CA2001538A1 - Application of glucanase to control industrial slime - Google Patents

Abstract

Case 4339 APPLICATION OF GLUCANASE TO CONTROL INDUSTRIAL SLIME

Abstract of the Disclosure A method of attacking and removing microbial slime in slime covered surfaces and maintaining a slime-free surface as in exposed cooling tower surfaces and in waste water treatment and paper making. This method comprises utilizing an enzyme blend in 2 to 100 parts per million (ppm) of beta-glucanase, alpha-amylase and protease. Glucanase has been found specifically to digest microbial slime and reduce microbial attachment and biofilm. A specific combination of polysaccharide degrading enzymes is a ratio of 2 parts beta-glucanase to 1 alpha-amylase to 1 protease utilized in 2-100 parts per million. Broadly, the alpha-amylase must be at least 1 and the protease may vary from .5 to 1 part.

Description

Case 4339 PPLIC~TION OF GLUC~N~SE TO CONTROL INDUSTRI~L SLIME
Backqround of the Invention The present invention relates to glucanase enzyme systems for treating microbially produced extracelLular polymers, present or which build up on surfaces of cooling water towers and in paper making broke water. Such extracellular polymers plus microbial cells are also known as biofilm or microbial slime.
~ icrobially produced extracellular polymers can build up, retard heat transfer and restrict water flow through cooling water systems. Controlling slime-forming bacteria by applying toxic chemicals is becoming increasingly unacceptable due to environmental problems. In addition, the efficacy of the toxicants is ~inimized by the slime itself, since the extracellular polysaccharide enveloping microorganisms are largely impenetrable.
Toxicants cannot adequately control large populations of attached bacteria and they are effective mainly against floating microorganisms. Qlthough surfactants and dispersants which penetrate and help loosen slime can enhance the activity of toxicants~ they are nonspecific and may have deleterious effects on the industrial process.
This invention describes the use of glucanase which has the advantage of being both specific and non-toxic. The approach is designed to ~a) enchance the removal of slime where it has formed, ~b) prevent the build-up of slime, and ~c) improve the efficacy of biocides against sessile bacteria. The gluconase specifically attacks the slime layer surrounding the bacteria.

Consequently, the microorganisms become planktonic--harmless in terms of biofilm production--and are rendered susceptible to biocides. The enzymes also act to maintain a clean surface (see Figure h and remarks).

