CA1274442A - Slime control in industrial waters using enzymes - Google Patents
Slime control in industrial waters using enzymesInfo
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- CA1274442A CA1274442A CA000539655A CA539655A CA1274442A CA 1274442 A CA1274442 A CA 1274442A CA 000539655 A CA000539655 A CA 000539655A CA 539655 A CA539655 A CA 539655A CA 1274442 A CA1274442 A CA 1274442A
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- alginase
- slime
- enzyme
- activity
- waters
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Abstract
ABSTRACT
The invention describes a biocidal composition useful intreating industrial process waters to prevent and control the growth of surface slimes in waters which contain Pseudomonas aeruginosa species, which composition contains a biocidally active amount of an alginase enzyme in a suitable carrier.
The invention describes a biocidal composition useful intreating industrial process waters to prevent and control the growth of surface slimes in waters which contain Pseudomonas aeruginosa species, which composition contains a biocidally active amount of an alginase enzyme in a suitable carrier.
Description
` ~ 4'-~4~ 1 Field of the Inventio_ ¦
The invention is in the field of slime control.
SLIME
The ability of bacteria to produce copious amounts of extracellular polysaccharide has long been recognized as a major problem in industrial process waters. The function of this polysaccharide capsule has not been determined, but research indicates that the extracellular capsule or slime layer may provide protection, or enhance the transport of nutrients, or both. Several conditions common to industrial process waters encourage slime production. Many of the bacteria found in industrial waters are sessile; the nutrient gradient or turbulence or any number of other factors promotes their attachment to surfaces; the formation of the slime layer assures a firm attachment; low levels in nitrogen and high levels of carbon compounds generally encourage slime production.
The traditional approach to controlling slime-forming bacteria has been the application of toxic chemicals. Toxicants reduce the population of microorganisms and hence reduce the production of slime. Toxic chemicals almost always present an environmental hazard, and the effectiveness of the toxicants is reduced by the slime itself. The extracellular polysaccharide surrounding the attached bacteria is largely water insoluble and impenetrable. The findings that slime production by certain bacteria is related to their resistance to harmful substances supports this view that extracellular polysaccharide impedes the action of toxicants. The toxicant can not adequately control large populations of fixed bacteria, it is mostly effective 1;~74"4;~ 1 against floating bacteria. Surfactants and dispersants, which increase the water solubility and penetrability of slime, can enhance the activity of toxicants; they are, however, nonspecific and may have deleterious effects on the industrial process in question.
NUTRIENTS
The composition of the extracellular polysaccharide can vary with the species of bacteria; it is sometimes dependant on the types of nutrients available, again depending on the species.
~ acteria prefer certain substrates; they will seek out and preferentially absorb certain nutrients.
The normal paper mill contains many disaccharides and carboxylic acids. The typical kraft softwood pulp contains 75-80 percent cellulose, 5 percent lignin, 3-5 percent mannan, 5-8 percent xylan, and about 2 percent hexuronic anhydride. The pulping process releases some carboxylic forms of the mannan (polymannose to mannuronic acid) and xylan (polyxylose to guluronic acid). Starch (polyglucose), if greatly hydrolyzed, will release some disaccharides and carboxylic acids. Amino acids are not often available in paper mills, except perhaps when casein is used. As mentioned previously, these low levels of nitrogen compared to carbon encourage extracellular polysaccharide production.
On the whole, the Paper mill presents the bacteria predominantly with carbohydrates such as mannan, xylan, and ¦glycans. From mill to mill, the nutrients probably vary most in quantity rather than in type.
~ ~74~4~ 1 BACTERIA
In some bacteria, nutrients induce a certain slime; this is most often the case with bacteria that produce homopolysaccharides such as levans and dextrans. Even under the conditions described above, where the bacteria have available to them very similar nutrients, these bacteria may produce different slimes from mill to mill. For this reason, levan, for example, cannot be considered to be ubiquitous in process waters.
Other bacteria, in particular those which produce heteropolysaccharides, tend to produce the same extracellular polysaccharide under most conditions.
The bacteria which are the greatest producers of slime in the paper mill are the gram-negative rod-shaped aerobes of such genera as Pseudomonas, Aerobacter, Klebsiella and Flavobacterium. Of this group, Pseudomonas aeruginosa is one of the most common and difficult to control.
