ENZYMATIC DEGRADATION OF PLUTONIUM-CONTAMINATED CELLULOSE PRODUCTS

Enzyme solutions produced for commercial purposes unrelated to waste management have the potential for reducing the volume of wastes in streams containing cellulose, lipid and protein materials. For example, we have shown previously that cellulases used in denim production and in detergent formulations are able to digest cellulose-containing sorbents and other cellulose-based wastes contaminated either with crude oil or with uranium. This presentation describes the use of one such enzyme preparation (Rapidase , manufactured by Genencor, Rochester, NY) for the degradation of cotton sorbents intentionally contaminated with low levels of plutonium. This is part of a feasibility study to determine if such treatments have a role in reducing the volume of low level and transuranic wastes to minimize the amount of radionuclide-contaminated waste destined for costly disposal options. INTRODUCTION Waste streams from National Laboratories with historic and current missions involving radionuclides used in the defense and nuclear energy industries present disposal problems. Plutonium, americium, neptunium and uranium, among other radioactive elements, contaminate these wastes. The problems they present include the potential for radiation poisoning since most have extremely long half lives, and there is potential for use in terrorist activities. Not only must weapons-grade plutonium be guarded from improper release from these facilities, but low level (LLW) and transuranic (TRU) wastes also must be disposed of in ways that prevent improper contact with living organisms and the environment. Such low level and transuranic wastes include items resulting from the production, testing, and storage (and now disassembly) of nuclear weapons. At Los Alamos National Laboratory (LANL) and other Department of Energy (DOE) facilities, work regularly is performed in glove boxes where plutonium (Pu) and other radionuclide residues can contaminate items within the boxes or on their internal surfaces. Normal maintenance of these glove boxes is done by cleaning with laboratory wipes and cheesecloth. which then must be handled as TRU or LLW and disposed of in special landfills or other repositories. LANL’s Plutonium Facility currently generates approximately 2500 kg of combustible-type material per year, and a portion of that is cellulose-based material WM'99 CONFERENCE, FEBRUARY 28 MARCH 4, 1999 Currently TRU waste products are slated for disposal at the WIPP (Waste Isolation Pilot Plant) facility located in New Mexico, but opposition from various groups has prevented the use of this completed facility, so the materials remain on the governmental defense reservations. Even when (if) this or a similar facility can begin accepting TRU waste, it will be expensive to store such items in perpetuity. The same argument can be made for the disposal of LLW in appropriate facilities. Therefore, any technology that can reduce the volume of such wastes would greatly reduce the cost of disposal and the associated risk of potential human and environmental contamination. The use of industrial-strength enzyme solutions developed and marketed for a variety of other applications may be useful in the reduction in volume of radionuclide-contaminated wastes. Unlike many enzymes used in research projects, most industrial enzyme preparations are able to withstand (and function under) harsh conditions of use such as low or high pHs and in the presence of detergents or heavy metals. A technology based on the use of industrial cellulases developed to treat oil-saturated cotton sorbents in the presence of seawater also was found to be useful for degrading uranium-contaminated cotton fabrics, releasing the uranium into the enzyme solution (1,2). The present project was intended to demonstrate the feasibility of this process for treatment of cellulose-containing materials in LANL's laboratory waste stream. Dr. Pat Unkefer and associates at LANL used a microbially-generated cellulase to initiate degradation of Putreated cotton prior to destroying the material by a sewage sludge consortium (3). They reported no significant effect of Pu on biodegradation. The current report describes the use of Rapidase, an industrially-available cellulase, to degrade lanthanide and Pu-contaminated cellulose substrates. To be determined was whether these heavy metals would inhibit the activity of the commercially-available enzyme. Also to be determined was whether the Pu remained with the digested cotton residue or if the radioactivity was released into the enzyme solution following enzyme treatment. MATERIALS AND METHODS The commercial enzyme used for this studies was Rapidase (Genencor, Rochester, NY). A five per cent solution of the commercial liquid was prepared in McIlvaine's citratephosphate buffer (4) to produce a final solution with a pH of 4.5. Cotton substrates (0.5g/flask) consisted of raw cotton (not treated by scouring and bleaching) that had