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Examples of prior art single enzyme formulations are: those found in 3,773,623, Hatcher, Economics Laboratories~ Inc., ~here the slime formulation in industrial water such as white water from pulp and paper mills is retarded by controlling amounts of enzyme levan hydrolase.
Qlso, 4,055,~67, Christensen ~Nalco) describes a slime and an industrial process whereby slime can be dispersed and prevented by treating said slime with a few ppm of the en~yme, Rhozyme HP-150, a pentosanase-hexosanase and 3,824,184, Hatcher (Economics Laboratories, Inc.) describes a slime formation contro11ed by intentionally adding to industrial water the controlled amounts of enz~me levan hydrolase.
~ dditionally, 4,684,469, Pedersen et al. (Qccolab, Inc.) discloses a method of a two-component biocidal composition suitable for controlling slime. The preparation consists of a biocide and a polysaccharide deqrading enzYme.
~ s to the biocides, generally methylene-bis-thiocyanate has been preferred. Other operable biocides include chlorophenate compounds, such as pentachlorophenates and trichlorophenates;
organomercurial compounds, such as phenylmercuric acid; carbamate compounds, such as methyldithiocarbamates, ethylenebisdithiocarbamates, and dimethyldithiocarbamates;
carbonate compounds such as cyanodithioimidocarbonates;
thiocyanates such as chloroethylene thiocyanate compounds; and other biocides such as bromo-hydroxyacetophenone compounds, benzothiazole compounds, ethylene diamine compounds, nitrilopropionamides, bromopropionamides, bromo-acetoxybutenes.
bromopropanolaldehyde compounds, bis-trichloromethyl sulfones bimethyl hydantoin compounds, and the like mixtures of biocides can also be used.
The biocide methylene-bis-thiocyanate has proven to be particularly effective in the context of this invention, as has combination of dimethyldithiocarbamate and disodium ethylenebisdithiocarbamate.
The advantages of the glucanase composition over the use Ot biocides to control bacteria are that the biocides constitute toxicants in the system and pollution problems are ever present.
The advantage of the present formulatirn over the formulation of a single enzyme plus biocide i5 that the single enzyme attacks only one narrow band of carbohydrate polymers whereas the present invention improves the range of attack by combining activities of a beta-glucanase and an alpha-amylase along with the basic protease, broadly attacking the carbohydrate polymer and protein surrounding the bacteria. ~ specific formulation embodying ratios, far the present use of multiple enzyme preparations, is 2 parts beta-glucanase, I part alpha-amylase, and 1 part protease.
In this formulation, the alpha-amylase is at least 1 and can be slightly over 1 part. The orotease which is set at 1 may actually be .S to I part, and the beta-glucanase is set at Z
parts ~ preferred composition is 2 parts beta-glucanase, I part alpha-amylase and 1 part protease. In the composition cerulase may be substituted for beta-glucanase.
In general, glucanase is used in a dosage of 2 to 100 ppm and may be from 2 to lO~parts per million. The glucanase can be obtained from many chemical suppliers such as ~merican Cyanamid, Betz, Beckman, Dearborn Chemical, Economics Laboratory, Inc.
Merck, Nalco, Vineland Chemical! and the like.
The concentration of glucanase required for effectiveness in this invention varies greatly and can depend upon the conditions such as temperature and pH of the water, the microbial count and the type of industrial water being treated. The lower and upper limits of the required concentrations will substantially depend upon the specific enzyme or combination of enzymes used. For example, a highly effective glucanase can require a concentration of mainly about 1 or 2 parts glucanase to one million parts industrial water in the context of this invention, or may require a minimum concentration of 80 or 100 ppm.
In contrast to the prior art, this formulation is both more specific and non-toxic. In view of this invention and in comparison with the prior art, it can be said that the present composition has the same over target polymers but digests them more efficiently because of the enzyme activities of glucanase in the mixture of [with?~ alpha-amylase, and the protease.
~oreover, the beta-glucanase is a unique enzyme component which allows this efficiency to take place. The alpha-amylase and the 20~1~3~3 protease nick the microbial slime and allow the beta-glucanase access to digest the slime exopolymer more effectively.
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It is noted as a matter of general mechanisms, that the alpha-amylase alone does not give slime protection or remove slime. It attacks the alpha-linkage between glucose molecules.
It nicks the outside of the slime molecule, so that the beta-glucanase can enter and attack said carbohydrate molecule. The protease attacks extracellular protein molecules.
~ ~j Up to this time, enzvme treatment of industrial slime or slime polvmer made by bacteria consisted of a single enzyme, for :~
`. e~ample levanase. Levanase would break down a polymer of levan into its subunits ~fructose). However, after the levanase would be used on the slime levan. resistant bacteria would still remain to proliferate. Further applications of levanase were .
~i ineffective because the polymer it attacks was no longer present.
~` The levan polymer would be gone~ but other 51 ime polymers would still be there and the bacteria would flourish. Although other enzyme preparations have been used in the marketplace, for '~''.'~ ' i: example ED~, a levan hvdrolyzer ~Sunoco), there has been no , .:
-~ combination of enzymes that would actually attack polymer made b~

Pseudomonas bacteria and other bacteria ln the field, such as ~, Klebsiellal ~cinetobacter. Flavobacterium, Enterobacter, and Aerobacter, which were rich in glucose, mannose and gulose sugars :.:, arranged in polymers.

Now, in a generalized process and in response to the prior ., '~ art above, the present invention has taken a clear culture of : ~
Pseudomonas bacteria and made them produce a slime polymer in a ~` low substrate environment. Second, the invention has taken a composite of microorganisms from the field blended with and grown together both at the laboratory and under field conditions.
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simulated cooling tower water and utility water.
The results indicate that the maximum removal of carbohydrate layers from pending bacteria has occurred. Thus utili2ing glucanase has a superior result, especially if the enzyme utilization was found to be useful in the very prevalent Pseudomonas bacteria.
variety of enzymes were utilized in testing against Pseudomonas bacteria. From 42 preparations of enzymes, three ~&~313 types of enzymes were found to be effective on slime produced by Pseudomonas bacteria. First, alpha-amylase was found to attack bacterial slime. Second, protease has been found also to have an effect on bacterial slime. Then it was found that a combination enzyme treatment with amylase. glucanase, and protease was effective in removinq the biofilm.

Brief Descril~ion of the Drawi~s Figure ~ is a plan view of the biofilm reactor sYstem.
Figure 2 is a graph indicating biofilm removal due to biodispersant, cellulase. and a mixture of alpha-amylase, beta-glucanase, protease in a 1:2:1 ratio.
Figure 3 is a plan view of the microbial fouling reactor system.
Figure 4 i5 a graph showing biofilm mass versus time for glucanase treatment and biodispersant.
Figure S is a graph showing pressure cdrop versus time for glucanase treatment and biodispersant.
Figure ~ is a graph showing the results of a treatment of enzyme versus biocide in a Microbial Fouling Reactor experiment.

