Enzymatic catalyzed biocide system
BACKGROUND OF INVENTION 
     The present invention relates generally to a biocidal composition for use in industrial process streams and more particularly to an enzymatic catalyzed biocide system for pulp and paper mill process streams. 
     Microorganisms, particularly bacteria, are known to contaminate industrial process waters and may create slime deposits of biological origin. This is particularly true in the case of pulp and paper mill process streams where slime deposits formed by microorganisms are a common occurrence. Such deposits give rise to many problems including fouling or plugging of pipes and filters, and of greater significance, breakouts of spots and the like in the paper produced with consequent loss of production and work stoppages for clean up. These and other problems produced by slime deposits have resulted in the extensive utilization of biocides to prevent the growth of microorganisms in pulp and paper process waters. 
     Materials which have heretofore enjoyed widespread use in such applications include chlorine and a wide variety of proprietary chemical biocides. Other physical and chemical methods for slime control including gamma radiation, filtration and ozonation have been successfully used, but only in small scale applications, because such methods are costly and difficult to scale up to commercial size industrial processes. The use of chlorination to prevent the growth of microorganisms is also limited both by its cost effectiveness and the fact that chlorine is an active chemical that may react in solution before its full biocidal function is achieved. Meanwhile, other biocides suffer from odor problems and various hazards with respect to storage, use and handling, and the fact that most biocides are selective in their activity. As a consequence, the use of known biocidal systems involves careful selection and deployment, and the continuous or frequent addition to a plurality of points in the process stream. 
     SUMMARY OF INVENTION 
     It is an object of the present invention to provide a chemical biocide system capable of killing a wide spectrum of microorganisms for disinfecting industrial process waters. 
     It is a further object to provide a chemical biocide system that is particularly useful for controlling the growth of slime deposits in pulp and paper mill process waters. 
     Unlike the known proprietary biocides available, the biocide system of the present invention is characterized as an enzymatically catalyzed process that may utilize one or more components of the process stream to be treated. While the mechanism of the invention is not completely understood, it is based on the observation that peroxidase enzymes in the presence of hydrogen peroxide will oxidize phenolic compounds to quinones or hydroquinones that are toxic to microorganisms. This reaction produces an antibacterial activity that is a function of the type of quinone produced and the degree of phenolic polymerization. Since pulp and paper mill process streams and other industrial process streams are known to include phenolic compounds such as black liquor and other lignin derived phenolics, the present invention is capable of using one or more of the components of the process stream to be treated. Where the process stream is deficient in oxidizable phenolic compounds, these may be added as needed. The present invention also contemplates the use of other microbial or plant dehydrogenase enzymes such as laccase, and other oxidizable substrates including halide salts, e.g., potassium iodide and sodium chloride, to generate the antibacterial activity. 
     The enzymatically catalyzed biocide system disclosed herein is not toxic to humans, it is water soluble, it contains no additives that may be deleterious to industrial process water, and has demonstrated a biocidal activity with respect to a wide variety of microorganisms. In addition, tests have shown that the enzymatic biocide system disclosed herein is economical, and comparable in efficiency to the known proprietary chemical biocides. 
     In one embodiment of the present invention horseradish peroxidase and hydrogen peroxide alone generated an antibacterial activity in selected paper mill process waters that was equivalent to that of traditional biocides. The addition of guaiacol or other phenolic substrates such as neutral black liquor (pH 7) or acid lignin to such waters generated a significantly higher antibacterial activity. Thus, black liquor and other lignin derived phenolics which are naturally occurring components of pulp and paper mill process streams, provide an inexpensive source for the mono and diphenolic compounds which are required to produce an optimum level of antibacterial activity according to the present invention. 
     The use of peroxidase enzymes with various monophenolic compounds to generate antibacterial activity is known with respect to the generation of host defense mechanisms in plants and animals. However, applicant is the first to suggest and apply the same enzymatic reaction to an industrial process water disinfection biocide. 
     DETAILED DESCRIPTION 
     The enzymatically catalyzed biocide system of the present invention is effective even at low concentrations for controlling the growth of a wide variety of microorganisms. Such microorganisms may comprise bacteria selected from the group consisting of aerobic bacteria, e.g., Pseudomonas, Azotobacter, Staphylococcus and Bacillus; facultative anaerobic bacteria, e.g., Escherichia, Flavobacterium, Chromobacterium, Nitrobacter, Hyphomicrobrium and Sphaerotilus; and anaerobic bacteria, e.g., Desulfovibrio, Clostridium and Veillonella. In addition, the biocide system is useful in controlling the growth of and killing actinomycetes, fungi and yeasts. Moreover, the antibacterial activity and cost of the enzymatic biocide disclosed herein has been found to be comparable to that of proprietary chemical biocides presently on the market. 
     The preferred dehydrogenase enzyme for practicing the present invention is from the peroxidase family and more specifically is a plant peroxidase, e.g., from horseradish, turnip, etc. However in the event that hydrogen peroxide is not the oxidant of choice, the enzyme laccase could be substituted for horseradish peroxidase. Where laccase is used, the preferred oxidant is oxygen. Meanwhile, since the biocide system disclosed herein requires the presence of an oxidizable substrate, several sources are contemplated. For instance, a survey of useful phenolic substrates includes catechol, guaiacol, acid lignin, black liquor or resorcinol. In addition, a halide salt such as potassium iodide or sodium chloride may be used as an oxidizable substrate. 
     Peroxidase enzymes are fairly ubiquitous, occurring in higher plants, yeasts, molds, bacteria and mammals. They catalyze the dehydrogenation of a large number of organic compounds such as phenols and aromatic amines, hydroquinones and hydroquinoid amines, especially benzidine derivatives. Meanwhile, horseradish peroxidase is a protein material of about 40,000 MW (molecular weight) and is derived from a renewable raw material, namely the horseradish plant. The horseradish peroxidase enzyme has a long shelf life and is thermotolerant, i.e., its catalytic activity remains high over a wide temperature and pH range on the order of from about 10-60 degrees C., and pH 4-11 respectively. 
     Horseradish peroxidase and other peroxidases have been used to generate antibacterial activity in plants and animals with peroxidase-catalyzed dehydrogenation products. In fact, most peroxidases seem to be capable of wide spectrum antimicrobial activity, including activity towards yeasts, fungi and viruses. However, the present invention represents the first attempt to utilize such catalyzed dehydrogenation products for the disinfection of industrial process waters either under static or dynamic conditions. 
     The invention herein will be better understood with reference to the following examples. However, it is to be understood that the examples are intended to be illustrative only and not in any way limiting of the invention as a whole. 
    
