# Patent

## Title
Method for reducing cyanuric acid in recreational water systems

## Abstract

The present invention provides compositions and methods of reducing cyanuric acid levels in recreational water systems.

## Background
RELATED APPLICATIONS
This application claims priority to and benefit of U.S. Provisional Application No. 62/073,335, filed in Oct. 31, 2014 and U.S. Provisional Application No. 62/101,741, filed on Jan. 9, 2015, the contents of which are hereby incorporated by reference in their entireties.


INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
The contents of the text file named “BIOW-013/001US_Sequence Listing_ST25.txt”, which was created on May 12, 2015 and is 3 KB in size, are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
The present invention relates to a method for treating recreational water systems with compositions comprising an oxidizable carbon species and micro-organisms, in order to reduce cyanuric acid levels in said water systems.
BACKGROUND OF THE INVENTION
Recreational water systems such as swimming pools, spas, hot tubs, and jetted tubs, are commonly treated with chlorinated derivatives of cyanuric acid (1,3,5-triazine-2,4,6(1H,3H,5H)-trione) in order to disinfect the water and maintain sanitary conditions. The action of these chlorinated cyanuric acid derivatives, typically referred to by the trade names di- or trichlor, is attributed to the generation of free chlorine as HOCl and OCl— arising from the hydrolytic equilibria of the various chlorinated species (O'Brien et al., 1974). When used in this way there is a gradual accumulation of residual cyanuric acid in the water (Downes et al., 1984). As the level of cyanuric acid rises, free chlorine's ability to act as a disinfectant is weakened due to increased complexation of chlorine. Above about 50 ppm cyanuric acid, the time it takes to kill bacteria in chlorinated water increases versus similarly treated water without cyanuric acid. In heated systems, such as hot tubs and spas, at even moderate levels of cyanuric acid the amount of time it takes chlorine to kill a common pathogen such as pseudomonas aeruginosa can be as much as one hundred times as long as similar systems without cyanuric acid.
A 2007 study by the United States Centers for Disease Control and Prevention (Shields et al., 2007) revealed that cyanuric acid significantly diminishes chlorine's ability to inactivate chlorine-resistant porotozoan and cryptosporidium. Based on these findings several state and local Departments of Health have issued recommendations to the recreational water industry that cyanuric acid levels not exceed 30 ppm.
It is a common practice in the recreational water industry to reduce excess cyanuric acid levels by partially draining pools, tubs, spas, holding tanks, etc., and refilling with fresh water. This is a labor intensive and costly solution, particularly in areas affected by prolonged drought such as Southern California where the cost to replenish a typical 20,000 gallon swimming pool with fresh water is prohibitively high. Accordingly, a need exists in the recreational water industry for compositions and methods to reduce excess cyanuric acid levels that do not require a draining and replenishing
SUMMARY OF THE INVENTION
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In cases of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing significant denitrification was observed within the first 24 hours of dosing the water with the composition of the invention.
FIG. 2 is a series of figures showing increased water clarity after dosing the water with the composition of the invention.
FIG. 3 is a graph showing decreased cyanuric acid with the composition of the invention.
FIG. 4 shows results from MS-MS analysis of in-vitro samples of cyanuric acid treated with the compositions of the invention.
FIG. 5 are growth curves showing that organisms can use cyanuric acid as a nitrogen source for growth.
FIG. 6 illustrate that Composition B from Example 1 degrades cyanuric acid.
FIG. 7 illustrates that with Composition B from Example 1 degrades cyanuric acid in water from public swimming pools
FIG. 8 illustrates that pools from southern California treated with Composition B from Example 1 have a drop in cyanuric acid concentration over time after treatment.
FIG. 9 shows the results from Florida field trials using Composition B from Example 1.
FIG. 10 shows the results comparing Compositions A, B, and C from Example 1 in actual swimming pools.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides compositions and methods for augmenting the treatment of commercial, public, and private recreational water systems such as swimming pools, spas, hot tubs, jetted tubs or the like. The composition an methods result in increased clarity of the water, decreased nitrate concentrations, decreased cyanuric acid levels, decreased biological oxygen demand (BOD), decreased total suspended solids (TSS), decreased total Kjeldahl nitrogen (TKN) and decreased fats, oils and grease (FOG) in the water. In specific embodiments, the compositions and methods are used to reduce cyanuric acid levels in recreational water systems where cyanuric acid stabilized chlorine is used as part of the routine disinfection and sanitization protocol.
In some aspects the compositions are microbial compositions. The microbial compositions are in powdered, dried form. Alternatively, the microbial compositions are in liquid form. For example, the composition includes non-pathogenic bacteria with the ability to degrade cyanuric acid.
In certain embodiments the bacteria are derived from the genus Bacillus, Lactobacillus, Pseudomonas, or Moorella. In other embodiments the composition contain a mixture of Bacillus or Lactobacillus In other aspects the microbial compositions contain a mixture of Bacillus and Lactobacillus bacteria. In various aspects the mixture contains at least one to seven different strains of Bacillus. The mixture contains at least one to four different strains of Lactobacillus. Optionally, the microbial compositions further include an oxidizable carbon source and/or a mixture of inorganic minerals commonly used to stimulate microbial growth.
In another aspect the composition contain containing an oxidizable carbon source and a mixture of inorganic minerals. The oxidizable carbon source is water soluble or water dispersible.
In some aspects the composition comprises at least 50%, preferably at least 75%, and most preferably at least 90% by weight of a water soluble or water dispersible oxidizable carbon source.
Preferred strains of Bacillus include for example, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium, Bacillus coagulans, or Paenibacillus polymyxa. Preferred Lactobacillus stains includes for example, Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, or Bifidobacterium animalis. 
In a preferred embodiment the Bacillus is subspecies Mojavenis. In particularly preferred embodiments, the Bacillus is Bacillus subtilis strain 34KLB (SEQ ID NO: 1):







