# US 10,869,484 B2 · Chlorella-based composition for application to plants and methods of its preparation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,869,484 B2 - Document ID: US10869484B2 - Status: Granted - Kind: B2 - Application number: 16/430,200 - Filed: 2019-06-03 - Published: 2019-09-26 - Granted: 2020-12-22 - Earliest claimed: 2014-12-16 - Assignees: Heliae Development, LLC - Applicants: Heliae Development, LLC - Inventors: Sandip Shinde; Stephen Ventre; Nicholas Donowitz; Michael Rohlfsen; Laura Carney - Web page: https://patentgrep.com/patent/US10869484B2 ## CPC - A01N 63/00: Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates (containing compounds of determined constitution A01N27/00 - A01N59/00; unicellular algae A01N65/03) - A01N 65/00: Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof (containing compounds of determined constitution A01N27/00 - A01N59/00) - A01N 65/03: Algae - C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00) - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Methods of preparing a liquid Chlorella based composition comprising pasteurization and stabilization of a low concentration of mixotrophic or non-mixotrophic Chlorella whole cells are disclosed. The liquid composition can be used to enhance the emergence and growth of plants in low concentration and low frequency soil, foliar, seed soak, capillary action, and hydroponic applications. ## Claims ### Claim 1 A method of preparing a whole cell Chlorella liquid plant growth treatment composition that enhances a characteristic of a plant compared to a composition comprising non-pasteurized Chlorella cells, lacking the whole pasteurized Chlorella cells, comprising: heating a culture comprising the whole Chlorella cells in a liquid culture medium at a temperature in the range of 50-90° C.; cooling the heated composition, wherein the heating and cooling steps pasteurize the whole Chlorella cells in the composition, making them suitable in the enhancement of a plant compared to a composition comprising non-pasteurized Chlorella cells, lacking the whole pasteurized Chlorella cells; and adjusting the composition to a pH in the range of 3-5. ### Claim 2 (depends on 1) The method of claim 1 further comprising the step of adjusting the concentration of the whole Chlorella cells in the heated composition to be in the range of 5-30% whole Chlorella cells by weight. ### Claim 3 (depends on 2) The method of claim 2 further comprising the step of diluting the liquid composition to a concentration in the range of 10-11% whole Chlorella cells by weight. ### Claim 4 (depends on 2) The method of claim 2 further comprising the step of diluting the liquid composition to a concentration in the range of 0.001-0.400% whole Chlorella cells by weight. ### Claim 5 (depends on 2) The method of claim 2 further comprising the step of diluting the liquid composition to a concentration in the range of 1-150 mL/gallon. ### Claim 6 (depends on 1) The method of claim 1 wherein the culture comprising whole Chlorella cells in the liquid culture medium is heated for a time between 15-360 minutes. ### Claim 7 (depends on 1) The method of claim 1 wherein the composition is cooled to a temperature in the range of 35-45° C. ### Claim 8 (depends on 1) The method of claim 1 further comprising the step of adjusting the composition to a pH in the range of 3-5. ### Claim 9 (depends on 1) The method of claim 1 wherein the pH of the composition is adjusted by contacting the composition with an acid. ### Claim 10 (depends on 9) The method of claim 9 wherein the acid comprises phosphoric acid. ### Claim 11 (depends on 10) The method of claim 10 wherein the amount of phosphoric acid is in the range of 0.01-0.3% by weight of the composition. ### Claim 12 (depends on 1) The method of claim 1 further comprising the step of contacting the composition with a culture stabilizer suitable for plants. ### Claim 13 (depends on 12) The method of claim 12 wherein the culture stabilizer is selected from: potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, and any combination thereof. ### Claim 14 (depends on 1) The method of claim 1 wherein the heating and cooling steps render the whole Chlorella cells to be non-viable. ### Claim 15 (depends on 1) The method of claim 1 wherein the whole Chlorella cells are cultured in mixotrophic conditions. ### Claim 16 (depends on 15) The method of claim 15 