CA3082844A1 - Biomass compositions - Google Patents

Abstract

The present invention generally relates to agriculture and, more specifically, to biomass compositions and methods for decreasing bruising, increasing plant health, increasing soil health, increasing sweetness in fruits and/or decreasing needle-drop in conifer species.

Description

2 BIOMASS COMPOSITIONS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application 62/680,373, filed June 4, 2018, entitled BIOMASS COMPOSITIONS. The entire contents of the foregoing are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] 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.
BACKGROUND OF THE INVENTION

[0003] The growth of a plant is a complex physiological process involving inputs and pathways in the roots, shoots, and leaves. Whether at a commercial or home garden scale, growers are constantly striving to optimize the yield and quality of plants.
SUMMARY OF THE INVENTION

[0004] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description.
This Summary is not intended to identify key factors or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0005] 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 H5399 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.

[0006] In some embodiments, 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. In some embodiments, the liquid composition can be administered at a rate in the range of .25-2 gallons/acre. In some embodiments, the liquid composition can include between 100g-800g per acre of at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells.

[0007] In some embodiments, the contacting step can include a drip irrigation system and/or process.

[0008] In some embodiments, the liquid composition treatment can further include phosphoric acid and potassium sorbate. In some embodiments, the liquid composition treatment can further include citric acid.

[0009] In some embodiments, 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.

[0010] In some embodiments, the pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells are pasteurized for between 90-150 minutes.

[0011] In some embodiments, the microalgae includes only Chlorella cells, only Aurantiochytrium cells, or Chlorella cells and Aurantiochytrium acetophilum 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. For example, the shelf life can be increased by about 5%, 7%, 10%, 15%, or 20%.

[0012] In some embodiments, the microalgae includes only Chlorella cells, only Aurantiochytrium cells, or Chlorella cells and Aurantiochytrium acetophilum cells and the liquid composition is applied in an amount effective to increase water retention of the plant by at least 5% compared a substantially identical untreated plant.
For example, the water retention can be incrased by about 5%, 7%, 10%, 15%, or 20%.

[0013] In some embodiments, the microalgae includes only Chlorella cells, only Aurantiochytrium cells, or Chlorella cells and Aurantiochytrium acetophilum cells in an amount effective to reduce needle-drop of the plant by at least 5%
compared to a substantially identical untreated plant. For example, the needle-drop can be reduced by about 5%, 7%, 10%, 15%, or 20%.

[0014] In some embodiments, the liquid composition can include pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells in a ratio of 25:75.

[0015] In some embodiments, the Aurantiochytrium acetophilum HS399 cells have been subjected to an extraction process to remove oils from the Aurantiochytrium acetophilum HS399 cells.

[0016] Some embodiments of the invention can relate to a composition for enhancing at least one plant characteristic. The composition 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

[0017] In some embodiments, 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.

[0018] In some embodiments, the microalgae biomass can include whole biomass and/or residual biomass.

[0019] In some embodiments, the composition can include a first species of microalgae and a second species of microalgae. In some embodiments, 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.

[0020] In some embodiments, the first species of microalgae is Chlorella and the second species of microalgae is Aurantiochytrium. In some embodiments, the ratio of Chlorella and Aurantiochytrium is 25:75, 50:50 or 75:25.

[0021] In some embodiments, the Chlorella can be whole biomass and Aurantiochytrium is residual biomass or the Chlorella can be residual biomass and Aurantiochytrium is whole biomass.

[0022] 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.

[0023]
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.

[0024] In some embodiments, the microalgae species can include Chlorella, Schizochytrium, Thraustochytri urn, Oblongichytrium and/or species and/or strains of Aurantiochytrium, such as, for example, A. acetophilum HS399. In other embodiments, the microalgae species can include Botryococcus, Chlamydomonas, Scenedesmus, Pavlova, Phaeodactylum, Nannochloropsis, Spirlulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Tetraselmis, and/or the like.

[0025] In some embodiments, 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.

[0026] In some embodiments, the composition can include one species of microalgae. In some embodiments, 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.

