KR20010108263A - Marine antifouling methods and compositions - Google Patents

Abstract

The present invention relates to a marine antifouling composition and / or paint comprising a hydrolase (s), microorganism (s) or a mixture of hydrolase (s) and microbial (s), wherein such microorganisms or hydrolases Reduces fouling of the surface coated by the marine antifouling composition and / or paint. Such compositions and / or paints may include catalytically effective amounts of inorganic salts. The invention also relates to articles coated with the compositions and / or paints. Finally, the present invention reduces the fouling of the ship surface, reduces the corrosion of the ship, limits the absorption of water by the ship surface, reduces the drag coefficient of the ship surface, removes marine growth from the ship surface, A method for reducing fungal fungi on ship surfaces.

Description

Marine antifouling method and composition for marine antifouling {MARINE ANTIFOULING METHODS AND COMPOSITIONS}

Ship fouling has been annoying since humans first contacted the marine environment. Ship fouling, in which organisms adhere undesirably to the surface of the ship, occurs not only for navigational equipment such as the ship's hull and drive system, but also for other structures exposed to seawater. Such structures may include submarine communications equipment such as piles, ship markings, cables and pipes, bulkheads, cooling towers, and any submerged and operated appliances or structures.

The fouling phenomenon depends on a number of factors including light, structure and properties of the substrate, water flow, chemical factors, larval biological complexes, larvae density and composition of the larvae community, and the presence of surface films.

Surface film on the surface of the ship is of great interest because most of the marine larvae are more easily fixed to the filmed surface (D. J. Crisp, Chemosorbtion in Marine Organisms: Factors Influencing the Settlement of Marine Invertebrate Larvae, ed. P. T. Grant & A. M. Mackie, 177, 215, 1974). Surface films on such ship structures are produced by marine microorganisms almost immediately upon receipt of the structure (D. Kirchman et al.,Mar. Chem.27,201-217, 1989).

These microorganisms stimulate more basal adherents (see CE Zobell & EC Allen, The Significance of Marine Bacteria in the Fouling of Submerged Surfaces, J. Bact . 29, 230-251, 1935). Indeed, there appears to be a strong correlation between primary film formation and attachment of animals to the surface of the vessel, the researchers said (R. Mitchell & L. Young, The Role of Microorganisms in Marine Fouling, Technical Report No. 3). VS Office of Naval Research Contract No. N00014-67-A-0298-0026 NR-306-025, 1972).

Surface films can include extracellular carbohydrates and proteins exuded by marine microorganisms that can be used to adhere to the vessel's surface itself (A. Danielsson et al., On Bacterial Adhesion-the effect of certain enzymes). on adhered cells of a marine Pseudomonas sp ., Botanica Marina 20 , 13-17, 1977 and GGG Geesey et al., Microscopic Examination of Natural Sessil Bacterial Populations from an Alpine Stream, Can.J. Microbio . 23 , 1733- 1736, 1977). Protein adsorption to surfaces can have a significant impact on microbiological, chemical and biochemical processes occurring at the sea surface interface (DL Kirchman et al., Adsorption of Proteins to Surfaces in Seawater, Marine Chemistry 27 , 201-217, 1989). Such attachment provides an advantage to microorganisms in that they can retain organic nutrients that are constantly regenerated and supplied within physical conditions that contribute to the growth of microorganisms (JW Costerton et al., How Bacteria Stick, Scientific American 238 , 86-95, 1977).

However, fouling phenomena (i.e., undesirably attaching organisms to the surface of the ship) cause many problems. That is, if the ship structure is damaged, drag, weight and corrosion increase, the aesthetic appearance of the ship structure is degraded, and the maintenance costs associated with removing and repairing the soil material of the structure are increased. In addition, even a small number of oysters or equivalent organisms attached to the propeller of a boat can result in a very low propeller efficiency or cavitation problem.

In the shipping industry, attempts have been made to reduce fouling by adding various toxic substances such as mercury, tin and copper to the coatings of apparatus and structures. However, the use of these additives creates serious environmental problems. Additive-containing coatings are typically formulated to expose the environment to toxic substances embedded in the coating structure. This exposure diminishes toxic substances into the marine environment, reducing adhesion by crustaceans.

However, the toxicity of these substances is like double-edged swords; That is, these additives not only reduce adhesion by crustaceans but also have an adverse effect on the marine environment as a whole. Because of the environmental issues associated with the use of these additives, the US Environmental Protection Agency (EPA) has significantly limited the continued use of these compounds, especially tin and mercury. In addition, even when the use of these additives is allowed, frequent renewal is required (in some areas every six months), which is expensive to use. Thus, these toxic additives are expensive both in terms of resource and environmental destruction. In addition, marine microorganisms that attach to the seabed are immune to toxic substances and can effectively render them ineffective.

In light of this, there is a need for a method of antifouling of a ship and a composition for antifouling of a ship which does not use toxic additives which substantially harm the environment. After many experiments the inventors developed the concept of incorporating hydrolase and / or hydrolyzable microorganisms into marine coatings, which functions to limit fouling of undesirable vessels.

This inventor's approach offers significant advantages over prior attempts to solve the ship's fouling problem. For example, the methods of the present invention rely on hydrolase and / or hydrolysable live cells to prevent fouling by living organisms. Thus, the coatings of the present invention can be formulated to maintain antifouling efficacy while containing little toxic substances such as heavy metals. This avoids environmental issues associated with the use of heavy metal biocides.

In these embodiments of the invention, the microorganisms and / or hydrolytic enzymes are embedded in marine stable coatings such as epoxy, polyurethane or other coating materials by simple mixing. Microorganisms and / or hydrolytic enzymes may be used on any surface (eg paddles, propellers, hulls, cooling towers, etc.) to which the marine compositions and / or paints of the present invention may be bound. Therefore, the coatings and / or paints of the present invention can be widely applied.