ALGINASE
The production of alginase can be induced in certain microorganisms using a commercial source of sodium alginate.
This alginase also was found to have activity on the extracellular polysaccharide produced by Pseudomonas aeruginosa, a slime-forming bacteria common in process waters. In an experiment simulating the process waters of a paper mill, the application of this enzyme reduced the numbers of attached bacteria by hydrolyzing the polysaccharide slime which allows them to attach. The application of this enzyme thus reduces the formation of slime deposits through two mechanisms, by preventing attachment of bacteria and by promoting the detachment of bacteria. The planktonic bacteria are thought to be more susceptible to the action of toxic chemicals as well.
~ ~.~74~
INVENTION
Generally therefore, the invention comprises a method of ¦preventing and reducing bacterial slimes which contain IPseudomonas aeruginosa which form on surfaces in contact with I
¦industrial process waters which comprises treating said waters with a slime inhibiting amount of alginase. This invention also ¦includes the alginase as used.
¦ A number of microorganisms are known to produce ¦alginase, for example: ~
A
¦ Aeromonas sp. aeneckea polagla ¦ Alginovibrio aQuatilis Clostridium alginolyticum ¦ Azotobacter vinelandii Cytophaga diffluens ¦ Bacillus circulans Pseudomonas sp.
¦ These organisms can be found growing on bacterial slimes in natural habitats.
For the purposes of this study, two organisms, Beneckea pelagia and Bacillus circulans, were considered most suited to the production of an unpurified enzyme to treat industrial process waters.
Dosages Recirculating water systems require at least 10 units of alginase activity (as tested using the thiobarbituric acid assay) per gallon of recirculating water and may require as much as lOû-250 units per gallon in systems containing copious slime;
generally lû-100 units per gallon would adequately treat the process waters.
` ~.~7~
Materials and Methods Bacteria Strains and Growth Media Strains of Beneckea pelagia (ATCC75916) were maintained on marine agar (Difco). Batch cultures were grown in 4% marine broth supplemented with 0.5% sodium alginate in order to induce the production of alginase. Strains of Bacillus circulans (NRS1349) were maintained on trypticase soy agar (BBL) or on nutrient agar (Difco) supplemented with alginate. Batch cultures were grown in 0.1% yeast extract (Difco), 0.1% alginate and 30 mM
magnesium chloride. Mucoid strains of Pseudomonas aeruginosa (ATCC15442) were maintained on trypticase soy agar or on tryptone glucose extract agar (Difco). Samples of extracellular polysaccharide were isolated from Pseudomonas aeruginosa grown in tryptic soy broth (Difco).
Beneckea Pelagia Ferment tion Five liters of 4.0% marine broth supplemented with 0.5%
alginate were introduced into a Microferm fermentor. The broth was inoculated with Beneckea pelagia (ATCC25916), and the culture was grown at 25C with vigorous aeration and agitation. The optical density at 600 nm reached its optimum at about 4-5 days and thereafter remained fairly level. The cells were harvested, washed in û.5M KCL, and resuspended in the same. 50 mg lysozyme per 50 ml suspension lysed the cells. The solids were washed again and used as a crude enzyme e~tract in the KCl solution.
' ~ X
Enzyme Assay An indication of enzyme activity was first observed in the reduction of the viscosity of a 0.5% alginate solution.
Quantitative estimates of the activity of the alginase produced by Beneckea pela~ were made measuring the increase in absorbance at 230 nm with a Bausch & Lomb Spectronic spectrophotometer. Ths method, as described by Preiss and Ashwell (1961), is based upon the marked absorption peak at 230 nm of the unsaturated uronic acid portion of the intermediate oligosaccharides. Neither intact alginic acid nor the final monosaccharide end product is measured in this assay. One unit of activity is taken as an increase of 0.001 in optical density (at 230 nm) per minute at a specified temperature. A standard reaction contained 0.5% alginate and 10 nM Tris.
