Enzyme and Microbial Technology 30 (2002) 506–517 www.elsevier.com/locate/enzmictec

Production of small molecular weight catalysts and the mechanism of trinitrotoluene degradation by several Gloeophyllum species

David Newcombe, Andrzej Paszczynski*, Wioletta Gajewska, Mario Kro¨ger,1 Gregor Feis,1 Ronald Crawford

Environmental Biotechnology Institute, University of Idaho, Moscow, ID 83844-1052 USA and Department of Microbiology, Molecular Biology & Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA

Abstract The ability of Gloeophyllum species to produce dimethoxybenzoquinones (DMBQ), particularly 2,5-dimethoxyhydroquinone (2,5- DMHQ), and oxalic acid was investigated. The involvement of these compounds, along with hydrogen peroxide and Fe(III), in 2,4,6- trinitrotoluene (TNT) degradation was examined. Salicylic acid (SA) and phenol (PH) were used as probes to trap Fenton process-produced hydroxyl radicals in several of the investigated species. All the cultures degraded SA and PH readily. A low concentration of 2,3-dihydroxy benzoic acids was detected in cultures of only two Gloeophyllum species. TNT was rapidly transformed by G. trabeum, but ring-UL-14C- 14 TNT was not converted to CO2. Mass balance studies indicated that about 74% of the radioactivity from TNT remained in the culture supernatant. Analysis of culture extracts revealed several aromatic nitro-amines and nitro-aldehydes and their oligomeric coupling products formed by Schiff base reaction mechanism. 2,2,6,6-Tetramethyl-1-piperidynyloxide (TEMPO), a stable free radical, was used as a trap in in vitro reactions containing hydrogen peroxide, 2,5-DMHQ, and TNT. The coupling products of TEMPO and 2,5-DMHQ were detected, indicating semi-dimethoxy quinone radical formation. The in vitro Fenton-like reaction containing all above reactants except methoxyqui- nones produced degradation products from TNT similar to those extracted from G. trabeum cultures, suggesting that reduced oxygen species produced by Fenton-like reactions are involved in the transformation of TNT by brown-rot fungi such as G. trabeum. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction from two higher plants, Delbergia melanoxylon (Legu- minoseae) [4] and Acorus calamus (Araceae) [5], well Several recent advances have been made in our knowl- known for their medicinal value in Africa and India, respec- edge of the mechanisms that brown rot fungi use to degrade tively. 2,5-DMBQ was isolated from Lenzites thermophilla wood and other organic compounds. Paszczynski et al. [1] in 1950 by Burton [6] during investigations of the antibiotic found 2,5-dimethoxyhydroquinone and 4,5-dimethoxycat- thermophillin secreted by this . The structure of ther- echol (4,5-DMC) in a culture ex- mophillin (2,5-DMBQ) was not known at the time. Its tract. They suggested that these hydroquinones might be structure, along with that of 3,4-dimethoxy-8-hydroxyiso- involved in Fenton-like reactions that could participate in coumarin, was elucidated many years later by LeBlanc and the depolymerization of lignin. Dimethoxyhydroquinones Babineau [7]. (DMHQ) have been isolated previously and studied as an- Other researchers have reported that liquid cultures of G. timicrobial agents. 2,5-DMHQ was isolated from Polyporus trabeum accumulate low molecular weight aromatic com- fumosus [2] and from G. sepiarium [3]. However, the strong pounds in significant concentrations [8,9]. In related chem- antifungal activity of G. sepiarium was related to produc- ical studies, Parra et al. [10] demonstrated that a mixture of tion of oospolactone 3,4-dimethyl-8-hydroxyisocoumarin, 2,3-dihydroxybenzoic acid (2,3-DA) and 3,4-dihydroxyphe- rather than to 2,5-DMHQ. 2,5-DMHQ was also isolated nylacetic acid (3,4-DB) together with Fe(III) degraded di- oxane-extracted lignin and bleaching effluent to up to 80%, confirming the ability of dihydroxybenzenes to participate ϩ ϩ * Corresponding author. Tel.: 1-208-885-6580; fax: 1-208-885- in degradation of soluble modified lignin. In another labo- 5741. E-mail address: [email protected] (A.J. Paszczynski). ratory, 2,5-DMHQ was also isolated from G. trabeum and 1 University of Paderborn, Faculty of Chemistry and Chemical Engi- was used as the source of electrons for reduction of Fe(III) neering, 33098 Paderborn, Germany. and oxygen in an extracellular, hydroquinone-driven Fen-

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0141-0229(02)00014-5 D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517 507 ton-like reaction. Polyethylene glycol-4000 (PEG) was used Table 1 to examine the depolymerization potential of DMHQ/ Fungal strains used in this research

