# US 10,766,799 B2 · Method for improving quality of aquaculture pond water using a nutrient germinant composition and spore incubation method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,766,799 B2 - Document ID: US10766799B2 - Status: Granted - Kind: B2 - Application number: 15/907,682 - Filed: 2018-02-28 - Published: 2019-03-07 - Granted: 2020-09-08 - Earliest claimed: 2014-05-23 - Assignees: NCH Corporation - Applicants: NCH Corporation - Inventors: Charles Greenwald; Gabriel F. K. Everett; Judy Pruitt; Amanda Rosmarin; Jordan Church; Daniel Aberle; George Aboagye; Skylar Rae White; Haibo Cao; Christopher Zetena; Kelly Gillespie - Web page: https://patentgrep.com/patent/US10766799B2 ## CPC - C02F 3/348: {characterised by the way or the form in which the microorganisms are added or dosed} - A01K 61/13: Prevention or treatment of fish diseases - A01K 63/04: Arrangements for treating water specially adapted to receptacles for live fish - C02F 3/302: {Nitrification and denitrification treatment (C02F3/308 takes precedence)} - C02F 2101/105: {Phosphorus compounds} - C02F 2101/16: Nitrogen compounds, e.g. ammonia - C02F 2101/30: Organic compounds - C02F 2103/20: from animal husbandry - C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes) - A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS ## Abstract A method for improving the quality of pond water used in aquaculture applications by adding to the pond water active bacteria that are preferably germinated from spores on site using a nutrient-germinant composition and an incubation method for increased spore germination efficiency, in combination with a nitrification enhancement agent such as calcium carbonate or calcified seaweed, and an optional reaction surface area modifier such as calcified seaweed or plastic or metal particles or fragments. The nutrient-germinant composition comprises L-amino acids, D-glucose and/or D-fructose, a phosphate buffer, an industrial preservative, and may include bacteria spores (preferably of one or more Bacillus species) or they may be separately combined for germination. The incubation method comprises heating a nutrient germinant composition and bacteria spores, to a temperature range of 35° C. to 60° C. for around 2 to 60 minutes to produce an incubated bacteria solution that is discharged to the aquaculture application. ## Claims ### Claim 1 A method of adding bacteria to water used in an aquaculture application, the method comprising: providing a volume of nutrient germinant composition and a volume of bacteria spores, which may be premixed together as a nutrient spore composition or separate; optionally mixing a portion of the nutrient germinant composition and a portion of the bacteria spores if separate to form the nutrient spore composition; heating a portion of the nutrient spore composition to a temperature in a range of around 38° C. to 60° C. at or near a site of the aquaculture application; maintaining the temperature in the range for an incubation period of around 2 minutes to around 6 hours to form a batch of germinated bacteria solution; periodically repeating the heating and maintaining steps to form additional batches of germinated bacteria solution over the course of a treatment cycle; dispersing each batch of germinated bacteria solution into the water used in the aquaculture application; providing a nitrification enhancement agent; dispersing the nitrification enhancement agent in the water contemporaneously with at least one of the batches of germinated bacteria solution; and wherein the bacteria are useful for remediating the water by degrading organic waste and inhibiting the growth of pathogenic bacteria or the bacteria are probiotic for a species in the aquaculture application. ### Claim 2 (depends on 1) The method of claim 1 wherein the bacteria is selected from the group consisting of the genera Bacillus, Bacteroides, Bifidobacterium, Lueconostoc, Pediococcus, Enterococcus, Lactobacillus, Megasphaera, Pseudomonas and Propionibacterium. ### Claim 3 (depends on 2) The method of claim 2 wherein the bacteria are one or more species of Bacillus licheniformis and Bacillus subtilis. ### Claim 4 (depends on 2) The method of claim 2 wherein the probiotic bacteria is selected from the group consisting of Bacillus amylophilus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bacteroides