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EP3700342A1 - Compositions de biomasse - Google Patents

Compositions de biomasse

Info

Publication number
EP3700342A1
EP3700342A1 EP19731117.8A EP19731117A EP3700342A1 EP 3700342 A1 EP3700342 A1 EP 3700342A1 EP 19731117 A EP19731117 A EP 19731117A EP 3700342 A1 EP3700342 A1 EP 3700342A1
Authority
EP
European Patent Office
Prior art keywords
microalgae
composition
chlorella
plant
biomass
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19731117.8A
Other languages
German (de)
English (en)
Inventor
Laura Carney
Michael Miller
Amy RIAL
Connor Osgood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Heliae Development LLC
Original Assignee
Heliae Development LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Heliae Development LLC filed Critical Heliae Development LLC
Publication of EP3700342A1 publication Critical patent/EP3700342A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/03—Algae

Definitions

  • BIOMASS COMPOSITIONS The entire contents of the foregoing are hereby incorporated by reference.
  • the present invention generally relates to agriculture and, more specifically, to biomass compositions and methods for increasing plant health, increasing soil health, increasing fruit water retention, increasing plant shelf-life and/or decreasing needle-drop in conifer species.
  • Some embodiments of the invention relate to a method of enhancing a plant.
  • the method can include a step of administering to the plant, seedling, or seed a liquid composition treatment including a culture of microalgae.
  • the microalgae can include at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells in an amount effective to enhance at least one characteristic of a plant compared to a substantially identical untreated plant.
  • the characteristic can be improved shelf life, increased water retention, and/or diminished needle-drop.
  • “Substantially identical” refers to being the same as is practicable under the circumstances of a given test, as that a person of ordinary skill in the art would consider any actual differences to be insignificant in evaluating the validity of experimental results. As understood in the art, in real-world biological experiments these kinds of comparisons are always necessary and are accepted by those of ordinary skill in the art in view of the fact that there are no practical alternatives except often impractically large data sets and sample sizes.
  • the method can include contacting soil in the immediate vicinity of the plant, seedling, or seed with an effective amount of the liquid composition treatment.
  • the liquid composition can be administered at a rate in the range of .25-2 gallons/acre.
  • the liquid composition can include between l00g-800g per acre of at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells.
  • the contacting step can include a drip irrigation system and/or process.
  • the liquid composition treatment can further include phosphoric acid and potassium sorbate. In some embodiments, the liquid composition treatment can further include citric acid.
  • the pasteurized Chlorella cells are pasteurized at a temperature in the range of 65°C-90°C and the pasteurized Aurantiochytrium acetophilum HS399 cells are pasteurized at a temperature in the range of 65°C-75°C.
  • Aurantiochytrium acetophilum HS399 cells are pasteurized for between 90-150 minutes.
  • the microalgae includes only Chlorella cells, only
  • Aurantiochytrium cells, or Chlorella cells and Aurantiochytrium acetophilum HS399 cells and the liquid composition is applied in an effective amount to increase shelf life of the plant by at least 5% compared to a substantially identical untreated plant.
  • the shelf life can be increased by about 5%, 7%, 10%, 15%, or 20%.
  • the microalgae includes only Chlorella cells, only
  • the microalgae includes only Chlorella cells, only
  • the liquid composition can include pasteurized
  • the Aurantiochytrium acetophilum HS399 cells have been subjected to an extraction process to remove oils from the Aurantiochytrium acetophilum HS399 cells.
  • compositions for enhancing at least one plant characteristic can include a microalgae biomass comprising at least two species of microalgae.
  • the composition can cause synergistic enhancement of at least one plant characteristic.
  • the characteristic can be improved shelf life, increased water retention, and diminished needle-drop
  • the microalgae species can be selected from
  • Botryococcus Chlorella, Chlamydomonas, Scenedesmus, Pavlova, Phaeodactylum, Nannochloropsis, Aurantiochytrium, Spirulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Schizochytrium, Tetraselmi, and/or the like.
  • the microalgae biomass can include whole biomass and/or residual biomass.
  • the composition can include a first species of microalgae and a second species of microalgae.
  • the ratio of the first species of microalgae and the second species of microalgae is between 1:20 and 1:1. In some embodiments, the ratio of the first species of microalgae and the second species of microalgae is between 1:4 and 1:1.
  • the first species of microalgae is Chlorella and the second species of microalgae is Aurantiochytrium.
  • the ratio of Chlorella a d Aurantiochytrium is 25:75, 50:50 or 75:25.
  • the Chlorella can be whole biomass and
  • Aurantiochytrium is residual biomass or the Chlorella can be residual biomass and Aurantiochytrium is whole biomass.
  • Some embodiments of the invention relate to a method of plant enhancement that can include administering to a plant, seedling, or seed a composition treatment.
  • the composition treatment can enhance at least one plant characteristic synergistically.
  • the characteristic can be from improved shelf life, increased water retention, and diminished needle-drop.
  • Embodiments of the invention relate to a composition for enhancing at least one plant characteristic.
  • the composition can include a microalgae biomass that includes at least one species of microalgae.
  • the composition can include a microalgae biomass that includes at least two species of microalgae.
  • the composition can cause synergistic enhancement of at least one plant characteristic.
  • the microalgae species can include Chlorella,
  • the microalgae species can include Botryococcus, Chlamydomonas, Scenedesmus, Pavlova, Phaeodactylum, Nannochloropsis, Spirlulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Tetraselmis, and/or the like.
  • the microalgae biomass can include whole biomass and/or residual biomass.
  • Whole biomass includes substantially all components and fractions of the cells from which the whole biomass is derived.
  • Residual or extracted biomass can be any remaining biomass after extraction and/or removal of one or more components of a whole biomass.
  • the composition can include one species of microalgae.
  • the composition can include a first species of microalgae and a second species of microalgae.
  • the ratio of the first species of microalgae and the second species of microalgae can be between about 25:75, 50:50, or 75:25.
  • the first species of microalgae may be Chlorella and the second species of microalgae may be Aurantiochytrium acetophilum HS399.
  • the ratio of Chlorella and Aurantiochytrium acetophilum HS399 may range between about 25:75 to 75:25.
  • the ratio of Chlorella and Aurantiochytrium acetophilum HS399 may be about 25:75, 50:50, or 75:25.
  • the Chlorella is whole biomass a d Aurantiochytrium acetophilum HS399 is residual/extracted biomass.
  • the Aurantiochytrium acetophilum HS399 is whole biomass and Chlorella is residual/extracted biomass.
  • the Chlorella and Aurantiochytrium acetophilum HS399 are both whole biomass and in other embodiments the Chlorella and Aurantiochytrium acetophilum HS399 are both residual/extracted biomass.
  • Some embodiments of the invention relate to a method of plant enhancement comprising administering to a plant, seedling, seed, or soil the composition treatment, wherein the composition treatment enhances at least one plant characteristic.
  • the composition is applied when the plant is under salt stress conditions, temperature stress conditions, and/or the like.
  • Embodiments of the invention relate to a method of plant enhancement comprising administering a composition treatment comprising at least one microalgae species to soil.
  • the administering can be by soil drench at the time of seeding.
  • the method can include growing the plant to a transplant stage.
  • the method can include transferring the plant at the transplant stage from an initial container to a larger container or a field, or the like.
  • the plant at the transplant stage has at least one enhanced plant characteristic.
  • the enhanced plant characteristic can be improved root density, improved root area, enhanced plant vigor, enhanced plant growth rate, enhanced plant maturation, and/or enhanced shoot development. After the transfer, the plant may have at least one enhanced plant characteristic.
  • the composition treatment can include at least one microalgae species such as Botryococcus, Chlorella, Chlamydomonas, Scenedesmus, Pavlova, Phaeodactylum, Nannochloropsis, Aurantiochytrium, Spirlulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Schizochytrium, Tetraselmis, and/or the like.
  • the microalgae composition may be applied to the soil of the fruiting plant by drenching the soil initially at the time of transplant and then subsequently every two weeks (once every 14 days) after transplant until harvest.