been cleaned of stem and leaf trash by treatment in a Shirley analyzer (British Cotton Industry Research Association, Patent No. 404888) and afterward referred to as Shirleyed cotton, or 0.5g of the other cotton substrates being tested (including cheesecloth and paper that had been powdered in a Wiley cutting mill to pass a 20-mesh screen). In the typical laboratory-scale experiments, a total volume of 75ml liquid plus 0.5g cotton substrate were treated for 6 days at 45C. The cellulose substrate was added to freshly prepared enzyme solution at the beginning of each experiment. Experimental and control (minus enzyme) flasks were incubated on a gyrotary shaker (New Brunswick Scientific) at 45C; alternately, incubation was conducted on a stirring hotplate where the temperature was maintained at 45C. WM'99 CONFERENCE, FEBRUARY 28 MARCH 4, 1999 Substrate digestion due to enzyme treatment was evaluated by determining the dry weight of each substrate prior to and following enzyme treatment. Residual substrate collected by filtration was dried to a constant weight at 100C. Per cent residual substrate is reported as both the range of values from three replicates and the mean value, unless otherwise noted in the tables. Residual glucose levels were determined by high pressure liquid chromatography (HPLC) using a Dionex DX 500 HPLC System with an AS 5300 autosampler. A gradient eluent of 500mM sodium acetate/200mM sodium hydroxide was used. Five mL of spent enzyme solution recovered from each flask following filtration to remove the residual cellulosic substrate was adjusted to pH 6-7 with 0.3g (Na2)3(PO4)2 and heated for approximately 2hr in a 70C waterbath to coagulate the protein. A 1mL sample of the heated and cooled supernatant fluid was diluted volumetrically 1:100 with 18.2 megaohm water. A portion of this solution was filtered through a 0.2μM filter into an autosampler vial from which 25μL was drawn for HPLC analysis. Glucose concentrations were calculated from the results as mg/mL of the supernatant fluid recovered after heating the residual enzyme solution. The effect of the following were evaluated to determine if they inhibited or slowed the digestion process: glucose, sucrose and nitrate concentrations (0.1 to 1.0M), and the presence of cerium oxide, cerium carbonate, praseodymium oxide or plutonium oxide. Liquid scintillation counting (Packard Instrument Company) or alpha spectroscopy by conventional methods were used to determine the radioactivity. These techniques both are based on the measurement of internal radioactivity due to the alpha particle emission from the plutonium-239 isotope. RESULTS Initial experiments were performed with various forms of cotton and cellulose-based manufactured items to define the conditions needed by the enzyme to degrade both the noncontaminated and the contaminated items. In previous studies testing the ability of cotton to be degraded by cellulases (1,2) a 5% solution of Rapidase in pH 4.5 McIlvaine’s buffer was found to be an effective way to digest raw cotton contaminated with diesel fuel, crude oil or uranium. The optimum temperature was found to be 45C, although degradation occurred between 25 and 60C. Agitation resulted in faster degradation than static treatment, with 200 rpm on a gyrotary shaker being optimal. Degradation of roughly 50% of the substrate occurred in 6 days under these conditions which were used to set the baseline for our studies here. The ability of the enzyme solution to degrade unused materials such as paper and cheesecloth (of the type that is used as sorbing wipes in the glove box line) that eventually contribute to the LANL waste stream was tested. Other experiments included determining the effect of various concentrations of sugars (glucose and sucrose) on the degradation process since the activity of one of the cellulase enzymes (cellobiase) is known to be inhibited by increasing concentrations of glucose. Further, the effect of added nitrate salts was tested since Pu is soluble in HNO3 and nitrates can be found in Pu-containing waste streams and thus potentially present on wipes used to clean glove boxes. Cotton contaminated with lanthanides, then with plutonium was tested to determine if actinides inhibited the ability of the enzyme to degrade raw cotton. The distribution of the plutonium between the residual substrate and enzyme solution following degradation of the cotton was determined. WM'99 CONFERENCE, FEBRUARY 28 MARCH 4, 1999 In the current study, the dry weight of residual substrate was used to determine the effectiveness of the enzyme treatment (per cent reduction in dry weight). Residual substrate was collected by filtration followed by drying to a constant weight at (100C). Residual reducing glucose was determined by HPLC analyses. Residual PuO2 was determined by liquid scintillation counting and alpha spectroscopy. The results from the six experiments are listed below. In the first experiment, cellulose-based material was exposed to 5% enzyme solution fo


