2254
Ind. Eng. Chem. Res. 1993,32, 2254-2258
Reusable Immobilized Enzyme/Polyurethane Sponge for Removal and Detoxification of Localized Organophosphate Pesticide Spills Patrick L. Havenst and Howard F. Rase' Department of Chemical Engineering, The University of Texas, Austin, Texas 78712
During the course of study on immobilization of an enzyme, parathion hydrolase, that detoxifies organophosphate pesticides, a very effective and reusable cleanup sponge was developed. This sponge was made by incorporating the enzyme through free amino-acid groups into a polymerizing isocyanate-based polyurethane foam. By controlling various reaction variables, it became possible to produce an enzyme-containing foam of high liquid-retention capacity and excellent enzyme stability. This product, which should prove valuable for easy simultaneous cleanup and detoxification of small spills, was found effective for both solutions of parathion and a typical commercial pesticide formulation. Organophosphate pesticides are extensively used worldwide for a wide range of agricultural and household applications. Although toxic to nontarget organisms,they have the advantage of very short half-lives and do not constitute a long-term environmental threat. Their handling and use, however, must be exercised with care, and contaminated rinse water and spills must be promptly remedied. Havens and Rase (1990) have reported an extensive study of an enzyme that detoxifies organophosphate pesticides. This enzyme, parathion hydrolase, was obtained from an overproducing strain of bacteria, Pseudomonas d i m i n u t a , and was immobilized upon a variety of solid supports and foams. Successful and efficient detoxification of a variety of organophosphate pesticides was observed, and various portable systems for detoxification of rinsates from drum cleanup were proposed and tested. During the course of the work on foams the authors postulated a sponge containing immobilized enzyme and having high liquid holding capacity. Such a sponge could prove useful for cleanup and simultaneous detoxification of small localized spills if the immobilized enzyme proved stable and the sponge reusable. This postulate was confirmed by the development of a successful and reusable sponge as described in the followingsections.
Origins and Development of Efficient Detoxifying Enzyme Systems The study of the biodegradation of organophosphates has been developed over about the past 15 years. A concerted effort has been directed at isolating the organisms responsible for the degradation, identifying the specific enzymes carrying out the actual chemical change, understanding this enzyme system at the genetic level, and utilizing these enzymes for organophosphate waste remediation. Lichtenstein and Schulz (1964) were the first to observe metabolites from reduction and hydrolysis of parathion, methyl parathion, and malathion in soils. The lack of such degradation in sterilized soils indicated to them the involvementof microorganisms. Eight years later Munnecke and his associates began extensive studies that yielded a mixed bacterial culture adapted to utilizing parathion and p-nitrophenol as a sole carbon source. Munnecke (1976) also did extensive work on activity of the ensyme system for various commercially used orga-
nophosphates. The studies of Munnecke and others which led to the development of a Ps. diminuta strain MG(pCMS55)of exceptional activity are summarized in Table I. It is this overproducing strain that was used to obtain the enzyme extract for the successful sponges to be described.
Enzyme Immobilization Using Polyurethane Foam A relatively recent advance in enzyme immobilization involves cross-linking within a polyurethane foam matrix. Hypo1 is the trade name of a family of foamable polyurethanes produced by the Organic Chemicals Division of W. R. Grace and Company developed from work patented by Wood and Frisch (1979). The material is delivered as a one-component prepolymer resin. The resin is based on a multiple branched poly(ethy1ene oxide). The chains terminate in hydroxyl groups, which are capped with methylenediphenyl diisocyanate (MDI) or tolyl diisocyanate (TDI). The foaming reaction occurs by addition of approximately equal volumes of aqueous solution combined with a surfactant to the prepolymer. Properties such as foam density, cell structure, rate of wetting, and water retention can be modified by controlling the ratio of water to prepolymer, foamingtemperature and pH, and the type and amount of surfactant added to the aqueous phase. A wide range of foam characteristics is possible, ranging from very fine celled, soft foam to very coarse, open celled foam. Heavy loadings of additives can be included, including abrasives, chemicals for time release, and pharmaceuticals, which are then incorporated into the final sponge form (W. R. Grace, Co., 1986). The first step in the polymerization to the final foam form is a chain extension reaction (eq 1). The carbon RNCO + H,O
-
RNH, +.CO,
dioxide produced in this step is released as a gas. The mixture is viscous enough to retain the gas, forming the cellular structure of the foam. The NH2 group produced can then react further with isocyanate groups, extending and cross-linking the chains (eq 2). The polymer has been
RNH2
+
RNCO
-
0
II
R-N-C-N-R
I
H + Current address: DowElanco, North American Environmental Laboratory, 9410 North Zionsville Road, Indianapolis, IN 46268.