  • FIG. 1 is graph showing a comparison of the effect of several microalgae compositions on lettuce shoot biomass under no stress versus salt stress conditions, wherein the effects are observed in an increase in shoot biomass relative to the untreated control (UTC) and a seaweed commercial reference product;
  • FIG. 2 is a graph showing another comparison of the effect the several microalgae compositions of FIG. 1 on lettuce shoot biomass under no stress versus salt stress conditions, wherein the effects are observed in an increase in shoot biomass relative to the UTC and a seaweed commercial reference product;
  • FIG. 3 is a graph showing another comparison of the effect of the several microalgae compositions of FIG. 1 on lettuce shoot biomass under no stress versus salt stress conditions over three separate trials, wherein the effects are observed in an increase in shoot biomass over the UTC;
  • FIG. 4 is a graph showing a comparison of the effect of the several microalgae compositions of FIG. 1 on lettuce head biomass under no stress versus salt stress conditions over two separate trials, wherein the effects are observed in an increase in head biomass relative to a seaweed commercial reference product;
  • FIG. 5 is a graph showing a comparison of the effect of the several microalgae compositions of FIG. 1 on lettuce leaf biomass under ideal conditions over two separate trials, wherein the effects are observed in an increase in leaf biomass relative to a seaweed commercial reference product;
  • FIG. 6 is a graph showing a comparison of the effect of the several microalgae compositions of FIG. 1 on lettuce leaf biomass under ideal conditions over three separate trials, wherein the effects are observed in an increase in leaf biomass relative to a seaweed commercial reference product;
  • FIG. 7 is a graph showing a comparison of the effect of the several microalgae compositions of FIG. 1 on lettuce leaf biomass under constant salt stress conditions over three separate trials, wherein the effects are observed in an increase in leaf biomass relative to a seaweed commercial reference product;
  • FIG. 8 is a graph showing a comparison of the effect of the several microalgae compositions of FIG. 1 on lettuce head biomass under ideal conditions, wherein the effects are observed in an increase in head biomass relative to a seaweed commercial reference product;
  • FIG. 9 is a graph showing a comparison of the effect of the several microalgae compositions of FIG. 1 on lettuce head biomass under constant salt stress conditions, wherein the effects are observed in an increase in head biomass relative to a seaweed commercial reference product;
  • FIG. 10 is a graph showing the effects of a microalgae composition on growth of romaine lettuce transplants, wherein the effects are observed in an increase in leaf canopy size relative to the UTC;
  • FIG. 11 is a graph showing the effects of the microalgae composition of
  • FIG. 10 on growth of romaine lettuce transplants, wherein the effects are observed in an increase in shoot dry weight relative to the UTC;
  • FIG. 12 is a graph showing the effects of the microalgae composition of
  • FIG. 10 on the growth of romaine lettuce transplants, wherein the effects are observed in an increase in root area relative to the UTC;
  • FIG. 13 is a graph showing a comparison of the effects of two microalgae compositions on the growth of roma tomato, wherein the effects are observed in an increase in canopy cover relative to the UTC;
  • FIG. 14 is a graph showing the effects of a microalgae composition on the growth of cauliflower, wherein the effects are observed in an increase in canopy cover relative to the UTC;
  • FIG. 15 is a graph showing the effects of a microalgae composition on the growth and yield of bell peppers, wherein the effects are observed in an increase in marketable yield based on the number of cartons per acre relative to the UTC;
  • FIG. 16 is a graph showing the effects of the microalgae composition of
  • FIG. 15 on the growth and yield of bell peppers, wherein the effects are observed in an increase in marketable yield based on the count and weight of large and extra-large bell peppers per 4 plants relative to the UTC;
  • FIG. 17 is a graph showing the effects of a microalgae composition on the growth of snap beans, wherein the effects are observed in an increase in shoot biomass relative to the UTC;
  • FIG. 18 is a graph showing the effects of the microalgae composition of
  • FIG. 17 on the growth of snap beans, wherein the effects are observed in an increase in marketable yield relative to the UTC;
  • FIG. 19 is a graph showing the effects of a microalgae composition on the growth of snap peas, wherein the effects are observed in an increase in shoot biomass relative to the UTC;
  • FIG. 20 is a graph showing the effects of the microalgae composition of
  • FIG. 19 on the growth of snap peas, wherein the effects are observed in an increase in marketable yield relative to the UTC;
  • FIG. 21 is a graph showing the effects of the microalgae composition of
  • FIG. 19 on the growth of snap peas, wherein the effects are observed in an increase in pod count per plant over the UTC;
  • FIG. 22 is a graph showing the effects of a microalgae composition on the health of soil used to grow sweet corn, snap peas, and snap beans, wherein the effects are observed in the microbial community dissimilarity to the UTC;
  • FIG. 23 is a graph showing the effects of the microalgae composition of
  • FIG. 22 on soil health, wherein the effects are observed in an increase in beneficial bacteria in the soil relative to the UTC;
  • FIG. 24 is a graph showing the effects of a microalgae composition on soil health, wherein the effects are observed in an increase in active carbon in the soil relative to the UTC and a seaweed commercial reference product;
  • FIG. 25 is a graph showing the effects of the microalgae composition of
  • FIG. 24 on soil health, wherein the effects are observed in an increase in soil protein in the soil relative to the UTC;
  • FIG. 26 is a graph showing the effects of the microalgae composition of
  • FIG. 24 on soil health, wherein the effects are observed in an increase in soil aggregates greater than 1 mm in size relative to the UTC and a seaweed commercial reference product;
  • FIG. 27 is a graph showing the effects of a microalgae composition on soil health, wherein the effects are observed in an increase in active carbon in the soil relative to the UTC;
  • FIG. 28 is a graph showing the effects of the microalgae composition of
  • FIG. 27, on soil health wherein the effects are observed in an increase in soil protein in the soil relative to the UTC;
  • FIG. 29 is a graph showing the effects of the microalgae composition of
  • FIG. 27 on soil health, wherein the effects are observed in an increase in soil water holding capacity relative to the UTC;
  • FIG. 30 is a table showing the effects of a microalgae composition on strawberry quality after storage, wherein the effects are observed in a decrease in bruising and increases in appearance, aroma, flavor, and texture relative to the UTC;
  • FIG. 31 is a graph showing the effects of the microalgae composition of
  • FIG. 30 on strawberry quality after storage, wherein the effects are observed in an increase in post-storage marketability relative to the UTC;
  • FIG. 32 is a graph showing the effects of the microalgae composition of
  • FIG. 30 on strawberry quality after storage, wherein the effects are observed in an increase in consumer preference based on appearance, aroma, flavor, and texture relative to the UTC;
  • FIG. 33 is a graph showing a comparison of the effects of two microalgae compositions on strawberry shelf-life and post-harvest quality, wherein the effects are observed in an increase in post-harvest marketability relative to the UTC and a seaweed commercial reference product;
  • FIG. 34 is a graph showing a comparison of the effects of the two microalgae compositions of FIG. 33 on strawberry shelf-life and post-harvest quality, wherein the effects are observed in a decrease in severe and moderate bruising of the strawberries relative to the UTC and a seaweed commercial reference product;
  • FIG. 35 is a graph showing a comparison of the effects of the two microalgae compositions of FIG. 33 on strawberry shelf-life and post-harvest quality, wherein the effects are observed in an increase in consumer preference based on appearance, aroma, flavor, and texture relative to the UTC and a seaweed commercial reference product;
  • FIG. 36 is a graph showing a comparison of the effects of several microalgae compositions on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in fruit water loss relative to the UTC and a seaweed commercial reference product;
  • FIG. 37 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 36 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in bruising of the strawberries after 5 days relative to the UTC and a seaweed commercial reference product;
  • FIG. 38 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 36 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in bruising of the strawberries after 7 days relative to the UTC and a seaweed commercial reference product;
  • FIG. 39 is a table showing a comparison of the effects of several microalgae compositions on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in bruising, a decrease in fruit water loss, and an increase in berry firmness relative to the UTC and a microbial-based commercial reference product;
  • FIG. 40 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 39 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in fruit weight (water) loss relative to the UTC and a microbial-based commercial reference product;
  • FIG. 41 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 39 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in an increase in berry firmness relative to the UTC and a microbial-based commercial reference product;
  • FIG. 42 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 39 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in an increase in marketability based on bruising relative to the UTC and a microbial-based commercial reference product;
  • FIG. 43 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 39 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in bruising relative to the UTC and a microbial-based commercial reference product;
  • FIG. 44 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 39 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in an increase in consumer preference based on appearance, aroma, flavor, and texture relative to the UTC and a microbial-based commercial reference product;
  • FIG. 45 is a table showing a comparison of the effects of several microalgae compositions on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in fruit water loss, a decrease in bruising, and an increase in berry firmness relative to the UTC and a seaweed commercial reference product;
  • FIG. 46 is a continuation of the table shown in FIG. 45 ;
  • FIG. 47 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 45 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in fruit water loss relative to the UTC and a seaweed commercial reference product;
  • FIG. 48 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 45 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in an increase in berry firmness relative to the UTC and a seaweed commercial reference product;
  • FIG. 49 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 45 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in an increase in marketability relative to the UTC and a seaweed commercial reference product;
  • FIG. 50 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 45 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in bruising relative to the UTC and a seaweed commercial reference product;
  • FIG. 51 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 45 on strawberry growth, yield, and post-harvest quality, wherein the effects are observed in an increase in consumer preference based on appearance, aroma, flavor, and texture relative to the UTC and a seaweed commercial reference product;
  • FIG. 52 is a table showing a comparison of the effects of several microalgae compositions on espresso tomato growth, yield, and post-harvest quality, wherein the effects are observed in a decrease in fruit water loss relative to the UTC;
  • FIG. 53 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 52 on espresso tomato growth, yield, and post-harvest quality, wherein the effects are observed in an increase in fruit water retention capacity relative to the UTC;
  • FIG. 54 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 52 on espresso tomato growth, yield, and post-harvest quality, wherein the effects are observed in an increase in fruit water retention capacity at 10 days post-harvest relative to the UTC;
  • FIG. 55 is a graph showing a comparison of the effects of the several microalgae compositions of FIG. 52 on espresso tomato growth, yield, and post-harvest quality, wherein the effects are observed in an increase in fruit water retention capacity at 18 days post-harvest relative to the UTC;
  • FIG. 56 is a graph showing the effects of a microalgae composition on
  • FIG. 57 is a graph showing the effects of the microalgae composition of
  • FIG. 56 on Douglas fir tree preservation, wherein the effects are observed in a decrease in top-off volumes relative to the UTC and a commercial reference product.
  • Non-limiting examples of plant families that can benefit from such compositions include plants from the following: Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae, Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae, Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae, Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae (Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae, Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae, Rubiaceae, Papveraceae, Illiciaceae Grossul
  • the Solanaceae plant family includes a large number of agricultural crops, medicinal plants, spices, and ornamentals in its over 2,500 species. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Manoliopsida (class), Asteridae (subclass), and Solanales (order), the Solanaceae family includes, but is not limited to, potatoes, tomatoes, eggplants, various peppers, tobacco, and petunias. Plants in the Solanaceae can be found on all the continents, excluding Antarctica, and thus have a widespread importance in agriculture across the globe.
  • the Rosaceae plant family includes flowering plants, herbs, shrubs, and trees. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Magnoliopsida (class), Rosidae (subclass), and Rosales (order), the Rosaceae family includes, but is not limited to, almond, apple, apricot, blackberry, cherry, nectarine, peach, plum, raspberry, strawberry, and quince.
  • the Fabaceae plant family (also known as the Leguminosae) comprises the third largest plant family with over 18,000 species, including a number of important agricultural and food plants. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Manoliopsida (class), Rosidae (subclass), and Fabales (order), the Fabaceae family includes, but is not limited to, soybeans, beans, green beans, peas, chickpeas, alfalfa, peanuts, sweet peas, carob, and liquorice.
  • Plants in the Fabaceae family can range in size and type, including but not limited to, trees, small annual herbs, shrubs, and vines, and typically develop legumes. Plants in the Fabaceae family can be found on all the continents, excluding Antarctica, and thus have a widespread importance in agriculture across the globe. Besides food, plants in the Fabaceae family can be used to produce natural gums, dyes, and ornamentals.