INTRODUCTION
Waste streams from National Laboratories with historic and current missions involving radionuclides used in the defense and nuclear energy industries present disposal problems.Plutonium, americium, neptunium and uranium, among other radioactive elements, contaminate these wastes.The problems they present include the potential for radiation poisoning since most have extremely long half lives, and there is potential for use in terrorist activities.Not only must weapons-grade plutonium be guarded from improper release from these facilities, but low level (LLW) and transuranic (TRU) wastes also must be disposed of in ways that prevent improper contact with living organisms and the environment.Such low level and transuranic wastes include items resulting from the production, testing, and storage (and now disassembly) of nuclear weapons.At Los Alamos National Laboratory (LANL) and other Department of Energy (DOE) facilities, work regularly is performed in glove boxes where plutonium (Pu) and other radionuclide residues can contaminate items within the boxes or on their internal surfaces.Normal maintenance of these glove boxes is done by cleaning with laboratory wipes and cheesecloth.which then must be handled as TRU or LLW and disposed of in special landfills or other repositories.LANL's Plutonium Facility currently generates approximately 2500 kg of combustible-type material per year, and a portion of that is cellulose-based material Currently TRU waste products are slated for disposal at the WIPP (Waste Isolation Pilot Plant) facility located in New Mexico, but opposition from various groups has prevented the use of this completed facility, so the materials remain on the governmental defense reservations.Even when (if) this or a similar facility can begin accepting TRU waste, it will be expensive to store such items in perpetuity.The same argument can be made for the disposal of LLW in appropriate facilities.Therefore, any technology that can reduce the volume of such wastes would greatly reduce the cost of disposal and the associated risk of potential human and environmental contamination.
The use of industrial-strength enzyme solutions developed and marketed for a variety of other applications may be useful in the reduction in volume of radionuclide-contaminated wastes.Unlike many enzymes used in research projects, most industrial enzyme preparations are able to withstand (and function under) harsh conditions of use such as low or high pHs and in the presence of detergents or heavy metals.A technology based on the use of industrial cellulases developed to treat oil-saturated cotton sorbents in the presence of seawater also was found to be useful for degrading uranium-contaminated cotton fabrics, releasing the uranium into the enzyme solution (1,2).
The present project was intended to demonstrate the feasibility of this process for treatment of cellulose-containing materials in LANL's laboratory waste stream.Dr. Pat Unkefer and associates at LANL used a microbially-generated cellulase to initiate degradation of Putreated cotton prior to destroying the material by a sewage sludge consortium (3).They reported no significant effect of Pu on biodegradation.The current report describes the use of Rapidase TM , an industrially-available cellulase, to degrade lanthanide and Pu-contaminated cellulose substrates.To be determined was whether these heavy metals would inhibit the activity of the commercially-available enzyme.Also to be determined was whether the Pu remained with the digested cotton residue or if the radioactivity was released into the enzyme solution following enzyme treatment.