(1)
I
(2)
H
used in a wide range of products, with heavy emphasis on consumer products. Since no catalyst is used during
0888-5885/93/2632-2254$04.00/00 1993 American Chemical Society
Ind. Eng. Chem. Res., Vol. 32, No. 10,1993 2266 Table I. Summary of Major Studies on Hydrolysis of Organophorphates by Biological Systems fiidings authors and date adapted a mixed soil culture to use parathion and p-nitrophenol aa ita sole carbon source Hsieh and Munnecke (1972) identified seven bacterial isolatea (mostly peudmonade) in the mixed culture that were active in the Munnecke and Hsieh (1974) hydrolysis of parathion obtained high growth rate in continuous fermentor &r a 36-day enrichment period on both technical Munnecke and Hsieh (1975) and parathion-xylene emulsifiable concentrate; slightly alkaline conditions were moet favorable identified several metabolites and poetulated three metabolic pathways; proposed the existence of a Munnecke and Hsieh (1976) single enzyme responsible for the hydrolysis and found it to be isolated within the cella and not excreted significantlyinto the growth medium; crude enzyme extract from the cells WBB found to be stable up to 55 "C studied detoflication of commonly used organophosphateswith the same crude extract Munnecke (1976) covalently attached the extract to nonporous glaes and tested in a continuous packed column wing Munnecke (1977) parathion-containingwater; a half-life of 280 days was observed used controlled-pore silica for attaching the crude enzyme extract and observed 99 % removal of Munnecke (1979) parathion and no loss of activity for 70 days studied commercial-container residue detoxificationusing a lyophilized enzyme mixture with 94-97 % Munnecke (1980) removal of parathion residue studied degradation of concentrated Diazinon in soil using a crude enzyme extract and found 98% Barik and Munnecke (1982) hydrolysis in 24 h isolated a particular organisms capable of hydrolyzing parathion and identified it aa Pseudomonas Serdar et al. (1982) diminuta; cured cells with mitomycin C and isolated a plasmid containing the parathion gene cloned and expressed the gene and obtained one strain of Ps.diminuta MG(pCMS55) that exhibited Serdar and Gibson (1985) the highest activity Table 11. Hyml Foam Prowrties description resin surfactant open celled; brittle and rigid; low water FHP-5000 P-65 absorption f i e celled; soft; low water absorption FHP-4000 P-65 very fine pore structure; easily torn;low FHP-2002 P-65 water absorption open celled; somewhat soft; good water FHP-5000 P-85 absorption fine celled; soft; fair water absorption FHP-4000 P-85 very fine pore structure; extremely soft; FHP-2002 P-85 easily deformable; low water absorption similar to FHP-5000/P-85but slightly FHP-6000 L-62 fiier celled; somewhat soft; excellent water absorption f i e celled; very soft; did not retain shape FHP-4000 L-62 after squeezing; low water absorption very fine celled; soft; low water absorption FHP-2002 L-62
polymerization,the polymer system lends itselfto avariety of products for personal care and medicinal uses (W. R. Grace Co.,1984). A patent by Wood et al. (1982)describes the binding of enzymes to these isocyanate-based polyurethane foams. The patent was assigned to W. R. Grace, which developed the materials described in the patent into their Hypol and Hypol Plus urethane prepolymers. Enzymes are incorporated covalently in the foam by linkages through free amino groups present on the proteins. RNCO
+
H2N-E
-
0
II
R-N-C-N-E
I
H
I
(3)
H
Since the protein may be attached at more than one point, the enzyme is actually a cross-linked part of the polymer matrix. Wood and his co-workers describe the binding and use of several different enzymes in numerous examples in the patent. These include breakdown of (carboxymethyl)cellulose by cellulase, clarification of apple juice by pectinase, proteolysis of casein by trypsin, hydrolysis of urea by trypsin, hydrolysis of starch by amyloglucosidase, conversion of lactose to glucose by lactase, and conversion of penicillin G to 6-aminopenicillanic acid by penicillin amidase. The different examples involve various prepolymer formulations and foaming conditions. The en-
