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Extensive biodegradation of hexahydro-1,3,5 -trinitro-1,3,5 -triazine (RDX) by the white-rot fungus Phanerochaete chrysosporium in liquid and solid matrices was observed. Some degradation in liquid occurred under nonligninolytic conditions, but was approximately 10 times higher under ligninolytic conditions. Moreover, elimination was accounted for almost completely as carbon dioxide. No RDX metabolites were detected. The degradation rates in liquid appeared to be limited to RDX concentration in solution (approximately 80 mg/L), but degradation rates in soil were nonsaturable to 250 mg/kg. Manganese-dependent peroxidase (MnP) and cellobiose dehydrogenase (CDH) from P. chrysosporium, but not lignin peroxidase, were able to degrade RDX. MnP degradation of RDX required addition of manganese, but CDH degraded RDX anaerobically without addition of mediators. Attempts to improve biodegradation by supplementing cultures with micronutrients showed that addition of manganese and oxalate stimulated degradation rates...

When Phanerochaete chrysosporium was cultured using conditions which promote the expression of cellobiose dehydrogenase (CDH), but not the ligninolytic peroxidases, the fungus effectively solubilized and mineralized an insoluble, crosslinked polyacrylate and an insoluble polyacrylate/polyacrylamide copolymer. Addition of iron to the cultures increased CDH activity in the cultures and the rate and extent of solubilization and mineralization of both polymers. Solubilization of both polymers was observed when incubated with purified CDH, ferric iron and hydrogen peroxide.

Biodegradation of two superabsorbent polymers, a crosslinked, insoluble polyacrylate and an insoluble polyacrylate/ polyacrylamide copolymer, in soil by the white-rot fungus, Phanerochaete chrysosporium was investigated. The polymers were both solubilized and mineralized by the fungus but solubilization and mineralization of the copolymer was much more rapid than of the polyacrylate. Soil microbes poorly solublized the polymers and were unable to mineralize either intact polymer. However, soil microbes cooperated with the fungus during polymer degradation in soil, with the fungus solubilizing the polymers and the soil microbes stimulating mineralization. Further, soil microbes were able to significantly mineralize both polymers after solubilization by P. chrysosporium grown under conditions that produced fungal peroxidases or cellobiose dehydrogenase, or after solubilization by photochemically generated Fenton reagent. The results suggest that biodegradation of these polymers in soil is best under conditions that maximize solubilization.

White-rot fungi are known to degrade a wide range of xenobiotic environmental pollutants, including the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). TNT is first reduced by the fungal mycelium to aminodinitrotoluenes and diaminonitrotoluenes. In a second phase, reduced TNT metabolites are oxidatively transformed and mineralized. The extracellular oxidative enzyme of the ligninolytic system of these fungi includes the lignin peroxidases (LiP) and the manganese-dependent peroxidases (MnP). In the present study, we have shown that a cell-free enzymatic system containing fast protein liquid chromatography (FPLC)-purified LiP (H8) from the white-rot fungus Phanerochaete chrysosporium was able to completely transform 50 mg/L of 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) in 1 and 48 h, respectively. Veratryl alcohol (VA), often described as a mediator in the LiP-catalyzed oxidative depolymerization of lignin, was not required for the enzymatic transformation of 2,4-DA-6-NT or 2-A-4,6-DNT. 2,4-DA-6-NT was also shown to be a competitive inhibitor of the LiP activity measured through the oxidation of VA. Experiments using 14C-U-ring labeled compounds showed that 2-A-4,6-DNT was converted to 2,2′-azoxy-4,4′,6,6′-tetranitrotoluene. No significant mineralization, measured by the release of 14CO2, was observed over 5 d.