MATERIALS AND METHODS
The commercial enzyme used for this studies was Rapidase TM (Genencor, Rochester, NY).A five per cent solution of the commercial liquid was prepared in McIlvaine's citratephosphate buffer (4) to produce a final solution with a pH of 4.5.
Cotton substrates (0.5g/flask) consisted of raw cotton (not treated by scouring and bleaching) that had been cleaned of stem and leaf trash by treatment in a Shirley analyzer (British Cotton Industry Research Association, Patent No. 404888) and afterward referred to as Shirleyed cotton, or 0.5g of the other cotton substrates being tested (including cheesecloth and paper that had been powdered in a Wiley cutting mill to pass a 20-mesh screen).
In the typical laboratory-scale experiments, a total volume of 75ml liquid plus 0.5g cotton substrate were treated for 6 days at 45 0 C. The cellulose substrate was added to freshly prepared enzyme solution at the beginning of each experiment.Experimental and control (minus enzyme) flasks were incubated on a gyrotary shaker (New Brunswick Scientific) at 45 0 C; alternately, incubation was conducted on a stirring hotplate where the temperature was maintained at 45 0 C. Substrate digestion due to enzyme treatment was evaluated by determining the dry weight of each substrate prior to and following enzyme treatment.Residual substrate collected by filtration was dried to a constant weight at 100 0 C. Per cent residual substrate is reported as both the range of values from three replicates and the mean value, unless otherwise noted in the tables.
Residual glucose levels were determined by high pressure liquid chromatography (HPLC) using a Dionex DX 500 HPLC System with an AS 5300 autosampler.A gradient eluent of 500mM sodium acetate/200mM sodium hydroxide was used.Five mL of spent enzyme solution recovered from each flask following filtration to remove the residual cellulosic substrate was adjusted to pH 6-7 with 0.3g (Na 2 ) 3 (PO 4 ) 2 and heated for approximately 2hr in a 70C waterbath to coagulate the protein.A 1mL sample of the heated and cooled supernatant fluid was diluted volumetrically 1:100 with 18.2 megaohm water.A portion of this solution was filtered through a 0.2µM filter into an autosampler vial from which 25µL was drawn for HPLC analysis.Glucose concentrations were calculated from the results as mg/mL of the supernatant fluid recovered after heating the residual enzyme solution.
The effect of the following were evaluated to determine if they inhibited or slowed the digestion process: glucose, sucrose and nitrate concentrations (0.1 to 1.0M), and the presence of cerium oxide, cerium carbonate, praseodymium oxide or plutonium oxide.Liquid scintillation counting (Packard Instrument Company) or alpha spectroscopy by conventional methods were used to determine the radioactivity.These techniques both are based on the measurement of internal radioactivity due to the alpha particle emission from the plutonium-239 isotope.

RESULTS
Initial experiments were performed with various forms of cotton and cellulose-based manufactured items to define the conditions needed by the enzyme to degrade both the noncontaminated and the contaminated items.In previous studies testing the ability of cotton to be degraded by cellulases (1,2) a 5% solution of Rapidase TM in pH 4.5 McIlvaine's buffer was found to be an effective way to digest raw cotton contaminated with diesel fuel, crude oil or uranium.The optimum temperature was found to be 45 0 C, although degradation occurred between 25 and 60 0 C. Agitation resulted in faster degradation than static treatment, with 200 rpm on a gyrotary shaker being optimal.Degradation of roughly 50% of the substrate occurred in 6 days under these conditions which were used to set the baseline for our studies here.
The ability of the enzyme solution to degrade unused materials such as paper and cheesecloth (of the type that is used as sorbing wipes in the glove box line) that eventually contribute to the LANL waste stream was tested.Other experiments included determining the effect of various concentrations of sugars (glucose and sucrose) on the degradation process since the activity of one of the cellulase enzymes (cellobiase) is known to be inhibited by increasing concentrations of glucose.Further, the effect of added nitrate salts was tested since Pu is soluble in HNO 3 and nitrates can be found in Pu-containing waste streams and thus potentially present on wipes used to clean glove boxes.Cotton contaminated with lanthanides, then with plutonium was tested to determine if actinides inhibited the ability of the enzyme to degrade raw cotton.The distribution of the plutonium between the residual substrate and enzyme solution following degradation of the cotton was determined.
In the current study, the dry weight of residual substrate was used to determine the effectiveness of the enzyme treatment (per cent reduction in dry weight).Residual substrate was collected by filtration followed by drying to a constant weight at (100 0 C).Residual reducing glucose was determined by HPLC analyses.Residual PuO 2 was determined by liquid scintillation counting and alpha spectroscopy.The results from the six experiments are listed below.
In the first experiment, cellulose-based material was exposed to 5% enzyme solution for 6 days at 45 o C. The material consisted of Shirleyed cotton, cotton balls, paper strips, powdered paper, cheesecloth, and strips of cotton blend fabric.The loss in dry weight of the various cellulose-containing materials following enzymatic degradation is listed in Table I.The effect of added glucose and sucrose on the amount of cotton degradation was examined.This was studied due to the glucose inhibition of cellobiase activity.Shirleyed cotton was used as the cellulose material.The glucose and sucrose concentrations were varied from 0.1 to 1.0M in the enzyme solutions.The results are listed in Table II.Prior to experiments with plutonium, the effect of lanthanides on cheesecloth degradation was examined.Cerium and praseodymium were added at varying concentrations (0.05, 0.1, 0.5 and 1.0mM) to the enzyme solution and cheesecloth.The results are shown in Table V.The effect of plutonium oxide (PuO 2 ) on the enzymatic digestion cheesecloth was examined.Plutonium oxide powder is a likely contaminant due to the inherent airborne particulate contamination that exists inside the glove box line.Three controls were included.One control had 0.5 g of cheesecloth with enzyme and no added plutonium.The second control contained only cheesecloth.The third control contained plutonium with no added enzyme.One experiment in triplicate was conducted.Following the experiment, the solutions were filtered and the plutonium activity was measured in both the enzyme solution (filtrate) and in the cheesecloth residue.The results are listed in Table VI.