Bacillus subtilis strain 24KLB


 


(SEQ ID NO: 1)


AGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCTTAGA


 


AAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTC


 


ACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTC


 


ACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAA


 


GGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATT


 


CCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATT


 


TGTGRGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTG


 


TAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCC


 


CCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAA


 


TGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACA


 


TCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCC


 


GAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAA


 


GGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGG


 


CCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGG


 


AGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGGCGGAAACCCCCTAACAC


 


TTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCG


 


CTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTT


 


CGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAA


 


TTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCC


 


CGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCC


 


TTTACGCCCAATAAtTCCGGACAACGCTTGCCACCTACGTATTACCGCGGC


 


TGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCC


 


GCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCC


 


GAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTG


 


CGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGT


 


CCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGT


 


GAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTG


 


GTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCC


 


GGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACC


 


CACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCT


 


GTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCC


 


ATGAACAAACTCTAAGGGCGAATTCTGCAGATATCCATCACACTGGCGGCC


 


GCTCGAGCATGCATCTAGAGGGCCCAATCGCCCTAT






In some aspects the microbial composition comprises a mixture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus pumilus. In another aspect the microbial composition comprises a mixture of Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum. 
In some aspects the composition comprises at least 50%, preferably at least 75%, and most preferably at least 90% by weight of a water soluble or water dispersible oxidizable carbon source. In some embodiments the composition comprises at least 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or 100% by weight of a water soluble or water dispersible oxidizable carbon source.
Suitable water soluble oxidizable carbon sources include carbohydrates, proteins, polysaccharides or mixtures thereof. In preferred embodiments the water soluble carbon source comprises glucose, dextrose, fructose, erythrose, arabinose, ribose, deoxyribose, galactose, mannose, sucrose, lactose, maltose, dextrin, maltodextrin, glycerol, sorbitol, xylitol, inulin, trehalose, low molecular weight starches, modified starches, cellobiose, modified celluloses, amino acids, water soluble peptides, or mixtures thereof.
Suitable water dispersible carbon sources include emulsified fats and oils. In certain preferred embodiments the water dispersible carbon source comprises soy lecithin, emulsified vegetable oil or mixtures thereof. Other embodiments include mixtures of water soluble and water dispersible oxidizable carbon sources.
Suitable minerals include disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, magnesium sulfate, calcium sulfate, magnesium chloride, calcium chloride, and iron(III) chloride. The minerals comprise between 1 to 50%, 10 to 50%, 20 to 50%, 30 to 50% or 40 to 50% by weight of the composition. Preferably, the minerals comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% by weight of the composition
A preferred composition according to the invention includes about 50% by weight of a water soluble or water dispersible oxidizable carbon source and about 50% by weight of a mixture of inorganic minerals. Preferably, the water soluble or water dispersible oxidizable carbon source is dextrose and the minerals include disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, and iron(III) chloride.
Another preferred composition according to the invention includes at least 94% by weight of a water soluble or water dispersible oxidizable carbon source and the remainder by weight of a microbial mixture. The microbial mixture is a mixture of Bacillus, Lactobacillus or both. In some aspects the microbial composition comprises a mixture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus. In another aspect he microbial composition comprises Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis Bacillus pumilus, Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum. 
A first preferred Bacillus mixture includes 10% by weight Bacillus licheniformis, 30% by weight Bacillus pumilus, 30% by weight Bacillus amyloliquefaciens and 30% by weight Bacillus subtilis. (referred to herein as Bacillus Mix #1)
A second preferred Bacillus mixture includes equal weights of Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens and Bacillus subtilis. (referred to herein as Bacillus Mix #2)
A preferred Lactobacillus mixture includes equal weights of Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum. (referred to herein as Lactobacillus Mix #1)