ruminocola, Bacteroides ruminocola, Bacterioides suis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium thermophilum, Enterococcus cremoris, Enterococcus diacetylactis, Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis, Enterococcus thermophiles, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Leuconostoc mesenteroides, Megasphaera elsdennii, Pediococcus acidilacticii, Pediococcus cerevisiae, Pediococcus pentosaceus, Propionibacterium acidipropionici, Propionibacterium freudenreichii, and Propionibacterium shermanii. ### Claim 5 (depends on 1) The method of claim 1 wherein the nutrient germinant composition comprises: an L-amino acid; one or more buffers comprising a phosphate buffer, HEPES, Tris base, or a combination thereof; an industrial preservative; optionally D-glucose, or optionally D-fructose, or optionally both D-glucose and D-fructose; and optionally a source of potassium ions. ### Claim 6 (depends on 5) The method of claim 5 wherein the L-amino acid is L-alanine, L-asparagine, L-valine, L-cysteine, a hydrolysate of soy protein, or a combination thereof. ### Claim 7 (depends on 5) The method of claim 5 wherein the nutrient germinant composition comprises around 17.8 g/L to 89 g/L total of one or more L-amino acids. ### Claim 8 (depends on 5) The method of claim 5 wherein the nutrient germinant composition and spores are premixed and the nutrient spore composition further comprises spores of a Bacillus species and a germination inhibitor. ### Claim 9 (depends on 8) The method of claim 8 wherein the germination inhibitor or preservative comprises sodium chloride, D-alanine, or a combination thereof. ### Claim 10 (depends on 9) The method of claim 9 wherein the premixed nutrient spore composition comprises around 29 g/L to 117 g/L sodium chloride. ### Claim 11 (depends on 9) The method of claim 9 wherein the premixed nutrient spore composition comprises around 8 g/L to 116 g/L D-alanine. ### Claim 12 (depends on 7) The method of claim 7 wherein the phosphate buffer comprises around 10-36 g/L of monosodium phosphate and around 30-90 g/L of disodium phosphate. ### Claim 13 (depends on 1) The method of claim 1 wherein the nutrient germinant composition, or nutrient spore composition if premixed, is a concentrated liquid comprising: around 8.9-133.5 g/L of one or more L-amino acids; around 0.8-3.3 g/L total of one or more industrial preservatives; around 40-126 g/L total of one or more phosphate buffers, around 15-61 g/L Tris base, or around 32.5-97.5 g/L HEPES, or a combination thereof; optionally around 18-54 g/L of D-glucose, D-fructose, or a combination thereof; and optionally around 7.4-22.2 g/L of KCl. ### Claim 14 (depends on 13) The method of claim 13 further comprising: adding a diluent to the nutrient germinant composition, or nutrient spore composition if premixed, prior to or during heating; and mixing the diluted nutrient-germinant composition and bacteria spores, or diluted nutrient spore composition, during the incubation period. ### Claim 15 (depends on 14) The method of claim 14 wherein the concentration of the diluted nutrient-germinant composition is around 0.1% to 10%. ### Claim 16 (depends on 1) The method of claim 1 wherein the nitrification enhancement agent increases the alkalinity of the water, provides increased surface area for biofilms of nitrifying bacteria to grow, or both. ### Claim 17 (depends on 1) The method of claim 1 wherein the nitrification enhancement agent is calcium carbonate, calcified seaweed, or both. ### Claim 18 (depends on 17) The method of claim 17, wherein the nitrification enhancement agent is provided in the form of prills, pellets or particles. ### Claim 19 (depends on 16) The method of claim 16 further comprising providing and dispersing an additional surface area modifier in the water contemporaneously with at least one batch of the germinated bacteria solution. ### Claim 20 (depends on 19) The method of claim 19 wherein the surface area modifier is selected from the group consisting of particles or fragments of plastic or metal. ### Claim 21 (depends on 1) The method of claim 1 wherein the nitrification enhancement agent comprises an alkalinity enhancing agent and wherein the alkalinity enhancing agent is dispersed in the water contemporaneously with one batch of