F
Update on the Status of Biological Processes

INTRODUCTION

The status of biological treatment processes for chemical agents and munitions was reviewed in the report Alternative Technologies for the Destruction of Chemical Agents and Munitions (Alternatives report; NRC, 1993a). It was determined that biological treatment may be potentially applicable to chemical agents but not to energetics, metal parts, or dunnage for the current stockpile configuration. Additional information indicates that some studies have been initiated for the use of biological treatment for energetics (DOD, 1993). Thus, the applicability of biological processes to chemical agents may include the following scenarios:

  1. direct biological detoxification of chemical agent;

  2. biodegradation of the products from a primary chemical agent detoxification process; and

  3. treatment of extracted energetic materials.

Direct biological detoxification of chemical agents may be based on specific enzymatic processes that would be implemented either as purified enzymes, nonviable whole-cell extracts, immobilized cells, or growing cell systems. Primary chemical agent detoxification processes that may be followed by biological treatment include direct biological detoxification (Bio-Bio), chemical neutralization (Chem-Bio), and wet air oxidation (WAO-Bio). The treatment of energetic materials may involve solubilization of solid material followed by one of the three processes mentioned above.

Considerations for the development and application of each of these potential processes must include the status of current research and the flexibility of the projected process for application to the actual stockpile configuration. The purpose of this appendix is to provide an update on the status of each of these potential applications.

Most of the research discussed in the following material is at an early exploratory and proof-of-concept level aimed at enzyme hydrolysis of GB and



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Recommendations for the Disposal of Chemical Agents and Munitions F Update on the Status of Biological Processes INTRODUCTION The status of biological treatment processes for chemical agents and munitions was reviewed in the report Alternative Technologies for the Destruction of Chemical Agents and Munitions (Alternatives report; NRC, 1993a). It was determined that biological treatment may be potentially applicable to chemical agents but not to energetics, metal parts, or dunnage for the current stockpile configuration. Additional information indicates that some studies have been initiated for the use of biological treatment for energetics (DOD, 1993). Thus, the applicability of biological processes to chemical agents may include the following scenarios: direct biological detoxification of chemical agent; biodegradation of the products from a primary chemical agent detoxification process; and treatment of extracted energetic materials. Direct biological detoxification of chemical agents may be based on specific enzymatic processes that would be implemented either as purified enzymes, nonviable whole-cell extracts, immobilized cells, or growing cell systems. Primary chemical agent detoxification processes that may be followed by biological treatment include direct biological detoxification (Bio-Bio), chemical neutralization (Chem-Bio), and wet air oxidation (WAO-Bio). The treatment of energetic materials may involve solubilization of solid material followed by one of the three processes mentioned above. Considerations for the development and application of each of these potential processes must include the status of current research and the flexibility of the projected process for application to the actual stockpile configuration. The purpose of this appendix is to provide an update on the status of each of these potential applications. Most of the research discussed in the following material is at an early exploratory and proof-of-concept level aimed at enzyme hydrolysis of GB and

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Recommendations for the Disposal of Chemical Agents and Munitions VX. Direct biological treatment of mustard is not possible. Enzyme-based processes for both GB and VX would have to be available for practical application to the stockpile configuration. Currently, proof of concept has been demonstrated only for GB. Engineering development appears premature. Biological treatment of reaction products from chemical agents, including GB VX, and mustard, appears however to have a reasonable chance for early application. DIRECT BIOLOGICAL TREATMENT OF CHEMICAL AGENTS Mustard Mustard (H, HD, HT) is a strong vesicant and DNA mutagen that destroys cellular membranes and enzymes. Development of a biological process for direct treatment of mustard agents is extremely unlikely because of their generic xenobiotic characteristics. No recent information has been reported on biological processes for direct destruction of mustard agents. GB and VX Several enzyme systems have been reported that are capable of degrading GB and other neurotoxic fluorophosphonates to varying extents and at differing rates (NRC, 1993a). The initial enzyme-catalyzed hydrolysis of GB results in the production of hydrogen fluoride and monoisopropyl methylphosphonate. In addition, several enzyme and cellular systems have been identified that are capable of cleaving the P—C bond; however, their direct applicability to GB or VX destruction has not been evaluated. At the time of the Alternatives report, critical limitations of enzyme systems to date were (1) tentative reported results indicating enzyme degradation activity on VX, (2) the absence of reaction rate data, and (3) lack of demonstration of proof of principle for practical process and reactor designs based on the reported enzyme systems. However, recent enzyme characterization and laboratory development of treatment processes based on specific enzyme systems have been initiated and several positive results reported. It has been reported that hydrolysis of VX with specific P—S bond hydrolysis was observed with the organophosphorus hydrolase (OPH) enzyme from soil bacteria (Harvey et al., 1993b; Kolakowski et al., 1993). The specific activity of the OPH enzyme in studies at Edgewood Research, Development and Engineering Center (ERDEC), Aberdeen, Maryland, on VX was between 0.5 and 1.0 µmol VX hydrolyzed per minute per milligram of protein.