The use of microorganisms in addition to hydrolase has additional benefits. For example, when a composition and / or paint of the present invention is inoculated with a beneficial microorganism, the microorganism may secrete substances that enhance hydrolytic enzymes that may be added to the coating and / or paint, such as additional hydrolytic enzymes. have. Refurbishment may continue in a robust fashion for the expected lifetime of the composition and / or paint or until the microbial colony disintegrates in the marine environment. Alternatively, beneficial microorganisms may over-compete with the seabed microorganisms on the surface of the vessel to reduce fouling.

The present invention relates to a method of antifouling of a ship, and a coating and composition for ship antifouling.

1 is a view showing the test results for the upper layer panels 1 to 4 and the control group having a coating according to the present invention.

Figure 2 shows the test results for the upper layer panels 5 to 7 with the coating according to the invention and the control.

3 shows the test results for upper layer panels 8 to 10 and the control group with the coating according to the invention.

Figure 4 shows the test results for the lower layer panels 1 to 4 with the coating according to the invention and the control.

5 shows the test results for lower layer panels 5 to 7 with the coating according to the invention and for the control.

Figure 6 shows the test results for the lower layer panels 8 to 10 with the coating according to the invention and the control.

Accordingly, the present invention relates to methods, compositions and paints that substantially avoid one or more of the above problems.

In order to achieve these and other advantages in accordance with the present invention as embodied and broadly described, the present invention includes one or more hydrolase, one or more microorganisms, or a mixture of one or more hydrolase and microorganisms, wherein the hydrolysis A marine antifouling composition for reducing fouling of a vessel surface coated with an enzyme, microorganism or mixture with a marine antifouling composition.

The present invention also includes a coating composition suitable for the shipbuilding industry, and at least one hydrolase, at least one microorganism, or at least one hydrolase and a mixture of microorganisms, wherein the hydrolase, microorganism or mixture is a marine antifouling paint. The present invention relates to a paint for ship antifouling, which reduces fouling of a ship's surface coated with water.

The present invention also relates to a method of reducing fouling of a vessel surface, a product coated with an antifouling composition or an antifouling paint, a method of reducing vessel corrosion, and a method of limiting water absorption of a vessel surface.

Another aspect of the invention relates to a method for removing marine growth from a vessel surface, a method of reducing the propensity of cavitation of a propeller under load, and a method for reducing fungal fungi on a vessel surface.

Another aspect of the present invention relates to a marine antifouling composition and paint comprising an inorganic salt present in a catalytically effective amount.

The present invention encompasses one or more hydrolases, one or more microorganisms, or a mixture of one or more hydrolases and microorganisms, wherein the hydrolase, microorganisms or mixtures reduce fouling of the vessel surface coated with the composition for ship antifouling. It relates to a marine antifouling composition. In addition, the present invention is a marine product coated with such a composition; And coating the ship surface with the composition, wherein the method relates to a method of reducing the fouling of the ship surface by coating the ship surface with a composition for ship antifouling which reduces the fouling of the coated ship surface.

Another aspect of the invention comprises a coating composition suitable for the shipbuilding industry, and at least one hydrolase, at least one microbe, or a mixture of at least one hydrolase and a microbe, wherein the hydrolase, microbe or mixture is It is a ship antifouling paint that reduces the fouling of the ship's surface coated with the ship antifouling paint. In addition, the present invention is a marine product coated with the paint; And coating the surface of the ship with the paint, wherein the method relates to reducing the contamination of the surface of the ship by coating the surface of the ship with a paint that reduces the contamination of the coated ship surface.

Another aspect of the invention is a method of reducing vessel corrosion, comprising coating the vessel surface with a composition for marine antifouling to form one or more films that reduce the adsorption of corrosive molecules to the surface. Also disclosed are methods in which the composition inhibits surface corrosion and intergranular corrosion.

Another aspect of the present invention is a method of reducing vessel corrosion, comprising coating the surface of the vessel with a marine antifouling paint to form one or more films that reduce the adsorption of corrosive molecules to the surface. In another aspect of the present invention, a method is disclosed in which a paint inhibits surface corrosion and grain boundary corrosion.

Another aspect of the invention is to limit the absorption of water by the surface of a vessel, comprising coating the surface with a marine antifouling composition or marine antifouling paint to form a film to reduce the porosity of the surface. That's how.

Another aspect of the present invention is a method of reducing the drag coefficient of a ship surface, comprising coating the surface with a ship antifouling composition or a ship antifouling paint. The present invention also relates to a method for using a marine antifouling composition or a marine antifouling paint, wherein a surfactant capable of acting as a humectant is produced by the microorganisms in the composition or paint.

Another aspect of the present invention is a method for removing marine growth from a vessel surface, comprising coating the vessel surface with a composition for ship antifouling or paint for ship antifouling. Another aspect of the present invention is a method of using a marine antifouling composition or a marine antifouling paint, wherein the marine growth is a hard or soft growth. Another aspect of the present invention is a method of using a marine antifouling composition or a marine antifouling paint, wherein the hydrolase or microorganism or mixture attacks the exudates of existing growth and causes the release of hard and soft growths.

Another aspect of the present invention is a marine antifouling composition or a marine antifouling paint containing an inorganic salt present in a catalytically effective amount. Another aspect of the present invention is a method of reducing the propensity of a propeller cavitation under load, comprising coating the propeller surface with a marine antifouling composition or marine antifouling paint. Another aspect of the present invention provides a method for coating a ship surface with a composition for ship antifouling to form one or more films that reduce the absorption or attachment of fungal fungi to the surface or inhibit the growth of fungal fungi on the ship surface. A method of using a composition for ship antifouling to reduce fungal fungi on a ship surface, comprising the step.