  • the Poaceae plant family supplies food, building materials, and feedstock for fuel processing. Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Liliopsida (class), Commelinidae (subclass), and Cyperales (order), the Poaceae family includes, but is not limited to, flowering plants, grasses, and cereal crops such as barely, com, lemongrass, millet, oat, rye, rice, wheat, sugarcane, and sorghum. Types of turf grass found in Arizona include, but are not limited to, hybrid Bermuda grasses (e.g., 328 tifgm, 419 tifway, tif sport).
  • hybrid Bermuda grasses e.g., 328 tifgm, 419 tifway, tif sport.
  • the Vitaceae plant family includes flowering plants and vines.
  • the Vitaceae family includes, but is not limited to, grapes.
  • botanists view the beginning of maturation as starting at when the first true leaf emerges beyond the cotyledon stage, as the cotyledons are already pre formed in the seed prior to germination. Some botanists see maturation as a long phase that proceeds until full reproductive potential has been achieved.
  • Important in the production of fruit from plants is the yield and quality of fruit, which can be quantified as the number, weight, color, firmness, ripeness, sweetness, moisture, degree of insect infestation, degree of disease or rot, degree of sunburn of the fruit.
  • a method of treating a plant to directly improve the characteristics of the plant, or to indirectly enhance the chlorophyll level of the plant for photosynthetic capabilities and health of the plant's leaves, roots, and shoot to enable robust production of fruit is therefore valuable in increasing the efficiency of marketable production.
  • Marketable and unmarketable designations can apply to both the plant and fruit, and can be defined differently based on the end use of the product, such as but not limited to, fresh market produce and processing for inclusion as an ingredient in a composition.
  • the marketable determination can assess such qualities as, but not limited to, color, insect damage, blossom end rot, softness, and sunburn.
  • the term“total production” can incorporate both marketable and unmarketable plants and fruit.
  • the ratio of marketable plants or fruit to unmarketable plants or fruit can be referred to as“utilization” and expressed as a percentage.
  • the utilization can be used as an indicator of the efficiency of the agricultural process as it shows the successful production of marketable plants or fruit, which will be obtain the highest financial return for the grower, whereas total production will not provide such an indication.
  • Microalgae can be grown in hetero trophic, mixotrophic, and phototrophic conditions.
  • Culturing microalgae in heterotrophic conditions comprises supplying organic carbon (e.g., acetic acid, acetate, glucose) to cells in an aqueous culture medium comprising trace metals and nutrients (e.g., nitrogen, phosphorus).
  • Culturing microalgae in mixotrophic conditions comprises supplying light and organic carbon (e.g., acetic acid, acetate, glucose) to cells in an aqueous culture medium comprising trace metals and nutrients (e.g., nitrogen, phosphorus).
  • Culturing microalgae in phototrophic conditions comprises supplying light and inorganic carbon (e.g., carbon dioxide) to cells in an aqueous culture medium comprising trace metals and nutrients (e.g., nitrogen, phosphorus).
  • the microalgae cells can be harvested from a culture and used as whole cells in a liquid composition for application to seeds and plants, while in other embodiments the harvested microalgae cells can be subjected to downstream processing and the resulting biomass or extract can be used in a dried composition (e.g., powder, pellet) or a liquid composition (e.g., suspension, solution) for application to plants, soil, or a combination thereof.
  • downstream processing comprise: drying the cells, lysing the cells, and subjecting the harvested cells to a solvent or supercritical carbon dioxide extraction process to isolate an oil or protein.
  • the extracted (i.e., residual) biomass remaining from an extraction process can be used alone or in combination with other microalgae or extracts in a liquid composition for application to plants, soil, or a combination thereof.
  • the resulting biomass is transformed from a natural whole state to a lysed condition where the cell is missing a significant amount of the natural components, thus differentiating the extracted microalgae biomass from that which is found in nature.
  • Excreted products from the microalgae can also be isolated from a microalgae culture using downstream processing methods.
  • microalgae include at least 60% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 50% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 40% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 30% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 20% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 10% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 5% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 1% of the active ingredient sources of the composition. In some embodiments, the composition lacks any detectable amount of any other active ingredient source other than microalgae.
  • microalgae biomass, excreted products, or extracts can also be mixed with biomass or extracts from other plants, microalgae, macroalgae, seaweeds, and kelp.
  • microalgae biomass, excreted products, or extracts can also be mixed with fish oil.
  • Non-limiting examples of other plants, macroalgae, seaweeds, and kelp fractions that can be combined with microalgae cells can include species of Lemna, Gracilaria, Kappaphycus, Ascophyllum, Macrocystis, Fucus, Laminaria, Sargassum, Turbinaria, and Durvilea.
  • the extracts can include 2% or less by volume of the composition. In some embodiments, the extracts can include 1% or less by volume ofthe composition.
  • microalgae refers to microscopic single cell organisms such as microalgae, cyanobacteria, algae, diatoms, dinoflagellates, freshwater organisms, marine organisms, or other similar single cell organisms capable of growth in phototrophic, mixotrophic, or hetero trophic culture conditions.
  • microalgae biomass, excreted product, or extracts can also be sourced from multiple types of microalgae, to make a composition that is beneficial when applied to plants or soil.
  • microalgae that can be used in the compositions and methods of the present invention include microalgae in the classes: Eustigmatophyceae, Chlorophyceae, Prasinophyceae, Haptophyceae, Cyanidiophyceae, Prymnesiophyceae, Porphyridiophyceae, Labyrinthulomycetes, Trebouxiophyceae, Bacillariophyceae, and Cyanophyceae.
  • the class Cyanidiophyceae includes species of Galdieria.
  • the class Chlorophyceae includes species of Haematococcus, Scenedesmus, Chlamydomonas, and Micractinium.
  • the class Prymnesiophyceae includes species of Isochrysis and Pavlova.
  • the class Eustigmatophyceae includes species of Nannochloropsis.
  • the class Porphyridiophyceae includes species of Porphyridium.
  • the class Labyrinthulomycetes includes species of Schizochytrium and Aurantiochytrium.
  • the class Prasinophyceae includes species of Tetraselmis.
  • the class Trebouxiophyceae includes species of Chlorella and Botryococcus.
  • the class Bacillariophyceae includes species of Phaeodactylum.
  • the class Cyanophyceae includes species of Spirulina.
  • Chaetoceros sp. Chlamydomonas sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolate, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var.
  • Chlorella kessleri Chlorella lobophora
  • Chlorella luteoviridis Chlorella luteoviridis var. aureoviridis
  • Chlorella luteoviridis var. lutescens Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var.
  • Taxonomic classification has also been in flux for organisms in the genus Schizochytrium. Some organisms previously classified as Schizochytrium have been reclassified as Aurantiochytrium, Thraustochytrium, or Oblongichytrium. See Yokoyama et al. Taxonomic rearrangement of the genus Schizochytrium [sensu lato] based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thrausochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen.
  • the culturing process differs from the culturing process that microalgae experiences in nature.
  • intervention by human operators or automated systems occurs during the non-axenic mixotrophic culturing of microalgae through contamination control methods to prevent the microalgae from being overrun and outcompeted by contaminating organisms (e.g., fungi, bacteria).
  • Contamination control methods for microalgae cultures are known in the art and such suitable contamination control methods for non-axenic mixotrophic microalgae cultures are disclosed in W02014/074769A2 (Ganuza, et al.), hereby incorporated by reference.
  • the impact of the contaminating microorganisms can be mitigated by suppressing the proliferation of containing organism populations and the effect on the microalgal cells (e.g., lysing, infection, death, clumping).
  • the microalgae culture produced as a whole and used in the described inventive compositions differs from the culture that results from a microalgae culturing process that occurs in nature.
  • the microalgae culture can also include cell debris and compounds excreted from the microalgae cells into the culture medium.
  • the output of the microalgae mixotrophic culturing process provides the active ingredient for composition that is applied to plants for improving yield and quality without separate addition to or supplementation of the composition with other active ingredients not found in the mixotrophic microalgae whole cells and accompanying culture medium from the mixotrophic culturing process such as, but not limited to: microalgae extracts, macroalgae, macroalgae extracts, liquid fertilizers, granular fertilizers, mineral complexes (e.g., calcium, sodium, zinc, manganese, cobalt, silicon), fungi, bacteria, nematodes, protozoa, digestate solids, chemicals (e.g., ethanolamine, borax, boric acid), humic acid, nitrogen and nitrogen derivatives, phosphorus rock, pesticides, herbicides,
  • the microalgae can be previously frozen and thawed before inclusion in the liquid composition.
  • the microalgae may not have been subjected to a previous freezing or thawing process.
  • the microalgae whole cells have not been subjected to a drying process.
  • the cell walls of the microalgae of the composition have not been lysed or disrupted, and the microalgae cells have not been subjected to an extraction process or process that pulverizes the cells.
  • microalgae whole cells are not subjected to a purification process for isolating the microalgae whole cells from the accompanying constituents of the culturing process (e.g., trace nutrients, residual organic carbon, bacteria, cell debris, cell excretions), and thus the whole output from the microalgae culturing process comprising whole microalgae cells, culture medium, cell excretions, cell debris, bacteria, residual organic carbon, and trace nutrients, is used in the liquid composition for application to plants.
  • the microalgae whole cells and the accompanying constituents of the culturing process are concentrated in the composition.
  • the microalgae whole cells and the accompanying constituents of the culturing process are diluted in the composition to a low concentration.
  • the microalgae whole cells of the composition are not fossilized.
  • the microalgae whole cells are not maintained in a viable state in the composition for continued growth after the method of using the composition in a soil or foliar application.
  • the microalgae base composition can be biologically inactive after the composition is prepared.
  • the microalgae base composition can be substantially biologically inactive after the composition is prepared.
  • the microalgae base composition can increase in biological activity after the prepared composition is exposed to air.
  • a liquid composition can include low concentrations of bacteria contributing to the solids percentage of the composition in addition to the microalgae cells.
  • bacteria found in non-axenic mixotrophic conditions can be found in W02014/074769A2 (Ganuza, et al.), hereby incorporated by reference.