    
     In the following examples, samples of papermachine white water (a general term for all waters of a paper mill which have been separated from the stock or pulp suspension), were aseptically taken from several papermachines and stored at 4 degrees C. until used. Horseradish peroxidase having an activity of 250 purpurogallin units/mg was purchased commercially from Sigma Chemical Company, St. Louis, Mo. The horseradish peroxidase was prepared as 50 Units/ml stock solution and stored in the dark at 4 degrees C. until used. Activity of the diluted stock was measured daily during the tests. 
     EXAMPLE I 
     The enzymatically catalyzed biocide system consisted of 60 μl horseradish peroxidase (5 Units/ml) and 0.14 μl hydrogen peroxide and 1 mM guaiacol for enhancement where indicated. These reagents were added to a test tube, followed by the addition of 0.3 ml E. coli ATCC 25922 bacterial suspension and white water for a total volume of 3.0 ml. Horseradish peroxidase (HRP) enzyme activity was always greater than 95%. E. coli bacteria were added to increase the number of bacteria already present in the non-sterile white water to a measurable level in the assay. The assay reaction mixtures were incubated 30  minutes at 35 Degrees C. and the number of viable bacteria after incubation determined in triplicate by the spread plate technique on nutrient agar plates. 
     
                       TABLE I                                                     
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Antibacterial Activity of HRP                                             
White Water Samples    Treatment (% Kill)                                 
Papermachine (PM)                                                         
                HRP    HRP + 1 mM Guaiacol                                
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#5              94.6   94.5                                               
#8              92.9   79.4                                               
#1              24.1   26.3                                               
#2              0      50                                                 
#1              11.3   36.7                                               
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     The data in Table 1 shows excellent antibacterial activity of the enzymatic catalyzed biocide system in #5 and #8 papermachines (PM). These samples were taken from a papermachine used for manufacturing bleached board. However, the antibacterial activity of samples taken from PM&#39;s #1 and #2 making kraft paper were unacceptably low. The addition of 1 mM Guaiacol as an oxidizable substrate resulted in an increase in the antibacterial activity only in #1 and #2 PM&#39;s. The inhibition of bacterial activity in the latter papermachine systems was found to be due to the presence of material of particle size greater than 1.2 μm which inactivated the enzyme. The data also shows that significant differences in the antibacterial activity of the biocide system may occur from time to time due to the presence of enzyme poisons such as sulfide or other reducing agents which sequester the oxidation products of the enzyme reaction. In the examples where no guaiacol was added, the phenolic substrate relied on for antibacterial activity of the biocide system was the black liquor contamination, from the pulping process, normally found in papermachine process water. 
     EXAMPLE II 
     In another experiment using the test procedure described above for Example I, enhancement of the antibacterial activity of the HRP biocide system was measured using different quantities of guaiacol. The results are shown in Table II. 
     