Another preferred composition according to the invention includes at least 94% by weight of a water soluble or water dispersible oxidizable carbon source and about at least 0.1 to 1%, 0.1 to 2%, 0.1 to 3%, 0.1 to 4%, 0.1 to 5% of Bacillus Mix# 1 and/or of Bacillus Mix# 2. Preferably the composition comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% of Bacillus Mix# 1 and/or of Bacillus Mix# 2. In some embodiments the composition also includes about at least 0.1 to 1%, 0.1 to 2%, 0.1 to 3%, 0.1 to 4%, 0.1 to 5% of Bacillus 34KLB. Preferably the composition comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% 1%, 2%, 3%, 4% or 5% of Bacillus 34KLB.
In yet another preferred composition according to the invention includes at least 94% by weight of a water soluble or water dispersible oxidizable carbon source and about at least 0.1. to 1%, 0.1 to 2%, 0.1 to 3%, 0.1. to 4%, 0.1 to 5% of Bacillus Mix# 1 and Bacillus Mix# 2 and about at least 1 to 5% Lactobacillus Mix #1. Preferably the composition comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% of Bacillus Mix# 1 and Bacillus Mix# 2 and about 1%, 2%, 3% 4% or 5% of Lactobacillus Mix #1.
The levels of the bacteria to be used according to the present invention will depend upon the types thereof. It is preferred that the present product contains bacteria in an amount between about 105 and 1011 colony forming units per gram.
The microbial compositions according to the invention may be produced using any standard fermentation process known in the art. For example, solid substrate or submerged liquid fermentation under conditions which are optimized for growth of each organism. The fermented cultures can be mixed cultures or single isolates.
In some embodiments the bacteria are anaerobically fermented in the presence of carbohydrates. Suitable carbohydrates include inulin, fructo-oligosaccharide, and gluco-oligosaccharides.
When the cell density of the fermentation reaches about 1011−1012 cfu/g, the individual bacteria are harvested. The bacteria may be harvested by any known methods in the art. For example the bacteria are harvested by filtration or centrifugation.
The bacteria are dried by any method known in the art. For example the bacteria are air dried, or dried by freezing in liquid nitrogen followed by lyophilization.
The compositions according to the invention have been dried to moisture content less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, or 1%. Preferably, the composition accord the invention has been dried to moisture content less than 5%.
In some embodiments the dried powder is ground to decrease the particle size. The bacteria are ground by conical grinding at a temperature less than 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° C., 1° C., 0° C., or less. Preferably the temperature is less than 4° C.
For example the particle size is less than 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 microns. Preferably, the freeze dried powder is ground to decrease the particle size such that the particle size is less than 800 microns. Most preferred are particle sizes less than about 400 microns. In most preferred embodiments, the dried powder has a mean particle size of 200 microns, with 60% of the mixture in the size range between 100-800 microns. In various embodiments the freeze dried powder is homogenized.
In various embodiments the microbial compositions are mixed with an inert carrier such anhydrous dextrose, dextrose monohydrate, dendritic salt, rice bran, wheat bran, oat bran, soybean meal, rice hulls, or a mixture thereof.
The inert carrier is at a concentration of at least 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. Preferably, the inert carrier is at a concentration of about between 75-95% (w/w).
In other aspects the bacterial compositions contain an organic emulsifier such as, for example, soy lecithin. The organic emulsifier is at a concentration of about 1%, 2%, 3%, 4%, 5%, 5, 7%, 8%, 9% or 10%. Preferably, the organic emulsifier is at a concentration of between 2 to 5% (w/w).
In other aspects the microbial compositions are mixed in equal proportion and added to an oxidizable carbon source. Typically, the final concentration of bacteria in the finished composition ranges from 105 to 1011 cfu/g.
Further, if desired, the microbial compositions may be encapsulated to further increase the probability of survival; for example in a sugar matrix, fat matrix or polysaccharide matrix.
Importantly, the compositions of the invention fully disperses upon the addition to water and unlike other water treatment microbial compositions the compositions do not require a pre-activation of the bacteria, prior to use.
The compositions of the invention are used to treat both fresh and salt water commercial, municipal, industrial, and residential swimming pools, spas, hot tubs, jetted tubs and the like.
An aqueous solution of the dry composition according to the invention can be employed to increase clarity of the water, decrease nitrate concentrations, cyanuric acid levels, biological oxygen demand (BOD), total suspended solids (TSS), total Kjeldahl nitrogen (TKN) and fats, oils and grease (FOG) in body of water. The compositions of the invention may also be used to treat swimming pools and the like to remove scum and reduce algae.
Solutions of the composition can be pumped into the system to be treated or sprayed onto the surface, or into the airspace surrounding the material, or applied to a filter or other solid support through which the water to be cleaned is passed. The dry material can be mixed into a slurry or solution at the point of application and applied in a similar manner.
The invention includes methods for treating a recreational water system by contacting the water system with a composition having an oxidizable carbon source. The carbon source is water soluble or water dispersible. The method results in decreased cyanuric acid levels.