Bacillus Circulans Fermentation Cultures were grown in Erlenmeyer flasks in a shaker bath held at 35C. The broth inoculated with Bacillus circulans (NRS 1349) consisted of 0.1% yeast extract, 0.1%
alginate, and 30 mM magnesium chloride. The cells were harvested after about one to two days, when the culture reached maximum turbidity. The culture was vigorously agitated using a Vortex mixer. The samples were then centrifuged to remove the cells.
The cells were discarded and the supernatant was tested for enzyme activity.
~f~d~
Enzyme Assay The thiobarbituric acid test, as modified by Weissbach and Hurwitz (1958), was used. This method is based on the formation of compounds giving rise to beta-formylpyruvic acid when subjected to periodate oxidaton. The uronic acid-portion of the intermediate oligosaccharides are cleaved by periodate to form betaformylpyruvate; intact alginic acid does not react. A
unit is defined as that amount of enzyme required to yield 1 micromole of beta-formylpyruvate under the stated incubation conditions. 0.01 micromole of beta-formylpryruvate gives an optical density reading of 0.290 at 549 nm.
A sample in 0.20 ml was added at time zero to 0.25 ml of 0.025 N HI04 in 0.125 N H2S04. After twenty minutes at room temperature, 0.50 ml of 2% sodium arsenite in 0.5 N HCl was added with shaking, and the solution was permitted to stand two minutes. Two ml of 0.03% thiobarbituric acid were added and, after stirring, the mixture was heated at 100C for 10 minutes. The mixture was cooled, and the optical density was measured at 548 nm in the Bausch & Lomb Spectronic 2000 sDectrophotometer .
A typical reaction mixture consisted of 0.25 ml enzyme supernatant, 0.25 ml distilled water, and 0.5 ml of a solution containing 0.5% alginate, 10 mM magnesium chloride, and 10 mM
Tris. The 0.2 ml samples were tested at 0, 15, 30, and 60 minutes.
I ~.~74~4~ ~
Enzyme Evaluation The slime rig is a standard method for the evaluation of the activity of toxicants and dispersants against attached bacteria. (Michalski et al. "A Method for Determining the Effect of Dispersants in Slime Control Performance", TAPPI 46, No. 2:
167a-172a tFebruary, 1963). Whitewater from a paper mill is recirculated: the whitewater spills over a weir to flow over a piece of leached basswood; the water is then pumped back to the weir to maintain a continuous circulation. The whitewater is inoculated with a microorganism typical of industrial process waters. As the water recirculates, the bacteria attach to the board and begin to produce extracellular polysaccharides; soon the board is covered with slime.
Two experiments were set up to test the effect of alginase. In the first, enzyme was added to a slime rig, the board of which was already coated with slime. In the second, enzyme was introduced continuously from the moment that the whitewater was inoculated with Pseudomonas aeruginosa ATCC
15442. In both cases, the rigs were treated each day, with approximately 50 ml of enzyme supernatant from Bacillus circulans (vide: Fermentation). A control without enzyme was run simultaneously. The whitewater of the control and of the experimental were tested using the thiobarbaritic acid assay.
Samples which showed an increase in optical density at 548 nm over the control were recorded as (+). A sample of enzyme was tested to assure that the enzyme itself did not produce a reading. The activity of the enzyme was also observed visually.
~44~
In these results reference is made to the figures in which Figure 1 shows the activity of alginase isolated from Bacillus pelagia, and Figure 2 shows similar data for Bacillus circulans.
Results Figure 1 shows the activity of the alginase isolated from Bacillus ~la~ia. The reaction mixture contained 1 ml of enzyme (from Beneckea pelagia) and 1 ml of 5% sodium alginate (O) or 1 ml of 5% bacterial exopolysaccharide (~ ). The con-trols tested (O) included 1 ml of enzyme with 1 ml distilled water, and 1 ml of 5% sodium alginate and 1 ml of 5% bacterial expolysaccharide, diluted together with 2 ml of distilled water.
The activity was measured as the change in optical density at 230 nm. This enzyme has about equal activity on a commercial source of sodium alginate and on an extracellular polysaccharide isolated from Pseudomonas aeruginosa. Various controls were tested, in particular enzyme alone and alginate alone; no change in optical density was observed.