[0027] In some embodiments, the first species of microalgae may be Chlorella and the second species of microalgae may be Aurantiochytrium acetophilum HS399. In some embodiments, the ratio of Chlorella and Aurantiochytriurn acetophilum HS399 may range between about 25:75 to 75:25. For example, the ratio of Chlorella and Aurantiochytrium acetophilum HS399 may be about 25:75, 50:50, or 75:25. In some embodiments, the Chlorella is whole biomass and Aurantiochytrium acetophilum is residual/extracted biomass. In some embodiments, the Aurantiochytriurn acetophilum HS399 is whole biomass and Chlorella is residual/extracted biomass. In some embodiments, 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.

[0028] 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.
In some embodiments, the composition is applied when the plant is under salt stress conditions, temperature stress conditions, and/or the like.

[0029]
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. In some embodiments 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, Aura ntiochytrium, Spirlulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Schizochytrium, Tetraselmis, and/or the like. In some of the embodiments and Examples below, 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.

[0030] To the accomplishment of the foregoing and related ends, the following description and annexed drawings set forth certain illustrative aspects and implementations. These are indicative of but a few of the various ways in which one or more aspects may be employed. Other aspects, advantages and novel features of the disclosure will become apparent from the following detailed description when considered in conjunction with the annexed drawings.
BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present application is further detailed with respect to the following drawings. These figures are not intended to limit the scope of the present application, but rather, illustrate certain attributes thereof.

[0032] 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;

[0033] 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;

[0034] 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;

[0035] 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;

[0036] 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;

[0037] 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;

[0038] 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;

[0039] 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;

[0040] 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;

[0041] 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;

[0042] 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;

[0043] 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;

[0044] 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;

[0045] 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;

[0046] 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;

[0047] 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;

[0048] 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;

[0049] 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;

[0050] 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;

[0051] 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;

[0052] 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;

[0053] 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;

[0054] 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;

[0055] 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;

[0056] 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;

[0057] 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 lmm in size relative to the UTC and a seaweed commercial reference product;

[0058] 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;

[0059] 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;

[0060] 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;

[0061] 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;

[0062] 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;

[0063] 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;

[0064] 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;

[0065] 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;

[0066] 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;

[0067] 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;

[0068] 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 days relative to the UTC and a seaweed commercial reference product;

[0069] 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;

[0070] 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;

[0071] 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;

[0072] 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;

[0073] 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;

[0074] 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;

[0075] 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;

[0076] 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;

[0077] FIG. 46 is a continuation of the table shown in FIG. 45;

[0078] 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;

[0079] 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;

[0080] 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;

[0081] 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;

[0082] 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;

[0083] 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;

[0084] 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;

[0085] 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 days post-harvest relative to the UTC;

[0086] 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;

[0087] FIG. 56 is a graph showing the effects of a microalgae composition on Douglas fir tree preservation, wherein the effects are observed in a decrease in fallen needles relative to the UTC and a commercial reference product;

[0088] 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.
DETAILED DESCRIPTION OF THE INVENTION

[0089] The description set forth below in connection with the appended drawings is intended as a description of presently preferred embodiments of the disclosure and is not intended to represent the only forms in which the present disclosure may be constructed and/or utilized. The description sets forth the functions and the sequence of steps for constructing and operating the disclosure in connection with the illustrated embodiments. It is to be understood, however, that the same or equivalent functions and sequences may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of this disclosure.

[0090] Many plants can benefit from the application of liquid compositions that provide a bio- stimulatory effect. 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 Grossulariaceae, Myrtaceae, Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae), Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae, Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae, Piperaceae, Proteaceae, and Cannabaceae.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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, corn, 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).

[0095] The Vitaceae plant family includes flowering plants and vines.
Taxonomically classified in the Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision), Magnoliophyta (division), Magnoliopsida (class), Rosidae (subclass), and Rhammales (order), the Vitaceae family includes, but is not limited to, grapes.

[0096]
Particularly important in the production of fruit from plants is the beginning stage of growth where the plant emerges and matures into establishment.
A method of treating a seed, seedling, or plant to directly improve the germination, emergence, and maturation of the plant; or to indirectly enhance the microbial soil community surrounding the seed or seedling is therefore valuable starting the plant on the path to marketable production. The standard typically used for assessing emergence is the achievement of the hypocotyl stage, where a stem is visibly protruding from the soil. The standard typically used for assessing maturation is the achievement of the cotyledon stage, where two leaves visibly form on the emerged stem. Some 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.