Solutions of the composition can be pumped into the system to be treated or sprayed onto the surface, or into the airspace surrounding the material, or applied to a filter or other solid support through which the water to be cleaned is passed. The dry material can be mixed into a slurry or solution at the point of application and applied in a similar manner.
The water system is dosed at a concentration range of about 0.01 to 100 ppm, preferably from 0.1 to 10 ppm.
The term “microbial, bacteria” or microbes” as used herein, refers to micro-organisms that confer a benefit. The microbes according to the invention may be viable or non-viable. The non-viable microbes are metabolically-active. By “metabolically-active” as used herein is meant that they exhibit at least some respiration or residual enzyme, or secondary metabolite activity characteristic to that type of microbe.
By the term “non-viable” as used herein is meant a population of bacteria that is not capable of replicating under any known conditions. However, it is to be understood that due to normal biological variations in a population, a small percentage of the population (i.e. 5% or less) may still be viable and thus capable of respiration and/or replication under suitable growing conditions in a population which is otherwise defined as non-viable.
By the term “viable bacteria” as used herein is meant a population of bacteria that is capable of respirating and/or replicating under suitable conditions in which respiration and/or replication is possible. A population of bacteria that does not fulfill the definition of “non-viable” (as given above) is considered to be “viable”.
The term “recreational water system” as used herein is meant to include swimming pools, spas, hot tubs, jetted tubs or the like, and includes both salt water and fresh water systems.
“Treating” as used herein means inoculating water with an oxidizable carbon source and/or microbes designed to enhance efficient degradation of organic matter, cyanuric acid or both.
The compositions of the invention are manufactured by any method suitable for production of bacterial compositions. Preferably, mixtures of bacteria containing Bacillus, Lactobacillus, Pseudomonas, or Moorella., are manufactured by individually aerobically or anaerobically fermenting each organism; harvesting each organism; drying the harvested organisms, grinding the dried organisms to produce a powder combining each of the organisms into the final mixture. The Bacillus organisms are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium, Bacillus coagulans, or Paenibacillus polymyxa. The Lactobacillus comprises Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, or Bifidobacterium animalis. The mixture has a moisture content of less than about 5%; and a final bacterial concentration of between about 105-1011 colony forming units (CFU) per gram of the composition.
Unless stated otherwise, all percentages mentioned in this document are by weight based on the total weight of the composition.
A better understanding of the present invention may be given with the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.
EXAMPLES
Example 1
Preparation of the Microbial Species
The microbial species of the present invention may be made by any of the standard fermentation processes known in the art. In the following examples, both solid state and submerged liquid fermentation processes are described.
Solid State Fermentation—Bacillus Species
Individual purified isolates of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium, Bacillus coagulans, and Paenibacillus polymyxa were grown-up in separate fermenters using standard aerobic submerged liquid fermentation protocols. The individual organisms were recovered from the fermenters via centrifugation, mixed together in equal proportions on a weight basis, then added to the following mixture: 1 part inulin, 2.2 parts isolated soy protein, 8 parts rice flour with 0.25% w/w sodium chloride, 0.045% w/w Calcium carbonate, 0.025% w/w Magnesium sulphate, 0.025% w/w Sodium phosphate, 0.012% w/w Ferrous sulphate and 29.6% water. This mixture was allowed to ferment for up to 5 days at 30° C. Upon completion of the fermentation, the entire mixture was freeze dried to a moisture content less than 5%, ground to an average particle size of 295 microns, with 60% of the product in the size range between 175-840 microns, and homogenized. The final microbial concentration of the powdered product is between 109 and 1011 CFU/g.
Submerged Liquid Fermentation—Bacillus Species
Individual starter cultures of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium, Bacillus coagulans, and Paenibacillus polymyxa are grown according to the following general protocol: 2 grams nutrient broth, 2 grams AmberFerm (yeast extract), and 4 grams Maltodextrin are added to a 250 ml Erlenmeyer flask. 100 milliliters distilled, deionized water was added and the flask stirred until all dry ingredients were dissolved. The flask was covered and placed for 30 min. in an Autoclave operating at 121° C. and 15 psi. After cooling, the flask was inoculated with 1 ml of one of the pure microbial strains. The flask was sealed and placed on an orbital shaker at 30° C. Cultures were allowed to grow for 3-5 days. This process was repeated for each of the micro-organisms in the mixture. This process provided starter cultures of each organism which were then used to prepare larger scale fermentations.
Individual fermenters were run under aerobic conditions at pH 7 at the temperature optimum for each species:














Microbe
Temperature Optimum









Bacillus subtilis
35° C.



Bacillus amyloliquefaciens
30° C.



Bacillus licheniformis
37° C.



Bacillus coagulans
37° C.



Bacillus megaterium
30° C.



Bacillus pumilus
32° C.



Paenibacillus polyniyxa
30° C.










Solid State Fermentation—Lactobacillus 
Individual purified isolates of Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, and Bifidobacterium animalis were grown-up in separate fermenters using standard anaerobic submerged liquid fermentation protocols. The individual organisms were recovered from the fermenters via centrifugation, mixed together in equal proportions on a weight basis, then added to the following mixture: 1 part inulin, 2.2 parts isolated soy protein, 8 parts rice flour with 0.25% w/w sodium chloride, 0.045% w/w Calcium carbonate, 0.025% w/w Magnesium sulphate, 0.025% w/w Sodium phosphate, 0.012% w/w Ferrous sulphate and 29.6% water. This mixture was allowed to ferment for up to 5 days at 30° C. Upon completion of the fermentation, the entire mixture was freeze dried to a moisture content less than 5%, ground to an average particle size of 295 microns, with 60% of the product in the size range between 175-840 microns, and homogenized. The final microbial concentration of the powdered product is between 109 and 1011 CFU/g.
Submerged Liquid Fermentation—Lactobacillus 
Individual, purified isolates of Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, and Bifidobacterium animalis were grown-up in separate fermenters using standard anaerobic submerged liquid fermentation protocols. After fermentation the individual cultures were filtered, centrifuged, freeze dried to a moisture level less than about 5%, then ground to a mean particle size of 295 microns, with 60% of the product in a size range between 175-840 microns. The individual dried microbial cultures were then mixed in equal proportion by weight to obtain the microbial composition of the present invention. The final microbial concentration of the mixed powdered product is between 109 and 1011 CFU/g.
Example 2
Formulation of Swimming Pool Treatment Products
The following formulations were prepared by dry blending the ingredients in a ribbon blender (all percentages are by weight):