The activity of the enzyme produced by Bacillus circulans is shown in Figure 2. The reaction mixture consisted .
of 0.25 ml enzyme (from Bacillus circulans), 0.25 ml distilled water, and 0.5 ml of a solution containing 0.5~ sodium alginate (O) or 0.5% bacterial exopolysaccharide ( ~), 10 mM MgC12 and 10 mM Tris. Control consisting of each reactant individually (O) were also tested. The thiobabituric acid assay was used, and activity recorded as the change in optical density at 548 nm. This enzyme has been characterized as an alginase which functions as an mannuronidase, the molecular weight of which is 40,000. The enzyme was found to have about equal acti-vity on sodium alginate and on extracellular polysaccharide from Pseudomonas aeruginosa. The thiobarbituric acid assay was used.
The co~trols tested were enzyme alone and each of the poly-saccharides alone. The controls exhibited no activity.
The enzyme was then tested in a study simulating paper mill conditions. Three rigs were inoculated with Pseudomonas aeruginosa; the first two rigs were left untreated, the third was ¦
¦treated with approximately 50 ml of alginase (produced by ¦Bacillus circulans) each day from the moment of inoculation. The ¦growth of slime on the first two rigs was evident within ¦twenty-four hours; the slime appeared as a layer of whitish ¦gelatinous material. The third rig, treated with enzyme, showed no evidence of slime adherinq to the board. The total count of bacteria in each rig twenty-four hours after inoculation was ¦about 5 x 106 organisms per ml.
¦ On the second day, three samples of the recirculating whitewater in each rig were assayed using the thiobarbituric acid method. Two control rigs gave the same reading. This reading was recorded as no enzyme activity. A reading higher in magnitude (by at least 0.580 OD548) was recorded as positive enzyme activity. The third rig showed positive enzyme activity.
¦ On the third day, the second rig was treated with enzyme '50 ml). The first rig continued as a control. The third rig ~as continuously treated.
On the fourth day, samples of the whitewater of each rig ! were assayed as before. The control rig showed no activity. The ¦second rig showed positive enzyme activity as did the third rig.
l The third rig remained clean of slime throughout the ¦experiment. The aPplication of enzyme to the second rig where ¦slime had already formed did not completely clean the board but ¦it did change the appearance and the attachment of the slime.
¦The slime became less continuous; pieces broke free. An increase ¦in water flow easily cleaned much of the slime from the board.
¦The slime on the control was much more firmly a~tached. Table I
¦summaries these results.
~ 4~
¦ Table I summarizes the results of the application of ¦ enzyme produced by Bacillus circulans. Rig 1 is the control.
¦ Rig 2 shows the effect of enzyme on existing slime. Rig 3 shows ¦ the effect of enzyme in preventing the build-up of slime. Enzyme ¦ treatment (~) refers to the addition of 50 ml of enzyme ¦ supernatant obtained as desc.ibed previous. Enzyme activity was ¦ measured using the thiobarbituric acid assay. Tne ~ refers to an ¦ optical density reading at 548 nm which exceeds the control (-) ¦ by at least 0.590.
4~
TABLE I
Day _ O 1 2 3 4 Rig 1 Enzyme Treatment~
Enzyme Activity: - _ Observations: No Slime Slime Slime Slime Slime Rig 2 Enzyme Treatment: - - - + +
Enzyme Activity: - +
Observations:No Slime SlimeSlimeSlime Less Continous Slime Rig 3 Enzyme Treatment: + + ~ + +
Enzyme Activity: + +
Observations: No Slime No Slime No Slime No Slime No Slime .
The invention is in the field of slime control.
SLIME
The ability of bacteria to produce copious amounts of extracellular polysaccharide has long been recognized as a major problem in industrial process waters. The function of this polysaccharide capsule has not been determined, but research indicates that the extracellular capsule or slime layer may provide protection, or enhance the transport of nutrients, or both. Several conditions common to industrial process waters encourage slime production. Many of the bacteria found in industrial waters are sessile; the nutrient gradient or turbulence or any number of other factors promotes their attachment to surfaces; the formation of the slime layer assures a firm attachment; low levels in nitrogen and high levels of carbon compounds generally encourage slime production.