DISCUSSION
Industrial enzyme preparations are able to dissolve natural organic polymers such as proteins (proteases), lipids (lipases) and cellulose (cellulases).Some uses for proteases include leather tanning, the production of soy sauce and use in contact lens cleaning solutions (5).Lipases are used in drain cleaners to dissolve the grease which entraps materials to form clogs. Cellulases are added to detergent formulations to digest grass and other stains, they are used in the finishing of denim (to provide and improved "hand" or soft feeling to the material), and for the ex situ disposal of sorbents used to clean up oil spills (2).
In the previous study which tested the ability of biodegradable oil sorbents to be degraded by cellulases, a 5% solution of Rapidase TM in pH 4.5 McIlvaine's buffer was found to be an effective way to digest raw cotton contaminated with either diesel fuel or crude oil.The optimum temperature for digesting 0.5g cotton in 100mL solution was 45 0 C, although degradation occurred between 25 and 60 0 C. Agitation resulted in faster degradation than static treatment.The current study is an extension of this work in which we tested the ability of this combination of enzyme and conditions to degrade cellulose-based material contaminated with radioactive actinides.
Cellulases are composed of a series of enzymes with activity against different portion of the cellulose molecule.The kinetics of cellulose degradation and the types of cellulase enzymes are reviewed by Haigler and Weimer (6).Endoglucanases break internal glucose-glucose bonds within the cellulose polymer.Exoglucanases remove individual glucose molecules from the ends of the cellulose chain and cellobiases cleave di-glucose units (cellobiase) into individual glucose molecules.With the exception of the pectins and waxes on raw cotton, the entire cellulose molecule theoretically can be degraded into individual glucose units.In practice, as the glucose concentration increases, the cellobiase enzyme activity is reduced.However, the structure of the cellulose fiber can be entirely destroyed by enzyme treatment, if carried out for a sufficient period of time.In our previous studies, treatment for 48 hr destroyed the structure of the mat of fibers which had been reduced to tiny individual filaments unable to hold crude oil or diesel fuel any longer.For these oils, the major means whereby cotton acts as a sorbent is by entrainment rather than adsorption onto the surface of the fiber or absorption into its lumen.For actinides, the mechanism of sorption is not known, but we have shown in this study, and in a previous one (1), that radioactive particles are no longer associated totally with the fiber residue as some of the uranium and plutonium were detected in the enzyme solution.In the present study however, most of the activity was found on the residue indicating an association with the fibers.
Since enzymes are protein catalysts and subject to inhibition by heavy metals, salts and other agents, the effect nitrate salts, nitric acid, lanthanides and actinides (as PuO 2 ) on the process was of primary interest because these materials are in the LLW and TRU waste streams, and may affect the potential for using cellulase to reduce the volume of wastes.In addition, since one of the enzyme components of cellulase (cellobiase) is inhibited by increasing concentrations of glucose, the effect of adding glucose and sucrose also was tested on the degradation process.
The intent of this study was to determine the feasibility of using cellulases to reduce the volume of cellulose-containing, actinide-contaminated, materials such as those that might be encountered at a facility with a military or nuclear energy mission.Testing of treatment parameters at a laboratory scale was in preparation for scale-up experiments.The following items previously were found to be significant to the digestion process, and were evaluated or used in this study: physical state of the substrate (the greater the surface area, the more rapid and complete the digestion; the more non-cellulosic components, the greater the residue following enzyme treatment), a temperature of 45 0 C, pH 4.5 using a citrate-phosphate buffer, agitation and a standard treatment time of 6 days.
The first set of experiments compared the ability of the enzyme solution to degrade handginned raw cotton and raw cotton that had been mechanically cleaned of stem and leaf trash (Shirleyed cotton) with scoured and bleached cotton balls (100% cotton), 100% cotton fabric, 50/50 cotton/polyester blend fabric and typical cellulose-containing items that wind up as Pucontaminated wastes such as cheesecloth wipes and paper.The latter items ordinarily would be slated for disposal in the WIPP site.In these experiments (Table I), it was found that, based on residual dry weights of the substrates, Shirleyed raw cotton and the raw cotton standard were approximately equal with regard to the amount degraded by the enzyme treatment after 6 days of treatment (~ 45 per cent degradation).Scoured and bleached cotton balls were degraded more completely than the raw cottons.Since residual waxes or other non-cellulose materials found on raw cottons are not present on scoured and bleached cotton, the additional residual weight in the raw cottons samples likely is due to the materials removed by scouring and bleaching.Fabric prepared from 100% cotton was degraded more completely than a 50/50 blend of cotton/polyester, as would be anticipated since cellulases do not degrade polyesters.Cheesecloth and 100% cotton fabric degradation was nearly equivalent to that of scoured and bleached cotton balls (100% cotton), as would be anticipated from the composition of these materials.