[0097]
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.

[0098] To achieve such improvements in emergence, maturation, and yield of plants, a method to treat such seeds and plants, and soil with a low-concentration microalgae-based composition, in a dried or liquid solution form was developed.
Microalgae can be grown in heterotrophic, 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).

[0099] In some embodiments, 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. Non-limiting examples of 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. In some embodiments, 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. By subjecting the microalgae to an extraction process 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.

[0100] In some embodiments, microalgae can be the predominant active ingredient source in the composition. In some embodiments, the microalgae population of the composition can include whole biomass, substantially extracted biomass, excreted products (e.g., EPS), extracted protein, or extracted oil.
In some embodiments, microalgae include at least 99% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 95% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 90% of the active ingredient sources of the composition. In some embodiments, microalgae include at least 80% of the active ingredient sources of the composition.

In some embodiments, microalgae include at least 70% of the active ingredient sources of the composition. In some embodiments, 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.

[0101] In some embodiments, microalgae biomass, excreted products, or extracts can also be mixed with biomass or extracts from other plants, microalgae, macroalgae, seaweeds, and kelp. In some embodiments, 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.
In further embodiments, the extracts can comprise, but are not limited to, liquid extract from a species of Kappaphycus. In some embodiments, the extracts can include 50% or less by volume of the composition. In some embodiments, the extracts can include 40% or less by volume of the composition. In some embodiments, the extracts can include 30% or less by volume of the composition. In some embodiments, the extracts can include 20% or less by volume of the composition. In some embodiments, the extracts can include IO% or less by volume of the composition. In some embodiments, the extracts can include 5% or less by volume of the composition. In some embodiments, the extracts can include 4%
or less by volume of the composition. In some embodiments, the extracts can include 3% or less by volume of the composition. In some embodiments, the extracts can include 2% or less by volume of the composition. In some embodiments, the extracts can include I% or less by volume ofthe composition.

[0102] The term "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 heterotrophic culture conditions.

[0103] In some embodiments, 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. Non-limiting examples of 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.

[0104] Non-limiting examples of microalgae genus and species that can be used in the compositions and methods of the present invention include:
Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var.
taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Aurantiochytrium sp., Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var.
subsalsum, 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. auxenophila, 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.
umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var.
ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var.
viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Galdieria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis a .ff galbana, Isochrysis galbana, Lepocinclis, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis camerae, Pleurochrysis dentate, Pleurochrysis sp., Porphyridium sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

[0105] Analysis of the DNA sequence of the strain of Chlorella sp. described in the specification was done in the NCBI 18s rDNA reference database at the Culture Collection of Algae at the University of Cologne (CCAC) showed substantial similarity (i.e., greater than 95%) with multiple known strains of Chlorella and Micractinium. Those of skill in the art will recognize that Chlorella and Micractinium appear closely related in many taxonomic classification trees for microalgae, and strains and species may be re-classified from time to time.
Thus, for references throughout the instant specification for Chlorella sp., it is recognized that microalgae strains in related taxonomic classifications with similar characteristics to the reference Chlorella sp. strain would reasonably be expected to produce similar results.

[0106]
Additionally, 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 lsensu latol based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thrausochytriaceae, Labyrinthulomycetes):
emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience (2007) 48:199-211. Those of skill in the art will recognize that Schizochytrium, Aurantiochytrium, Thraustochytrium, and Oblongichytrium appear closely related in many taxonomic classification trees for microalgae, and strains and species may be re-classified from time to time.
Thus, for references throughout the instant specification for Schizochytrium, it is recognized that microalgae strains in related taxonomic classifications with similar characteristics to Schizochytrium would reasonably be expected to produce similar results.

[0107] By artificially controlling aspects of the microalgae culturing process such as the organic carbon feed (e.g., acetic acid, acetate), oxygen levels, pH, and light, the culturing process differs from the culturing process that microalgae experiences in nature. In addition to controlling various aspects of the culturing process, 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. By intervening in the microalgae culturing process, 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). Thus, through artificial control of aspects of the culturing process and intervening in the culturing process with contamination control methods, 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.