The traditional approach to controlling slime-forming bacteria has been the application of toxic chemicals. Toxicants reduce the population of microorganisms and hence reduce the production of slime. Toxic chemicals almost always present an environmental hazard, and the effectiveness of the toxicants is reduced by the slime itself. The extracellular polysaccharide surrounding the attached bacteria is largely water insoluble and impenetrable. The findings that slime production by certain bacteria is related to their resistance to harmful substances supports this view that extracellular polysaccharide impedes the action of toxicants. The toxicant can not adequately control large populations of fixed bacteria, it is mostly effective 1;~74"4;~ 1 against floating bacteria. Surfactants and dispersants, which increase the water solubility and penetrability of slime, can enhance the activity of toxicants; they are, however, nonspecific and may have deleterious effects on the industrial process in question.
NUTRIENTS
The composition of the extracellular polysaccharide can vary with the species of bacteria; it is sometimes dependant on the types of nutrients available, again depending on the species.
~ acteria prefer certain substrates; they will seek out and preferentially absorb certain nutrients.
The normal paper mill contains many disaccharides and carboxylic acids. The typical kraft softwood pulp contains 75-80 percent cellulose, 5 percent lignin, 3-5 percent mannan, 5-8 percent xylan, and about 2 percent hexuronic anhydride. The pulping process releases some carboxylic forms of the mannan (polymannose to mannuronic acid) and xylan (polyxylose to guluronic acid). Starch (polyglucose), if greatly hydrolyzed, will release some disaccharides and carboxylic acids. Amino acids are not often available in paper mills, except perhaps when casein is used. As mentioned previously, these low levels of nitrogen compared to carbon encourage extracellular polysaccharide production.
On the whole, the Paper mill presents the bacteria predominantly with carbohydrates such as mannan, xylan, and ¦glycans. From mill to mill, the nutrients probably vary most in quantity rather than in type.
~ ~74~4~ 1 BACTERIA
In some bacteria, nutrients induce a certain slime; this is most often the case with bacteria that produce homopolysaccharides such as levans and dextrans. Even under the conditions described above, where the bacteria have available to them very similar nutrients, these bacteria may produce different slimes from mill to mill. For this reason, levan, for example, cannot be considered to be ubiquitous in process waters.
Other bacteria, in particular those which produce heteropolysaccharides, tend to produce the same extracellular polysaccharide under most conditions.
The bacteria which are the greatest producers of slime in the paper mill are the gram-negative rod-shaped aerobes of such genera as Pseudomonas, Aerobacter, Klebsiella and Flavobacterium. Of this group, Pseudomonas aeruginosa is one of the most common and difficult to control.
ALGINASE
The production of alginase can be induced in certain microorganisms using a commercial source of sodium alginate.
This alginase also was found to have activity on the extracellular polysaccharide produced by Pseudomonas aeruginosa, a slime-forming bacteria common in process waters. In an experiment simulating the process waters of a paper mill, the application of this enzyme reduced the numbers of attached bacteria by hydrolyzing the polysaccharide slime which allows them to attach. The application of this enzyme thus reduces the formation of slime deposits through two mechanisms, by preventing attachment of bacteria and by promoting the detachment of bacteria. The planktonic bacteria are thought to be more susceptible to the action of toxic chemicals as well.
~ ~.~74~
INVENTION
Generally therefore, the invention comprises a method of ¦preventing and reducing bacterial slimes which contain IPseudomonas aeruginosa which form on surfaces in contact with I
¦industrial process waters which comprises treating said waters with a slime inhibiting amount of alginase. This invention also ¦includes the alginase as used.
¦ A number of microorganisms are known to produce ¦alginase, for example: ~
A
¦ Aeromonas sp. aeneckea polagla ¦ Alginovibrio aQuatilis Clostridium alginolyticum ¦ Azotobacter vinelandii Cytophaga diffluens ¦ Bacillus circulans Pseudomonas sp.
¦ These organisms can be found growing on bacterial slimes in natural habitats.
For the purposes of this study, two organisms, Beneckea pelagia and Bacillus circulans, were considered most suited to the production of an unpurified enzyme to treat industrial process waters.
Dosages Recirculating water systems require at least 10 units of alginase activity (as tested using the thiobarbituric acid assay) per gallon of recirculating water and may require as much as lOû-250 units per gallon in systems containing copious slime;
generally lû-100 units per gallon would adequately treat the process waters.