                       TABLE II                                                    
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Antibacterial Activity of HRP + Guaiacol                                  
Treatment   White Water Samples                                           
Guaiacol    (PM) (% Kill)                                                 
(mM)        #5     #8          #1   #2                                    
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0           --     52.5        0    11.3                                  
0.0001      0      86.8        --   --                                    
0.01        0      91.0        57.2 39.8                                  
0.1         19.9   97.6        49.3 36.5                                  
1           80.7   100         2.8  4.3                                   
10          97.4   100         38.6 46.1                                  
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     As shown in Table II, the addition of guaiacol appears to enhance the antibacterial activity of the biocide system at least for the bleached board water samples. The presence of a sequestering agent in the raw water samples from PM&#39;s #1 and #2 limited the amount of antibacterial activity in these samples to no more than about 50% kill. In each of the samples, the black liquor concentration was assumed to be constant and as before the normal microflora of these samples were enriched with the addition of 0.3 ml E. coli ATCC 26922 bacteria suspension. 
     EXAMPLE III 
     Process water samples were taken at several sites from papermachines used to manufacture bleached board, fine papers and kraft paper. A total of nineteen samples were tested to determine the antibacterial activity of the HRP-hydrogen peroxide biocide system in process waters of widely varying composition. Approximately 50 ml of each process water sample was warmed to 37 degrees C. for 60 minutes prior to use. Each sample was added to two sterile flasks, one as a control and the other for treatment. All flasks received 0.1 ml of the E. coli ATCC 25922 suspension in order to obtain a viable assay. Control flasks contained only the sample and an E. coli inoculum. The HRP treated flasks received sample, 200 μl horseradish peroxidase (50 U/ml), 100 μl hydrogen peroxide and E. coli suspension. After the addition of all reagents and E. coli  suspension, the flasks were incubated for 30 minutes with shaking at 37 degrees C. The number of viable bacteria after 30 minutes incubation were determined by serial dilution of the samples onto nutrient agar spread plates. The % kill was calculated by comparing the number of surviving bacteria in the treated flasks with the bacteria in the control flasks. The results are shown in Tables III, IV and V. 
     
                       TABLE III                                                   
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Bleached Board Papermachine Process Waters                                
Sample           % Kill                                                   
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Shower Water     97.8                                                     
Hardwood Stock   75.3                                                     
Softwood Stock   44.0                                                     
Broke            34.2                                                     
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                       TABLE IV                                                    
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Fine Paper Papermachine Process Waters                                    
Sample           % Kill                                                   
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Raw Water        99.9                                                     
Broke Thickener  98.0                                                     
Tray Water       95.4                                                     
Raw Stock        91.4                                                     
Saveall Slurry   73.5                                                     
Shower Water     63.9                                                     
Broke Chest      8.2                                                      
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                       TABLE V                                                     
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Kraft Papermachine Process Waters                                         
Sample            % Kill                                                  
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Raw Water         9.3                                                     
Shower Water      70.4                                                    
Tray Water        66.1                                                    
Broke Thickener   60.7                                                    
Sized White Water 41.3                                                    
Clipping Hydropulper                                                      
                  39.2                                                    
201 Chest         35.8                                                    
203 Chest         16.5                                                    
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     As shown in Tables III, IV and V, antibacterial activities ranged from a low of about 8.2% to a high of about 99.9%, with the lowest activities generally associated with high consistency, poorly mixed pulp slurries. The high antibacterial activity in some of the samples, i.e., the fine paper papermachine process waters, is believed to be due in part to an oxidation reaction of the peroxidase enzyme with organo-halides or chloride salts generated by pulp bleaching or chlorination of the raw water. 
     EXAMPLE IV 
     In another test, the effect of several monophenolic compounds on the short term (30 minutes) killing of bacteria, and long term (3 hours) inhibition of bacterial growth by the HRP biocide system was determined. The while water samples used were taken from kraft papermachine process waters containing a mixed population of bacteria such as Pseudomonas, Bacillus, Desulfovibrio and Flavobacterium. In this assay, a 1.0 ml white water sample was added to 50 ml of nutrient broth and incubated overnight at 37 degrees C. with shaking. The resulting bacteria were harvested by centrifugation at 7000 rpm and resuspended in an equal volume of white water. These bacteria were then added to white water samples in order to fortify the total number of active indigenous bacteria. The assays were completed as set forth in Example III. The results are set forth in Table VI. 
     