[0108] During the mixotrophic culturing process 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, insecticides, enzymes, plant fiber (e.g., coconut fiber).

[0109] In some embodiments, the microalgae can be previously frozen and thawed before inclusion in the liquid composition. In some embodiments, the microalgae may not have been subjected to a previous freezing or thawing process.
In some embodiments, 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. The 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. In some embodiments, the microalgae whole cells and the accompanying constituents of the culturing process are concentrated in the composition. In some embodiments, 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. In some embodiments, 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. In some embodiments, the microalgae base composition can be biologically inactive after the composition is prepared. In some embodiments, the microalgae base composition can be substantially biologically inactive after the composition is prepared. In some embodiments, the microalgae base composition can increase in biological activity after the prepared composition is exposed to air.

[0110] In some embodiments, a liquid composition can include low concentrations of bacteria contributing to the solids percentage of the composition in addition to the microalgae cells. Examples of 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. In some embodiments, the composition includes an aerobic plate count of 40,000-400,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 40,000-100,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 100,000-200,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 200,000-300,000 CFU/mL. In some embodiments, the composition includes an aerobic plate count of 300,000- 400,000 CFU/mL.

[0111] In some embodiments, 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). In some embodiments, 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. In some embodiments, the supplemented nutrient is not uptaken, chelated, or absorbed by the microalgae. In some embodiments, the concentration of the supplemental nutrient can include 1-50 g per 100 g of the composition.

[0112] A liquid composition comprising microalgae can be stabilized by heating and cooling in a pasteurization process. As shown in the Examples, the inventors found that 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. In other embodiments, liquid compositions with whole cells or processed cells (e.g., dried, lysed, extracted) of microalgae cells may not need to be stabilized by pasteurization. For example, 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.

[0113] In some embodiments, 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.

[0114] In some embodiments, 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 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 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.

[0115] After the step of heating or subjecting the liquid composition to high temperatures is complete, the compositions can be cooled at any rate to a temperature that is safe to work with. In one non-limiting embodiment, the composition can be cooled to a temperature in the range of 35-45 C. In some embodiments, the composition can be cooled to a temperature in the range of 36-44 C. In some embodiments, the composition can be cooled to a temperature in the range of 37-43 C. In some embodiments, the composition can be cooled to a temperature in the range of 38-42 C. In some embodiments, the composition can be cooled to a temperature in the range of 39-41 C. In further embodiments, 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.

[0116] In some embodiments, 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.

[0117] In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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.

[0118] In some embodiments, 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. In some embodiments, 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.

[0119] In another non-limiting embodiment, correlating the application of the microalgae biomass or extract on a per plant basis using the assumption of 15,000 plants per acre, the composition application rate of 1 gallon per acre is equal to about 0.25 mL per plant = 0.025 g per plant = 25 mg of microalgae biomass or extract per plant. 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. In some embodiments, 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. In some of the embodiments and Examples below, the applications were performed using a 10% solids solution by weight microalgae composition. For these field trials, the rates are indicated in gal/acre and the amount of carrier water would be determined according to user preference. For field trials, the application rate may range between 0.25 gal/acre ¨ 2 gal/acre. In the field trials, where the application rate of the microalgae composition is 0.25 gal/acre, the equivalent expressed in total grams of solid microalgae would be 100g 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 160g 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.

[0120] Overall, as shown in the embodiments and Examples below, the microalgae composition may comprise between 100g-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 gallons of liquid carrier volume/acre or less than 100 gallons of liquid carrier volume/acre.

[0121] In some embodiments, 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.
Such 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. In some embodiments, the stabilizing means are suitable for plants and do not inhibit the growth or health of the plant. In the alternative, 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.

[0122] In some embodiments, 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.

[0123] In some embodiments, 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.

[0124] In some embodiments, 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.

[0125] 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 W02017/218896A1 (Shinde et al.) entitled Microalgae-Based Composition, and Methods of its Preparation and Application to Plants, which is incorporated herein in full by reference. In one or more embodiments, the microalgae composition may comprise approximately 10%40.5% w/w of Chlorella microalgae cells. In one or more embodiments, 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. For example, in one or more embodiments, 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. As a further example, in one or more embodiments where it is desired to use an OMRI (Organic Materials Review Institute) certified organic composition, 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. In one or more embodiments, the pH of the microalgae composition may be stabilized to between 3.0-4Ø

[0126] In some embodiments and Examples below, 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%-1.5%
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.