` ~.~7~
Materials and Methods Bacteria Strains and Growth Media Strains of Beneckea pelagia (ATCC75916) were maintained on marine agar (Difco). Batch cultures were grown in 4% marine broth supplemented with 0.5% sodium alginate in order to induce the production of alginase. Strains of Bacillus circulans (NRS1349) were maintained on trypticase soy agar (BBL) or on nutrient agar (Difco) supplemented with alginate. Batch cultures were grown in 0.1% yeast extract (Difco), 0.1% alginate and 30 mM
magnesium chloride. Mucoid strains of Pseudomonas aeruginosa (ATCC15442) were maintained on trypticase soy agar or on tryptone glucose extract agar (Difco). Samples of extracellular polysaccharide were isolated from Pseudomonas aeruginosa grown in tryptic soy broth (Difco).
Beneckea Pelagia Ferment tion Five liters of 4.0% marine broth supplemented with 0.5%
alginate were introduced into a Microferm fermentor. The broth was inoculated with Beneckea pelagia (ATCC25916), and the culture was grown at 25C with vigorous aeration and agitation. The optical density at 600 nm reached its optimum at about 4-5 days and thereafter remained fairly level. The cells were harvested, washed in û.5M KCL, and resuspended in the same. 50 mg lysozyme per 50 ml suspension lysed the cells. The solids were washed again and used as a crude enzyme e~tract in the KCl solution.
' ~ X
Enzyme Assay An indication of enzyme activity was first observed in the reduction of the viscosity of a 0.5% alginate solution.
Quantitative estimates of the activity of the alginase produced by Beneckea pela~ were made measuring the increase in absorbance at 230 nm with a Bausch & Lomb Spectronic spectrophotometer. Ths method, as described by Preiss and Ashwell (1961), is based upon the marked absorption peak at 230 nm of the unsaturated uronic acid portion of the intermediate oligosaccharides. Neither intact alginic acid nor the final monosaccharide end product is measured in this assay. One unit of activity is taken as an increase of 0.001 in optical density (at 230 nm) per minute at a specified temperature. A standard reaction contained 0.5% alginate and 10 nM Tris.
Bacillus Circulans Fermentation Cultures were grown in Erlenmeyer flasks in a shaker bath held at 35C. The broth inoculated with Bacillus circulans (NRS 1349) consisted of 0.1% yeast extract, 0.1%
alginate, and 30 mM magnesium chloride. The cells were harvested after about one to two days, when the culture reached maximum turbidity. The culture was vigorously agitated using a Vortex mixer. The samples were then centrifuged to remove the cells.
The cells were discarded and the supernatant was tested for enzyme activity.
~f~d~
Enzyme Assay The thiobarbituric acid test, as modified by Weissbach and Hurwitz (1958), was used. This method is based on the formation of compounds giving rise to beta-formylpyruvic acid when subjected to periodate oxidaton. The uronic acid-portion of the intermediate oligosaccharides are cleaved by periodate to form betaformylpyruvate; intact alginic acid does not react. A
unit is defined as that amount of enzyme required to yield 1 micromole of beta-formylpyruvate under the stated incubation conditions. 0.01 micromole of beta-formylpryruvate gives an optical density reading of 0.290 at 549 nm.
A sample in 0.20 ml was added at time zero to 0.25 ml of 0.025 N HI04 in 0.125 N H2S04. After twenty minutes at room temperature, 0.50 ml of 2% sodium arsenite in 0.5 N HCl was added with shaking, and the solution was permitted to stand two minutes. Two ml of 0.03% thiobarbituric acid were added and, after stirring, the mixture was heated at 100C for 10 minutes. The mixture was cooled, and the optical density was measured at 548 nm in the Bausch & Lomb Spectronic 2000 sDectrophotometer .
A typical reaction mixture consisted of 0.25 ml enzyme supernatant, 0.25 ml distilled water, and 0.5 ml of a solution containing 0.5% alginate, 10 mM magnesium chloride, and 10 mM
Tris. The 0.2 ml samples were tested at 0, 15, 30, and 60 minutes.