zymes were derived from various sources, including purified lyophilized preparations, crude extracts, and fermentation broths. The authors found no enzyme which could not be bound, and all the bound enzymes they tried retained their activity to some degree. They used the foams in batch operations by immersing pieces of foam in substrate solutions, as well as in a continuous fashion by packing the foams into columns. Kitto (1987)has described the incorporation of the enzyme carbonic anhydrase into a Hypol foam. This enzyme acts to transfer carbon dioxide from gas to a liquid. The immobilized foam has possible application to underwater breathing apparatus to remove carbon dioxide from a diver's expelled breath.
Immobilized Parathion Hydrolase on Hypol Polymer Supports The Hypol polyurethane foams differ in many respects from more conventional supports, the most significant being that the enzyme actually becomes a part of a crosslinked polymer matrix. This cross-linking of the enzyme should impart considerable stability to the immobilized enzyme, allowing a long lifetime for the enzyme-support formulation. The final form of the polymer is a foam whose characteristics can be adjusted by altering the conditions under which the polymerization reaction takes place. One of the most important of these conditions is the addition of surfactants to the aqueous phase. The surfactants affect the final foam's cell structure, wetability, and water holding capacity (Table 11). Parathion hydrolase immobilized on Hypol was studied for two possible applications: first, as a column packing for use in a system as described by Havens (1990);second, as an enzyme "sponge" for localized spill cleanup. The sponge concept would fill a presently unfilled niche to detoxify the small spills inevitably generated during product pouring, mixing, and filling. The sponge would soak up a spill on a mixing pad or building floor and then would be sealed in a plastic bag to carry out the hydrolysis reaction. On completion of the reaction, the sponge could be wrung into an appropriate container and reused or could be simply thrown away as nonhazardous waste. Experimental Methods and Materials Cell Growth. Culture plates (15% tryptone, 0.5% yeast extract,0.5% NaC1,1.5% agar, 50pg/mL kanamycin) were
2256 Ind. Eng. Chem. Res., Vol. 32, No. 10, 1993
inoculated with cells from frozen glycerol suspensions of
Ps.dirninuta MG(pCMS55). After overnight incubation at 32 "C, single colonieswere transferred to 250-mL shake flasks containing 50 mL of TYE medium (15% tryptone, 0.5% yeast extract, 0.5% NaC1, 50 pg/mL kanamycin). Following overnight incubation at 32 "C, these flasks were used to inoculate a 1- or 1.5-L fermenter, which was maintained at 32 "C, pH 7. Fermentation was continued until the cells were at the upper log growth phase. Cells were harvested by centrifugation (8000g, 20 min, 4 "C) and resuspended in 1 mL of phosphate buffer (50 mM, pH 8.5) per gram of cells. The cells were then disrupted by sonication and centrifuged again (600OOg), yielding the crude cell extract as the supernatant. Partial purification of the extract was obtained by ammonium sulfate precipitation (20-40% cut), and the salt was removed by dialysis against 50 mM phosphate buffer, pH 8.5. Enzyme Assays. Standard assays were carried out by pipeting 20 pL of protein solution into a quartz spectrophotometer cuvette and adding 400 pL of parathion solution which had been prepared from reference standard material in methanol (US EPA Pesticide and Industrial Chemicals Repository using Tris buffer as diluent (50mM, pH 8.5). The production of a hydrolysis product of parathion, p-nitrophenol, was followed by tracking the increase in absorbance at the 410-nm wavelength. Since the hydrolysis stoichiometryis one-to-one, the appearance of the phenol corresponds to the disappearance of parathion. The concentration of p-nitrophenol was determined by comparison with a standard absorbance curve. Regression upon the initial linear portion of the concentration vs time curve yielded an initial rate in nanomoles of parathion per microliter per minute. Protein assays were carried out using premixed Coomassie Blue reagent according to the manufacturer's (Pearce Chemical, No. 23200) instructions. The rate was divided by the amount of protein to yield a specific activity, defined as nanomoles of parathion consumed per minute per milligram of protein. Analysis of chlorpyrifos was accomplished using reversed-phase high-performance liquid chromatography (HPLC). Alinear gradient of 5050 methanokwater (+1% acetic acid) to 100% methanol over 30 min on a Waters pBondapack C-18 column at 1 mL/min flow eluted chlorpyrifosat 28.3 min and trichloropyridinol at 17.3min. A calibration curve was prepared by injecting known concentrations of chlorpyrifos. Making the Sponges. A rough mold was prepared by lining a