EX~MPLES
Example I
Preliminary activity screening of about forty enzyme candidates was carried out using slimed microscope slides which were treated with the enzyme candidates in small, stirred, sterile beakers. The test slides were prepared in a slime generation box using a colony isolate of Pseudomonas or a composite of field microorganisms known to produce extracellula(-polymers in industr-ial waters. 8acteria were propagated in tryptic soy broth (TS8) and were enumerated on tryptone glucose extract ~TGE). ~nhydrous dextrose ~D-glucose) was used to supplement the TS8 nutrient.

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Enzyme digestion rates were determined at 1, 2 and 4-hour intervals by assessing biofilm removal from the slides visually.
Enzyme candidates showing promising activity in this screening test were explored more fully as below.

E _ ple II
The nine most promising carbohydratases and proteases from the screening test were subjected to further examination using a ~iofilm Removal Reactor (~RR) which simulates water-tube fouling in field applications. The reactor is shown schematically in Figure 1. The reactor tubes were first slimed by exposure to slime-forming bacteria in circulated minimal sùbstrate for a 72-hour period.
Each of the candidate enzymes was tested in the reactor at a level of 100 ppm for a 24-hour- period under the conditions shown in Table I. The removal of biofilm in the 9RR was measured in terms of the percent decrease in biomass resulting from enzYme treatment of the fouled system. The results for the mixed protease-carbohydratase are shown in Figure 2 and Table II. For these tests, the reactor tubes were dried overnight at bO degrees C and weighed; then cleaned, dried and reweighed to obtain the recorded gravimetric data.
Further tests of these enzymes were conducted in a ~icrobial Fouling Rea~tor (~FR)~ a similar apparatus which also provides for a measure of pressure drop across the slimed reactor tubes as a criterion of fouling. The apparatus is shown in Figure 3 and the experimental conditions are listed in Table I. The experimental procedure for the biomass measurements was generall~
similar to that used in ~RR, above. except that the biomass is measured several times during the course of the experiment. In addition, the effectiveness of the enzyme treatment is measured by the decrease in pressure drop across the slimed tubes of the reactor as well as by visual observation in the sight glass section. Figures 4 and 5 show the results of tests of the mixed enzyme compared to a polyol biodispersant.

Five of the enzyme preparations tested in the 8iofilm Removal Reactor were effective in controlling slime. These are tabulated in Table II with their relative effectiveness. Of these, the qlucanase was clearly the best performer. This enzyme composite is a combination of one protease and two carbohydratases, namely alPha-amylase and beta-g1ucanase. It was found to be effective In digesting slime la~ers produced by cultures of pure and ml ecl strains of bacteria. ane commerciall available mixed en~,me composition is shown in the table to give 37'~, biomass removal In ~he time period of the test.
The ~iofilm Rer~o al Reactor ~Figure 1) results are also depicted in Figure 2. In the oiofilm remo~al experiments. the enzyme cellulase remo~,ed r'3''. of the biomass ~b2 mn,XcmZ after treatment as opposerl to 80 mg~cm2 before treatment) in 24 hour-,.
The l:2:1 combinatlon of alpha-amylase, beta-glucanase. and protease enzymes remo,ed 3,'~/. of the biofilm in the same time frame. The contl-ol (b!ank), which was untreated, continued to increase in biomass ~5%. For comparison. a non-enzymatic chemical biodispersant essentially checked overall develoDment of biofilm but did not remove any biomass. Therefore, the multiple enzyme approach was the best ~37'~.). -The biomass removal results in the ~FR experiment agreed essentially with 37% removal between 72.5 and 96.5 hr ~Figure 4).
The pressure drop data ~Figure 5) in the same ~MFR) experiment support this finding.

EX~MPLE III
Focusing on the ml ed enzyme, further l~lrP .tudies were conducted to deter~lne the effect of pH or 1--; effecti~eness ir biofilm removal. r-- ~luc3nase was teste~1 i.lnq a pol~ol biodispersant as 3 c~ ol ln single-c~cle ~,n~etic tap water with DH maintainecl 3t '.-,, 8.5 or 9Ø rhe results are summarized in Table III. The glucanase was effective up to pH ~.
The efficacy of the glucanase is also compared to that of the dispersant at neutral and alkaline pH's in Table III.