Now, preferred embodiments of the present invention are described in detail by way of examples.

Protective coatings and / or paints of the present invention containing microorganisms and / or hydrolase may function in a variety of ways. One possible mechanism of action for the present invention is that a harmless microbial population can be used as part of the coating. These harmless microorganisms can reduce fouling by selecting unnecessary organisms of "over-competition". These harmless microorganisms can act to selectively and selectively remove critical nutrients such as organic compounds or food source microorganisms from microfilm of water on the surface of ship structures. Alternatively, such microorganisms can act by exuding antibiotics or other compounds to slow the growth of basal attachment organisms. In this way, these harmless microorganisms effectively reduce the growth of basal microorganisms (such as fungal fungi) and further colonize by marine larvae.

Coatings and / or paints may function by directly attacking the surface film and destroying the polymer structure through hydrolysis of the proteins and polysaccharides of the film. This ultimately blocks the chain when large quantities of marine organisms (such as bacteria, fungi, oysters, etc.) accumulate on the ship's hull. This attack may be accomplished by using extracellular enzymes that degrade the carbohydrates and proteins that make up the surface film. We tested this mechanism using skim milk and corn starch as model substrates to determine the activity of two major hydrolytic enzymes, proteases and alpha-amylases. Alternatively, the coating and / or paint may function by varying the surface tension of the vessel surface to which the coating and / or paint is applied. This change in surface tension can prevent the surface from colonizing by undesirable marine organisms.

The present invention has utility for the following crustaceans and other marine hard growths:

Tubeworms : polychaetes, Annelida door, Eunicea subfamily , Serpulidae family ;

Mussels: bivalve Ryu, Ruth Mall car (Mollusca) statement, funny terry loop Pia (Pteriomorphia) (Mytilidae) in the subclass, the US and the Antilles;

Oysters: Bivalve , Moluscamun , Pteriomopia subclass, Osteridae ;

Clams: the bivalve flow, mole loose kamun, blurred Tero donta (Hterodonta) subclass, Venetian Florida (Veneridae) and;

Lichen beetles: Lichen beetles, Bryozoa doors, Anasca subfamily and Ascophora subfamily, Schizoporella genus; And

Oyster: Crustacean, Arthropoda , Crustacea .

In addition, the present invention has utility for soft growth that can hinder the efficiency of the hull form, destroy the base of the vessel structure, generally shorten the life span of the equipment and skyrocket operating costs. Examples of such soft growths include:

Seaweed (plants): Padina and Codium ;

Moss bug (animal): Bugula Neretina ;

Hydroworm (animal): Obelia ;

Savelides (animals); And

Delaya Marina ( Marium ): Zibria .

The methods and compositions disclosed herein can be used on a variety of surfaces, including but not limited to boat hulls, ship markings, bulkheads, piles, inlets, floors, roofs, and shingles. For example, the methods and compositions can be used to minimize fouling of vessel labels. These labels constitute various categories of suspended solids and are mostly damaged by the accumulation of marine growth.

Similarly, the present methods and compositions can be used for bulkheads of ships. Accumulation of marine growth in the bulkhead structure damages the bulkhead structure over time. In addition, growth causes aesthetically unpleasant and dangerous significant short-term effects. Moreover, the coarse abrasive properties of hard growth can cause major damage to the instrument.

Similarly, the present invention can be used to minimize the blockage of heat exchangers, evaporators, condensers and fire extinguishers due to fouling by marine growth so that maintenance costs for all categories of ship structures can be significantly reduced.

The compositions and / or paints according to the invention may comprise various hydrolase enzymes, but the invention may be practiced without such hydrolase enzymes. Examples of suitable enzymes include proteases, amylases, and other hydrolytic enzymes known in the art. The hydrolase selected serves to prevent or reduce adhesion by marine organisms that are unnecessary or undesirable. The hydrolase chosen should be able to survive and thrive in the marine environment to be exposed.

The compositions and / or paints according to the invention may comprise a variety of microorganisms, but the invention may be practiced without these microorganisms. Suitable micro-organisms are included in the two other microorganisms known in Bacillus (Bacillus), Escherichia (Escherichia), Pseudomonas (Pseudomonas) or art. The microorganisms chosen serve to prevent or reduce attachment by unnecessary or undesirable marine organisms. The microorganism selected should be able to survive and thrive in the marine environment to be exposed.

The compositions and / or paints according to the invention comprise the enzymes and / or microorganisms in an amount effective to reduce the growth of unnecessary or undesirable microorganisms. Such compositions and / or paints may exist in various forms, including paints, lacquers, pastes, laminates, epoxies, resins, waxes, gels, glues, and other forms known to those skilled in the art. The composition and / or paint may be a polymer, oligomer or monomer, and may contain a crosslinking agent or a curing accelerator as necessary. Such compositions and / or paints may contain other additives in addition to those mentioned above in order to achieve the purpose known to those skilled in the art. Such other additives include preservatives, pigments, dyes, fillers, surfactants and other additives known to those skilled in the art.

The compositions and / or paints according to the invention may comprise a polymer resin base, but the invention may be practiced without such a base or with a base of different materials. The compositions and / or paints may be applied in a single coating or in multiple coatings.