small open box with aluminium foil. One to two percent (w/v) of surfactant was added to a volume of protein solution in buffer. Then an approximately equal or a slightly greater weight of Hypol Plus FHP-5000 (W. R. Grace, 1986) was placed in a plastic beaker, and the protein solution was added and stirred vigorously with a glass rod. When the mixture reached the ucreamnstage (less than 1min), it was poured evenly into the mold and allowed to polymerize. After the reaction was complete, the skin was trimmed from the block with a band saw. Assay of Sponge Activity. A Beckman DU-50 spectrophotometer was calibrated a t 410 nm on Tris buffer (pH 8.5). Munnecke (1976) found that alkaline conditions promoted high levels of enzyme activity. A block of sponge was placed in a beaker, a volume of substrate solution was added to the beaker, and the sponge was squeezed to absorb the solution. The sponge was then squeezed periodically at noted time intervals so that small aliquots of solution could be removed for analysis. With parathion solutions, absorbance at 410 nm was measured and the aliquots were
readsorbed into the sponge. Solutions containing chlorpyrifos were analyzed by HPLC.
Results Comparison of Foam Formulation Physical Properties. Three different Hypol prepolymers were studied as possible supports for application of parathion hydrolase: FHP-2002 (TDI based),FHP-4000 (MDI based), and FHP-5000 (MDI based). The three resins were foamed with three surfactants from the Pluronic series (BASF Corporation), Pluronic P-65, P-85, and L-62, dissolved in the aqueous phase (50 mM phosphate buffer, pH 8.5) at the 1-2% level by weight. The Pluronic series was recommended by W. R. Grace to give a wide range of foam properties. The results of this series of experiments are summarized in Table 11. It can be seen from these results that the properties of the foams can indeed vary widely. For the sponge configuration, a formulation exhibiting high water absorption and good physical strength is desired while a more rigid foam would be better for continuous operation. Characteristics of Immobilized Enzyme/Foam Formulations. The next step was to immobilize enzyme upon the resin and test for activity. Crude cell extract was immobilized upon FHP-5000 without surfactant. This experiment was performed to test if the protein solution itself had any surfactant action. The resulting foam was very fine celled and soft, quite different from previous FHP-5000 formulations, indicating that the proteins had poor surfactant action. The block of foam was chopped into small pieces and packed into a 2.5-cm-diameter column, and parathion solution was pumped through the column in recycle. There was a great deal of wall flow and bypassing observed and conversion rate was very low. Similar results were obtained with several other foam formulations. Compared to the immobilized enzyme on controlled-pore glass previously described (Havens and Rase, 1991),the Hypol polymer foams are a greatly inferior column packing support. As an absorbent sponge, however, the foams merited further study. Experiments were designed to determine the feasibility of an enzyme foam/sponge for soaking up and detoxifying small spills. FHP-5000 with L-62 surfactant was found to be the best formulation for this application. P-85 was rejected for use because experiments showed it deactivated the enzyme, seemingly irreversibly. Among all the formulations studied, the FHP-5000/L-62 exhibited properties most similar to a conventional cellulose sponge, easily able to soak up and hold as much as 10 times its own weight in water. Enzymatic activity was retained and the sponges immobilized up to 5 mg of protein per gram of prepolymer resin. The resin was incorporated into the aqueous phase manually, resulting in some nonuniformity of cell structure. This problem can easily be remedied by using commercially available urethane foaming and molding equipment (the Martin Sweets Company of Louisville, KY, manufactures equipment specifically designed for Hypol foams). A representative experiment designed to determine in a preliminary way the detoxifying action of an FHP-50001 L-62 sponge preparation is presented graphically in Figure 1. This particular sponge weighed 5.5 g and contained about 2.9 mg of protein per gram of prepolymer resin. It can be seen that the sponge was able to reduce the parathion concentration from 13.5 ppm to about 1 ppm in about 30 min. This initial concentration is within the concentration range expected for rinsate waters.