wherein the whole Chlorella cells are cultured in axenic mixotrophic conditions. ### Claim 17 (depends on 15) The method of claim 15 wherein the whole Chlorella cells are cultured in non-axenic mixotrophic conditions. ### Claim 18 (depends on 1) The method of claim 1 wherein the whole Chlorella cells have been subjected to a drying process or the whole Chlorella cells have not been subjected to a drying process. ### Claim 19 (depends on 1) The method of claim 1 wherein the composition comprises phytohormones selected from: abscisic acid, abscisic acid metabolites, cytokinins, auxins, gibberellins, and any combination thereof. ### Claim 20 (depends on 1) The method of claim 1 wherein the plant characteristic is selected from: seed germination rate, seed germination time, seedling emergence, seedling emergence time, seedling size, plant fresh weight, plant dry weight, utilization, fruit production, leaf production, leaf formation, thatch height, plant health, plant resistance to salt stress, plant resistance to heat stress, plant resistance to heavy-metal stress, plant resistance to drought, maturation time, yield, root length, root mass, color, insect damage, blossom end rot, softness, fruit quality, and sunburn. ### Claim 21 (depends on 1) The method of claim 1 wherein the whole Chlorella cells are cultured in non-mixotrophic conditions. ### Claim 22 (depends on 21) The method of claim 21 wherein the whole Chlorella cells are prepared through heterotrophy. ### Claim 23 (depends on 21) The method of claim 21 wherein the whole Chlorella cells are prepared through phototrophy. ### Claim 24 (depends on 1) The method of claim 1 wherein the liquid treatment composition is supplemented with at least one of nitrogen, phosphorus, and potassium in a concentration of at least 1% of the composition. ### Claim 25 (depends on 15) The method of claim 15 wherein the mixotrophic conditions comprises supplying an organic carbon source to the culture. ### Claim 26 (depends on 25) The method of claim 25 wherein the organic carbon source is one of acetic acid and acetate and is supplied to the culture in a feed via a pH auxostat system. ### Claim 27 (depends on 15) The method of claim 15 wherein the mixotrophic conditions comprises culturing the whole Chlorella cells with a dissolved oxygen concentration of between 0.1-4 mg/L. ### Claim 28 (depends on 15) The method of claim 15 wherein the mixotrophic conditions comprises supplying light to the culture as an energy source. ### Claim 29 A method of preparing a whole cell Chlorella liquid plant growth treatment composition that enhances a characteristic of a plant compared to a composition comprising non-pasteurized Chlorella cells, lacking the whole pasteurized Chlorella cells, comprising: heating a culture comprising the whole Chlorella cells in a liquid culture medium at a temperature in the range of 50-90° C.; and cooling the heated composition, wherein the heating and cooling steps pasteurize the whole Chlorella cells in the composition, making them suitable in the enhancement of a plant compared to a composition comprising non-pasteurized Chlorella cells, lacking the whole pasteurized Chlorella cells, wherein the composition comprises phytohormones selected from: abscisic acid, abscisic acid metabolites, cytokinins, auxins, gibberellins, and any combination thereof. ### Claim 30 (depends on 29) The method of claim 29 further comprising the step of adjusting the concentration of the whole Chlorella cells in the heated composition to be in the range of 5-30% whole Chlorella cells by weight. ### Claim 31 (depends on 29) The method of claim 29 wherein the composition is heated for a time period in the range of 15-360 minutes. ### Claim 32 (depends on 29) The method of claim 29 wherein the whole Chlorella cells are pasteurized in a culture having a concentration greater than 11% by weight of the whole Chlorella cells, at between 55-65° C. for between 90-150 minutes, and wherein the culture is then diluted to 10-11% whole Chlorella cells by weight and cooled to between 35-45° C. ### Claim 33 (depends on 30) The method of claim 30 further comprising the step of diluting the liquid composition to a concentration in the range of 0.001-0.400% whole Chlorella cells by weight. ### Claim 34 (depends on 30) The method of claim 30 further comprising the step of diluting the liquid composition to a concentration in the range of 1-150 