In addition, the inventors have added that some inorganic salts (such as NaCl, CaCl ₂ , MgSO _4, etc.) enhance the catalytic hydrolysis of both liquid and solid state (resin landfill) alpha-amylases (available from Genencor). Was observed. Although calcium chloride is involved as a cofactor in the alpha-amylase catalysis, the amount required for activation (about 60 ppm) is much less than the amount used in the epoxy resin formulations provided in the examples below. Therefore, such an inorganic salt can be added in a catalytically effective amount. The catalytically effective amount is greater than the amount of inorganic salt required for activation. Catalytic effects are described in more detail in Examples 7 and 8 below.

Various aspects of the invention are now demonstrated in the following examples.

Example 1

A series of experiments were conducted to verify that an enzyme encapsulated in a suitable marine coating or paint retained its enzyme properties. Substrates used to measure the activity of two major enzymes, protease and alpha-amylase, were skim milk and corn starch, respectively. As discussed above, these substrates each exhibited test substrates for the protein and polysaccharide portions of the target glycoprotein described as an initial component of the biofouling process.

Plastic or glass bottles (volume 100 ml) were used as enzyme reactors. Enzymes were obtained from Genencor International, Inc. (Rochester, NY). The enzymes tested were:

Desize 160 (alpha-amylase-liquid);

Maxamyl CXT 5000 (alpha-amylase-encapsulated);

Purafect 2000G (protease-encapsulation); And

Barracks 15,000 CXT (alpha-amylase-liquid).

In all assays, encapsulated enzymes were added directly to the indicated coatings. When examining the activity of the liquid enzyme, the enzyme was first added to calcium chloride. Since water interferes with the curing process, calcium chloride has been included as an absorbent to facilitate mixing with the coating. It was later found that addition of CaCl ₂ and other salts increased starch hydrolytic activity.

The following assay was used to determine proteolytic activity. Vinegar (diluted acetic acid) precipitates milk protein from solution. Thus, the enzymatic hydrolysis of the milk protein was monitored by exposing the milk protein solution to the protease and adding acetic acid to the drawn sample over time. The decrease in the amount of precipitate is a measure of enzymatic activity and expressed as hydrolysis rate.

Unless otherwise specified, enzyme activity analysis was performed by adding 25 ml of 1: 4 aqueous diluted skim milk (diluted to contain 0.94% protein). Activity was quantified compared to a control without enzymes or cells. Sufficient acid was added to precipitate all the proteins present. The amount of precipitate concentrated by sedimentation was used as a volume measure of the amount of protein present in the milk solution before hydrolysis. Enzyme activity was determined by comparing the amount of acid precipitated protein present before and after exposure to the indicated enzyme or cell. Absence of acid precipitated protein is shown by 100% hydrolysis by the addition enzyme.

Starch hydrolase activity was measured by adding a suspension of corn starch in water and mixing to achieve viscosity approach solidification (12 g / 10 ml of water). Water and starch were added to alpha-amylase suspended in water or embedded in solidified epoxy. The reaction to fully liquefy the solidified starch suspension was designated as complete (100%) hydrolysis. In the reaction of decreasing viscosity by adding alpha-amylase, the activity was approximately equal to the resistance to manually stirred paddles. Although this activity measurement was passive, it was reproducible compared to standard controls such as samples containing water and starch without enzymes.

400 mg of encapsulated protease (Purafect 2000G, commercially available from Genenco International Inc., Rochester, NY, USA) is a 2-inch ribbon epoxy and curing agent (2,4,6-tri (dimethylaminomethyl) phenol, USA And commercially available from ITW Brands, Udal, Illinois. The resin / enzyme mixture was used to coat the inner bottom of a plastic bottle with a liquid volume of 100 ml and a diameter of 2 inches (50 mm). The mixture was cured for 16 hours. Diluted skim milk was added to the reactor as shown in Table 1 below and incubated at room temperature for 5 hours. Samples were removed and diluted hydrochloric acid was added to precipitate unhydrolyzed protein and tested for proteolytic activity:

Test mixture Hydrolysis Rate (%) Epoxy sole 0 Epoxy + Furafect 2000G 100

Thus, the encapsulated protease (Purafect 2000G) retained its enzymatic activity after being embedded in epoxy resin glue.

Example 2

Except for using tap water instead of seawater as skim milk diluent, the enzyme activity of the embedded protease (Furafect 2000G) was measured according to the procedure of Example 1. The same result as in Example 1 was obtained. Therefore, the use of tap water instead of seawater had no effect on the activity of the proteolytic enzymes encapsulated in the epoxy resin.

Example 3

0.5 inch (12.5 mm) diameter plastic beads were coated with an epoxy / enzyme mixture according to Example 1. Skim milk diluted to 12% protein was added to each reactor. After incubation at ambient temperature for 4 hours, the samples were removed and diluted acetic acid was precipitated by addition of dilute acetic acid. The results are shown in Table 2 below:

Test substance Hydrolysis Rate (%) Control (no enzyme) 0 Epoxy + Furafect 2000G 100 Puraffect 2000G Coated Bead 80

Thus, reactors containing epoxy / enzyme coatings or enzyme coated beads on the bottom inner surface have been demonstrated to exhibit proteolytic hydrolysis completely or almost completely.

Example 4

The enzymatic reactor prepared according to Example 1 by coating the bottom inner surface of the plastic bottle with epoxy / enzyme (protease) was tested for hydrolysis activity 24 hours and 28 days after preparation to check stability over time. The results are shown in Table 3 below:

Test substance Hydrolysis Rate (%) Control (no enzyme) 0 Epoxy + Furafect 2000 G (24 hours) 100 Epoxy + Furafect 2000G (28 days) 80

Epoxy and enzymes were still only active after 28 days at ambient temperature.