Ind. Eng. Chem. Res., Vol. 32,No. 10,1993 2251
20
10
0
30
time, min 0.05
0.04
3s
Age 0.03
Q
0
0.M
O
*
1 day zdays 3days 51days
0.01
0.00 0
50
100
150
time, min 0.05
0.04
Age
0.03 0
0.02
*
1 day 2days 3days 51 days
0.01 0.00
0
20
40
60
80
100
1000
2000
3000
4000
5000
6000
time, minutes Figure 3. Sponge test with 0.12% Dursban EC.
Figure 1. Hypol sponge run with parathion solution.
3
0
40
120
time, min
Figure 2. Hypol sponges: immobilized enzyme stability over time: (A, top) sponge stored in buffer for days indicated; (B, bottom) sponge stored dry for days indicated.
The question of stability of the enzyme immobilized in the foam formulations is an issue of paramount importance to the eventual successfulapplication of the sponge device. It would be ideal if the sponges required no special handling or storage conditions and were readily available for use in the event of a spill. To examine this issue, two pieces of sponge from the same immobilization batch were studied over a 50-day time period. One sponge was stored between runs in buffer under refrigeration, while the other piece was allowed to dry out a t room temperature. Both pieces were thoroughly rinsed in water at the conclusion of each detoxificationrun before being returned to storage. Figure 2 shows the results of these experiments. It can be seen from these plots that the sponge stored wet showed a lower activity loss. However, the dried sponge did retain a large part of its activity, showing that it may be practical to supply the sponges dry, without any special storage requirements. Further experiments would be useful to compare these results with a sponge stored wet at room temperature (not done in this study). In addition, these results show that the sponges are reusable, at least a t the concentrations studied here.
Tests with Commercial Pesticide Formulations. In order for the detoxifying sponge to be a useful device, it must not be sensitive to the other compounds present in commercial pesticide formulations, including organic solvents, emulsifiers, and detergents. This was studied in preliminary fashion by subjecting a sponge sample to a solution of Dursban (trademark of the Dow Chemical Co.) EC containing 0.12 % chlorpyrifos active ingredient, about 1/2 normal spray concentration. Dursban is widely used on a variety of grain, vegetable, and ornamental crops. An HPLC method was used for analysis. Ten milliliters of Dursban solution was applied to a 5.6-cm3 piece of detoxifying foam. Samples were taken periodically as before and analyzed. Figure 3 shows the result. It can be seen from this plot that the sponge was capable of significantly reducing the amount of active ingredient in the commercial formulation of chlorpyrifos in the presence of inert ingredients and organic diluents. Again, the concentration used here is high compared to rinsate waters. However, in the case of spills of actual spray mix, these results are directly comparable. A rather long period of time would be required for complete hydrolysis of the organophosphate in the sponge. Higher loadingsof enzyme of the sponges could perhaps be a solution to this problem. Long term stability toward commercial formulations was not studied. Discussion The preliminary results presented in this section show that the detoxifying sponge may be a viable answer to the problem of small, localized spills of organophosphate pesticides. It can be visualized that sponges would be supplied by the pesticide manufacturer with every drum of spray concentrate as a show of "product stewardship". Instructions would be included to soak up spills with the device, seal up the sponge in a liquid-tight plastic bag, and then wait a period of time before disposal of the sponge. The device could also be sold to spray operators and smallscale commercial and home users. It is possible that mass production of such a sponge could realize sufficient savings on enzyme production to yield an economically viable sponge for multiple use.