                       TABLE VI                                                    
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Antibacterial Activity of HRP System                                      
With Several Phenolic Compounds                                           
Kraft Papermachine Process Water                                          
Phenolic      Phenolic     % Kill                                         
Compound      Concentration                                               
                           0.5 hour 3.0 hours                             
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Catechol      10.sup.-2 M  99.85    99.97                                 
              10.sup.-3 M  99.85    99.94                                 
Guaiacol      10.sup.-2 M  99.85    99.97                                 
              10.sup.-3 M  29.2     36.88                                 
Neutral Black Liquor                                                      
               5 μg/ml  38.36    40.43                                 
              10 μg/ml  43.85    41.61                                 
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     The data in Table VI suggest that the peroxidase system amended with various monophenolic compounds is effective against a mixed population of bacteria indigenous to a mill process water stream with no apparent regrowth of bacteria after extended periods of incubation. The practical implications of these findings are that the peroxidase-monophenolic system can be used for long term killing and inhibition of diverse population of bacteria in a paper mill process water stream. 
     EXAMPLE V 
     Another experiment was designed to test the antibacterial activity of the HRP biocide system during a typical papermaking process after treatment of the pulp with the enzymatic biocide. The Figure of drawing included herewith shows a schematic representation of a typical papermachine. The pulp tanks each contained about 6×10 6  bacteria/ml prior to the HRP treatment. Each tank containing 400 l of virgin pulp received 10 U/l of HRP, 2.04 mg/l Hydrogen peroxide and 3.1 mg/l guaiacol. After addition of the HRP reagents the pulp was mixed for 30 minutes and then used for papermaking. Antibacterial activity (% kill) was measured for supply tank and white water samples taken every 5 minutes during a 45 minute production run. The results are given in Table VII. 
     
                       TABLE VII                                                   
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Antibacterial Activity of HRP System                                      
             Supply Tank                                                  
                       White Water                                        
Time         % Kill    % Kill                                             
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5            94.2      98.3                                               
10           95.8      97.3                                               
15           99.5      99.6                                               
20           99.5      99.9                                               
25           98.8      98.9                                               
30           99.5      99.6                                               
35           97.6      98.9                                               
40           99.0      97.1                                               
45           97.9      98.7                                               
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     These results show that the antibacterial activity in both the supply tank and white water were consistently high during the entire production run. Thus, the antibacterial activity generated by the addition of HRP into the pulp tank appears to carry over significantly to the rest of the system. 
     EXAMPLE VI 
     A second papermachine trial was conducted during paper production to determine the antibacterial activity of the HRP biocide system under stressful conditions, i.e., with constant input of large numbers of bacteria and with minimal biocide/bacterium contact time. HRP, guaiacol and hydrogen peroxide were continuously added to the mix tank for a period of about 20 minutes (or about 2.5 white water recycles) followed by a 20 minute period of no addition. The addition rates were 5.0 ml/min HRP, 6.1 ml/min Hydrogen peroxide and 20 ml/min guaiacol.* Samples were taken every 5 minutes from the supply tank, mix tank and white water tank. The results of this experiment are tabulated in Table VIII. 
    
     
                       TABLE VIII                                                  
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Antibacterial Activity of HRP System                                      
       Supply Tank   Mix Tank  White Water                                
Time   % Kill        % Kill    % Kill                                     
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5      0             28.4      0                                          
10     11.4          43.9      0                                          
15     86.2          77.8      38.9                                       
20     81.4          73.6      66.1                                       
25     46.3          64.6      15.1                                       
30     19.9          41.9      35.8                                       
35     44.3          50.0      42.2                                       
40     60.2          43.9      43.9                                       
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 *Solution concentrations 20 U/ml; 0.72 mg/ml; 0.025 mM                   
 
    
     These results show that the % kill of bacteria in all three process waters increased after 5-10 minutes to a maximum of 65-86% kill between about 15 to 20 minutes time. Equilibrium of the HRP biocide system was apparently attained in the mix tank and supply tank before it was established in the white water as evidenced by the earlier antibacterial activity maxima and higher rates of kill. However, these experiments show that the antibacterial activity of the HRP reaction products is enhanced with extended contact time. Thus the biocide system disclosed herein will be most effective when applied to small working volumes having maximum residence time. 
     It will, therefore, be appreciated that the present invention provides an effective control for regulating the growth of a wide variety of bacteria, fungi and yeasts in process water systems. The invention utilizes an enzymatically catalyzed system which employs a microbial or plant dehydrogenase enzyme such as peroxidase or laccase. Thus, whereas only particular embodiments of the invention have been described herein for purposes of illustration, it will be evident to those skilled in the art that numerous variations in the details disclosed may be made without departing from the invention as defined in the appended claims.