  • a live bacteria count can be determined using methods known in the art such as plate counts, plates counts using Petrifilm available from 3M (St. Paul, Minnesota), spectrophotometric (turbidimetric) measurements, visual comparison of turbidity with a known standard, direct cell counts under a microscope, cell mass determination, and measurement of cellular activity.
  • Live bacteria counts in a non-axenic mixotrophic microalgae culture can range from 104 to 109 CFU/mL, and can depend on contamination control measures taken during the culturing of the microalgae.
  • the level of bacteria in the composition can be determined by an aerobic plate count which quantifies aerobic colony forming units (CFU) in a designated volume.
  • the composition includes an aerobic plate count of 40,000-400,000 CFU/mL.
  • the composition includes an aerobic plate count of 40,000-100,000 CFU/mL.
  • the composition includes an aerobic plate count of 100,000-200,000 CFU/mL.
  • the composition includes an aerobic plate count of 200,000-300,000 CFU/mL.
  • the composition includes an aerobic plate count of 300,000- 400,000 CFU/mL.
  • the microalgae based composition can be supplemented with a supplemental nutrient such as nitrogen, phosphorus, or potassium to increase the levels within the composition to at least 1% of the total composition (i.e., addition of N, P, or K to increase levels at least 1-0-0, 0-1-0, 0-0-1, or combinations thereof).
  • a supplemental nutrient such as nitrogen, phosphorus, or potassium to increase the levels within the composition to at least 1% of the total composition (i.e., addition of N, P, or K to increase levels at least 1-0-0, 0-1-0, 0-0-1, or combinations thereof).
  • the microalgae composition can be supplemented with nutrients such as, but not limited to, calcium, magnesium, silicon, sulfur, iron, manganese, zinc, copper, boron, molybdenum, chlorine, sodium, aluminum, vanadium, nickel, cerium, dysprosium, erbium, europium, gadolinium, holmium, lanthanum, lutetium, neodymium, praseodymium, promethium, samarium, scandium, terbium, thulium, ytterbium, and yttrium.
  • the supplemented nutrient is not uptaken, chelated, or absorbed by the microalgae.
  • the concentration of the supplemental nutrient can include 1-50 g per 100 g of the composition.
  • a liquid composition comprising microalgae can be stabilized by heating and cooling in a pasteurization process.
  • the active ingredients of the microalgae based composition maintained effectiveness in at least one characteristic of a plant after being subjected to the heating and cooling of a pasteurization process.
  • liquid compositions with whole cells or processed cells (e.g., dried, lysed, extracted) of microalgae cells may not need to be stabilized by pasteurization.
  • microalgae cells that have been processed, such as by drying, lysing, and extraction, or extracts can include such low levels of bacteria that a liquid composition can remain stable without being subjected to the heating and cooling of a pasteurization process.
  • the composition can be heated to a temperature in the range of 50-130°C. In some embodiments, the composition can be heated to a temperature in the range of 55-65°C. In some embodiments, the composition can be heated to a temperature in the range of 58-62°C. In some embodiments, the composition can be heated to a temperature in the range of 50-60°C. In some embodiments, the composition can be heated to a temperature in the range of 60- 90°C. In some embodiments, the composition can be heated to a temperature in the range of 70-80°C. In some embodiments, the composition can be heated to a temperature in the range of 100-150°C. In some embodiments, the composition can be heated to a temperature in the range of 120-130°C.
  • the composition can be heated for a time period in the range of 1-150 minutes. In some embodiments, the composition can be heated for a time period in the range of 110-130 minutes. In some embodiments, the composition can be heated for a time period in the range of 90-100 minutes. In some embodiments, the composition can be heated for a time period in the range of 100- 110 minutes. In some embodiments, the composition can be heated for a time period in the range of 110-120 minutes. In some embodiments, the composition can be heated for a time period in the range of 120-130 minutes. In some embodiments, the composition can be heated for a time period in the range of 130-140 minutes. In some embodiments, the composition can be heated for a time period in the range of 140- 150 minutes. In some embodiments, the composition is heated for less than 15 min. In some embodiments, the composition is heated for less than 2 min.
  • the compositions can be cooled at any rate to a temperature that is safe to work with.
  • the composition can be cooled to a temperature in the range of 35-45°C.
  • the composition can be cooled to a temperature in the range of 36-44°C.
  • the composition can be cooled to a temperature in the range of 37- 43 °C.
  • the composition can be cooled to a temperature in the range of 38-42°C.
  • the composition can be cooled to a temperature in the range of 39-41 °C.
  • the pasteurization process can be part of a continuous production process that also involves packaging, and thus the liquid composition can be packaged (e.g., bottled) directly after the heating or high temperature stage without a cooling step.
  • the composition can include 5-30% solids by weight of microalgae cells (i.e., 5-30 g of microalgae cells/100 mL of the liquid composition). In some embodiments, the composition can include 5-20% solids by weight of microalgae cells. In some embodiments, the composition can include 5- 15% solids by weight of microalgae cells. In some embodiments, the composition can include 5-10% solids by weight of microalgae cells. In some embodiments, the composition can include 10-20% solids by weight of microalgae cells. In some embodiments, the composition can include 10-20% solids by weight of microalgae cells. In some embodiments, the composition can include 20-30% solids by weight of microalgae cells. In some embodiments, further dilution of the microalgae cells percent solids by weight can occur before application for low concentration applications of the composition.
  • the composition can include less than 1% by weight of microalgae biomass or extracts (i.e., less than 1 g of microalgae derived product/100 mL of the liquid composition). In some embodiments, the composition can include less than 0.9% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.8% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.7% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.6% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.5% by weight of microalgae biomass or extracts.
  • the composition can include less than 0.4% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.3% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.2% by weight of microalgae biomass or extracts. In some embodiments, the composition can include less than 0.1 % by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.0001 % by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.001 % by weight of microalgae biomass or extracts. In some embodiments, the composition can include at least 0.01% by weight of microalgae biomass or extracts.
  • the composition can include at least 0.1 % by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.0001-1 % by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.0001-0.001% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.001-.01% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.01-0.1% by weight of microalgae biomass or extracts. In some embodiments, the composition can include 0.1-1% by weight of microalgae biomass or extracts.
  • an application concentration of 0.1% of microalgae biomass or extract equates to 0.04 g of microalgae biomass or extract in 40 mL of a composition. While the desired application concentration to a plant can be 0.1% of microalgae biomass or extract, the composition can be packaged as a 10% concentration (0.4 mL in 40 mL of a composition). Thus, a desired application concentration of 0.1% would require 6,000 mL of the 10% microalgae biomass or extract in the 100 gallons of water applied to the assumption of 15,000 plants in an acre, which is equivalent to an application rate of about 1.585 gallons per acre.
  • a desired application concentration of 0.01% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.159 gallons per acre. In some embodiments, a desired application concentration of 0.001% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.016 gallons per acre. In some embodiments, a desired application concentration of 0.0001% of microalgae biomass or extract using a 10% concentration composition equates to an application rate of about 0.002 gallons per acre.
  • the water requirement assumption of 100 gallons per acre is equal to about 35 mL of water per plant. Therefore, 0.025 g of microalgae biomass or extract in 35 mL of water is equal to about 0.071 g of microalgae biomass or extract per 100 mL of composition equates to about a 0.07% application concentration.
  • the microalgae biomass or extract based composition can be applied at a rate in a range as low as about 0.001-10 gallons per acre, or as high as up to 150 gallons per acre.
  • the applications were performed using a 10% solids solution by weight microalgae composition.
  • the rates are indicated in gal/acre and the amount of carrier water would be determined according to user preference.
  • the application rate may range between 0.25 gal/acre - 2 gal/acre.
  • the equivalent expressed in total grams of solid microalgae would be lOOg microalgae/acre; wherein the application rate of the microalgae composition is 0.4 gal/acre, the equivalent expressed in total grams of solid microalgae would be l60g microalgae/acre; where the application rate of the microalgae composition is 0.5 gal/acre, the equivalent expressed in total grams of solid microalgae would be 200g microalgae/acre; where the application rate of the microalgae composition is 1.0 gal/acre, the equivalent expressed in total grams of solid microalgae would be 400g microalgae/acre; and where the application rate of the microalgae composition is 2.0 gal/acre, the equivalent expressed in total grams of solid microalgae would be 800g microalgae/acre.
  • the microalgae composition may comprise between l00g-800g per acre, as it is common practice for growers to use between 100-250 gallons of liquid carrier volume/acre. It should be clearly understood, however, that modifications to the amount of microalgae per acre may be adjusted upwardly or downwardly to compensate for greater than 250 gallons of liquid carrier volume/acre or less than 100 gallons of liquid carrier volume/acre.
  • stabilizing means that are not active regarding the improvement of plant germination, emergence, maturation, quality, and yield, but instead aid in stabilizing the composition can be added to prevent the proliferation of unwanted microorganisms (e.g., yeast, mold) and prolong shelf life.
  • inactive but stabilizing means can include an acid, such as but not limited to phosphoric acid or citric acid, and a yeast and mold inhibitor, such as but not limited to potassium sorbate.
  • the stabilizing means are suitable for plants and do not inhibit the growth or health of the plant.
  • the stabilizing means can contribute to nutritional properties of the liquid composition, such as but not limited to, the levels of nitrogen, phosphorus, or potassium.
  • the composition can include between 0.5- 1.5% phosphoric acid. In other embodiments, the composition may comprise less than 0.5% phosphoric acid. In some embodiments, the composition can include 0.01- 0.3% phosphoric acid. In some embodiments, the composition can include 0.05- 0.25% phosphoric acid. In some embodiments, the composition can include 0.01- 0.1% phosphoric acid. In some embodiments, the composition can include 0.1 -0.2% phosphoric acid. In some embodiments, the composition can include 0.2- 0.3% phosphoric acid. In some embodiments, the composition can include less than 0.3% citric acid.
  • the composition can include 1.0-2.0% citric acid. In other embodiments, the composition can include 0.01-0.3% citric acid. In some embodiments, the composition can include 0.05-0.25% citric acid. In some embodiments, the composition can include 0.01-0.1% citric acid. In some embodiments, the composition can include 0.1 -0.2% citric acid. In some embodiments, the composition can include 0.2-0.3% citric acid.