FeIII/O2 in vivo and in vitro [11]. The substantial depoly- Fungus ATCC no. Source merization of PEG was demonstrated; however, PEG as a Gloeophyllum trabeum 12678, 46150 Univ. of Maine Collection simple aliphatic homopolymer did not truly represent the 32892 Univ. of Maine Collection native lignocellulose heteropolymer, which is far more re- Gloeophyllum abietinum * USDA Forest Products Lab sistant to degradation. Gloeophyllum mexicanum 64431 ATCC In the research reported here we used the model com- * USDA Forest Products Lab pound TNT, which is more difficult to degrade than PEG. Gloeophyllum subferrugineum * USDA Forest Products Lab Gloeophyllum striatum 24597 ATCC TNT-transforming organisms, TNT degradation pathways, Gloeophyllum carbonarium 64503 ATCC and the fate of TNT and other organonitro compounds in the Postia placenta 11538 Univ. of Maine Collection environment have been well studied [12–17] and standards * No accession number found. of various expected metabolites are readily available. Some of the most extensive mineralization of TNT by a biological system has been observed in cultures of the white rot fungus P. chrysosporium growing under lignolytic con- acid, and five produced methoxyquinones, especially 2,5- ditions [18]. The non-specific lignin-degrading enzymes DMHQ, when grown on malt extract medium or mineral produced by P. chrysosporium catalyze the oxidation of media. New complexes of oxalic acid and iron were iden- many xenobiotic compounds [19]. The mechanisms of TNT tified using electrospray ionization tandem mass spectrom- transformation by P. chrysosporium may be similar to those etry (ESI-MS/MS). In vitro Fenton-like reactions produced of brown rotters since both types of fungi exist in nature in degradation products from TNT similar to those found in similar environments and both degrade wood. P. chrysospo- fungal cultures. rium initiates TNT degradation by reduction of a nitro group, followed by oxidation and subsequent degradation of 2. Materials and methods the aromatic ring to CO2 [20–22]. Reductive steps are thought to be catalyzed in a stepwise fashion by an aromatic nitroreductase [21,22]. This activity in P. chrysosporium 2.1. Fungal strains was found to be membrane-bound in two studies [23,24] and soluble in another [20]. Mineralization of TNT by P. The fungal strains used in this research and their sources chrysosporium has been found to be correlated with expres- are listed in Table 1. sion of lignolytic activity, including production of various peroxidases. 2.2. Reagents Recent research points toward the importance of manga- nese peroxidase in the oxidation of reduced TNT metabo- All chemicals, unless specified otherwise, were pur- lites. Scheibner and Hofrichter [25,26] showed that cell-free chased from Sigma-Aldrich Chemical Co. (St. Louis, MO) preparations of manganese peroxidase from cultures of at the highest purity available. Ring-UL-14C-TNT was pre- Nematoloma frowardii and Stropharia rugosoannulata pared by two-step nitration from ring-UL-14C-toluene (spe- transformed TNT, 2-amino-4,6-dinitrotoluene (2-ADNT), cific activity, 58.9 ␮Ci/mmol; radiochemical purity, Ͼ99% 4-amino-2,6-dinitrotoluene (4-ADNT), and 2,6-diamino-4- measured by high-performance liquid chromatography nitrotoluene (2,6-DANT) to unknown metabolites. Further- (HPLC) and liquid scintillation counting [28]. Non-labeled more, these researchers noted that the presence of reduced TNT was prepared from 2,4-dinitrotoluene as described by thiols like glutathione or the amino acid cysteine consider- Lewis, et al. [28]. The TNT metabolites 2ADNT, 4ADNT, ably enhanced the rate and extent of TNT mineralization. In 2,4DANT, 4,4Ј-azoxy-2,2Ј,6,6Ј-tetranitrotoluene, and 2,2Ј- similar experiments with manganese peroxidase purified azoxy-4,4Ј,6,6Ј-tetranitrotoluene were obtained from R. from Phlebia radiata, Van Aken et al. [27] described the Spanggord (SRI International, Menlo Park, CA). ability of these enzymes to oxidatively attack and mineral- Standards of 2,5-DMHQ and 4,5-dimethoxy-1,4-ben- ize TNT through a free radical mechanism. This observation zenediol were synthesized from 2,5-dimethoxy-1,4-benzo- is relevant to our research because brown rotters are be- quinone and 4,5-dimethoxy-1,2-benzoquinone obtained lieved to employ extracellular Fenton-like radical-based re- from TCI (Tokyo, Japan). Typically, 200 mg of the benzo- actions as their main extracellular degradation process. quinone was placed in a 20-ml glass vial and 3 ml of 95% This paper examines the ability of the brown rot fungus ethanol added. The solution was stirred, 100 mg sodium Gloeophyllum trabeum to transform TNT. The roles of borohydride was added, and the vial was capped. The cap dimethoxyquinones, oxalic acid, Fe(III), and hydrogen per- was opened briefly at short intervals to release accumulated oxide in the transformation of TNT are examined. We hydrogen. When the solution became white and efferves- studied eight Gloeophyllum species for production of me- cence ceased, the reaction was considered complete. Next, 5 thoxyquinones and oxalic acid. All strains produced oxalic ml of deoxygenated water was added to the reaction mix- 508 D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517