I ~.~74~4~ ~
Enzyme Evaluation The slime rig is a standard method for the evaluation of the activity of toxicants and dispersants against attached bacteria. (Michalski et al. "A Method for Determining the Effect of Dispersants in Slime Control Performance", TAPPI 46, No. 2:
167a-172a tFebruary, 1963). Whitewater from a paper mill is recirculated: the whitewater spills over a weir to flow over a piece of leached basswood; the water is then pumped back to the weir to maintain a continuous circulation. The whitewater is inoculated with a microorganism typical of industrial process waters. As the water recirculates, the bacteria attach to the board and begin to produce extracellular polysaccharides; soon the board is covered with slime.
Two experiments were set up to test the effect of alginase. In the first, enzyme was added to a slime rig, the board of which was already coated with slime. In the second, enzyme was introduced continuously from the moment that the whitewater was inoculated with Pseudomonas aeruginosa ATCC
15442. In both cases, the rigs were treated each day, with approximately 50 ml of enzyme supernatant from Bacillus circulans (vide: Fermentation). A control without enzyme was run simultaneously. The whitewater of the control and of the experimental were tested using the thiobarbaritic acid assay.
Samples which showed an increase in optical density at 548 nm over the control were recorded as (+). A sample of enzyme was tested to assure that the enzyme itself did not produce a reading. The activity of the enzyme was also observed visually.
~44~
In these results reference is made to the figures in which Figure 1 shows the activity of alginase isolated from Bacillus pelagia, and Figure 2 shows similar data for Bacillus circulans.
Results Figure 1 shows the activity of the alginase isolated from Bacillus ~la~ia. The reaction mixture contained 1 ml of enzyme (from Beneckea pelagia) and 1 ml of 5% sodium alginate (O) or 1 ml of 5% bacterial exopolysaccharide (~ ). The con-trols tested (O) included 1 ml of enzyme with 1 ml distilled water, and 1 ml of 5% sodium alginate and 1 ml of 5% bacterial expolysaccharide, diluted together with 2 ml of distilled water.
The activity was measured as the change in optical density at 230 nm. This enzyme has about equal activity on a commercial source of sodium alginate and on an extracellular polysaccharide isolated from Pseudomonas aeruginosa. Various controls were tested, in particular enzyme alone and alginate alone; no change in optical density was observed.
The activity of the enzyme produced by Bacillus circulans is shown in Figure 2. The reaction mixture consisted .
of 0.25 ml enzyme (from Bacillus circulans), 0.25 ml distilled water, and 0.5 ml of a solution containing 0.5~ sodium alginate (O) or 0.5% bacterial exopolysaccharide ( ~), 10 mM MgC12 and 10 mM Tris. Control consisting of each reactant individually (O) were also tested. The thiobabituric acid assay was used, and activity recorded as the change in optical density at 548 nm. This enzyme has been characterized as an alginase which functions as an mannuronidase, the molecular weight of which is 40,000. The enzyme was found to have about equal acti-vity on sodium alginate and on extracellular polysaccharide from Pseudomonas aeruginosa. The thiobarbituric acid assay was used.
The co~trols tested were enzyme alone and each of the poly-saccharides alone. The controls exhibited no activity.
The enzyme was then tested in a study simulating paper mill conditions. Three rigs were inoculated with Pseudomonas aeruginosa; the first two rigs were left untreated, the third was ¦
¦treated with approximately 50 ml of alginase (produced by ¦Bacillus circulans) each day from the moment of inoculation. The ¦growth of slime on the first two rigs was evident within ¦twenty-four hours; the slime appeared as a layer of whitish ¦gelatinous material. The third rig, treated with enzyme, showed no evidence of slime adherinq to the board. The total count of bacteria in each rig twenty-four hours after inoculation was ¦about 5 x 106 organisms per ml.
¦ On the second day, three samples of the recirculating whitewater in each rig were assayed using the thiobarbituric acid method. Two control rigs gave the same reading. This reading was recorded as no enzyme activity. A reading higher in magnitude (by at least 0.580 OD548) was recorded as positive enzyme activity. The third rig showed positive enzyme activity.
¦ On the third day, the second rig was treated with enzyme '50 ml). The first rig continued as a control. The third rig ~as continuously treated.