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Recommendations for the Disposal of Chemical Agents and Munitions At the recent ERDEC Scientific Conference on Chemical Defense Research (November 16-19, 1993), Kolakowski reported on OPH characterization and increased turnover numbers as a function of metal-ion cofactor with the OPH derived from Pseudomonas diminuta. OPH was evaluated for its effectiveness in P—S bond cleavage of VX and related analogues, most of which are commercially available pesticides. Two forms of the enzyme, one with cobalt and the other with zinc as cofactor, were allowed to react with millimolar concentrations of substrate in dilute aqueous media maintained at pH 8.5 and 25ºC. Hydrolysis rates were determined by following the formation of free SH groups. Small, but measurable rates of hydrolysis were observed for VX and several analogues studied, with the activity of OPH (Co2+) usually being 5 to 10 times greater than that of OPH (Zn2+). Spontaneous hydrolysis was negligible. First-order kinetics were observed in all cases, and turnover numbers ranged from a high of 5 per minute for VX to a low of 0.1 per minute for the analogue IBP1. OPH is the only enzyme known that hydrolyzes the P—S bond of VX. Other researchers have reported on the ability to express OPH activity from promoter 1 from Cochliobolus heterostrophus and the trpC terminator from Aspergillus nidulans in the native soil fungus Gliocladium virens (Dave et al., 1993a, b). Various opd+ transformants displayed differing levels of enzymatic activity, and Western blot analysis of mycelial extracts from transformants confirmed the expression of a mature processed form (lacking the membrane targeting signal) of the enzyme in the fungus. Harvey et al. (1993b) reported that purified OPH(Co2+) enzyme was able to hydrolyze 0.77- to 1.4-molar concentrations of munitions-grade GB to less than 6 parts per million (ppm) in 1 hour at room temperature. The initial GB concentration was a 5:1 or 10:1 aqueous dilution of chemical agent directly removed from the stockpile. The major stockpile impurity ethyl-GB (approximately 2 percent of the total) was also hydrolyzed. The Biotechnology Division at ERDEC is accelerating its search for new enzymes and microorganisms capable of degrading both U.S. and Russian stockpile agents. The research team has recently cloned an opd gene that encodes the Alteromonas JD6.5 enzyme at highly expressed levels (˜5 percent of cell protein in Escherichia coli). This enzyme has excellent hydrolytic activity with GB (˜300 µmol/min/mg; Cheng et al., 1993a). In addition, Cheng et al. (1993b) have purified an even more active enzyme from Alteromonas undina. The specific activity on GD (soman)2 is 1,850 µmol/min/mg at 25ºC, pH 7.2, and GB activity is good. Studies are currently under way to clone the gene for this enzyme into E. coli. 1   O, O-diisopropyl-S-benzylphosphothiate (insecticide Kitazin P) 2   GD is O-pinacolyl methylphosphonofluoridate.

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Recommendations for the Disposal of Chemical Agents and Munitions In summary, the above results indicate that enzyme-based processes capable of both GB and VX hydrolysis may be developed into practical applications. (However, the reported rate of VX hydrolysis is much too slow for practical application.) The OPH enzyme has been characterized and cloned into various viable host cells. Furthermore, the capability of the enzyme to work directly on stockpile agent has been demonstrated. An immobilized whole-cell reactor would limit required enzyme production and purification, eliminate biomass increase, and reduce permanent complications. Major milestones remaining include increased activity (turnover rates) on VX and reactor engineering and evaluation. The time required for development of enzymes with increased activity on VX is not predictable because of the nature of the basic research required. BIOLOGICAL TREATMENT FOLLOWING NEUTRALIZATION Mustard Chemical hydrolysis of mustard under alkaline conditions results in the formation of thiodiglycol as the primary reaction product and numerous other tentatively identified dechlorinated reaction products. Biological treatment of the reaction products from mustard hydrolysis was indicated in the Alternatives report as one of the more promising applications of biological processes to the agent stockpile. Subsequent laboratory investigations have demonstrated biodegradation of hydrolysis products from HD (Harvey et al., 1993c). Investigations of appropriate reactor design for treatment of mustard hydrolysis products have been initiated using thiodiglycol (TDG) as a model substrate. Theoretical studies evaluating different potential reactor configurations indicated that substantial reductions in required reactor volume can be achieved through the use of a fed-batch reactor rather than a continuous stirred tank reactor with cell recycle or two stirred tanks in series design (Sines et al., 1993). Laboratory studies of biological treatment process design have recently been initiated. Some limited thiodiglycol substrate inhibition was indicated in preliminary experiments at substrate concentrations greater than 120 mM. However, this inhibition was very mild, and cells were capable of growth and degradation at much higher thiodiglycol concentrations. The optimal concentration for a bioreactor may be around 120 mM. Bioreactor studies utilizing cryoimmobilized Alcaligenes species capable of degrading thiodiglycol have been initiated at the Edgewood Research, Development and Engineering Center.

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Recommendations for the Disposal of Chemical Agents and Munitions GB and VX Biological treatment of reaction products from chemical neutralization of GB and VX was considered in the Alternatives report as a potentially viable process subsequent to chemical neutralization. This is being pursued at the Edgewood Research, Development and Engineering Center, but only very preliminary results have been reported. Cultures have been isolated that utilize ethylmethylphosphonic acid (EMPA) and pinacolyl alcohol as sole sources of carbon for growth. EMPA is produced in stoichiometric amounts from either enzymatic or chemical cleavage of the P—S bond of VX. Pinacolyl alcohol is the alcohol product of GD hydrolysis. Efforts are under way to identify organisms involved and to characterize these processes. BIOLOGICAL TREATMENT FOLLOWING WET AIR OXIDATION Application of wet air oxidation as a primary treatment process for chemical agents would result in the formation of aqueous solutions containing acetic acid and other low molecular weight organic compounds. Biological treatment of waste streams similar to those anticipated from wet air oxidation has been applied broadly at full scale. Process development for the specific wastewater stream produced by wet air oxidation would still be required.