  • the composition can include less than 0.5% potassium sorbate. In some embodiments, the composition can include 0.01-0.5% potassium sorbate. In some embodiments, the composition can include 0.05-0.4% potassium sorbate. In some embodiments, the composition can include 0.01-0.1% potassium sorbate. In some embodiments, the composition can include 0.1-0.2% potassium sorbate. In some embodiments, the composition can include 0.2-0.3% potassium sorbate. In some embodiments, the composition can include 0.3-0.4% potassium sorbate. In some embodiments, the composition can include 0.4-0.5% potassium sorbate.
  • the present invention involves the use of a microalgae composition.
  • microalgae compositions methods of preparing liquid microalgae compositions, and methods of applying the microalgae compositions to plants are disclosed in WO2017/218896A1 (Shinde et al.) entitled Microalgae-Based Composition, and Methods of its Preparation and Application to Plants, which is incorporated herein in full by reference.
  • the microalgae composition may comprise approximately 10%-10.5% w/w of Chlorella microalgae cells.
  • the microalgae composition may also comprise one of more stabilizers, such as potassium sorbate, phosphoric acid, ascorbic acid, sodium benzoate, citric acid, or the like, or any combination thereof.
  • the microalgae composition may comprise approximately .3% w/w of potassium sorbate or another similar compound to stabilize its pH and may further comprise approximately .5- 1.5% w/w phosphoric acid or another similar compound to prevent the growth of contaminants.
  • the microalgae composition may comprise 1.0-2.0% w/w citric acid to stabilize its pH, and may not contain potassium sorbate or phosphoric acid.
  • the pH of the microalgae composition may be stabilized to between 3.0-4.0.
  • the microalgae composition may be referred to as PHYCOTERRA ® .
  • the PHYCOTERRA ® Chlorella microalgae composition is a microalgae composition comprising Chlorella.
  • the PHYCOTERRA ® Chlorella microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at between 65°C - 75°C for between 90 - 150 minutes, adding potassium sorbate and phosphoric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration.
  • the PHYCOTERRA® Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the PHYCOTERRA ® Chlorella microalgae composition may comprise between approximately 0.3% potassium sorbate and between approximately .5%-l.5% phosphoric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88.2%-89.2% water. It should be clearly understood, however, that other variations of the PHYCOTERRA ® Chlorella microalgae composition, including variations in the microalgae strains, variations in the stabilizers, and/or variations in the % composition of each component may be used and may achieve similar results.
  • the microalgae composition may be an OMRI certified microalgae composition referred to as PhycoTerra Organic ® (previously known as TERRENE ® ).
  • the OMRI certified PhycoTerra Organic ® shall be referred to hereinafter as PT-0 for brevity.
  • PT-0 Chlorella microalgae composition is a microalgae composition comprising Chlorella.
  • the microalgae composition may be an OMRI certified microalgae composition referred to as OMRI certified PhycoTerra ® Organic Chlorella pasteurized at 65°C microalgae composition or as PT- 065.
  • PT-0 Chlorella pasteurized at 65°C microalgae composition is a microalgae composition comprising Chlorella.
  • PT-0 Chlorella pasteurized at 65°C microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at 65°C for between 90 - 150 minutes, adding citric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration.
  • PT-0 Chlorella pasteurized at 65°C microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells.
  • PT-0 Chlorella pasteurized at 65°C microalgae composition may comprise between approximately 0.5% - 2.0% citric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88-89.5% water. It should be clearly understood, however, that other variations of PT-0 Chlorella pasteurized at 65°C microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, and/or variations in the % composition of each component may be used and may achieve similar results.
  • the microalgae composition may be an OMRI certified microalgae composition referred to as PT-0 Chlorella pasteurized at 90°C microalgae composition or as PT-O90.
  • PT-0 Chlorella pasteurized at 90°C microalgae composition is a microalgae composition comprising Chlorella.
  • PT- O Chlorella pasteurized at 90°C microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at 90°C for between 90 - 150 minutes, adding citric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration.
  • PT-0 Chlorella pasteurized at 90°C microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells.
  • PT-0 Chlorella pasteurized at 90°C microalgae composition may comprise between approximately 0.5% - 2.0% citric acid to stabilize the pH of the Chlorella to between 3.0- 4.0 and 88-89.5% water. It should be clearly understood that other variations of PT-0 Chlorella pasteurized at 90°C microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, and/or variations in the % composition of each component may be used and may achieve similar results.
  • the microalgae composition may be referred to as Aurantiochytrium acetophilum HS399 whole biomass (WB) or HS399 WB.
  • the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition is a microalgae composition comprising Aurantiochytrium acetophilum HS399.
  • the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition treatments were prepared by growing the Aurantiochytrium acetophilum HS399 microalgae in non-axenic acetic acid supplied heterotrophic conditions, increasing the concentration of Aurantiochytrium acetophilum HS399 using a centrifuge, pasteurizing the concentrated Aurantiochytrium acetophilum HS399 at between 65°C- 75°C for between 90-150 minutes, adding approximately 0.3% w/w of potassium sorbate and between approximately .5-1.5% phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilum HS399 to between 3.0-4.0, and then adjusting the whole biomass to a desired concentration.
  • the microalgae composition may be referred to as Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed).
  • the Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition is a microalgae composition comprising Aurantiochytrium acetophilum HS399.
  • the Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition treatments were prepared by growing the Aurantiochytrium acetophilum HS399 microalgae in non-axenic acetic acid supplied heterotrophic conditions, increasing the concentration of Aurantiochytrium acetophilum HS399 using a centrifuge, pasteurizing the concentrated Aurantiochytrium acetophilum HS399 at between 65°C-75°C for between 90-150 minutes, adding approximately 0.3% w/w of potassium sorbate and between approximately .5%-l.5% phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilum HS399 to between 3.0-4.0, and then adjusting the whole biomass to a desired concentration.
  • the Aurantiochytrium acetophilum HS399 microalgae cells were concentrated from the harvest, they were washed; i.e. diluted with water in a ratio of 5:1 and centrifuged again in order to remove dissolved material and small particles.
  • WB washed whole biomass
  • the microalgae composition may be referred to as Aurantiochytrium acetophilum HS399 extracted biomass (EB) or HS399 EB.
  • the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition is a microalgae composition comprising Aurantiochytrium acetophilum HS399.
  • the Aurantiochytrium acetophilum HS399 extracted biomass (EB) treatments were prepared by growing the Aurantiochytrium acetophilum HS399 microalgae in non-axenic acetic acid supplied heterotrophic conditions, increasing the concentration of Aurantiochytrium acetophilum HS399 using a centrifuge, pasteurizing the concentrated Aurantiochytrium acetophilum HS399 at between 65°C-75°C for between 90-150 minutes, adding approximately 0.3% w/w of potassium sorbate and between approximately .5%-l.5% phosphoric acid to stabilize the pH of the Aurantiochytrium acetophilum HS399 to between 3.0-4.0, processing the Aurantiochytrium acetophilum HS399 with an oat filler in an expeller process to lyse the cells and separate oil from the residual biomass, and then adjusting the residual biomass to a desired concentration.
  • EB extracted biomass
  • the microalgae composition may be referred to as a combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition or 25% Chlorella : 75% HS399 WB.
  • the combination 25% Chlorella : 75% HS399 whole biomass (WB) microalgae composition is a microalgae composition comprising Chlorella and Aurantiochytrium acetophilum HS399.
  • the Chlorella microalgae cells were cultured in outdoor pond reactors in non-axenic acetic acid supplied mixotrophic conditions and the concentration of Chlorella was increased using a centrifuge.
  • the Aurantiochytrium acetophilum HS399 cells were cultured in non- axenic acetic-acid supplied heterotrophic conditions and the concentration of HS399 was increased using a centrifuge.
  • the concentrated Chlorella cells were then combined with the concentrated HS399 whole biomass cells and adjusted to the desired concentration of 25% Chlorella : 75% HS399 whole biomass (WB).
  • the combination 25% Chlorella : 75% HS399 whole biomass (WB) microalgae composition was then pasteurized at between 65°C-75°C for between 90- 150 minutes and then stabilized by adding approximately 0.3 % w/w of potassium sorbate and between approximately .5%-l.5% phosphoric acid to stabilize the pH of the 25% Chlorella : 75% HS399 whole biomass (WB) microalgae composition to between 3.0-4.0.
  • the microalgae composition may be referred to as GP2C.
  • the GP2C Chlorella microalgae composition comprised Chlorella.
  • the GP2C Chlorella microalgae composition treatments were prepared by growing the Chlorella in non-axenic acetic acid supplied mixotrophic conditions, increasing the concentration of Chlorella using a centrifuge, pasteurizing the concentrated Chlorella at between 65°C-75°C for between 90-150 minutes, adding potassium sorbate and phosphoric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration.
  • the GP2C Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, the GP2C microalgae composition may comprise between approximately 0.3% potassium sorbate and between approximately 05%-l.5% phosphoric acid to stabilize the pH of the Chlorella to between 3.0-4.0 and 88.2%-89% water. It should be clearly understood, however, that other variations of the GP2C Chlorella microalgae composition, including variations in the microalgae strains, variations in the stabilizers, and/or variations in the % composition of each component may be used and may achieve similar results.
  • the microalgae composition may be referred to as a combination 25% Chlorella : 75% HS399 extracted biomass (EB) microalgae composition, a 50% Chlorella : 50% HS399 extracted biomass (EB) microalgae composition, a 75% Chlorella : 25% HS399 extracted biomass (EB) microalgae composition, or a combination GP2C:399 microalgae composition.
  • the combination GP2C:399 microalgae composition comprises Chlorella and Aurantiochytrium acetophilum HS399 extracted biomass (EB).