2.4. Extraction and analysis of fungal supernatant

Mature fungal cultures and non-inoculated controls were extracted and analyzed for TNT metabolites and other or- ganic compounds. Typically, a culture was filtered through a glass wool filter to remove mycelia, and the filtrate was extracted twice with equal volumes of methylene chloride by shaking for 5 min. Prior to the second extraction 1 ml of

4N H2SO4 was added to the aqueous fraction. The organic fractions were combined, and approximately5gofNa2SO4 were added. The suspension was passed through a Whatman #2 filter (Whatman, Clifton, NJ), and the filtrate dried under a gentle stream of nitrogen. During this step care was taken to minimize exposure to moisture. Once completely dry, the concentrate was resuspended in 500 ␮l of acetonitrile for Fig. 1. Dry weights of mycelium from 50-ml cultures of 4-week-old G. trabeum cultures in the presence of various concentrations of TNT. GC-MS or ESI-MS and ESI-MS/MS analysis. GC-MS analysis of the extracts was performed with a Hewlett-Packard (HP) series II 5890 gas chromatograph equipped with a capillary fused-silica column (30 m ϫ 0.25 ture, and the pH was adjusted to 7 with HCl. The sample mm) coated with CP-SIL 8CB MS (Chrompack, Middel- was extracted five times with equal volumes of ether under burg, The Netherlands). The injector temperature was set at a stream of N2. The N2-saturated ether fraction was then 250°C, and the GC-MS interface was set at 280°C. Chro- dried at room temperature under a vacuum in a Brinkman matographic separations were achieved under a linear tem- (Westbury, NY) model Rotovapor R rotating evaporator. perature gradient from 100 to 200°C at a rate of 5°C/min After all the solvent was removed, the flask was transferred and then from 200 to 300°Cat20°C/min. Samples (1 to 5 to a lyophilizer (Labconco Lyph Lock 4.5, Labconco Corp, ␮l) were injected via an automatic injector (HP-7673). A Kansas City, MO) to remove traces of water. The 2,4- HP quadrupole MS (5989A) controlled by HP MS Chem- DMHQ was weighed and dissolved under anaerobic condi- station software (PC version) was used for MS and analysis tions (10% H2, 90% N2) in acetonitrile for analysis of purity under the following conditions: repeller, 7 V; emission, 300 by HPLC and gas chromatography/mass spectrometry (GC- V; electron energy, 70 eV. The source temperature was MS) (see below for methods). 250°C, and the quadrupole temperature was 125°C. The scan parameters were 30 to 350 or 30 to 750 m/z. Interpre- 2.3. Culture conditions tation of the MS spectrum was aided by the Wiley and National Institute of Standards and Technology library of All fungal strains were grown in malt extract broth mass spectra stored in the Chemstation database (approxi- (2–3% w/v) or in a defined medium originally developed for mately 200,000 spectra). P. chrysosporium [29] and containing 1% glucose, 0.5 mM Fungal culture filtrates were analyzed for TNT, metabo- NH NO , and 0.1 mM MnCl . This medium was modified lites, and methoxyquinones directly on an HP 1090 HPLC 4 3 2 ϫ for growth of brown rot fungi by including 0.5 mM aspar- equipped with a 150 mm 1.00 mm phenyl-hexyl column agine and 0.5 mM arginine [30]. It was buffered with 10 (Phenomenex, Torrance, CA) and an ultraviolet-visible mM 2,2Ј-dimethylsuccinic acid sodium salt at a pH of 4.5. (UV/Vis) diode array detector. Samples were eluted using a gradient mobile phase containing 18 m⍀ water (A) and Malt medium was prepared by adding 25–30 g malt extract 0.1% formic acid in methanol (B). The method was as (Difco Laboratories, Detroit, MI) to 1000 ml of water. Static follows: flow rate, 0.65 ml/min; injection volume 20 ␮l; cultures were established at 24°Cor30°C in the dark by 30% B (0 min) to 100% B (at 20 min, held for 10 min) inoculating a 150-ml Erlenmeyer flask containing 50 ml of followed by a return to initial conditions (at 30 min) for 5 medium or a 1-liter Roux flask containing 150 ml of me- min to rinse and equilibrate the column; peak detection was dium. Two methods were used to inoculate flasks. The first at 254 nm, 260 nm, 285 nm, and 299 nm, with peak- 2 consisted of removing approximately 2 cm of mycelial mat actuated scanning from 200–600 nm. Salicylic acid, phe- from a 6-week-old liquid culture. After this material was nol, catechol, hydroquinone, and isomers of dihydroxyben- added, it was broken into small pieces by shaking the flasks zoic acid were also detected in the fungal culture for about 20 s. The second method utilized a Brinkman supernatants with HPLC. The same phenyl-hexyl column model PT 10/35 homogenizer (Brinkman Instruments, was used, and the samples were eluted using a isocratic