Example 5

The following resins were tested in place of the previously used Devcon epoxy:

1) PC-11 Heavy Epoxy Resin Glue (commercially available from Protective Painting Company) 2) Polypoxy Epoxy Resin Compositions (Underwater Patch Compound 7055 from Pettit Paint Company, Rockaway, NJ) 3) Gel-Coat Polyester resin composition (white gel coat available from Clear Coat Corp., Florida, USA) 4) Bondo Polyester resins and fiberglass materials (commercially available from Dynatron / Bondo Corporation)

All resin / enzyme mixtures were prepared as described in Example 1 and 100 mg of Furaffect 2000G was used as a proteolytic enzyme source. After 2 hours, samples were removed from each reaction mixture. The hydrolysis rate was measured as described in Example 1. The results are shown in Table 4 below:

Test substance Hydrolysis Rate (%) Control (no epoxy or enzyme) 0 PC-11 70 Polyoxy 90 Gel-coat 50 Fiberglass 95

Example 6

Liquid enzymes were studied as components of enzyme-resin formulations. In previous experiments, only protein hydrolysis was considered as a means of hydrolyzing glycoproteins believed to be primary agents in the course of fouling by organisms. In this experiment, both proteolytic and starch hydrolase were tested. Puraffect 4000L (0.5 ml) or Decsize 160 (0.5 ml) was first added to 4.0 g of CaCl ₂ and then mixed with Devcon 5 Minute epoxy to reduce the amount of free water added with the enzyme. . As a result, free water inhibited the solidification of the epoxy resin.

Starch suspension was added to reaction vessels 2 and 4. Diluted milk was added to reaction vessels 1 and 3. After incubation for 2 hours at ambient temperature, the degree of hydrolysis of each mixture was observed according to the method described in Example 1. The compositions and results are shown in Table 5 below:

Test substance result Reaction vessel Epoxy protease Epoxy amylase Starch hydrolysis rate (%) Milk hydrolysis rate (%) No. 1 - - - 0 No. 2 - - 0 - No. 3 0 - - 100 No. 4 - 0 100 -

The reaction mixture in Reaction Vessel 3 was clarified and unreacted even with the addition of dilute acetic acid, indicating that the milk protein was fully hydrolyzed by the CaCl ₂ absorbed enzyme / resin formulation. The reaction mixture containing CaCl ₂ and water was viscous and difficult to swell. The mixture containing the enzyme resin was watery and easy to pour. In each case liquid enzymes have been demonstrated that can be used in epoxy resin formulations. Both starch and protein were hydrolyzed by epoxy / liquid enzyme preparations. Hydrolysis of starch (polysaccharide) or protein was not hindered by adding CaCl ₂ to the enzyme resin formulation.

Example 7

As discussed above, the effect of adding an inorganic salt to the enzyme-epoxy resin formulation was studied to examine how that salt affects the activity of alpha-amylase. This study was first prompted by questions about the effects of calcium chloride desiccants on enzymatic reactivity. Calcium ions are known to be involved in alpha-amylase activity and function at concentrations of about 60 ppm as cofactors. Therefore, the effect of calcium chloride addition on the activity of liquid alpha-amylase was examined.

Sample containers were prepared according to the method of Example 1. To each vessel was added 12 mg of starch together with water and calcium chloride as indicated below. The aqueous mixture was homogenized by stirring. The percent hydrolysis of the mixture was measured as described in Example 1. The results are shown in Table 6 below:

Vessel H ₂ O CaCl ₂ Desize 160 (alpha-amylase) Hydrolysis Rate (%) No. 1 10 ml 0 0.5 ml 10 No. 2 10 ml 1 mg 0.5 ml 80 No. 3 10 ml 2 mg 0.5 ml 80 No. 4 10 ml 4 mg 0.5 ml 90 No. 5 10 ml 8 mg 0.5 ml 0 ^* Excess calcium chloride produces an elastic, flexible and moldable polymer. This suggests an interaction between starch and divalent ions.

The catalytic activity of liquid alpha-amylase has been shown to be greatly improved by adding calcium chloride in an amount greater than that required for simple activation of the enzyme. Another mechanism for improving alpha-amylase catalytic activity appears to be indicated.

Example 8

Other inorganic salts were also examined to see if they improved the catalytic activity of alpha-amylase. Two solutions were chosen: sodium chloride, a neutral monovalent salt, and magnesium chloride, a divalent acid salt capable of forming a hydrate.

The following results were obtained using the procedure described in Example 7:

Vessel MgSO ₄ NaCl Desize 160 Hydrolysis Rate (%) No. 1 - - - 0 No. 2 - - 0.5 ml 10 No. 3 0.5 mg - 0.5 ml 30 No. 4 1.0 mg - 0.5 ml 30 No. 5 2.0 mg - 0.5 ml 70 No. 6 4.0 mg - 0.5 ml 90 No. 7 - 0.5 mg 0.5 ml 50 No. 8 - 1.0 mg 0.5 ml 90 No. 9 - 2.0 mg 0.5 ml 90 No. 10 - 4.0 mg 0.5 ml 100

Example 9

Instead of using encapsulated or liquid enzymes as the enzymatic active source, either growth cells or resting cells can be used. For example, liquid suspensions of spores or growing cells of various uses capable of producing alpha-amylases and / or proteases have been prepared by Sybron Corporation.

Therefore, the spores and cell suspensions of Sibron embedded in the gel-coat were examined. Calcium chloride was included in the epoxy resin mixture because the reaction vessel was used to test the activity of both alpha-amylase and protease in the same reactor. Enzymatic activity of alpha-amylase and protease was compared to the activity of cell and spore suspensions.

The spore suspension used was commercially available from Sybron Chemicals Inc., Salem Kessler Mill Road 111, Virginia, USA, and contains a spore form of Bacillus polymyxa . It was B +.