  • the Chlorella microalgae cells were cultured in outdoor pond reactors in non-axenic acetic acid supplied mixotrophic conditions and the concentration of Chlorella was increased using a centrifuge; the Aurantiochytrium acetophilum HS399 microalgae cells were cultured in non-axenic acetic acid supplied heterotrophic conditions, the concentration of HS399 was increased using a centrifuge, and the HS399 cells were then processed with an oat filler in an expeller process to lyse the cells and separate oil from the residual biomass.
  • the concentrated GP2C Chlorella whole biomass microalgae cells and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae cells were blended together to the ratios of 50:50, 25:75, and 75:25, then pasteurized at between 65°C-75°C for between 90-150 minutes and then stabilized by adding approximately 0.3% w/w of potassium sorbate and between approximately .5%-l.5% phosphoric acid to stabilize the pH of the 25% Chlorella : 75% HS399 extracted biomass (EB) microalgae composition to between 3.0-4.0.
  • Greenwater Polyculture may be prepared by beginning with a culture of Scenedesmus microalgae that is left outdoors in an open pond and harvested continuously over a year.
  • the culture may comprise anywhere from less than 50% Scenedesmus to greater than 75% Scenedesmus and the concentration varies throughout the year.
  • Other algae may colonize in the GWP as well as other bacteria and microorganisms.
  • the composition is a liquid and substantially includes of water.
  • the composition can include 70-99% water.
  • the composition can include 85-95% water.
  • the composition can include 70-75% water.
  • the composition can include 75-80% water.
  • the composition can include 80-85% water.
  • the composition can include 85- 90% water.
  • the composition can include 90- 95% water.
  • the composition can include 95-99% water.
  • the liquid nature and high-water content of the composition facilitates administration of the composition in a variety of manners, such as but not limit to: flowing through an irrigation system, flowing through an above ground drip irrigation system, flowing through a buried drip irrigation system, flowing through a central pivot irrigation system, sprayers, sprinklers, and water cans.
  • the liquid composition can be used immediately after formulation, or can be stored in containers for later use.
  • the composition can be stored out of direct sunlight.
  • the composition can be refrigerated.
  • the composition can be stored at 1-10°C.
  • the composition can be stored at l-3°C.
  • the composition can be stored at 3- 50°C.
  • the composition can be stored at 5-8°C.
  • the composition can be stored at 8-10°C.
  • administration of the liquid composition to soil, a seed or plant can be in an amount effective to produce an enhanced characteristic in plants compared to a substantially identical population of untreated seeds or plants.
  • Such enhanced characteristics can include accelerated seed germination, accelerated seedling emergence, improved seedling emergence, improved leaf formation, accelerated leaf formation, improved plant maturation, accelerated plant maturation, increased plant yield, increased plant growth, increased plant quality, increased plant health, increased fruit yield, increased fruit sweetness, increased fruit growth, and increased fruit quality.
  • Non-limiting examples of such enhanced characteristics can include accelerated achievement of the hypocotyl stage, accelerated protrusion of a stem from the soil, accelerated achievement of the cotyledon stage, accelerated leaf formation, increased marketable plant weight, increased marketable plant yield, increased marketable fruit weight, increased production plant weight, increased production fruit weight, increased utilization (indicator of efficiency in the agricultural process based on ratio of marketable fruit to unmarketable fruit), increased chlorophyll content (indicator of plant health), increased plant weight (indicator of plant health), increased root weight (indicator of plant health), increased shoot weight (indicator of plant health), increased plant height, increased thatch height, increased resistance to salt stress, increased plant resistance to heat stress (temperature stress), increased plant resistance to heavy metal stress, increased plant resistance to drought, increased plant resistance to disease, improved color, reduced insect damage, reduced blossom end rot, and reduced sun bum.
  • Such enhanced characteristics can occur individually in a plant, or in combinations of multiple enhanced characteristics.
  • a liquid composition can be administered before the seed is planted. In some embodiments, a liquid composition can be administered at the time the seed is planted. In some embodiments, a liquid composition can be applied by dip treatment of the roots. In some embodiments, a liquid composition can be administered to plants that have emerged from the ground. In some embodiments, a liquid or dried composition can be applied to the soil before, during, or after the planting of a seed. In some embodiments a liquid or dried composition can be applied to the soil before or after a plant emerges from the soil.
  • the volume or mass of the microalgae based composition applied to a seed, seedling, or plant may not increase or decrease during the growth cycle of the plant (i.e., the amount of the microalgae composition applied to the plant will not change as the plant grows larger).
  • the volume or mass of the microalgae based composition applied to a seed, seedling, or plant can increase during the growth cycle of the plant (i.e., applied on a mass or volume per plant mass basis to provide more of the microalgae composition as the plant grows larger).
  • the volume or mass of the microalgae based composition applied to a seed, seedling, or plant can decrease during the growth cycle of the plant (i.e., applied on a mass or volume per plant mass basis to provide more of the microalgae composition as the plant grows larger).
  • the administration of the composition may comprise contacting the foliage of the plant with an effective amount of the composition.
  • the liquid composition may be sprayed on the foliage by a hand sprayer, a sprayer on an agriculture implement, or a sprinkler.
  • the composition can be applied to the soil.
  • the rate of application of the composition at the desired concentration can be expressed as a volume per area.
  • the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 10-50 gallons/acre.
  • the rate of application of the liquid composition in a foliar application can comprise a rate in the rage of 10-15 gallons/acre.
  • the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 15-20 gallons/acre.
  • the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 20-25 gallons/acre.
  • the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 25-30 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 30-35 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 35-40 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 40-45 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 45-50 gallons/acre.
  • the rate of application of the liquid composition in a soil or foliar application can comprise a rate in the range of 0.01-10 gallons/acre. In some embodiments, the rate can be 0.12-4%. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 0.01-0.1 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil or foliar application may comprise a rate in the range of 0.1- 1.0 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 0.25-2 gallons/acre.
  • the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 1-2 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 2-3 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 3-4 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 4-5 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application may comprise a rate in the range of 5-10 gallons/acre. [0145] In some embodiments, the v/v ratio of the composition can be between
  • the v/v ratio can be between 0.01-25%.
  • the v/v ratio of the composition can be between 0.03-10%.
  • the frequency of the application of the composition can be expressed as the number of applications per period of time (e.g., two applications per month), or by the period of time between applications (e.g., one application every 21 days).
  • the plant can be contacted by the composition in a foliar application every 3-28 days.
  • the plant can be contacted by the composition in a foliar application every 4-10 days.
  • the plant can be contacted by the composition in a foliar application every 18-24 days.
  • the plant can be contacted by the composition in a foliar application every 3-7 days.
  • the plant can be contacted by the composition in a foliar application every 7-14 days.
  • the plant can be contacted by the composition in a foliar application every 14-21 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 21-28 days. In some embodiments, the soil or plant can be treated with the composition once per planting. In some embodiments, the soil or plant can be treated with the composition one time every cutting/harvest.
  • Foliar application(s) of the composition generally begin after the plant has become established, but can begin before establishment, at defined time period after planting, or at a defined time period after emergence from the soil in some embodiments.
  • the plant can be first contacted by the composition in a foliar application 5-14 days after the plant emerges from the soil.
  • the plant can be first contacted by the composition in a foliar application 5-7 days after the plant emerges from the soil.
  • the plant can be first contacted by the composition in a foliar application 7-10 days after the plant emerges from the soil.
  • the plant can be first contacted by the composition in a foliar application 10-12 days after the plant emerges from the soil.
  • the plant can be first contacted by the composition in a foliar application 12-14 days after the plant emerges from the soil.
  • the administration of the composition can include contacting the soil in the immediate vicinity of the planted seed with an effective amount of the composition.
  • the liquid composition can be supplied to the soil by injection into a low volume irrigation system, such as but not limited to a drip irrigation system supplying water beneath the soil through perforated conduits or at the soil level by fluid conduits hanging above the ground or protruding from the ground.
  • the liquid composition can be supplied to the soil by a soil drench method wherein the liquid composition is poured on the soil.
  • the composition can be diluted to a lower concentration for an effective amount in a soil application by mixing a volume of the composition in a volume of water.
  • the percent solids of microalgae sourced components resulting in the diluted composition can be calculated by the multiplying the original concentration in the composition by the ratio of the volume of the composition to the volume of water.
  • the grams of microalgae sourced components in the diluted composition can be calculated by the multiplying the original grams of microalgae sourced components per 100 mL by the ratio of the volume of the composition to the volume of water.
  • the rate of application of the composition at the desired concentration can be expressed as a volume per area.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 50- 150 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 75-125 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 50-75 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 75-100 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 100-125 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 125-150 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 10-50 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 10- 20 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 20-30 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 30- 40 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 40-50 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 0.01-10 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 0.01-0.1 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 0.1 -1.0 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 1-2 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 2-3 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 3-4 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 4-5 gallons/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 5-10 gallons/acre.
  • the rate of application of the liquid composition in a soil application can include a rate in the range of 2-20 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 3.7- 15 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 2-5 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 5-10 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 10-15 liters/acre. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 15-20 liters/acre.
  • FIGURE 10 shows the average leaf canopy area (cm 2 ) of the romaine lettuce transplants 14 days after seeding.
  • Table 5 below details the various treatment concentrations of the microalgae composition that were applied to the romaine lettuce transplants and the resulting average leaf canopy area for each concentration.
  • the increased leaf canopy area (cm 2 ) of the romaine lettuce transplants shows that the microalgae composition enhances early plant establishment.
  • FIGURE 11 shows the shoot dry weight gain of the romaine lettuce transplants as a percent of the UTC (i.e. no PT-0 Chlorella microalgae composition added) 35 after seeding.
  • Table 6 below details the various treatment concentrations of the PT-0 Chlorella microalgae composition that were applied to the romaine lettuce transplants, the resulting average shoot dry weight (mg), and the resulting % UTC average shoot dry weight (mg) for each concentration.
  • the PT-0 Chlorella microalgae composition was applied at 3% and 5% following seeding, there were significant increases (29% and 25%, respectively) in shoot biomass compared to the UTC at 35 days after seeding.
  • FIGURE 12 shows the root area (cm 2 ) of the romaine lettuce transplants as a percent of the UTC.