Westbury, NY) to liquefy the mycelial mat from a 50-ml gradient containing 75% phosphate (A): 6.80 g KH2PO4,1 ␮ Ͼ 4-week-old culture, and 200 l of the suspension were ml H3PO4 in 1000 ml of deionized water (resistivity 18 transferred to flasks containing 50 ml of medium. megaohm-centimeter), and 25% acetonitrile in water (B) at D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517 509

Fig. 2. Concentration (mg/l) of —F— trinitrotoluene, —E— 2-amino-4,6-dinitrotoluene, —— 4-amino-2,6-dinitrotoluene, —ƒ— 2,4-diamino-6- nitrotoluene in the supernatant of G. trabeum cultures over time. The concentration of nitrogen in the mineral salts medium was 1.2 mM (A), 5 mM (B), or 15 mM (C). A 3% malt medium was also used (D). On day 18 TNT was added a second time at a level of 20 mg/l. Sampling on day 19 occurred 12 h after the addition of TNT. a flow rate of 0.65 ml/min; injection volume 20 ␮l. Absorp- The instrument was calibrated using a polyethylene glycol tion detection was at 230 nm with peak-actuated scanning solution. All spectra were an average of 10–15 scans. from 190–600 nm. 2.6. Analysis of 14C radiolabeled compounds 2.5. ESI-MS and ESI-MS/MS mass spectrometry

Radiolabeled CO2 was captured in culture flasks Analyses of TNT, TNT degradation products, methoxy equipped with hanging cups containing 1 ml 1N NaOH [31]. quinones, oxalic acid, oxalic acid metal complexes, and At regular intervals the NaOH was removed and the cup radical trap products were completed using a Micromass rinsed twice with equal volumes of deionized water. The Quattro II mass spectrometer (Altrincham, England) NaOH and rinses were combined and added to a 20-ml equipped with an electrospray ionization probe, two quad- scintillation vial containing 15 ml of Biosafe scintillation rupole analyzers, and a hexapole collision cell. The ion cocktail (Research Products International, Inc., Mount Pros- source was operated in the negative- or positive-ion mode. pect, IL). Supernatant samples (1 ml) were removed from Samples were delivered into the source at a flow rate of 5 the flasks and added directly to 15 ml of scintillation cock- ␮l/min using a syringe pump (Harvard Apparatus, South tail. Fungal hyphae were removed by filtration, rinsed with Natick, MA). A potential of 2.5 kV was applied to the water, and added to an equal volume (approximately 40 ml) electrospray needle. The sample cone was kept at an aver- of tissue solubilizer (NEN Research Products, Boston, MA) age of 15 V and the counter electrode, skimmer, and RF lens and incubated for 24 h at 50°C. Aliquots (1 ml) of the potentials were tuned to maximize the ion beam for a given solubilized tissue solution were then added to 15 ml of solvent. Argon was used as a collision gas during daughter Ready Organic scintillation cocktail (Beckman Instruments, analysis. The source temperature was kept constant at 80°C. Fullerton, CA). All samples were analyzed with a Tri-Carb 510 D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517

Table 2 Degradation of TNT and acetophenone by G. trabeum and P. chrysosporium

(%) 14C-TNT (%) 14C-Acetophenone

14 14 Filtrate CO2 Hyphae Filtrate CO2 Hyphae P. chrysosporium 30 Ϯ 1.2† 10 Ϯ 1.2 60 Ϯ 4.8 10 Ϯ 0.1 15 Ϯ 0.3 70 Ϯ 5.6 G. trabeum 74.1 Ϯ 2.8 2.7 Ϯ 0.3 15.6 Ϯ 3.6 *ND 4.1 Ϯ 0.8 ND

* Data not collected. † Data were collected after four weeks of incubation and are represented as the percentage of total radiolabel added Ϯ standard deviation. Heat-killed and non-inoculated cultures were run in tandem as controls (data not shown). The amount of total TNT added to cultures of P. chrysosporium (4 mg/l) was less than the amount added to cultures of G. trabeum (20 mg/l).

