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LOW-TEMPERATURE, LIQUID-PHASE PROCESSES 128 Biologically based technologies used as alternatives to inchaeration in destroying the current agent stockpiles are assessed below. The discussion is limited to the potential for destroying purified chemical agent stored in bulk containers or collected through the disassembly of chemical weapons. Direct Destruction of GB and VX Technology description. Enzyme-based systems that can directly degrade GB have been identified from numerous systems (Table 6-2). The initial, enzyme-catalyzed hydrolysis of GB would result in the production of hydrogen fluoride (HF) and mono-isopropyl methylphosphonate. Several microbial strains capable of hydrolyzing VX have reportedly been isolated, but none has been characterized to date (Harvey and DeFrank, 1992). Several enzyme and cellular systems have been identified that are capable of cleaving the P-C bond and degrading the methylphosphonate products of agent hydrolysis. Microbial cultures capable of degrading the relatively nontoxic phosphonic acids are described in the section below on the biodegradation of the reaction products from chemical processing of GB and VX. The main issues for enzyme-or microbial-based hydrolysis of the chemical nerve agents are not the intrinsic capability of biological systems to detoxify the agents, but rather system integration questions: ⢠What is the greatest degree of destruction that can practically be achieved, and what is the associated system efficiency? ⢠What are the impacts of impurities and stabilizing agents in the chemical warfare agent stockpiles? ⢠How much aqueous dilution will be necessary if biological processing requires solubilization with a solvent or another biological product? ⢠What volume of biomass and neutralization salts will be produced by biological destruction, and what are the characteristics of and management considerations for these components? ⢠What, if any, metabolic products result from biological treatment of materials containing significant amounts of trace compounds? (See the section on chemical processes for trace by-products.) ⢠Do the various physical states and chemical impurities of the stockpile materials interfere with the biological processes? ⢠Are the kinetics of the biological processes adequate for practical scaleup of degradation? ⢠What types of analytical and process control systems must be developed to monitor the processing streams.?
LOW-TEMPERATURE, LIQUID-PHASE PROCESSES 129 TABLE 6-2 Enzymes Capable of Degrading Organophosphorus Neurotoxins Substrate Source (protein) P-O P-F P-X Genetics Reference Human serum A P,C GD>GB pac cloned Hasset et al. (1991); Furlong et al. (1991); Gan et al. (1991); Smolen et al. (1991) Human serum B P,C GB>GD pac cloned Hasset et al. (1991); Furlong et al. (1991); Gan et al. (1991); Smolen et al. (1991) Rabbit serum P GB>GD pac cloned Furlong et al (1991); Zimmerman and Brown (1986) Rat serum - GD>GB>DFP GA - de Jong et al. (1989) Rat liver P GB>GD>DFP GA - Little et al. (1989) Hog kidney - GD>DFP>GB GA - Mazur (1946); Hoskin (1990) Sheep serum P DFP - - Main (1960); Mackness and Walker (1981) Pig liver P GB>GD>DFP GA - Whitehouse and Ecobichon (1975) Squid nerve - DFP>GD>GB - cloned Hoskin (1990); Ward (1991) Squid muscle - GD>DFP - - Hoskin (1990) Clam foot - DFP>GD - - Landis (1991); Anderson et al. (1988) T. thermophila - DFP>GD - - Landis and DeFrank (1991); Landis et al. (1987) B. sterothermophilus NPEPP GD>GB - - DeFrank and Cheng (1991); DeFrank (1991) Ps. diminuta P,C+ GB>DFP>GD VX cloned Dumas et al. (1989a,b); McDaniel et al. (1988); Lewis et al. (1988); Serdar et al. (1989) E. coli - GD>GB>DFP - - Zech and Wigand (1975) Thermophilic bacteria - GD - - Chettur et al. (1988) Halophilic bacteria - GD>GB GA cloned DeFrank and Cheng (1991) Substrates: PâO bond: P, paraoxon; C, coumaphos; NPEPP, 7-nitrophenyl ethyl (phenyl) phosphinate; +, others. PâF bond: GB, Sarin; GD, Soman; DFP, diisopropyl fluorophosphate; M, mipafox. PâX bonds: GA, Tabun (P-CN bond); pac, phenyhcetate (P-C bond); VX (P-S bond). The > sign indicates which agent is used more effectively by the substrates. Source: Based on Dave et al. (1993).
LOW-TEMPERATURE, LIQUID-PHASE PROCESSES 130 The following sections address the scientific principles of primary detoxification, availability of appropriate biological systems, and systems issues. Biological systems. Numerous biological systems have defined enzyme-based capabilities for detoxification of nerve agents (Table 6-2). Several degradative gene systems have been dolled and subjected to genetic manipulation (Pseudomonas, halophilic bacteria, mammalian serum enzymes, and squid nerve enzyme). Several degradative enzymes (from squid nerve, soil bacteria, halophilic bacteria, and mammalian serum paroxonases) have been purified and to various degrees their interactions with G agents (GA, GB, and GD) characterized. The diversity and substrate preferences of agents treatable by enzyme-based systems are indicated in Table 6-2. In addition, numerous other biological systems have been shown to possess degradative capabilities, although the genes and enzymes have not been extensively detailed with multiple substrates and their specificity for GB and VX has not been determined (Mounter et al., 1955; Attaway et al., 1987; Mulbry and Karns, 1989a,b). Bulk liquid GB has been directly hydrolyzed at laboratory scale by defined enzyme-based systems. Several biological systems that have been used to degrade GB are also applicable to other G agents. Only a few of the enzymatic reactions identified in these various biological systems (Pseudomonas, the halophilic bacterium Altermonas , and squid nerve ganglion) have been well characterized with G agents; most of the others represent preliminary whole cell identification of biochemical capabilities. The direct use of biochemical degradation for G agents was first suggested for the squid enzyme in 1982 (Hoskin and Roush, 1982). The enzymes from Pseudomonas and squid have been purified and immobilized in active form on various matrices (Hoskin and Roush, 1982; Caldwell and Raushel, 1991), suggesting a potential for their use in bioreactor development. The organophosphate hydrolyzing systems of soil bacteria (the opd gene) have been successfully used in field studies to detoxify the insecticide coumaphos, a neurotoxic surrogate for the G agents (Kearney et al., 1988). Although the enzyme involved is also capable of GB hydrolysis (Dumas et al., 1990), the coumaphos studies address neither the actual substrate concentrations that could feasibly be used in direct treatment of agents nor the effect of stockpile contaminants on the activity and stability of these enzymes. These biological systems need further R&D before being used in biodegradation technologies (see Engineering Prospects).