Cell suspensions used are commercially available from Sibronn Chemicals, Inc., 25143 Salem Kessler Mill Road 111, Virginia, USA, Bacillus subtilis , Pseudomonas Aeruginosa , Pseudomonas Aeruginosa , Pseudomonas putida ( Pseudomonas putida , Pseudomonas fluoresens and Escherichia hermanii microorganisms, including the growth forms of the microorganisms.

A sample vessel was prepared according to the method of Example 1 and the percent hydrolysis of starch and milk solution was measured using the procedure described in Example 1:

Milk hydrolysis rate (%) Starch hydrolysis rate (%) 1 Bio P 100 0 2 bio B + 100 0 3 size 160 0 90 4 Furaffect 2000G 100 50 5 Barracks 15,000 0 90 6 milk control 0 - 7 starch control - 0

Although alpha-amylase activity was not detected in this assay using spores or growing cells, cell or spore suspensions are expected to provide this activity.

The activity of the protease (Purafect 2000G) on starch suggested that the enzyme was contaminated by alpha-amylase.

Example 10

Both encapsulated alpha-amylase and liquid alpha-amylase embedded in polyester resins and glass fiber materials (as shown in Example 5, above) were tested for starch hydrolysis catalytic activity. Magnesium sulfate was added to both encapsulated alpha-amylase and liquid alpha-enzyme preparations. Each enzyme was added at a concentration approximately normalizing its activity.

The reaction vessel was prepared according to the procedure of Example 5 and the results are shown in Table 9 below:

Reaction vessel MgSO ₄ (Liquid) des 160 Encapsulated Barracks 5000 Liquid Bark Mill 15,000 No. 1 5.0 mg - - - No. 2 - - - - No. 3 0.5 ml - - No. 4 - ^* 2.0 mg - No. 5 - - - 0.5 ml No. 6 5.0 mg 0.5 ml - - No. 7 5.0 mg - ^* 2.0 mg - No. 8 5.0 mg - - 0.5 ml * 0.1 ml of H ₂ O was added before embedding in resin.

The vessel was cured overnight. 10 ml of water and 11.5 g of starch were added to each vessel and the mixture was homogenized by stirring. After 60 minutes the percent hydrolysis was measured according to the procedure of Example 1 and the results are shown in Table 10 below:

Sample Hydrolysis Rate (%) One 10 2 10 3 10 4 0 5 10 6 50 7 80 8 70

Example 11

To explore the concept of using microorganisms to protect the seabed from fouling by marine organisms, fiberglass plates were coated with a mixture of microorganisms embedded in several different coating materials as described in Table 11 below. The manufacturing process is as follows:

Twenty-one panels made of fiberglass were sanded by hand using 60 grit sandpaper and wiped with xylene and paper towels to remove residue. The long panel has dimensions of 17 7/8 x 5 7/8 x 1/8 inch thick (454 mm x 149 mm x 3.2 mm), and the short panel is 13 7/8 x 5 7/8 x 1/8 inch It has dimensions of 352 mm x 148 mm x 3.2 mm.

The microorganisms according to Table 11 below were added to the resin base and blended by hand shaking. The unit of amount specified in Table 11 below was 100 times ounce. Control panel "C" was not coated with the test composition.

Dura Shine is a liquid polymer disclosed in US Pat. No. 5,073,407 and commercially available from Howe Labs, Eden, NY, USA. Turtle Wax Finish 2001 Liquid (Finish 2001) is a urethane-containing silicone resin available from Turtle Wax, Chicago, Illinois, USA. Glidden Latex is a non-toxic acrylic latex creep paint commercially available from Glidden Paints, Jacksonville, FL.

Bio B + was obtained from Sibron Chemicals, Inc., 25143 Salem Kessler Mill Road 111, Virginia, USA. This mixture included spore forms of Bacillus polymix, Bacillus subtilis and Bacillus Lichenformis .

Bio P was also obtained from Sibronn Chemicals, Inc., 25143 Salem Kessler Mill Road 111, Virginia. This included growth forms of Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens and Escherichia hermani microorganisms.

The coating mixture was then applied via a synthetic paint brush by hand using a 2 inch brush for poly paint. The panel was aerated for 24 hours before another coating was applied. The panels were ventilated for 48 hours and wrapped in paper towels and transferred to the marine test site.

Marine Experiment Protocol Plate number Bio B + Bio P Dura Shine Finish 2001 Gliden Latex One ^* 25 25 200 - 2 - 50 200 - 3 50 - 200 - 4 25 25 - 200 5 - 50 - 200 6 50 - - 200 7 25 25 - - 200 8 - 50 - - 200 9 50 - - - 200 10 50 50 100 - 100 * 100 times ounce

Upon arrival, the panels were attached to PVC racks and suspended in a floating barge into the marine environment, where they were continuously immersed in seawater with appropriate algae. Plates were immersed in seawater for 4 months at two different levels (ie top layer = waterline, bottom layer = complete immersion). The amount of seaweed accumulated each month was measured and recorded in Table 12 below:

% Shield of seaweed Upper layer Lower layer Panel number ℃ Panel number ℃ One 2 3 4 5 6 7 8 9 10 One 2 3 4 5 6 7 8 9 10 January 20 50 30 60 25 25 30 35 30 35 40 January 3 2 One 70 65 70 40 25 10 15 10 February 15 25 20 40 15 15 30 30 30 35 35 February 3 2 2 45 40 45 35 30 15 20 15 In March 12 20 12 15 7 7 35 30 25 35 35 In March 4 3 3 20 25 30 40 35 30 20 25 April 3 3 2 2 One One 35 35 25 40 30 April 5 5 5 7 2 5 45 40 35 30 30 * = Control plate (untreated)

Table 12 shows the panel area percentages fouled by seaweed on monthly (January to April) and panel by panel (1 to 10 and control) for the upper and lower layer racks. The results are also shown in FIGS. 1-6.