2100TR scintillation counter (Packard Instrument company, ciated with the hyphal fraction. Furthermore, most (Ͼ70%) Inc., Downers Grove, IL). of the radioactivity was found in the aqueous fraction of the culture (Table 2). In the same incubation period, P. chryso- sporium was able to mineralize about 10% of TNT, and 3. Results about 60% of the 14C was associated with hyphae. Both cultures were also incubated with ring-UL-14C-acetophe- 3.1. Transformation of TNT none. Four percent of the added radioactivity was recovered 14 from cultures of G. trabeum as CO2. In a similar experi- G. trabeum (Table 1) was able to grow in minimal ment P. chrysosporium removed more than 85% of the medium augmented with TNT and to tolerate fairly high (35 radioactivity from the medium and released about 15% of 14 mg/l) concentrations of TNT (Fig. 1). However, concentra- the label as CO2. The comparison between these two fungi tions of TNT at 40 mg/l significantly inhibited the hyphal showed that G. trabeum has limited ability compared to P. growth. Comparisons of cultures grown in the presence of chrysosporium to metabolize the aromatic ring, and ring 30 mg/l TNT to control cultures without TNT revealed an substituents have little effect on this process. enhancement of hyphal growth by this amount of TNT (Fig. 1). G. trabeum cultures grown for 2 weeks in the minimal 3.2. Radical trapping with TEMPO salts medium and then spiked with TNT transformed the TNT within 2 days (Fig. 2). Different concentrations of The compound 2,2,6,6-tetramethyl-1-piperidynyloxide nitrogen in the culture had no effect on TNT degradation (TEMPO), a stable free radical, was employed as a trap (Fig. 2A, B, C, D). The transient accumulation of 2-amino- within in vitro reactions containing Fe(II), 2,5-DMHQ, and and 4-aminodinitrotoluenes was observed during growth in TNT. The coupling product of TEMPO and 2,5-DMHQ was all four media (Fig. 2). Small amounts of 2,4-DANT were detected (Fig. 3). The known biological transformation observed during growth in mineral media, but not in malt products of TNT were never observed in this reaction mix- extract medium. This metabolite was not always detectable ture (data not shown). The presence or absence of oxygen by HPLC in the culture supernatant, but was detectable by did not influence this reaction. GC/MS after extraction and concentration. In order to follow the fate of TNT in the cultures, similar experiments were run by growing G. trabeum for 2 weeks in Table 3 a nitrogen-limited (1.2 mM) minimal medium with subse- Production of 2,5-dimethoxybenzoquinone and 4,5-dimethoxy-1,2- quent addition of ring-UL-14C-TNT (Table 2). After four benzoquinone (DMBQs) by Gloeophyllum strains weeks of incubation, less than 3% of added TNT was Strain Concentration of DMBQ (mg/l) Production 14 14 mineralized to CO2 and about 15% of the C was asso- HPLC analysis confirmation by GC/MS Day 14 Day 21 Day 28 G. abietinum 000 Ϫ G. carbonarium 000 Ϫ G. subferrugineum 3.5–8.5† 5.3–13.0 5.3–12.8 ϩ G. striatum 3.3–7.4 2–3.5 1.0–1.37 ϩ G. mexicanum 0 1.0–1.1 1.0 ϩ G. protractum 0 1.0 1–4.9 ϩ G. trabeum 2.4–10.0 9.4–20.0 11.0–21.0 ϩ G. sepiarium 1.0 1.0–3.4 1.7–7.0 ϩ Fig. 3. Structures of a semi-quinone radical, TEMPO, and their coupling product observed during in vitro experiments employing TEMPO as a † The data are given as ranges due to wide fluctuations of quinone radical trap. concentration observed in cultures. D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517 511

Fig. 4 A. Degradation of salicylic acid (initial concentration 150 mg/l) by cultures of: —F— G. striatum; G. mexicanum; —Œ— G. subferrugineum; —E— G. sepiarium; —ᮀ— G. carbonarium; —‚— G. protractum; —}— G. abietinum; —ƒ— G. trabeum. B. Degradation of phenol (initial concentrations 150 mg/l) by cultures of: —F— G. trabeum; —■— G. sepiarium; —Œ— G. striatum; ⅐⅐⅐ ⅐⅐ ⅐G. subferrugineum.