On the fourth day, samples of the whitewater of each rig ! were assayed as before. The control rig showed no activity. The ¦second rig showed positive enzyme activity as did the third rig.
l The third rig remained clean of slime throughout the ¦experiment. The aPplication of enzyme to the second rig where ¦slime had already formed did not completely clean the board but ¦it did change the appearance and the attachment of the slime.
¦The slime became less continuous; pieces broke free. An increase ¦in water flow easily cleaned much of the slime from the board.
¦The slime on the control was much more firmly a~tached. Table I
¦summaries these results.
~ 4~
¦ Table I summarizes the results of the application of ¦ enzyme produced by Bacillus circulans. Rig 1 is the control.
¦ Rig 2 shows the effect of enzyme on existing slime. Rig 3 shows ¦ the effect of enzyme in preventing the build-up of slime. Enzyme ¦ treatment (~) refers to the addition of 50 ml of enzyme ¦ supernatant obtained as desc.ibed previous. Enzyme activity was ¦ measured using the thiobarbituric acid assay. Tne ~ refers to an ¦ optical density reading at 548 nm which exceeds the control (-) ¦ by at least 0.590.
4~
TABLE I
Day _ O 1 2 3 4 Rig 1 Enzyme Treatment~
Enzyme Activity: - _ Observations: No Slime Slime Slime Slime Slime Rig 2 Enzyme Treatment: - - - + +
Enzyme Activity: - +
Observations:No Slime SlimeSlimeSlime Less Continous Slime Rig 3 Enzyme Treatment: + + ~ + +
Enzyme Activity: + +
Observations: No Slime No Slime No Slime No Slime No Slime .
Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of preventing and reducing bacterial slimes which contain Pseudomonas aeruginosa, which form on surfaces in contact with industrial process waters, which method comprises treating said waters with a slime inhibiting amount of alginase.
2. The method of Claim 1 where the industrial process water is a water used in the manufacture of paper and the algin-ase in an unpurified alginase derived from the fermentation of the bacteria Beneckea pelagia or Bacillus circulans.
3. The method of Claim 1 wherein the slime inhibiting amount of alginase is sufficient to provide at least 10 units of alginase activity (as tested using the thiobarbituric acid assay) per gallon of treated process water.
4. The method of Claim 3 wherein the slime inhibiting amount of alginase is sufficient to provide from about 10 to about 250 units of alginase activity per gallon of treated process water.
5. The method of Claim 4 wherein the slime inhibiting amount of alginase is sufficient to provide from about 10 to about 100 units of alginase activity per gallon of treated pro-cess water.
6. A composition for controlling slime in industrial pro-cess water comprising alginase formed by the fermentation of Beneckea pelagia or Bacillus circulans.
7. A composition for treating slime in industrial waters comprised of:
a. lysed, washed cells of the bacteria Beneckea pelegia, said cells having alginase activity when mixed with commercially-prepared or naturally occurring alginic acid and tested by increase in optical density of 235 nm; and b. water.
a. lysed, washed cells of the bacteria Beneckea pelegia, said cells having alginase activity when mixed with commercially-prepared or naturally occurring alginic acid and tested by increase in optical density of 235 nm; and b. water.
8. The composition of Claim 5, further comprising potassium chloride.
9. A composition for treating slime in industrial waters comprised of the extracellular supernatant of Bacillus circulans cells grown in broth, the supernatant of the said cells having alginase activity when mixed with commercially prepared or naturally occurring alginic acid and tested according to thio-barbituric acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US87493386A | 1986-06-16 | 1986-06-16 | |
| US874,933 | 1992-04-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1274442A true CA1274442A (en) | 1990-09-25 |
Family
ID=25364892
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000539655A Expired - Fee Related CA1274442A (en) | 1986-06-16 | 1987-06-15 | Slime control in industrial waters using enzymes |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1274442A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5238572A (en) * | 1993-01-25 | 1993-08-24 | Betz Laboratories, Inc. | Enzyme treatment for industrial slime control |
-
1987
- 1987-06-15 CA CA000539655A patent/CA1274442A/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5238572A (en) * | 1993-01-25 | 1993-08-24 | Betz Laboratories, Inc. | Enzyme treatment for industrial slime control |
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