[0127] In some embodiments and Examples below, 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. PT-0 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 citric acid to stabilize the pH of the Chlorella, and then adjusting the whole biomass treatment to the desired concentration. PT-0 Chlorella microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells.
Furthermore, PT-0 Chlorella 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 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.

[0128] In some embodiments and Examples below, 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 microalgae composition may comprise approximately 10% w/w of Chlorella microalgae cells. Furthermore, 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.

[0129] In some embodiments and Examples below, 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-090. 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. Furthermore, PT-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.

[0130] In some embodiments and Examples below, the microalgae composition may be referred to as Aurantiochytrium acetophilum HS399 whole biomass (WB) or HS399 WB. The Aurantiochytriurn 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. It should be clearly understood that other variations of the Aurantiochytri urn acetophilum HS399 whole biomass (WB) 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.

[0131] In some embodiments and Examples below, the microalgae composition may be referred to as Aurantiochytriurn 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%-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. Once 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. It should be clearly understood that other variations of the Aurantiochytrium acetophilum HS399 washed whole biomass (WB washed) microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, variations in the washing method, and/or variations in the % composition of each component may be used and may achieve similar results.

[0132] In some embodiments and Examples below, 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%-1.5% 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. It should be clearly understood that other variations of the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the pasteurization temperature, variations in the extraction method, and/or variations in the % composition of each component may be used and may achieve similar results.

[0133] In some embodiments and Examples below, 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. For the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition, 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 Aura ntiochytrium 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%-1.5% phosphoric acid to stabilize the pH of the 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition to between 3.0-4Ø It should be clearly understood, however, that other variations of the combination 25% Chlorella: 75% HS399 whole biomass (WB) microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the order of the processing steps (blending, pasteurizing, stabilizing), and/or variations in the % composition of each component may be used and may achieve similar results.

[0134] In some embodiments and Examples below, 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%-1.5% 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.

[0135] In some embodiments and Examples below, 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). For the combination GP2C:399 microalgae composition, 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%-1.5% phosphoric acid to stabilize the pH of the 25%
Chlorella: 75%
HS399 extracted biomass (EB) microalgae composition to between 3.0-4Ø It should be clearly understood, however, that other variations of the combination GP2C:399 microalgae composition, including variations in the microalgae strains, variations in the stabilizers, variations in the order of the processing steps (blending, pasteurizing, stabilizing), and/or variations in the % composition of each component may be used and may achieve similar results.

[0136] In some embodiments and Examples below, a Greenwater Polyculture (GWP) treatment may be used. 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.

[0137] In some embodiments, the composition is a liquid and substantially includes of water. In some embodiments, the composition can include 70-99%
water.
In some embodiments, the composition can include 85-95% water. In some embodiments, the composition can include 70-75% water. In some embodiments, the composition can include 75-80% water. In some embodiments, the composition can include 80-85% water. In some embodiments, the composition can include 85-90% water. In some embodiments, the composition can include 90- 95% water. In some embodiments, 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.

[0138] In some embodiments, the liquid composition can be used immediately after formulation, or can be stored in containers for later use. In some embodiments, the composition can be stored out of direct sunlight. In some embodiments, the composition can be refrigerated. In some embodiments, the composition can be stored at 1-10 C. In some embodiments, the composition can be stored at 1-3 C.
In some embodiments, the composition can be stored at 3- 50 C. In some embodiments, the composition can be stored at 5-8 C. In some embodiments, the composition can be stored at 8-10 C.

[0139] In some embodiments, 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.

[0140] In some embodiments, 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.

[0141] In some embodiments, 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). In some embodiments, 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). In some embodiments, 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).

[0142] In one non-limiting embodiment, the administration of the composition may comprise contacting the foliage of the plant with an effective amount of the composition. In some embodiments, the liquid composition may be sprayed on the foliage by a hand sprayer, a sprayer on an agriculture implement, or a sprinkler. In some embodiments, the composition can be applied to the soil.