3.3. Production of DMBQ by cultures DMBQ. When the level of DMBQ was observed over time in cultures of G. trabeum with and without added TNT, the In our HPLC analyses both quinones, 2,5-dimethoxy- DMBQ concentration initially dropped significantly in cul- 1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone, tures with TNT added (data not shown). After an incubation were eluted in a single peak; this fraction was called time of 10 days the levels of DMBQ in both cultures were 512 D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517

Table 4 GC and MS data for organic compounds observed during experiments

Compound GC Retention Time Mass spectrum m/z [relative intensity (%)] 2,4,6-trinitrotoluene (culture extract) 22.16 51 (10); 63 (27); 89 (35); 134 (13); 210 (100) 2-amino-4,6-dinitrotoluene (culture extract) 27.39 52 (57); 78 (97); 104 (57); 180 (100); 197 (71) 4-amino-2,6-dinitrotoluene (culture extract) 25.90 52 (56); 78 (62); 104 (74); 180 (100); 197 (49) 2,4-diamino-6-nitrotoluene (culture extract) 25.17 41 (37); 94 (55); 121 (57); 150 (32); 167 (100) 2,5-dimethoxy-1,4-benzoquinone (culture extract) 18.03 53 (34); 69 (100); 95 (22); 139 (34); 168 (6) 2,5-dimethoxy-1,4-hydroquinone (culture extract) 14.15 53 (17); 112 (28); 127 (57); 155 (100); 170 (74) salicylic acid (culture extract) 10.39 64 (34); 92 (89); 120 (100); 138 (54) ϩ 2,3-dihydroxy-benzoic ( CH3) (culture extract) 17.30 52 (17); 80 (28); 108 (20); 136 (100); 168 (35) 2,4,6-trinitrotoluene (standard) 22.12 51 (20); 63 (48); 89 (45); 134 (14); 210 (100) 2-amino-4,6-dinitrotoluene (standard) 27.39 52 (64); 78 (100); 104 (40); 180 (65); 197 (49) 4-amino-2,6-dinitrotoluene (standard) 26.06 52 (39); 78 (54); 104 (70); 180 (100); 197 (48) 2,4-diamino-6-nitrotoluene (standard) 25.30 41 (18); 94 (48); 121 (45); 150 (33); 167 (100) 2,5-dimethoxy-1,4-benzoquinone (standard) 18.17 53 (36); 69 (100); 95 (26); 139 (36); 168 (5) 2,5-dimethoxy-1,4-hydroquinone (standard) 14.12 53 (18); 112 (20); 127 (44); 155 (100); 170 (84) salicylic acid (standard) 8.04 64 (14); 92 (58); 120 (100); 138 (44) ϩ 2,3-dihydroxy-benzoic ( CH3) (standard) 17.57 52 (25); 80 (33); 108 (34); 136 (100); 168 (35)

not significantly different. However, when cultures were conditions (Fig. 5A). At pH 4.5 ferric and ferrous com- spiked with 20 mg/l TNT again, the level of DMBQ was plexes were clearly visible (Fig. 5B). In addition, a double significantly greater than in non-spiked controls (data not charged peak of Fe(IV)O, coordinated by two oxalates (m/ shown). Six of eight strains produced DMBQs when grown z ϭ 148), was observed. The structures of complexes were on malt extract media (Table 3). The concentrations ranged confirmed using MS/MS daughter analyses (data not from 1 mg/l for G. mexicanum to 13 mg/l for G. subferrug- shown). ineum and 21 mg/l for G. trabeum. 3.6. Analysis of TNT degradation products by Fenton-like 3.4. Determination of hydroxyl radical in fungal cultures reactions containing oxalic acid

Solutions of salicylic acid were added to 2-week-old Solid TNT (50 mg/l) was added to the reaction mixture cultures to detect the presence of hydroxyl radicals. HPLC of 3 mM oxalic acid, 1 mM FeCl3, and 10 mM H2O2 the 50. analysis showed that all of the cultures examined degraded The pH of the solution was adjusted to 4.5 with NaOH. salicylic acid (Fig. 4A). In addition, the presence of 2,3- After 24 hours the reaction mixture was analyzed using dihydroxy benzoic acid was observed in G. striatum and G. ESI-MS/MS. Numerous organic products were tentatively protractum (Table 4). The presence of this hydroxylation identified as shown in Fig. 6 using daughter analyses. product is an indication of hydroxyl radical in solution [32]. In a similar experiment phenol was used as the hydroxyl radical trap (Fig. 4). The four cultures of the Gloeophyllum 4. Discussion species examined readily degraded phenol, but hydroqui- none and catechol that might be expected as products of The complex structure and chemical composition of hydroxyl radical reactions [33] were not observed in direct lignocellulosic biopolymers have significant influences on analysis of culture filtrates with HPLC. their degradation in the environment. Fungi are the only organisms known capable of complete degradation of fully 3.5. Chelation of iron by oxalic acid lignified wood. The mechanisms by which these microor- ganisms initiate their attack on wood is not fully understood.