White-rot fungi have the ability to degrade lignin, a biopolymer in wood and woody plants which is resistant to attack by most microorganisms.1 Lignin is a complex, three-dimensional, nonrepeating polymer. White-rot fungi can degrade lignin using an extracellular, rather nonspecific, free-radical based biodegradation system.’ Because this degradation system can oxidize and reduce a wide variety of chemicals, it is rather nonspecific and can also degrade a wide variety of environmental pollutants 3,4 The degradation system is multicomponent, somewhat complex and unique, and somewhat redundant 3,5 That is, there may be more than one way to catalyze the same reaction or type of reaction,5 and there may be some very unusual ways to accomplish reactions through the involvement of chemicals produced by the fungi during secondary metabolism. The degradation of lignin occurs during secondary metabolism, when the fungi are limited in some nutrient.6–8 They obtain no energy from the degradation of lignin. The carbon substrate for the fungi is cellulose, but the fungi must degrade the surrounding lignin to gain access to the cellulose7 This seems to give these fungi the ability to degrade a wide variety of otherwise quite recalcitrant environmental pollutants3,4,9 or potential pollutants, including synthetic polymers.10

Quinones which are produced during the mineralization of lignin and xenobiotics by the white rot fungus Phanerochaete chrysosporium were reduced by a plasma membrane redox system of the fungus. Both intracellular enzymes and the plasma membrane redox system were able to reduce 1,l-benzoquinone. However, no quinone reductase activity was observed with the extracellular culture fluid. The intracellular reductase activity had a pH optimum between 6.0 and 7.0 and a K m of 150 μM. Reduction of 1,4-benzoquinone by the plasma membrane redox system had a pH optimum between 7.5 and 8.5 and exhibited saturation kinetics ( K m = 11 μM, V max = 16 nmol/min/mg mycelia dry weight). Ferricyanide totally inhibited the quinone reduction until the ferricyanide was completely reduced by the membrane. Radicals (chlorpromazine and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)) that can be generated by the lignin peroxidases were also reduced by the plasma membrane redox system. Reduction of the ABTS cation radical also totally inhibited quinone reduction until the radical was completely reduced. Finally, quinone reduction rates were identical after the reduction of ferricyanide, ABTS cation radical, or quinone, suggesting that the plasma membrane redox system may actually protect the fungus from oxidative damage from free radicals generated by the lignin degrading system.

A transplasma membrane redox system of Phanerochaete chrysosporium was studied using ferricyanide, a membrane-impermeable electron acceptor. Rates of reduction were dependent upon initial ferricyanide concentration and mycelial mass. Specific activities of 12 ± 2 nmol/min/mg mycelia (dry wt) were consistently obtained using nutrient-sufficient mycelia at pH 8.0 and 10 mM ferricyanide. Upon nutrient limitation (either carbon or nitrogen), activity decreased. Reduction was inhibited by carbonyl cyanide m -chloromethoxyphenyl hydrazone, 2,4-dinitrophenol, and sodium azide but not by potassium cyanide at 100 nmol/mg mycelia. Ferricyanide reduction and proton export rates increased with pH above the physiological pH for the fungus. The stimulation in proton exported by the addition of ferricyanide was equal to the rate of ferricyanide reduced at pH 8.0 when Hepes buffer was used. The relevance of these findings with regard to the physiological pH optimum of the fungus and the metabolism of pollutants by this fungus is discussed.

Trinitrotoluene (TNT) has become the predominant explosive worldwide. TNT wastes generated during TNT production and munitions manufacturing, as well as improper storage, use, and disposal have led to contamination of the environment (67, 94). Due to the solubility of TNT in water (~ 125 ppm at 20°C) large volumes of contaminated waste water can be generated during TNT production and munitions loading operations. As much as 500,000 gallons of TNT-contaminated water have been generated per day by a single munitions plant (91). In the past, the contaminated water was typically collected in large lagoons. Repetitive use of the lagoons has left high residual concentrations of TNT in the sediment (42, 67). Other contamination sites include missile production facilities, mining sites, military firing ranges and sites where outdated explosives were burned. Continual leaching of TNT from these soils over the years has resulted in the contamination of groundwater (42, 67). Typical contaminated sites may contain average concentrations of 10,000 mg/kg TNT in soil and 100 ppm TNT in water (30).