Biodegradation of Nitro-Substituted Explosives by White-Rot Fungi: A Mechanistic Approach BENOi'T VAN AKEN Department of Civil and Environmental Engineering University of Iowa Iowa City, Iowa 52242 SPIROS N. AGATHOS Unit of Bioengineering Universitd Catholique de Louvain Louvain-la-Neuve, Belgium I. Introduction II. Biodegradation of Lignin by White-Rot Fungi A. Lignin B. Lignin-Degrading (Ligninolytic) Microorganisms C. Fungal Wood-Decaying Enzymes D. Ligninolytic Enzymes E. H202-Generating Systems F. Cooperation of Enzymes in Degradation of Lignocellulose G. Physiological Features Supporting Fungal Ligninolysis III. Low-Molecular-Weight Mediators and Free Radicals A. Veratryl Alcohol (VA) B. Manganese and Oxalate C. Free Radicals IV. Biodegradation of Nitro-Substituted Explosives TNT, RDX, and HMX A. TNT, RDX, and HMX as Environmental Pollutants B. Other Nitro-Substituted Compounds C. Toxicity of Nitro-Substituted Explosives D. Biodegradation of the Nitroaromatic Explosive TNT E. Biodegradation of Nitramines RDX and HMX E Field Experiments G. Mechanistic Considerations H. Nonligninolytic Transformations V. Conclusions References I. Introduction "Perhaps the easiest way to understand the non-specific ability of these [white-rot] fungi to degrade pollutants is to consider their ecological 'niche.' White-rot fungi are those organisms that are able to degrade lignin, the structural polymer found in woody plants (Barr and Aust, 1994)." I n 1985, a w e l l - k n o w n l i g n i n o l y t i c w h i t e - r o t f u n g u s , Phanerochaete chrysosporium, w a s r e p o r t e d to m i n e r a l i z e i n t o t h e i r m i n e r a l ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 48 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 0065-2164/01 $35.00 2 VANAKENANDAGATHOS components a variety of toxic pollutants recalcitrant to biodegradation by other microbes, e.g., dioxins, polychlorinated biphenyls (PCB), organochlorine compounds, and polyaromatic hydrocarbons (PAH) (Bumpus et al., 1985, Aust, 1990). Since that time, an ever-increasing number of research works have been published, focusing on the exceptional bioremediation capacities of white-rot fungi and their potential use in environmental biotechnologies. White-rot fungi are ligninolytic organisms, i.e., able to degrade lignin, a major constituent of wood. Lignin is a complex, insoluble, and randomly structured biopolymer, usually resistant to biological attack (Buswell and Odier, 1987). White-rot fungi produce powerful extracellular and nonspecific ligninolytic enzymes, i.e., lignin peroxidases (LIP), manganese-dependent peroxidases (MnP), and laccases, which allow them to break down a wide range of xenobiotic organic ecopollutants, including the nitro-substituted explosives 2,4,6-trinitrotoluene (TNT), hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX), and octahydro1,3,5,7-tetranitro-l,3,5,7-tetrazocine (HMX) (Barr and Aust, 1994). Best known for their explosive properties, nitroaromatic and nitroheterocyclic explosives such as TNT, RDX, and HMX are also harmful chemicals constituting a serious biological hazard for water and soils. Throughout most of the 20th century, production, use and destruction of ammunition stocks have led to an increasing contamination of military and industrial sites worldwide (Spain, 2000). TNT, RDX, and HMX are toxic for living organisms and usually recalcitrant to microbial degradation (Smock et al., 1976, Won et al., 1976). Fernando and Aust (1991) reported the capacity of the white-rot fungus Phanerochaete chrysosporium to par