Literature Cited Barik, S.; Munnecke, D. M. Enzymatic Hydrolysis of Concentrated Diazinon in Soil. Bull. Enuiron. Contam. Toxicol. 1982,29,235239.
Havens, P. L. Detoxification of Organophosphorus Pesticide Solutions: An Immobilized Enzyme System. Dissertation, The University of Texas, 1990.
2258 Ind. Eng. Chem. Res., Vol. 32,No. 10,1993 Havens, P. L.; Rase, H. F. Detoxification of Organophosphate Pesticide Solutions: ImmobilizedEnzyme Systems. In Hazardous Waste Management ZI; Tedder, D. W., Pohland, F. G., Eds.;ACS SymposiumSeries 468;American Chemical Society: Washington, DC, 1991;pp 261-281. Hsieh, D. P. H.; Munnecke, D. Accelerated Microbial Degradation of Concentrated Parathion; In Fermentation Technology Today; Terui, G., Ed.; Societyof FermentationTechnology: Tokyo,Japan, 1972;pp 551-554. Kitto, G. B. Personal communication, 1987. Lichtenstein, E. P.; Schultz, K. R. The Effects of Moisture and Microorganisms on the Persistance and Metabolism of Some Organophosphorous Insecticides in Soils, with Special Emphasis on Parathion. J. Econ. Entomol. 1964,57,618. Munnecke, D. M. Enzymic Hydrolysis of Organophosphate Insecticides, a Possible Pesticide Disposal Method. Appl. Enuiron. Microbiol. 1976,32,7. Munnecke,D. M. Properties of an Immobilized Pesticide-Hydrolyzing Enzyme. Appl. Enuiron. Microbiol. 1977,33,503. Munnecke, D. M. Hydrolysis of Organophosphate Insecticides by an ImmobilizedEnzyme System. Biotechnol. Bioeng. 1979,21,2247. Munnecke,D. M. EnzymicDetoxification of Waste Organophosphate Pesticides. J. Agric. Food Chem. 1980,28, 105. Munnecke, D. M.; Hsieh, D. P. H. Microbial Decontamination of Parathion and p-Nitrophenol in AqueousMedia. Appl. Microbiol. 1974,28,212.
Munnecke, D. M.; Hsieh, D. P. H. Microbial Metabolism of a Parathion-xylene Pesticide Formulation. Appl. Microbiol. 1975, 30,575. Munnecke, D. M.; Hsieh, D. P. H. Pathways of Microbial Metabolism of Parathion. Appl. Environ. Microbiol. 1976,31,63. Serdar, C. M.; Gibson, D. T. Enzymic Hydrolysis of Organophosphates: Cloning and Expression of a Parathion Hydrolase Gene from Pseudomonas diminuta. BiolTechnology 1985,3,567. Serdar, C. M.; Gibson, D. T.; Munnecke, D. M.; Lancaster, J. H. Plasmid Involvement in Parathion Hydrolysis by Pseudomonas diminuta. Appl. Environ. Microbiol. 1982,44, 246. W. R. Grace and Company. Hypol Marketeer; Lexington, MA, June 1984. W. R. Grace and Company. Hypol PlusTMLaboratory Procedures and Foam Formulations; Lexington, MA, 1986. Wood, L. L.; Frisch, K. C. Crosslinked Hydrophilic Foams and Method. U.S.Patent 4,137,200,Jan 30,1979. Wood,L. L.; Hartdegen, F. J.; Hahn,P. A. Enzyme Bound to Polyurethane. U.S.Patent 4,342,834,Aug 3, 1982.
Received for review November 3, 1992 Accepted April 12, 1993. Abstract published in Advance ACS Abstracts, August 15, 1993.