COMPOSITIONS















Ingredients
A
B
C
D
E
F
G
H





Microbial
5
5
10
10
25
25




Composition from










Example 1










Bacillus subtilis






1
10


34KLB produced










via submerged










liquid fermentation










according to










Example 1 B










Monohydrate
95

90

75

99
90


Dextrose










Nutri-Sure TM

95

90

75









Example 3
Denitrification of Swimming Pool Water
The composition of Example 2H was used to reduce the nitrate level in a swimming pool. 100 grams of the composition of Example 2H were added to the skimmer of a 15,000 gallon swimming pool with initial nitrate level of about 25 ppm. Significant denitrification was observed within the first 24 hours of dosing. FIG. 1.
Example 4
Water Clarification
The conditions of a typical swimming pool (chlorine level, temperature) were mimicked in the laboratory using 10 gallon aquaria fitted with a filter and circulation pump. The composition of Example 2F was evaluated for its ability to clarify the water after addition of 250 ppm Sunscreen. Results are in FIG. 2.
Significant clarification is noted in as little as 24 hours. Separate respirometer analysis with sunscreen as a substrate showed significant CO2 generation and O2 consumption when the composition of Example 2F is added.
Example 5
Biodegradation of Cyanuric Acid
The composition of Example 2E was evaluated for its ability to degrade cyanuric acid. A solution of 1 gram of the composition of Example 2E in 1 L of Deionized Water was prepared and allowed to stand for 40 hours at room temperature. 5 mls of this solution were then added to 500 mls of water containing minimal media and 50 ppm cyanuric acid. The degradation of cyanuric acid was followed by HPLC. Results are shown in FIG. 3
Example 6
Cyanuric Acid Control in Swimming Pools
The composition of Example 2E was evaluated in swimming pool applications. Testing was conducted in 6 fresh water swimming pools and 3 salt water pools in Southern California. 100 grams of the composition of Example 2E were added to each pool and the cyanuric acid level determined as a function of time using a commercially available cyanuric acid test kit. Results are summarized in Table 1:







TABLE 1







Cyanuric Acid Control in Swimming Pools











Cyanuric Acid at
Cyanuric Acid 
% 



T = 0
at 1 week
Reduction













Fresh Water Pool





Pool #1
100 ppm
57.5 ppm
42.5


Pool #2
100 ppm
  65 ppm
35


Pool #3
 50 ppm
  20 ppm
60


Pool #4
100 ppm
67.5 ppm
32.5


Pool #5
>125 ppm 
 100 ppm
>20


Pool #6
100 ppm
  65 ppm
35


Salt Water Pool





Pool #1
>100 ppm 
  40 ppm
>60


Pool #2
>200 ppm 
  60 ppm
>70


Pool #3
>500 ppm 
 120 ppm
>75









Example 7
Preparation of Cyanuric Acid Reducing Compositions
The following compositions were prepared (all percentages are by weight):

















Composition
Composition 
Composition
Composition
Composition
Composition 



A
B
C
D
E
F





















Dextrose
 100%
94.30%
95.0% 
97.0%

50%


Maltodex




94.0%



Bacillus Mix #
0.43%

 1.0%
0.44%




Bacillus Mix #
0.43%

 1.0%
0.44%




Lactobacillus

 4.40%


4.40%



Mix #1








B. Subtilis

 0.44%
 5.0%
 1.0%
0.72%



34KLB








Mineral Mix





50%









Where Bacillus Mix #1 comprises 10% by weight Bacillus licheniformis, 30% by weight Bacillus pumilus, 30% by weight Bacillus amyloliquefaciens, and 30% by weight Bacillus subtilis subspecies Mojavensis and has an activity ≧1011 cuf/g; Bacillus Mix #2 comprises equal weights of Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus subtilis with an activity ≧109 cfu/g; and, Lactobacillus Mix #1 comprises equal weights of Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum at a concentration ≧1010 cfu/g. The Bacillus subtilis 34KLB had an activity ≧1010 cfu/g.
The mineral mix comprises the following:














Mineral
Wt. %









KH2PO4
12%



K2HPO4
31%



Na2HPO4*2H2O
48%



CaCl2*2H2O
 5%



MgSO4*7H2O
 3%



FeCl3*6H2O 
 1%










Example 8
In Vitro Cyanuric Acid Reduction
Compositions A and B from Example 7 were tested for their ability to degrade cyanuric acid in vitro. Stock solutions of Cyanuric acid were prepared by dissolving 100 mg cyanuric acid in 1 liter of hot autoclaved DI water. Compositions A and B were dosed at 1 gram/L into separate 100 mls aliquots of the stock cyanuric acid solutions then incubated in an incubating shaker at 30° C./150 rpm. 10 ml aliquots were pulled from each sample at times 0, 3, 6, 8 and 24 hours then frozen to shut down microbial activity. The frozen samples were analyzed by MS-MS. Results are shown in FIG. 4:
As expected, in sterile systems, addition of an oxidizable carbon source alone (Composition A) is not sufficient to cause reduction of cyanuric acid. However, in combination with select microbial species (Composition B), 65% of the initial cyanuric acid is removed after 24 hours incubation.
Example 3
In-vivo Cyanuric Acid Reduction
Compositions A, B, and C from Example 7 were tested for their ability to reduce cyanuric acid in recreational swimming pools. For this analysis cyanuric acid levels were measured using a test kit common to the pool industry. The kit comprises a plastic tube into which a sample of pool water is added. A reagent solution comprising a low level of melamine is added to the pool water and the sample agitated for 1-2 minutes. Cyanuric acid levels are recorded visually using a dipstick calibrated between 20-100 ppm. 8 Ounces of the compositions from Example 7 were added into the skimmers of three separate pools. Results are shown below:















Pool #1
Pool #2
Pool #3









4.3 ppm 
3.7 ppm 
3.7 ppm



Composition A
Composition B
Composition C



Pool Capacity = 
Pool Capacity = 
Pool Capacity = 



14,000 gal.
16,000 gal.
16,000 gal.



82° F.
80° F.
85° F.



pH: 7.8
pH: 7.6
pH: 7.6



Initial CYA level = 
Initial CYA level = 
initial CYA level = 



+150 ppm
120 ppm
120 ppm



CYA level at 25 
CYA level at 
CYA level at 



hours = 55 ppm
25 hours = 65 ppm
25 hours = 75 ppm



% CYA 
% CYA 
% CYA 



Reduction >63%
Reduction >45%
Reduction >37%










The Compositions were further tested for their ability to reduce cyanuric acid in recreational swimming pools. Results are shown below:


