Fxample IV
~ n experiment was run on the Microbial Fouling Reactor and the results are shown in Figure 6. The experiment was designed to test whether the enzyme product of this invention would keep a surface clean. The conditions for the experiment differed in substrate concentration and treatment dosage (Table IV). The substrate concentration was low similar to substrate level in cooling water. The dose was eit~,er 51ug of biocide or enzyme product.
In Figure 6. the control or no treatment (-~-) curve indicates what biofilm growth is possible in low substrate conditions. The biocide curve indicates 100 ppm nonoxidizing biocide slugged in the reactor at days 6, 9, 13, 16, 20, 23, 28 and 31 caused losses in biofilm, as measured by decreases in pressure drop. The curve representing performance of the en7vme combination also indicates biofilm loss after each treatment.
Qfter 31 days the difference between the biocide-treated line and the untreated control was 2.4 inches (~ p = 2.4 in.); 2.7 inches using the enzyme blend. Fiqure 6 indicates that after treatments were stopped~ the biofilm in both lines grew.
The results were good. The experiment demonstrated that the enzymes controlled the biofilm growth very well over one month.
The enzyme blend, which is nontoxic, performed at least as well as the toxicant (nonoxidizing) biocides.

In the specification and claims glucanase is equivalent and equal to beta-glucanase.

2(3~3~3 TA~L~ I
Biofilm Removal Test Conditions Conditions Per APparatus Parameter BRR MFR
pH 8.5 7.5, 8.5 or 9.0a Temperature (C) 36+1b 33.0+1C

Make-up Water Synthetic Synthetic Chicago Tapd Chicago Tapd Substrate Concentration TSB 50 ppm 50 ppm D-glucose 50 ppm SO ppm Inoculum Field Field Composite Composite Growth Period 72 Hr p < 10 ine Treatment Enzyme Concentration 100 ppm 100 ppm Duration 24 Hr 24 Hr a pH setting depended on experiment.

b BRR temperature is consisten~ly 36+1C resulting from operation of recirculating pump.
c ~IFR tempera~ure is thermostatically controlled.
d Single cycle synthetic Chicago tap.
e MFR p of 10 inches occu~red at approximately 72 Hr.

~:0~38 TABLE II
Summary of BRR Studies at pH 8.5 Type of Enzyme ~ Remova Neutral Protease -lO.0 Alkaline Protease~/~ -50.0 \ Alkaline Protease~Z? 18.0 Debranching Enzyme -l9.0 Alkaline alpha-amylase21.5 Beta-glucanase ~l~ 0 Beta-glucanase ~2) 14.0 Cellulasea 23.0 Alpha-amylase, Beta-b 37.0 glucanase + Protease a Cellula~e attacks the beta-linkage between sugar molecules.
b Alpha-amylas~, beta-glucanase and neutral protease activities.

-- ~ 2~ LS~8 TABLE III
Effect of pH on Enzyme Treatment Performancea % Removal of Biofilmb pH Enzyme Biodispersant 7.5 48 3 8.5 35b lb 9.0 44b l1b a Performance is evaluated at 100 ppm enzyme, 20 ppm biodispersant concentration levels.
b Removal is an average of two experime~ts at 33 + 1C.

2~3L538 TABLE IV
Microbial Fouling Reactor Test Conditions for Biofilm Control Experiment Parameter Conditions pH 8.5 Temperature 33.0+1.0C
Make-up Water Synthetic Tap Water Substrate Concentration TSB 10 ppm D glucose 10 ppm Inoculum Field Composite Treatments Enzyme Concentration 150 ppm Duration Slug dose Frequency Twice per week Biocide Concentration 100 ppm Duration Slug dose Frequency . Twice per week

Claims (6)

1. A method of removing slime from slime-covered surfaces of cooling towers which comprises contacting said surfaces with an effective enzymatic amount of beta-glucanase.

2. A method of removing slime from slime covered surfaces which comprises contacting said slime surfaces with an effective amount of a beta-glucanase preparation consisting of a ratio of 2 parts beta-glucanase, 1 part alpha-amylase and 1 part protease and maintaining a slime-free surface.

3. A method of using a composite enzyme system consisting of beta-glucanase, alpha-amylase and protease to digest microbial slime and reduce microbial attachment and biofilm to maintain a slime-free surface.

4. A method of digesting slime from slime covered surfaces and wastewater treatment in systems which comprises treating said water with at least 80 ppm of beta-glucanase where the active ingredient to degrade the slime produced is an effective amount of beta-glucanase.

5. An enzyme blend effective in removal of microbial biofilm from slime covered surfaces and maintaining a slime-free surface in wastewater treatment which consists of 2 parts beta-glucanase, 1 part alpha-amylase and 1 part protease.

6. An enzyme blend effective in removal of microbial biofilm from slime covered surfaces and maintaining a slime-free surface in wastewater treatment which consists of 2 parts beta-glucanase, at least 1 and less than 2 parts alpha-amylase and .5 to 1 part protease.