mL/gallon. ### Claim 35 (depends on 29) The method of claim 29 further comprising the step of adjusting the composition to a pH in the range of 3-5. ### Claim 36 (depends on 29) The method of claim 29 further comprising the step of contacting the composition with a culture stabilizer suitable for plants, wherein the culture stabilizer is selected from: potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, and any combination thereof. ### Claim 37 (depends on 29) The method of claim 29 wherein the heating and cooling steps render the whole Chlorella cells to be non-viable. ### Claim 38 (depends on 29) The method of claim 29 wherein the whole Chlorella cells are cultured in mixotrophic conditions, the mixotrophic conditions comprising culturing the whole Chlorella cells in a suitable medium for a culture length of 7-14 days, at a temperature between 20-30° C., at a pH between 6.5-8.5, and a dissolved oxygen concentration can range between 0.1-4 mg/L. ### Claim 39 (depends on 29) The method of claim 29 wherein the whole Chlorella cells are cultured in non-axenic mixotrophic conditions. ### Claim 40 (depends on 29) The method of claim 29 wherein the whole Chlorella cells are cultured in axenic mixotrophic conditions. ### Claim 41 (depends on 38) The method of claim 38 wherein the mixotrophic conditions further comprises supplying an organic carbon source to the culture, wherein the organic carbon source is one of acetic acid and acetate and is supplied to the culture in a feed via a pH auxostat system. ### Claim 42 (depends on 29) The method of claim 29 wherein the whole Chlorella cells are cultured in non-mixotrophic conditions. ### Claim 43 (depends on 42) The method of claim 42 wherein the whole Chlorella cells are prepared through heterotrophy. ### Claim 44 (depends on 42) The method of claim 42 wherein the whole Chlorella cells are prepared through phototrophy. ### Claim 45 (depends on 29) The method of claim 29 wherein the liquid treatment composition is supplemented with at least one of nitrogen, phosphorus, and potassium in a concentration of at least 1% of the composition. ### Claim 46 (depends on 29) The method of claim 29 wherein the whole Chlorella cells have been subjected to a drying process or the whole Chlorella cells have not been subjected to a drying process. ### Claim 47 A method of preparing a whole cell Chlorella liquid plant growth treatment composition that enhances a characteristic in a plant compared to a composition comprising non-pasteurized Chlorella cells, lacking the whole pasteurized Chlorella cells, comprising: heating a culture comprising the whole Chlorella cells in a liquid culture medium at a temperature in the range of 50-90° C.; cooling the heated composition, wherein the heating and cooling steps pasteurize the whole Chlorella cells in the composition, making them suitable in the enhancement of a plant compared to a composition comprising non-pasteurized Chlorella cells, lacking the whole pasteurized Chlorella cells; and contacting the composition with a culture stabilizer suitable for plants, wherein the culture stabilizer is selected from: potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, and any combination thereof. ### Claim 48 (depends on 47) The method of claim 47 wherein the whole Chlorella cells are cultured in at least one of axenic mixotrophic conditions, non-axenic mixotrophic conditions, through heterotrophy, and through phototrophy. ### Claim 49 (depends on 47) The method of claim 47 wherein the whole Chlorella cells have been subjected to a drying process or the whole Chlorella cells have not been subjected to a drying process. ### Claim 50 (depends on 47) The method of claim 47 further comprising the step of adjusting the composition to a pH in the range of 3-5. ### Claim 51 (depends on 47) The method of claim 47 further comprising the step of adjusting the concentration of the whole Chlorella cells in the heated composition to be in the range of 5-30% whole Chlorella cells by weight. ### Claim 52 (depends on 51) The method of claim 51 further comprising the step of diluting the liquid composition to a concentration in the range of 10-11% whole Chlorella cells by weight. ### Claim 53 (depends on 47) The method of claim 47 wherein the liquid treatment composition is supplemented with at least one of nitrogen, phosphorus, and potassium in a concentration of at least 1% of the composition.