The complexes formed by oxalic acid with FeCl3 were The present leading hypothesis is that ligninolytic fungal examined at two different pHs using negative ESI-MS/MS. peroxidases and oxidases are too large to penetrate into

The solutions contained 3 mM oxalic acid and 1 mM FeCl3; intact wood, [34,35] so both classes, white rot and brown rot the pH was adjusted with NaOH. At pH 1.5 the expected fungi, use low molecular weight redox compounds (medi- hexacoordinated octahedral complex was observed with two ators) to launch their initial attack on intact wood cell walls. carboxylic groups of oxalic acid protonated. The other com- Research on brown rot decay mechanisms became focused plex observed contained two oxalate molecules and a single on the involvement of reduced species of oxygen generated ferric ion. In the second complex iron was most likely from hydrogen peroxide by chelated transition metal mole- additionally coordinated by two molecules of water. Re- cules after Koenigs proposed in 1974 the involvement duced iron complexes have not been observed under these H2O2-Fe(II) as a radical generating system in brown rot D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517 513

Fig. 5. Mass spectra of oxalic acid chelated Fe(III) at pH 1.5 (A) and pH 4.5 (B). The insets are the proposed structures, and the arrows point to their corresponding m/z fragment. decay [36]. Several groups have proposed experimental philic nature of the nitro group causes TNT to readily evidence that supports an oxygen radical-based degradation oxidize biological reductants, causing toxicity directly or by mechanism whereby brown rot fungi might attack wood formation of other reactive products such as nitroarene [1,9,11,37]. radicals [40]. In addition, the nitro groups draw electrons In research reported here we used TNT as a model from the aromatic ␲ bonds, effectively reducing the electron recalcitrant compound to study brown rot degradative abil- density of the conjugated aromatic system. As a result, TNT ities. TNT and related munitions compounds can cause liver is resistant to degradation via electrophilic attack by oxy- damage, react with hemoglobin, and also uncouple oxida- genases [41–43]. In order for TNT to be mineralized, or- tive phosphorylation in mammalian mitochondria. Their ganisms must first remove or transform the nitro groups. Its mutagenicity is also well documented [38,39]. The electro- abundance, persistence, and resistance to degradation make 514 D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517

Fig. 6. Mass spectra of compounds present during in vitro experiments containing oxalic acid (3 mM), FeCl3 (1 mM), H2O2 (10 mM), with solid TNT added to a final concentration of 50 ppm. Structures of detected compounds are presented near their m/z ions.

TNT one of the most intensely studied hazardous organo- was water-soluble material, and 16% was soluble in nitro compounds with respect to bioremediation. The ability methylene chloride and shown by HPLC analysis to be of white rot fungi to degrade nitroaromatics has been well more polar than TNT [46]. Higher concentrations of TNT documented [44,45]. In cultures of P. chrysosporium, ap- appear to inhibit TNT mineralization in this fungus. proximately 35% of the radioactivity from ring-labeled Spiker et al. [47] found that TNT was inhibitory to spore 14C-TNT added at a concentration of 1.3 mg/l was germination of P. chrysosporium at concentrations 14 trapped as CO2. Of the remaining radioactivity, 25% greater than 5 mg/l. This toxicity could be related to the