POOL
TYPE
TEMPERATURE
CHLORINE
ALKALINITY
PH
CYA
FILLER
GALLONS























1
Salt water
START: 55° 24 HR: 58°
START: 2.0
START: 100
START: 7.4
START: 70 3 HR: N/C
DE
20,000



Vinyl liner

24 HR: 1.0
24 HR: 90
24 HR: 7.4
24 HR: 50




2
Fresh water
START: 55° 24 HR: 59°
START: 5 
START: 120
START: 7.2
START: 140-150 3 HR: 
SAND
14,000



Vinyl liner

24 HR: 3.0
24 HR: 120
24 HR: 7.4
100-110 24 HR: 80




3
Salt water
START: 58° 24 HR: 53°
START: 3.0 
START: 90 
START: 7.2
START: 75-80 

20,000



Vinyl liner
48 HR: 55°
24 HR: 3.0 
24 HR: 90
24 HR: 7.4 
24 HR: 45-50







48 HR: 3.0
48 HR: 90
48 HR: 7.4
DAY 5: 35-40




6
Fiber Glass
START: 65° 3 HR: 85°
START: 3.0 
START: 100 
START: 7.6
START: 200+ 3 HR: 100
SAND
6,500




26 HR: 85° 65 HR: 65°
3 HR: 4.0 
3 HR: 120 
3 HR: 7.8 
22 HR: 80-90 26 HR: 70







72 HR: 4
72 HR: 100
72 HR: 7.8
72 HR: 50-70




7
Fiber Glass
START: 60° 3 HR: 62°
START: 2.0 
START: 80 
START: 7.4
START: 200+ 3 HR: N/C
SAND
5,000




24 HR: 58°
3 HR: 2.0 
2 HR: 80
3 HR: 7.2 
24 HR: 90 







24 HR: 2.0
24 HR: 80
24 HR: 7.4
48 HR: 70




8
Cement
START: 58° 24 HR: 58°
START: 5 
START: 120
START: 7.4
START: 300+
SAND
12,000





24 HR: 4
24 HR: 120 
24 HR: 7.4 
24 HR: 100-110







48 HR: 10
48 HR: 100
48 HR: 7.4
48 HR: 80-90




9
Commercial
START: 60° 21 HR: 62°
START: 1.0 
START: 120
START: 7.8
START: 180 
SAND
108,000



Gunnite
72 HR: 60°
21 HR: 2.0 
21 HR: 120 
21 HR: 7.8 
21 HR: 100







72 HR: 2
72 HR: 120
72 HR: 7
72 HR: 60









OTHER EMBODIMENTS
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

## Claims

1. A composition comprising
a. between 75-95% w/w of anhydrous dextrose or dextrose monohydrate;
b. a mixture of Bacillus bacterial species comprising Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus pumilus having a bacterial concentration of at least 1×106 colony forming units (CFU) per gram of the mixture, wherein each of the Bacillus species are individually fermented aerobically, dried and ground to an average particle size of about 200 microns, and wherein the Bacillus subtilis comprises Mojavensis; 
c. a mixture of Lactobacillus bacterial species comprising Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum having a bacterial concentration of at least 1×106 colony forming units (CFU) per gram of the mixture, wherein each of the Lactobacillus species are fermented anaerobically, dried, and ground to an average particle size of about 200 microns.

2. The composition of claim 1, wherein the bacterial species are non-pathogenic.

3. The composition of claim 1, wherein the Bacillus subtilis includes Bacillus subtilis 34 KLB.

4. The composition of claim 1, wherein each of the Lactobacillus bacterial species are present in equal amounts by weight.

5. The composition according to claim 1, wherein the composition further comprises an inorganic mineral that stimulates bacterial respiration and growth.

6. The composition according to claim 5, wherein the inorganic mineral is disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, magnesium sulfate, calcium sulfate, magnesium chloride, calcium chloride, or iron(III) chloride.

7. A method for reducing cyanuric acid concentration on recreational water systems comprising contacting a pool's filtration system with the composition of claim 1.

8. A composition comprising
a. between 75-95% w/w of anhydrous dextrose or dextrose monohydrate;
b. at least 1% w/w of a mixture containing Bacillus bacterial species comprising Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus pumilus, wherein each of the Bacillus species are individually fermented aerobically, dried and ground to an average particle size of about 200 microns, and wherein the Bacillus subtilis comprises Mojavensis; 
c. at least 4% w/w of a mixture containing Lactobacillus bacterial species comprising Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum, wherein each of the Lactobacillus species are fermented anaerobically, dried, and ground to an average particle size of about 200 microns.

9. The composition of claim 8, wherein a bacterial concentration of the Bacillus mixture is at least 1×106 colony forming units (CFU) per gram of the mixture and the bacterial concentration the Lactobacillus mixture is at least 1×106 colony forming units (CFU) per gram of the mixture.

10. The composition of claim 9, wherein at least 15% of the Bacillus CFUs are Bacillus subtilis 34 KLB.

11. A composition comprising:
a. at least 0.4% w/w/ of a first Bacillus mixture comprising Bacillus subtilis subsp mojavensis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus pumilus; 
b. at least 0.4% w/w/ of a second Bacillus mixture comprising Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus pumilus; 
c. at least 0.4% w/w/ of a mixture o Bacillus subtilis 34 KLB; and
d. at least 4% w/w of a Lactobacillus mixture comprising Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum.

12. The composition of claim 11, wherein the remainder of the composition comprise an oxidizable carbon source.

13. The composition of claim 12, wherein the oxidizable carbon source is anhydrous dextrose or dextrose monohydrate.

14. The composition of claim 11, wherein the Lactobacillus in the Lactobacillus mixture are present in equal proportions by weight.

15. The composition of claim 11, the wherein the Bacillus subtilis subsp mojavensis, the Bacillus amyloliquefaciens, and the Bacillus pumilus in the first Bacillus mixture are present in equal proportions by weight.