  • Table 7 below details the various treatment concentrations of the PT-0 Chlorella microalgae composition that were applied to the romaine lettuce transplants, the resulting average root area per plant (cm 2 ), and the % UTC average root area (cm 2 ) per plant (mg) for each concentration.
  • Romaine lettuce root growth was assessed as root area by image analysis of transplant plugs 35 days after seeding.
  • FIGURE 12 when the PT-0 Chlorella microalgae composition was applied once at seeding, there were significant increases in root growth compared to the controls at 0.5%, 2%, 3%, and 5% concentration rates.
  • the 2% and 5% PT-0 Chlorella microalgae composition treatments resulted in the highest root growth (26% and 25% increases, respectively).
  • a one-time soil application of the PT-0 Chlorella microalgae composition at 3- 5% at the time of seeding was significantly enhanced early plant establishment and accelerated both shoot and root development of romaine lettuce (var. Valley Heart) by greater than 25%.
  • This overall acceleration in plant growth by one-time application of the PT-0 Chlorella microalgae composition treatment may decrease required time from seeding to delivery-ready romaine transplants.
  • the PT-0 Chlorella microalgae composition was applied as soil drench at the time of seeding. Organic fertilizer was applied following first true leaf emergence once every 3-5 days for the remainder of the study. Plants were managed according to the table below. Relative to the control, the PT-0 Chlorella microalgae composition showed the greatest amounts of enhanced initial plant establishment, accelerated plant establishment, and canopy growth during the first 21 days after planting when applied once after seeding.
  • FIGURE 13 shows the leaf canopy area (cm 2 ) of the tomato transplants as a percent of the UTC at 21 days after seeding.
  • Table 8 below details the various treatment concentrations of the two microalgae compositions that were applied to the tomato transplants, the resulting average canopy area (cm 2 ) as a percent of UTC, and the resulting canopy cover per plant (cm 2 ) for each microalgae composition treatment.
  • the 5 % concentrations of the PT-0 Chlorella microalgae composition and the 2.5% and 5% concentrations of the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition increased early tomato canopy growth.
  • This overall acceleration in plant growth by one-time application of the PT-0 Chlorella microalgae composition treatment may decrease required growing time from seeding to delivery-ready tomato transplants.
  • FIGURE 14 shows the leaf canopy area (cm 2 ) of the cauliflower transplants as a percent of the UTC at 16 days after seeding.
  • Table 9 below details the various treatment concentrations of the PT-0 Chlorella microalgae composition that were applied to the cauliflower transplants, the resulting canopy area (cm 2 ) as a percent of UTC, and the resulting canopy cover per plant (cm 2 ) for each microalgae composition treatment.
  • the 3% and 5% concentrations of the PT-0 Chlorella microalgae composition accelerated cauliflower transplant establishment ⁇
  • This overall acceleration in plant growth by one-time application of the PT-0 Chlorella microalgae composition may decrease required growing time from seeding to delivery- ready cauliflower transplants.
  • FIGURE 18 shows that plants receiving the PT-0 Chlorella microalgae composition applied at 1 gal/A produced 7% higher marketable yield than those receiving only standard practice. Crop utilization was not affected. Yield was likely driven by the number of pods produced per plant which was numerically higher than the UTC and correlated more closely with marketable yield than individual pod weight.
  • a trial was conducted on snap peas in Paynesville, Minnesota to evaluate performance of the PT-0 Chlorella microalgae composition.
  • the trial was transplanted in late June 2016 and harvested in late August 2016 and harvested in late August 2016 (63 days) to evaluate the performance of the PT-0 Chlorella microalgae composition. All plots were managed according to standard practice (see Study Parameters below). Relative to standard practice alone, bi-weekly additions of the PT-0 Chlorella microalgae treatments increased shoot growth, number of pods produced per plant, and marketable pea yield.
  • FIGURE 19 shows that fresh shoot biomass was increased 8% when the PT-0 Chlorella microalgae composition was applied bi-weekly at the base of the plant at 4 gal/acre compared to standard practice only (UTC). No effect was observed on root biomass at these rates.
  • plants receiving the PT-0 Chlorella microalgae composition bi-weekly at or above 2 gal/acre produced 5-6 % higher marketable yield than those receiving only standard practice. Increased yield was partially due to more pods produced per plant (by 5-6%) rather than larger pods. This increase was statistically significant at 4 gal/acre. This may indicate faster time to flowering.
  • the PT-0 Chlorella microalgae composition had a significant impact on microbial community similarity.
  • Community dissimilarity analysis performed across all three types of crops and all application rates (1%, 2%, and 4%) indicated that the PT-0 Chlorella microalgae composition increased similarity among plots compared to the UTC (p 0.001).
  • the PT-0 Chlorella microalgae composition increased the abundance of beneficial soil bacteria.
  • Plots receiving the PT-0 Chlorella microalgae composition had at least a two-fold increase in abundances of two bacteria known to be beneficial to plants; Bacillus, a plant growth promoter and Nitrospira, a complete nitrifier.
  • Bacillus Bacillus
  • Nitrospira a complete nitrifier.
  • Phylogenetic analysis placed the unknown Bacillus sp. as B. megaterium (99% 425 bp amplicon). This strain is commonly used as a direct additive in microbial plant products to improve plant performance.
  • a third rhizosphere-associated bacterium, Gaiellales also increased in dominance.
  • biological and physical soil health indicators 1) active carbon - organic matter readily oxidized by soil microbes; 2) soil protein - organic nitrogen pool available to plants in soil; and 3) dry soil aggregate size distribution - indicator of soil quality (e.g. porosity, erosion, resistance, and root penetration).
  • the PT-0 Chlorella microalgae composition causes an increase in biological soil health.
  • FIGURE 24 shows that after one application of the PT-0 Chlorella microalgae composition (0.3-3%), active carbon consistently increased in the soil to a“High” health score within 5 days. This level sustained for at least 15 days.
  • a seaweed commercial reference was included for comparison and affected active carbon similarly to the PT-0 Chlorella microalgae composition when it was applied at label-recommended rates; however, the PT-0 Chlorella microalgae composition at 3% showed a significant advantage (p ⁇ 0.l) over the seaweed commercial reference in all three trials starting 5 days after application and the PT-0 Chlorella microalgae composition showed significant advantage over the seaweed commercial reference on Day 15 in Run 3 (p ⁇ 0.l).
  • soil protein was significantly increased during two trial runs within 15 days of a single application of the PT-0 Chlorella microalgae composition compared to the UTC. This response was observed in the same soil mix as discussed above, planted with tomatoes.
  • the PT-0 Chlorella microalgae composition causes a significant increase in physical soil quality.
  • the portion of soil aggregates that were greater than 1 mm in size increased for 15 days after the PT-0 Chlorella microalgae composition was applied at 3% and was higher than when just water (UTC) or the seaweed commercial reference product were applied.
  • Chlorella microalgae composition on soil health Soil was collected from a field planted with alfalfa from Gilbert, A classified as Antho sandy loam and diluted by 40% with a peat based soil ix and perlite to allow drainage. Quart pots were filled with soil and drenched bi-weekly with the PT-Oand drenched bi-weekly with PT-0 Chlorella microalgae composition (0.3-3% solution v/v in city water) or city water alone (UTC) starting on day 0. Pots were kept moist by watering every 2 days with city water.
  • Soil samples were collected before set-up, immediately following application and then every 15 days and assayed for the following biological and physical soil health indicators: 1) active carbon - organic matter readily oxidized by soil microbes; 2) soil protein - organic nitrogen pool available to plants in soil; and 3) total water holding capacity - improves nutrient delivery and soil microbial health.
  • FIGURES 27-28 PT-0 Chlorella microalgae composition causes an increase in biological soil health.
  • FIGURE 27 shows that after 2 applications of PT-0 Chlorella microalgae composition (0.3-3%), active carbon increased in the soil from a“Medium” to a“Very High” health score.
  • soil protein was“Very Low’ in the initial soil but increased to“medium” over 30 days compared to the UTC.
  • PT-0 Chlorella microalgae composition causes a significant increase in the soil’s water holding capacity.
  • Water holding capacity of treated soil 30 days after the experiment started was increased 6-12% compared to the initial soil sample.
  • Water holding capacity decreased over time for soil receiving water alone (UTC).
  • shelf-life quality may include characteristics or metrics such as, but not limited to, fruit water retention, firmness, and reduction of bruising. Improvement of these factors leads to higher marketability of the fruits.
  • fruits are stored after harvest at room temperature or in cold storage ( ⁇ 40°F) for 5-20 days depending on the trial. For consumer preference 80-100 respondents were recruited and polled for their preference of treated and untreated strawberries.
  • Shelf-life metrics described in the Examples below may include: fruit water-retention; fruit firmness; reduction of bruising; consumer preference for appearance, overall liking, aroma, texture, and flavor (hedonics); and Christmas tree needle loss and water usage.
  • Water-retention leads to a longer shelf-life due to the fact that the fruit maintains its water content longer post-harvest.
  • Fruit is harvested and weighed immediately and then weighed again after a period of time specified for each trial. The difference in weight is attributed to the amount of water lost during the storage period.
  • a penetrometer is used to measure the force that it takes to penetrate the fruit surface. A firmer fruit indicates longer shelf-life.
  • bruising stored fruit is assessed for appearance and bruising is scored as“slight,” “moderate,”“severe,” or“very severe.” Berries with no bruising, slight/light bruising, and moderate bruising are considered “marketable.” For determining consumer preference for appearance, overall liking, aroma, texture, and flavor (hedonics), a subset of treatments from 3 trials was shipped to a sensory lab for consumer testing. Reduced needle loss is an indicator of longer shelf-life of cut Christmas trees.
  • berries from a mid-season harvest were assessed for shelf-life quality after being shipped cold to the University lab and stored for 3 days at 34°F.
  • Post-storage marketability was determined by assessing the percent of stored berries with no to slight bruising, compared to those with moderate to severe bruising (unmarketable). Post-storage marketability was significantly improved for berries from plots treated with the microalgae composition.
  • berries from plots receiving the microalgae composition were preferred slightly more than those grown using local standard practice overall and for appearance, aroma, flavor, and texture when tested 2 days post-harvest.
