, both picric acid and TNT undergo two initial reductive hydrogenations. Novel insights into the genes, enzymes and metabolites involved in picric acid degradation make picric acid a suitable model for identifying and overcoming the bottleneck steps in complete degradation of TNT. Identification of conserved regions in the enzyme sequences and mutagenesis (gene shuffling or directed evolution) should facilitate the design of enzymes with improved or new activities for bottleneck metabolites of TNT degradation. Understanding the regulatory mechanisms of picric acid degradation may also open avenues for accomplishing the mineralization of TNT. It is conceivable that the enzymes for picric acid degradation possess (low levels of) activity for the analogous compounds of TNT degradation. Nonproductive TNT degradation may be due to too low an enzyme level because of an inactive regulator in the presence of TNT. Broadening the effector specificity of the regulator may improve productive TNT degradation.

2. Rodgers JD, Bunce NJ: Treatment methods for the remediation of • nitroaromatic explosives. Water Res 2001, 35:2101-2111. This paper lists all relevant technologies including the biological ones that are currently available to remediate TNT-contaminated areas. 3.

Peres CM, Agathos SN: Biodegradation of nitroaromatic pollutants: from pathways to remediation. Biotechnol Annu Rev 2000, 6:197-220.

4.

Nishino SF, Spain JC, He Z: Strategies for aerobic degradation of nitroaromatic compounds by bacteria: process discovery to field application. In Biodegradation of Nitroaromatic Compounds and Explosives. Edited by Spain JC, Hughes JB, Knackmuss HJ. Boca Raton: CRC Press LLC; 2000:7-61.

5.

Lenke H, Achtnich C, Knackmuss HJ: Perspectives of bioelimination of polynitroaromatic compounds. In Biodegradation of Nitroaromatic Compounds and Explosives. Edited by Spain JC, Hughes JB, Knackmuss HJ. Boca Raton: CRC Press LLC; 2000:91-126.

6. •

Rieger PG, Meier HM, Gerle M, Vogt U, Groth T, Knackmuss HJ: Xenobiotics in the environment: present and future strategies to obviate the problem of biological persistence. J Biotechnol 2002, 94:101-123. The authors critically discuss the perspectives and limitations to evolve and use specific organisms or consortia for the elimination of xenobiotic pollutants like TNT in the environment. 7.

Haderlein SB, Hofstetter TB, Schwarzenbach RP: Subsurface chemistry of nitroaromatic compounds. In Biodegradation of Nitroaromatic Compounds and Explosives. Edited by Spain JC, Hughes JB, Knackmuss HJ. Boca Raton: CRC Press LLC; 2000:311-356.

8.

Hawari J, Halasz A, Beaudet S, Paquet L, Zhou E, Spencer B, Ampleman G, Thiboutot S: Characterization of metabolites in the biotransformation of 2,4,6-trinitrotoluene with anaerobic sludge: role of triaminotoluene. Appl Environ Microbiol 1998, 64:2200-2206.

9. •

Pak JW, Knoke, KL, Noguera DR, Fox BG, Chambliss GH: Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C. Appl Environ Microbiol 2000, 66:4742-4750. This paper provides extensive chemical and spectroscopic analysis of reductive transformation products of TNT. Although the investigation is confined to transformations by a pure enzyme, the very complex chemical interactions that occur between highly reactive products demonstrate the problem of diverse metabolic misrouting. 10. Huang S, Lindahl PA, Wang C, Bennett GN, Rudolph FB, Hughes JB: 2,4,6-Trinitrotoluene reduction by carbon monoxide dehydrogenase from Clostridium thermoaceticum. Appl Environ Microbiol 2000, 66:1474-1478. 11. Riefler RG, Smets BF: Enzymatic reduction of 2,4,6-trinitrotoluene and related nitroarenes: kinetics linked to one-electron redox potentials. Environ Sci Technol 2000, 34:3900-3906. 12. Oh BT, Sarath G, Shea PJ: TNT nitroreductase from a Pseudomonas aeruginosa strain isolated from TNT-contaminated soil. Soil Biol Biochem 2001, 33:875-881. 13. Esteve-Nuñez A, Luchessi G, Philipps B, Schink B, Ramos JL: Respiration of 2,4,6-trinitrotoluene by Pseudomonas sp. strain JLR11. J Bacteriol 2000, 182:1352-1355. 14. Lewis TA, Ederer MM, Crawford RL, Crawford DL: Microbial transformation of 2,4,6-trinitrotoluene. J Ind Microbiol Biotechnol 1997, 18:89-96. 15. Rieger PG, Knackmuss HJ: Basic knowledge and perspectives on biodegradation of 2,4,6-TNT and related nitroaromatic compounds in contaminated soil. In Biodegradation of Nitroaromatic Compounds. Edited by Spain JC. New York: Plenum Press; 1995:1-8.

References and recommended reading

16. Esteve-Nuñez A, Ramos JL: Metabolism of 2,4,6-trinitrotoluene by Pseudomonas sp. JLR11. Environ Sci Technol 1998, 32:3802-3808.

Papers of particular interest, published within the annual period of review, have been highlighted as:

17.

• of special interest •• of outstanding interest 1. Esteve-Nuñez A, Caballero A, Ramos JL: Biological degradation of • 2,4,6-trinitrotoluene. Microbiol Mol Biol Rev 2001, 65:335-352. This is an up-to-date review that covers relevant literature in a very comprehensive and precise way.