germinated bacteria solution on a seasonal basis. ### Claim 22 (depends on 1) The method of claim 1 wherein the bacteria spores are in a separate spore composition comprising: one or more Bacillus species in spore form; about 0.002 to 5.0% by weight thickener; about 0.01 to 2.0% by weight total of one or more acids or salts of acids; and wherein the percentages are by weight of the spore composition. ### Claim 23 (depends on 5) The method of claim 5 wherein the bacteria spores are in a separate spore composition comprising: one or more Bacillus species in spore form; one or more acids or salts of acids; and a thickener. ### Claim 24 (depends on 23) The method of claim 23 wherein the spore Bacillus species are one or more of: Bacillus pumilus, Bacillus licheniformis, Bacillus amylophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus firmus, Bacillus megaterium, Bacillus mesentericus, Bacillus subtilis var. natto, or Bacillus toyonensis. ### Claim 25 (depends on 23) The method of claim 23 wherein the spore composition has a pH of around 4.5 to around 5.5. ### Claim 26 (depends on 23) The method of claim 23 wherein the acids or salts of acids are one or more of acetic acid, citric acid, fumaric acid, propionic acid, sodium propionate, calcium propionate, formic acid, sodium formate, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, or calcium sorbate. ### Claim 27 (depends on 23) The method of claim 23 wherein the spore composition comprises: about 0.002 to 5.0% by weight thickener; about 0.01 to 1% by weight total of one or more acids or salts of acids; and about 0.00005 to 3.0% by weight of a surfactant; wherein the percentages are by weight of the spore composition. ### Claim 28 (depends on 4) The method of claim 4 wherein the incubation period is around 2 minutes to around 5 minutes. ### Claim 29 (depends on 28) The method of claim 28 wherein the aquaculture application is a growing pond containing fish or eel. ### Claim 30 (depends on 1) The method of claim 1 wherein the incubation period is around 4 to 6 hours. ### Claim 31 (depends on 30) The method of claim 30 wherein the aquaculture application is a growing pond containing shrimp. ### Claim 32 (depends on 1) The method of claim 1 wherein the temperature is in a range of around 38° C. to 50° C. ### Claim 33 (depends on 1) The method of claim 1 wherein the temperature is in a range of around 41° C. to 44° C. ### Claim 34 (depends on 5) The method of claim 5 wherein the temperature is in a range of around 38° C. to 50° C. ### Claim 35 (depends on 5) The method of claim 5 wherein the temperature is in a range of around 41° C. to 44° C. ### Claim 36 (depends on 1) The method of claim 1 wherein the nutrient germinant composition does not include fructose or glucose. ### Claim 37 (depends on 5) The method of claim 5 wherein the nutrient germinant composition does not include fructose or glucose. ### Claim 38 (depends on 34) The method of claim 34 wherein the nutrient germinant composition does not include fructose or glucose. ### Claim 39 (depends on 35) The method of claim 35 wherein the nutrient germinant composition does not include fructose or glucose. ### Claim 40 (depends on 5) The method of claim 5 wherein the industrial preservative comprises methyl chloro isothiazolinone, methyl isothiazolinone, propylparaben, methylparaben, diazolidinyl urea, or a combination thereof. ### Claim 41 (depends on 13) The method of claim 13 wherein the one or more industrial preservatives comprise methyl chloro isothiazolinone, methyl isothiazolinone, propylparaben, methylparaben, diazolidinyl urea, or a combination thereof. ### Claim 42 (depends on 5) The method of claim 5 wherein the bacteria are one or more strains of Bacillus licheniformis and Bacillus subtilis. ### Claim 43 (depends on 34) The method of claim 34 wherein the bacteria are one or more strains of Bacillus licheniformis and Bacillus subtilis. ### Claim 44 (depends on 35) The method of claim 35 wherein the bacteria are one or more strains of Bacillus licheniformis and Bacillus subtilis. ### Claim 45 (depends on 35) The method of claim 35 wherein the bacteria comprise two strains of Bacillus licheniformis and one strain of Bacillus subtilis. ### Claim 46 (depends on 22) The method of claim 22 further comprising about 0.00005 to 3.0% by weight of a surfactant.