  • the microalgae composition reduced the effects of post-harvest shipping and storage on berry quality (e.g. less bruising and improved appearance, aroma, flavor, and texture).
  • microalgae compositions were shaken well before application and agitated while in chemigation tank to prevent solids from settling. All plots were managed according to the local standard practice (see Study Parameters below).
  • a second harvest was conducted for a sensory panel. Upon harvest, 100 berries of similar size and ripeness from the standard practice and 2-3 additional treatments were packed into clamshells and shipped to a University sensory lab for sensory panel evaluation. Clamshells were wrapped in bubble wrap and shipped on blue ice.
  • FIGURES 33-34 sixteen weeks after planting, strawberries were harvested and either kept in cold storage onsite or shipped overnight to the university lab where they were kept in cold storage. After a period of 4 days in storage, at both sites, berries were assessed for bruise severity and marketability. With few exceptions, all treatments improved marketability compared to standard practice by >20%. After shipping and storage, almost all treatments also improved marketability over the seaweed- based commercial reference product. Similar patterns were observed for the reduction of severe and moderate bruising. After shipping and storage, the PHYCOTERRA ® Chlorella microalgae composition and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition showed the most advantage.
  • EB extracted biomass
  • CA to evaluate performance of various microalgae compositions on strawberry growth, yield, and post-harvest berry quality; specifically, the PHYCOTERRA ® Chlorella microalgae composition, PT-O90 Chlorella microalgae composition, the Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, the Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition, and the combination 25% Chlorella : 75% HS399 whole biomass (WB) microalgae composition. All plots received standard local fertilization regimen used by the grower for this crop excluding biostimulants.
  • microalgae compositions were added in addition to standard fertilization.
  • Strawberry plants were transplanted to the field in early June 2017, according to local commercial practice.
  • the first product application was via drip irrigation at the time of transplanting and then every 14 days afterward until harvest.
  • the untreated control received the same amount of carrier water as other treatments at the time of each product application.
  • the microalgae compositions were shaken well before application and agitated, if possible, while in the chemigation tank to prevent solids from settling. Berries were harvested according to local commercial schedule. All plots were managed according to the local standard practice (see Study Parameters below). Raw data is shown in Table 11 below.
  • the commercial reference was applied first to the soil at a rate of 20 gal/acre.
  • the PT-065 microalgae composition was then added on top via drip irrigation.
  • the commercial reference was only applied 3 times per season, whereas the PT-065 microalgae composition was applied every 14 days until harvest.
  • Treatments included two versions of an OMRI certified Chlorella microalgae composition that differ by pasteurization temperature (PT-065 microalgae composition and PT-O90 microalgae composition), each tested alone and each tested in combination with the microbial-based commercial reference.
  • Strawberry plants (frigo) were transplanted to the field in June 2017, according to local commercial practice.
  • the first product application was via drip irrigation at the time of transplanting and then every 14 days afterward through to final harvest.
  • the untreated control received the same amount of carrier water as other treatments at the time of each product application.
  • the microalgae compositions were shaken well before application and agitated while in the chemigation tank in order to prevent solids from settling.
  • PT-065microalgae composition (1/2 gal/A; 11-40%), PT-065 microalgae composition alone (1/4 gal/A; 4-8%), and PT-O90 microalgae composition alone (1/4 gal/A, 2-9%) all showed an advantage over standard practice in 2 of 3 assessments.
  • PT-065 microalgae composition and PT-O90 microalgae composition showed an advantage increasing marketability in 2 of 3 assessments (5-30%).
  • PT-065 microalgae composition (1/4 gal/A) and the combination of the Commercial Reference + PT-O90 microalgae composition reduced severe bruising (10-30%) in 2 of 3 assessments. Compared to the commercial reference, multiple treatments showed a benefit.
  • PT-065 microalgae composition applied at 1 ⁇ 2 gal/A was preferred over standard practice for overall liking and flavor liking and generally performed similarly to the microbial-based commercial reference.
  • the commercial reference was preferred over standard practice for all attributes.
  • the microalgae compositions were added in addition to standard fertilization. Strawberry plants were transplanted to the field in July 2017, according to local commercial practice. The first product application was via drip irrigation at the time of transplanting and then every 14 days afterward through to final harvest. The untreated control received the same amount of carrier water as other treatments at the time of each product application.
  • the microalgae compositions were shaken well before application and agitated, if possible, while in the chemigation tank to prevent solids from settling. Berries were harvested according to local commercial schedule (twice per week during the fruiting season). All plots were managed according to the local standard practice (see Study Parameters below).
  • the PT-065 microalgae composition (1/4 gal/ A) and Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition (1/2 gal/ A) showed advantage in 4 of 5 assessments (4-41%); the seaweed-based commercial reference, the PHYCOTERRA ® Chlorella microalgae composition, and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition showed an advantage in 3 of 5 assessments.
  • the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition (1/2 gal/ A) showed an advantage for reducing water loss of stored berries (13-20% in 3 of 5 assessments).
  • the PT- 065 microalgae composition Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition (1/4 gal/A), Aurantiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, and the combination 25% Chlorella : 75% HS399 whole biomass (WB) microalgae composition all showed an advantage in skin firmness (4-20%) compared to standard practice.
  • the PHYCOTERRA ® Chlorella microalgae composition and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition showed an advantage in 3 of 5 assessments (4-14%).
  • microalgae compositions showed an advantage over the commercial reference for the first assessment (2-20%) and for the late season final assessment of PHYCOTERRA ® Chlorella microalgae composition (0.25 gal/A), the PT-065 microalgae composition (0.25 gal/A), and both rates of Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition (6-11%).
  • EB Aurantiochytrium acetophilum HS399 extracted biomass
  • the rockwool and coco block were treated with 25% strength nutrient solution (see Vegetative Recipe in Table 17 below) and watered with city water from 07/18/2017 to 10/13/2017; then switched with 25% fruiting nutrient solution (see Fruiting Recipe in Table 17 below) on 10/13/2017 until termination of the experiment.
  • the irrigation occurred every 20 min, each for a 1 min duration between 07: 15 and 17:30.
  • the microalgae compositions were added beginning at seeding and then every two weeks through the irrigation system in greenhouse.
  • An untreated control (UTC) was also included in the experiment and did not receive any microalgae composition during the entire time of the experiment but did receive the same nutrient media as all other treatments.
  • Microalgae compositions were applied at 38mL/gal (1% solution v/v).
  • the PT-0 microalgae composition treatment improved shelf life of espresso tomato fruits after harvest in comparison to the other three treatments (UTC, the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, and the combination 25% Chlorella: 75% HS399 extracted biomass (EB) microalgae composition).
  • UTC the Aurantiochytrium acetophilum HS399 extracted biomass
  • EB Aurantiochytrium acetophilum HS399 extracted biomass
  • Chlorella 75% HS399 extracted biomass
  • Tomatoes grown with the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition and the combination 25% Chlorella : 75% HS399 extracted biomass (EB) microalgae composition showed 8.2% and 18.2% increase of water retention respectively.
  • the PT-0 microalgae composition treatment showed a 19% increase of water retention compared to UTC.
  • the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition and the combination 25% Chlorella : 75% HS399 extracted biomass (EB) microalgae composition showed an increase of water holding capacity compared to UTC with 6.95% and 10%, respectively.
  • Example 17 [0230] Many products advertised as“Christmas tree preservatives” are sold at nurseries and garden centers where Christmas trees are sold during the Winter holiday season. Advertising claims for tree preservatives usually state that the products reduce “needle (leaf) drop” and preserve the freshness of cut Christmas trees. These products vary wildly in composition and often do not list active ingredients on labels. The PT-0 microalgae composition was evaluated against an untreated control and a commercial reference product for its potential to preserve the quality of cut Christmas trees.
  • Trees were installed in plastic tree stands with approximately 4’ spacing between each tree. The experiment was conducted in a climate-controlled warehouse space. A square perimeter was established around each tree to designate the collection zone for fallen needles. At initial setup, each stand was filled with 1.5L of solution (maximum volume after displacement from tree trunk).
  • the PT-0 microalgae composition was applied at 0.1%, 1.0% and 5.0%
  • Needles were placed in paper bags (separate for each replicate) and dried in a dehydrator at l60°F for at least one week before weighing. Three collections occurred for the fresh tree group and two for the stored tree group. Tree height (ft) and trunk circumference (ft) were measured at the end of the experiment.

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  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Agronomy & Crop Science (AREA)
  • Microbiology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

La présente invention concerne de manière générale l'agriculture et, plus particulièrement, des compositions de biomasse et des procédés permettant de diminuer les meurtrissures, d'augmenter la santé des plantes, d'augmenter la santé du sol, d'augmenter le pouvoir sucrant dans les fruits et/ou de diminuer la chute d'aiguilles dans des espèces de conifères.
EP19731117.8A 2018-06-04 2019-06-03 Compositions de biomasse Withdrawn EP3700342A1 (fr)

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CN104955937A (zh) 2012-11-09 2015-09-30 赫里开发公司 在非纯性兼养条件中培养微生物和用乙酸盐和/或氧化剂控制培养物中细菌污染的方法
MX392111B (es) * 2016-01-29 2025-03-21 Heliae Dev Llc Composiciones basadas en chlorella seca y metodos para el mejoramiento de las plantas.
WO2017218896A1 (fr) 2016-06-16 2017-12-21 Heliae Development, Llc Composition à base de microalgues, et ses procédés de préparation et d'application sur les plantes
WO2018052502A1 (fr) * 2016-09-15 2018-03-22 Heliae Development, Llc Compositions à base de microalgues ayant un effet bénéfique pour les plantes et procédés d'application
EP3512601A1 (fr) * 2016-09-15 2019-07-24 Heliae Development, LLC Compositions à base de microalgues ayant un effet bénéfique pour les plantes et procédés d'application
US20190218504A1 (en) * 2016-09-30 2019-07-18 Heliae Development Llc Methods of culturing aurantiochytrium using acetate as an organic carbon source
WO2019094715A1 (fr) * 2017-11-10 2019-05-16 Heliae Development, Llc Compositions de biomasse

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