Achtnich C, Fernandes E, Bollag JM, Knackmuss HJ, Lenke H: Covalent binding of reduced metabolites of 15N-TNT to soil organic matter during a bioremediation process analyzed by 15N NMR spectroscopy. Environ Sci Technol 1999, 33:4448-4456.

18. Lenke H, Warrelmann J, Daun G, Hund K, Sieglen U, Knackmuss HJ: Biological treatment of TNT-contaminated soil. 2. Biologically induced immobilization of the contaminants and full-scale application. Environ Sci Technol 1998, 32:1964-1971.

Bioelimination of trinitroaromatic compounds: immobilization versus mineralization Heiss and Knackmuss

19. Achtnich C, Sieglen U, Knackmuss HJ, Lenke H: Irreversible binding of biologically reduced metabolites of TNT to soil. Environ Toxicol Chem 1999, 18:2416-2423. 20. Peters D: Biologische Sanierung von Rüstungsaltlasten — Vergleich zweier alternativer Verfahren zur Sanieung TNT-kontaminierter Böden: Kompostierung und Bodensuspensionsverfahren [Diploma thesis]. Freiburg, Germany: University of Freiburg; 2001. [Title translation: Biological remediation of military sites —Comparison of two alternative processes for clean-up of TNMT-contaminated soils: composting versus bioslurrying.] 21. Knicker H, Bruns-Nagel D, Drzyzga O, von Löw E, Steinbach K: Characterization of 15N-TNT residues after an anaerobic/aerobic treatment of soil/molasses mixtures by solid-state 15N NMR spectroscopy. 1. Determination and optimization of relevant NMR spectroscopy parameters. Environ Sci Technol 1999, 33:343-349. 22. Knicker H, Achtnich C, Lenke H: Solid-state 15N NMR analysis of biologically reduced TNT in a soil slurry remediation. J Environ Qual 2001, 30:403-410. 23. Fritsche W, Scheibner K, Herre A, Hofrichter M: Fungal degradation of explosives: TNT and related nitroaromatic compounds. In Biodegradation of Nitroaromatic Compounds and Explosives. Edited by Spain JC, Hughes JB, Knackmuss HJ. Boca Raton: CRC Press LLC; 2000:213-238. 24. Banerjee HN, Verma M, Hou LH, Ashraf M, Dutta SK: Cytotoxicity of TNT and its metabolites. Yale J Biol Med 1999, 72:1-4.

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Compounds and Explosives. Edited by Spain JC, Hughes JB, Knackmuss HJ. Boca Raton: CRC Press LLC; 2000:127-143. Using a genetic approach, this paper describes an elegant method used to discover the enzymes involved in the initial reductive hydrogenation of picric acid. 29. Heiss G, Hofmann KW, Trachtmann N, Walters DM, Rouvière P, Knackmuss HJ: npd gene functions of Rhodococcus (opacus) erythropolis HL PM-1 in the initial steps of 2,4,6-trinitrophenol degradation. Microbiology 2002, 148:799-806. 30. Ebert S, Fischer P, Knackmuss HJ: Converging catabolism of 2,4,6-trinitrophenol (picric acid) and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A. Biodegradation 2001, 12:367-376. 31. Vorbeck C, Lenke H, Fischer P, Spain JC, Knackmuss HJ: Initial reductive reactions in anaerobic microbial metabolism of 2,4,6-trinitrotoluene (TNT). Appl Environ Microbiol 1998, 64:246-252. 32. French CE, Nicklin S, Bruce NC: Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase. Appl Environ Microbiol 1998, 64:2864-2868. 33. Heiss G, Knackmuss HJ: Analysis of the genes of various Actinobacteria encoding the initial hydride transfer to polynitroaromatics. Abstract Q-461 (p678) of the 101st General Meeting of the American Society for Microbiology. 2001 May 20-24, Orlando, FL.

25. Behrend C, Heesche-Wagner K: Formation of hydride–Meisenheimer complexes of picric acid (2,4,6-trinitrophenol) and 2,4-dinitrophenol during mineralization of picric acid by Nocardioides sp. strain CB 22-2. Appl Environ Microbiol 1999, 65:1372-1377.

34. Lenke H, Knackmuss HJ: Initial hydrogenation and extensive reduction of substituted 2,4-dinitrophenols. Appl Environ Microbiol 1996, 62:784-790.

26. Rajan J, Valli K, Perkins RE, Sariaslani FS, Barns SM, Reysenbach AL, Rehm S, Ehringer M, Pace NR: Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains. J Ind Microbiol 1996, 16:319-324.

35. Blasco R, Moore E, Wray V, Pieper D, Timmis K, Castillo F: 3-Nitroadipate, a metabolic intermediate for mineralization of 2,4-dinitrophenol by a new strain of a Rhodococcus species. J Bacteriol 1999, 181:149-152.

27.

36. Walters DM, Russ R, Knackmuss HJ, Pouviere PE: High-density sampling of a bacterial operon using mRNA differential display. Gene 2001, 273:305-315.

Ebert S, Rieger PG, Knackmuss HJ: Function of coenzyme F420 in aerobic catabolism of 2,4,6-trinitrophenol and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A. J Bacteriol 1999, 181:2669-2674.

28. Russ R, Walter DM, Knackmuss HJ, Rouviere PE: Identification of • genes involved in picric acid and 2,4-dinitrophenol degradation by mRNA differential display. In Biodegradation of Nitroaromatic

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Blehert DS, Fox BG, Chambliss GH: Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases. J Bacteriol 1999, 181:6254-6263.