These results demonstrate that the selection of appropriate microorganisms can achieve and maintain antifouling properties in coatings. For example, the Dura Shine coating had an average seaweed fouling area of 2.67% in the top layer compared to 30% for the control. Finish 2001 coatings had an average seaweed fouling area of 1.33% in the top layer compared to 30% for the control.

Tables 13 and 14 below show the area percentages fouled by enveloped moss as a representative example of a hard bottom adherent colony after a four month soaking period. Lichen bug growths were inhibited 33-100% by application of a coating surrounded by microorganisms. In addition, dark green raw film was observed to develop on the plate. Such live films are believed to reflect major microbial films that protect against soft and hard growth. This is consistent with the fact that the most effective coating formulations, Dura Shine and Finish 2001, have the highest area ratios masked by fresh film, which demonstrates the growth of added protective microbial colonies suspended in the coating.

Upper layer (4 months) Plate number Enveloped moss (%) Raw film (%) One One 80 2 One 70 3 One 85 4 0 75 5 One 65 6 0 75 7 One 55 8 3 55 9 2 65 10 5 25 C 5 55

Inferior layer (4 months) Plate number Enveloped moss (%) Raw film (%) One 3 85 2 One 75 3 3 80 4 3 85 5 2 65 6 3 75 7 5 35 8 5 40 9 7 40 10 12 25 C 7 55

Tables 15 and 16 below show the total area percentage of panels that remain antifouling (soft and hard growth) when examining the plates. Data are shown monthly (January to April) and panel by panel (1 to 10 and control) for the top layer and bottom layer racks:

Upper layer Plate number One 2 3 4 5 6 7 8 9 10 ℃ January 77 45 65 38 74 69 68 63 67 55 48 February 82 69 75 58 84 83 67 66 64 51 52 In March 82 70 79 81 90 90 61 64 69 47 53 April 91 84 86 93 95 96 58 55 66 29 59

Lower layer Plate number One 2 3 4 5 6 7 8 9 10 ℃ January 77 73 89 28 30 28 45 50 70 75 65 February 80 80 89 52 54 53 51 66 66 63 68 In March 82 82 87 76 69 67 43 61 51 54 62 April 86 89 84 86 91 89 35 42 47 28 56

These results show that remarkably good antifouling performance can be achieved by using the compositions and / or paints according to the invention as compared to untreated controls.

Example 12

The effect of the present invention on hard growth in the polyurethane resin mixture was tested. The test mixture contained various combinations of spores, enzymes and growing cells. The procedure used is as follows.

A panel was prepared according to the procedure of Example 11. The panels were then split longitudinally using plastic tape to provide two test surfaces per panel. Using 4 ounces of polyurethane resin, commercially available as Polyurethan Clear Gloss 603 from Behr Process Corp., Antana, Calif., As shown in Table 17. The coating mixture was prepared by adding 0.25 ounce of cells and / or enzyme each. The mixture was then mixed by hand and applied by hand to the panel as shown in Table 17. Spores, growth cells and / or enzymes used are the same as those used in Example 11.

Indentation number Plate number Bio P Bio B + Alpha-amylase Protease 49 Control - - - - 45 3A - - - 0.25 √ 38 3B - - 0.25 0.25 √ 37 4A 0.25 - 0.25 0.25 √ 38 4B 0.25 0.25 0.25 0.25 46 5A - 0.25 - - 51 5B 0.25 0.25 - - √ 28 6A 0.25 - - - √ 27 6B - - 0.25 - √ 28 7A 0.25 0.25 0.25 - √ 37 7B 0.25 0.25 - 0.25 √ = most effective mixture

The amount of spores, growing cells and / or enzymes is expressed in ounces.

After applying the coating the panels were suspended in PVC pipe racks for 24 hours to air dry. The finished panels were then transferred to test equipment. The panel was attached to the PVC pipe frame in the equipment. Attachment techniques using plastic ties resuspend the panels at the corners from PVC frame work. After all panels were attached to the PVC frame, the frames were suspended in water so that the panel lined up was approximately 6 inches below the water surface. The PVC frame was unwound and suspended in a floating breakwater. Thus, the panel maintained the same relative position in water throughout the experiment.

The panels were submerged for 3 months. During the first month, the panel was exposed to low speed water, about three knots, once a week. The panel was not dried at all. After exposure, the PVC pipe frame was suspended in water to its original depth.

The test was terminated after 4 months. At the end of the first month, the number of oyster lamps, mussels, oysters, moss worms and grass growths was recorded. The counted area was limited to three square inches from the top of the test panel. The results are shown in Table 17 above. The data shows that the average number of oysters in the test area was 38.5 and the average number of controls was 49. This represents a 21% reduction and demonstrates that the present invention is useful for inhibiting hard growth. Grass and moss masked about 40% of the test area. For the next three months, the growth of the test area remained essentially the same while the area of the control continued to grow (ie, laminar growth) even at a thickness of 3/8 inch above 100% surface. Moreover, soft growth continued to decrease over time in the test area and only 10% of the area had soft growth at the end of the test. This strongly demonstrates that the present invention is useful for inhibiting both soft and hard growth.

Example 13

The invention was carried out according to the following examples to further demonstrate the invention. In this example, two liquid polymers were examined instead of using an epoxy resin as a landfill for catalytically active cells and enzymes.

The test coating was blended by hand shaking. The components for each coating mixture are as shown in Table 18 below (specified measurements are in g). Finish 2001, Dura Shine, Bio B + and Bio P are the same as those described in Example 11. The protease used was Puraffect 4000L as described in Example 6.