Fig. 7. Proposed dimer formation by the spontaneous Schiff-base reaction between an amine and aldehyde derivative of TNT degradation in Fenton-like reactions. D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517 515 activity of TNT as an oxidant, since reduction to 2- or of eight Gloeophyllum strains examined produced me- 4-aminodinitrotoluenes relieved toxicity. thoxylated quinone isomers (Table 3). We did not find a Results of our mineralization experiment (Table 2) con- correlation between quinone production and TNT trans- firmed the superiority of P. chrysosporium over G. trabeum formation in G. trabeum, but secretion of methoxylated for both TNT and acetophenone degradation. However, quinones seems to be a common feature of the Gloeo- freshly inoculated cultures of G. trabeum were almost ten phyllum genus. times more resistant to high concentrations of TNT than a Salicylic acid and phenol were used as probes to trap similar culture of P. chrysosporium. We observed the typ- hydroxyl radicals in cultures of several of the investigated ical biotransformation products that are indicative of reduc- species. All of the cultures examined readily degraded tive degradation (Fig. 2), suggesting that the pathway salicylic acid and phenol. Low concentrations of 2,3- through 4-ADNT is the most favorable initial reduction dihydroxy benzoic acid was observed in the media ex- reaction. However, 2,4-DANT did not accumulate, and the tracts of cultures of G. striatum and G. protractum. This 2,4-DANT concentrations observed were much lower than product is indicative of the presence of hydroxyl radicals, those of the mono-amino products. This result implies that but the low concentration of this compound and its ab- there are reactions that compete with the reduction of the sence from cultures of other Gloeophyllum species sug- second nitro group to an amino group, or that 2,4-DANT is gest the involvement of mechanisms other than the hy- rapidly further transformed. It has been shown in previous droxyl radicals in these reactions. ESI-MS/MS results studies that oligomers can form by the reaction of hydroxy- (Fig. 6) showed a peak at m/z ϭ 260. Daughter fragmen- lamino and nitroso groups in the presence of oxygen, form- tation analyses revealed a fragmentation pattern consis- ing 4,4Ј,6,6Ј-tetranitro-2,2Ј-azoxytoluene or 2,2Ј,6,6Ј-tetra- tent with TNT (mass 227) substituted with OOH* (mass nitro-4,4Ј-azoxytoluene [12,22,48,49]. Cultures of G. 33). The pKa for hydroperoxy radical is 4.8, [51] and trabeum incubated with TNT maintained a dark brown should permit the existence of this species in our reac- color, which is consistent with polymer formation. How- tion, which had a pH ϭ 4.5. ever, standards of the azoxy compounds did not match any The coupling products of TEMPO and 2,5-dime- compounds observed in the culture supernatants and iden- thoxyquinone (2,5-DMHQ) were detected and are indica- tified by GC/MS. tive of semi-dimethoxyquinone radical formation. This ob- In an effort to determine the fate of the metabolites, other servation suggests that the presence of semiquinone radicals polymerization and condensation reactions were consid- in fungal cultures, but subsequent data do not support their ered. A Schiff-base formation occurs spontaneously be- direct involvement in TNT degradation. tween an aldehyde and an amine and is a well-known The research presented supports the involvement of re- reaction in biological systems [50]. In the current study the duced oxygen species, generated by Fenton-like reactions in key metabolites for such a reaction would be trinitrobenz- the degradation of TNT by brown rot fungi. We are now aldehyde (TNBA) or 4-amino-2,6-dinitrobenzaldehyde (4- conducting additional experiments to characterize the re- ADNBA). These compounds could undergo reactions at duced oxygen species involved in redox reactions in brown 15 either the amino or the aldehyde position, forming oli- rot cultures. We are using specifically labeled 2,4,6- NO2, gomers or even larger polymers (Fig. 7). Since we observed -CD3, and -ringD3 TNT, specific spin trap compounds, aminodinitrobenzaldehyde in culture supernatants of G. tra- hydrogen peroxide and tert-butyl hydroperoxide, and 18O- beum, such reactions are presumably likely to occur. Anal- labeled water and oxygen to address this fundamental ques- yses of G. trabeum cultures (Fig. 2, Table 4) and in vitro tion. reactions (Fig. 6) revealed several aromatic nitro-amines and nitro-aldehydes that could form the expected polymers. Any heteropolymer formed would be particularly difficult to Acknowledgments analyze, because there are numerous possibilities for the formation of condensed metabolites with 2,3,4 or more This research was supported by USDA/CSREES grant benzene ring cores, with different combinations of nitro- 99-35103-7997. and amino-substitutions. The small peaks (m/z 390–440) The authors thank Dr. Stefan Goszczynski for his valu- (Fig. 6) revealed daughter fragmentation patterns consistent able help with developing the methoxylated quinone reduc- with dimer structures. The similarities between TNT deg- ing method and Connie Bollinger for editorial assistance. radation products in these experiments and compounds found in fungal cultures suggest the involvement of reduced oxygen species produced by Fenton-like reactions in fungal cultures. References The in vitro experiments with 2,5-dimethoxy-1,4-hydro- [1] Paszczynski A, Crawford R, Funk D, Goodell B. De novo synthesis quinone, FeCl2 and TNT in reaction mixtures did not pro- of 4,5-dimethoxycatechol and 2,5-dimethoxyhydroquinone by the duce any of the detectable degradation products found in the brown rot fungus Gloeophyllum trabeum. Appl Environ Microbiol G. trabeum cultures (data not shown). Moreover, six out 1999;65:674–9. 516 D. Newcombe, et al. / Enzyme and Microbial Technology 30 (2002) 506–517

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