Regarding LANL wastes, the amount of cheesecloth degraded was approximately the same as that of raw cotton and 100% cotton fabric; shredded and milled paper were approximately equivalent in digestibility; therefore extensive pre-treatment of paper waste to increase its surface area should be unnecessary prior to enzyme treatment, Other LANL combustible waste items such as HEPA filters, Tyvek sleeves and Tyvek labcoats were not degraded (results not reported).Since these items are made from plastics, it was not expected that they would be degraded by treatment with cellulase.
Residual glucose, as determined by HPLC analyses for each of these substrates was approximately 10-fold less than the amount known to cause inhibition of digestion, as determined by adding varying concentrations of glucose and sucrose to the enzyme solution at the beginning of the experiment.Inhibition of the degradation process increased as the concentrations of both glucose and sucrose in the solutions increased (Table II).Added glucose or sucrose at concentrations of 0.1M resulted in a slight increase in the amount of residual substrate as compared to the control.Inhibition of enzyme activity was greater at 0.5M than 0.1M glucose and sucrose, whereas 1.0M concentrations of both glucose and sucrose nearly completely inhibited enzymatic digestion of the cotton substrate.It is likely that inhibition of digestion in the presence of sucrose is due to factors other than glucose accumulation in the enzyme solution since all sucrose solutions had residual glucose levels less than those of controls.
Since plutonium salts are soluble in nitric acid, it is possible that nitrates will be present in low concentrations in LLW and TRU waste streams.When the effects of added nitrate salts at concentrations from 0.1 to1.0M and nitric acid at 0.02M on substrate digestion were evaluated, it was found that increasing nitrate salt concentrations also inhibited enzyme activity and substrate degradation (Table III).Roughly three-fold decreases in degradation were observed at a concentration of 0.1M nitrate salt, regardless of whether KNO 3 or NaNO 3 was used.At 1.0M concentrations of both salts, inhibition was nearly complete.These results indicate nitrates in high concentrations likely will be a problem in degradation, but it is not known whether this is due to salt effects or specifically to the nitrate ion.Dilute HNO 3 had approximately the same effect as the nitrate salts at 0.1M, i.e., there was a slight decrease in the amount of cotton degraded, as compared to the control values.Higher concentrations of nitric acid were not tested to avoid any possibility of forming cellulose nitrate in the test system In anticipation of scale-up experiments, a comparison was made between the type of agitation that was used in these laboratory-scale experiments (gyrotory shaker) and stirring which is a more likely type of agitation that will be used in the testing of larger volumes.There was no substantial difference between the amount of residual substrate degraded between raw cotton and LANL cheesecloth when agitation was on the shaker, but more cheesecloth was degraded under the same conditions if the mixture was stirred and not shaken (Table IV).
Experiments with lanthanide surrogates of plutonium salts were performed prior to conducting the experiments with plutonium to test the effect of these heavy metals on the digestion of cellulose.When cereum carbonate Ce(CO 2 ) 3 , cerium oxide CeO 2, and praseodymium oxide PrO 3 at various concentrations (0.05, 0.1, 0.5 and 1.0mM), were added to the enzyme solution, enzymatic digestion of the cheesecloth was not inhibited by concentrations of up to 1.0mM (Table V).The speciation or location of these metals following the enzymatic treatment was not determined.
In the experiments that were conducted inside the Pu glove boxes (Table VI), it was found that enzymatic digestion of cheesecloth occurred in the presence of 0.0025% PuO 2 at 45 0 C.There was little effect on degradation with this level of Pu compared to experimental flasks with no added Pu, showing that the presence of plutonium at this level did not adversely affect the digestion process using this commercial source of cellulase.The solid residue (residual cellulose and filter) and the spent enzyme solution after removal of the residual substrate were tested for the presence of radioactivity to determine the fate of the plutonium following degradation.It was found that the majority of the plutonium was isolated with the residue on the filter, and only a minor component was located with the spent enzyme solution.This indicates that, should cellulase digestion be used to treat LLW and TRU cellulosic wastes, because the radioactivity remained with the solid residue, extensive treatment of the enzyme solution for the removal of residual radionuclides is likely to be unnecessary, and that enzymatic degradation of such wastes is a feasible option for reducing the volume of such materials.This study has therefore demonstrated the feasibility of using a commercially-available cellulase solution to reduce the volume of plutonium-contaminated LLW and TRU waste.possible losses of enzyme activity over time, appropriate configurations of the reaction vessels and issues regarding the disposal of spent enzyme solutions and residual substrate.
Whether other types of enzymes can contribute to reducing the disposal volume of contaminated items not normally of biological origin, such as plastics, rubber and other common organic components of LLW and TRU waste also is of interest.Although current industrial enzyme preparations which degrade proteins, carbohydrates and lipids probably would not be effective, it is possible that enzymes with different specificities, such as polyester depolymerases, would be an effective way to degrade radioactive wastes of organic, but nonbiological, origins.