The coating mixture was applied to a 1 × 1 1/2 inch cross section of the wood blade and air dried for 60 minutes. The coated wood blade was then washed with running water to remove the unattached coating. The coated blade was submerged in 25 ml of 1: 4 diluted skim milk for 2 hours. The hydrolysis rate was measured according to the method of Example 1. The results are shown in Table 18 below:

Sample Finish 2001 ^* Dura Shine ^* Protease ^* Bio B + ^* Bio P ^* Hydrolysis Rate (%) One 4.8 0 0.56 0 0 70 2 4.8 0 0 0.56 0 20 3 4.8 0 0 0 0.56 0 4 4.8 0 0.56 0.56 0 70 5 4.8 0 0.56 0 0.56 50 6 0 4.8 0.56 0 0 50 7 0 4.8 0 0.56 0 0 8 0 4.8 0 0 0.56 10 9 0 4.8 0.56 0.56 0 60 10 0 4.8 0.56 0 0.56 80 11 4.8 0 0 0 0 0 12 0 4.8 0 0 0 0 * = measured in g

It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the invention without departing from the spirit of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims (29)

Base materials selected from the group consisting of epoxy materials, polyurethane materials, polyester materials, fiberglass materials, silicone materials and acrylic materials; And One or more microorganisms that produce one or more starch hydrolase or proteolytic enzymes, and one or more starch hydrolase or proteolytic enzymes, mixed with the base material Wherein the enzyme and microorganism are present in an amount effective to reduce or prevent fouling of the vessel surface coated with the marine antifouling composition. A marine product coated with the composition of claim 1. A method of reducing fouling of a ship's surface comprising coating the ship's surface with a composition for ship antifouling according to claim 1 which reduces the fouling of the coated ship's surface. Paint base materials suitable for ships, selected from the group consisting of epoxy materials, polyurethane materials, fiberglass materials, polyester materials, silicone materials and acrylic materials; Pigments; And One or more microorganisms that produce one or more starch hydrolase or proteolytic enzymes, and one or more starch hydrolase or proteolytic enzymes, mixed with the paint base material Wherein the enzyme and microorganism are present in an amount effective to reduce or prevent fouling of the vessel surface coated with the marine antifouling paint. Marine products coated with the paint of claim 4. A method of reducing fouling of a ship's surface, comprising coating the ship's surface with a paint according to claim 4 which reduces the fouling of a coated ship's surface. A method of reducing corrosion of a vessel, comprising coating the surface of the vessel with the composition according to claim 1, wherein the composition forms one or more films that reduce the adsorption of corrosive substances on the vessel surface. The method of claim 7, wherein The composition inhibits surface corrosion and intergranular corrosion. A method of reducing corrosion of a ship, comprising coating the surface of the ship with the paint according to claim 4, wherein the paint forms one or more films that reduce the adsorption of the corrosive material on the surface of the ship. The method of claim 9, How paint inhibits surface and intergranular corrosion. A method of limiting adsorption of water by a vessel surface, comprising coating the vessel surface with the composition according to claim 1, wherein the composition forms a film to reduce the porosity of the surface. A method of limiting adsorption of water by a ship surface, comprising coating the ship surface with a paint according to claim 4, thereby reducing the porosity of the surface by forming a film. A method of reducing the drag coefficient of a ship surface comprising covering the ship surface with a composition according to claim 1. The method of claim 3, wherein A method of producing surfactants in which microorganisms act as wetting agents. A method of reducing the drag coefficient of a ship's surface comprising covering the ship's surface with a paint according to claim 4. The method of claim 6, A method of producing surfactants in which microorganisms act as wetting agents. The method of claim 1, A marine antifouling composition comprising an inorganic salt present in a catalytically effective amount. The method of claim 4, wherein A marine antifouling paint containing an inorganic salt present in a catalytically effective amount. A method of reducing the propensity of a cavitation under load, comprising covering the surface of the propeller with the marine defense composition of claim 1. A method of reducing the propensity of a propeller cavitation under load, comprising covering the surface of the propeller with the composition for marine antifouling of claim 4. A method for reducing fungal fungus on a ship's surface, comprising coating the ship's surface with the composition according to claim 1, wherein the composition forms one or more films that reduce the adsorption of fungal fungi on the ship's surface. Polymeric resin base materials; And One or more microorganisms that produce one or more starch hydrolase or proteolytic enzymes in the underlying material And wherein the microorganism is present in an amount effective to reduce or prevent fouling of the vessel surface coated with the marine antifouling composition. The method of claim 30, A composition for ship antifouling, wherein the polymer resin base material is selected from the group consisting of epoxy material, polyurethane material, polyester material, fiberglass material, silicone material and acrylic material. The method of claim 30, A composition for marine antifouling, wherein the microorganism also produces a surfactant that acts as a wetting agent. Paint base materials suitable for the shipbuilding industry; Pigments; And One or more microorganisms that produce one or more starch hydrolase or proteolytic enzymes in the paint base material Wherein the microorganisms are present in an amount effective to reduce or prevent fouling of the vessel surface coated with the marine antifouling paint. The method of claim 33, wherein Paint The antifouling paint selected from the group consisting of epoxy, polyurethane, polyester, glass fiber, silicone and acrylic materials. The method of claim 33, wherein Marine antifouling paints which also produce surfactants in which microorganisms act as wetting agents. The method of claim 30, Ship antifouling composition further comprising at least one starch hydrolase or proteolytic enzyme in the base material. The method of claim 33, wherein A marine antifouling paint further comprising at least one starch hydrolase or proteolytic enzyme in the paint base material.