SUMMARY AND CONCLUSIONS
A commercially available cellulase was able to reduce the volume of cellulose substrates contaminated with lanthanide surrogates and PuO 2 .Residual radioactivity remained primarily with the solid residue following digestion (undegraded cellulose plus filter).This indicates enzyme digestion of LLW and TRU wastes to reduce their volume is a promising technology.

ACKNOWLEDGMENTS
This project entitled Enzymatic Degradation of Plutonium-Contaminated Cellulose Products was prepared with the support of Subrecipient Agreement No. UTA95-0176 from The University of Texas at Austin, using funds provided by the U.S. Department of Energy and passed through the State of Texas -Office of the Governor.However, any opinions, findings, conclusions, or recommendations expressed herein are those of the authors and do not necessarily reflect the views of The University of Texas at Austin, the State of Texas -Office of the Governor or the U.S. Department of Energy.
We would like to acknowledge LANL for the use of their facilities and Dennis Padilla for the experimental work with plutonium.Thanks also are extended to Bob Wyatt of the International Textile Center at Texas Tech University for designing and performing the HPLC analyses.Special thanks go to Dr. Larry Avens of LANL for his advice and support for the duration of this project.

Table I .
Enzymatic degradation of cellulose-based material by cellulase

Table II .
The effect of added sugars on enzymatic degradation of Shirleyed cotton.Triplicates were conducted on all experiments.¨Athirdexperiment evaluated the effect of nitrates on the degradation of raw cotton.Sodium nitrate, potassium nitrate or nitric acid was added to the enzyme solution and the loss of cotton weight and residual glucose was monitored.The results are listed in TableIII.

Table III .
The effect of added nitrate on enzymatic degradation of cotton.Triplicates were conducted unless noted.2) not determined To examine the varied stirring conditions used in this study, the effect of various types of agitation on the degradation of cheesecloth or cotton was examined.Mixing / agitation was accomplished by a shaker table or magnetic stirring on a hotplate.The results are listed in TableIV.

Table IV .
The effect of agitation on enzymatic degradation of cotton.

Table V .
The effect of lanthanides on enzymatic degradation of cheesecloth.Triplicates were conducted on all experiments.2) Data point not comparable due to incorrect incubation temperature.

Table VI .
The effect of plutonium on enzymatic degradation of cheesecloth.The three reaction mixtures contained enzyme solution, cheesecloth and added PuO 2 .Plutonium activity was measured by both liquid scintillation counting and alpha spectroscopy.The average of the results are listed.2) Plutonium activity was measured by alpha spectroscopy.3) Results indicate that no weight loss was observed in the cheesecloth