[0143] The rate of application of the composition at the desired concentration can be expressed as a volume per area. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the range of 10-50 gallons/acre. In some embodiments, the rate of application of the liquid composition in a foliar application can comprise a rate in the rage of 10-15 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 15-20 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 20-25 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 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.

[0144] In some embodiments, 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.
In some embodiments, 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 0.001%-50%. The v/v ratio can be between 0.01-25%. The v/v ratio of the composition can be between 0.03-10%.

[0146] 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).
In some embodiments, the plant can be contacted by the composition in a foliar application every 3-28 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 4-10 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 18-24 days.
In some embodiments, the plant can be contacted by the composition in a foliar application every 3-7 days. In some embodiments, the plant can be contacted by the composition in a foliar application every 7-14 days. In some embodiments, 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.

[0147] 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. 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. In some embodiments, the plant can be first contacted by the composition in a foliar application 5-7 days after the plant emerges from the soil. In some embodiments, the plant can be first contacted by the composition in a foliar application 7-10 days after the plant emerges from the soil. In some embodiments, the plant can be first contacted by the composition in a foliar application 10-12 days after the plant emerges from the soil. In some embodiments, the plant can be first contacted by the composition in a foliar application 12-14 days after the plant emerges from the soil.

[0148] In another non-limiting embodiment, 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. In some embodiments, 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. In some embodiments, the liquid composition can be supplied to the soil by a soil drench method wherein the liquid composition is poured on the soil.

[0149] 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. Alternatively, 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.

[0150] The rate of application of the composition at the desired concentration can be expressed as a volume per area. In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 50-150 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 75-125 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 50-75 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 75-100 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 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 gallons/acre.

[0151] In some embodiments, 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-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.

[0152] In some embodiments, the rate of application of the liquid composition in a soil application can include a rate in the range of 0.01-10 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. In some embodiments, 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.

[0153] In some embodiments, 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.

[0154] Prior patent applications containing useful background information and technical details are PCT/US2017/053432 titled METHODS OF CULTURING
AURANTIOCHYTRIUM USING ACETATE AS AN ORGANIC CARBON SOURCE, filed on September 26, 2017;
PCT/U52015/066160, titled MIXOTROPHIC
CHLORELLA-BASED COMPOSITION, AND METHODS OF ITS PREPARATION
AND APPLICATION TO PLANTS, filed on December 15, 2015; and PCT/U52017/037878 and PCT/2017/037880, both applications titled MICROALGAE-BASED COMPOSITION, AND METHODS OF ITS PREPARATION AND
APPLICATION TO PLANTS, both filed on June 16, 2017. Each of these applications is incorporated herein by reference in its entirety.

[0155] It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto. All patents and references cited herein are explicitly incorporated by reference in their entirety.
Examples IMPROVED PLANT HEALTH
Example 1

[0156] An experiment was performed to compare the benefits of applying a combination GP2C:399 microalgae composition treatment comprising a blend of Chlorella whole biomass microalgae cells and Aurantiochytrium acetophilum H5399 extracted/residual biomass microalgae cells to plants to a treatment application of Chlorella whole biomass or Aurantiochytrium acetophilum H5399 residual biomass individually. Iceberg lettuce seedings were transplanted into individual pots of SUN GRO SUNSHINE Mix #4 growth substrate mix (soil) and grown in a greenhouse for two weeks under both no stress and salt stress of 60mM applied via drip irrigation on a daily basis throughout the experiment. At the time of transplanting seedlings from plug trays to quart pots 20 replicates of plants were treated with a 140mL application to the growth substrate through a drench application containing a treatment comprising Chlorella whole biomass microalgae cells only, Aurantiochytrium acetophilum H5399 extracted/residual biomass microalgae cells only, or a blend of Chlorella whole biomass microalgae cells and Aurantiochytrium acetophilum H5399 extracted/residual biomass microalgae cells as outlined in the tables below at a concentration of 0.03% or 0.3% v/v. For reference, replicates of an untreated control (UTC), a treatment with a 0.1% v/v commercially available seaweed commercial reference product, and a treatment comprising 0.03% or 0.3% v/v of an oat filler used in a solventless extraction process for separating oil from the residual Aurantiochytrium acetophilum H5399 biomass were also performed.

[0157] At the end of two weeks the lettuce plants were harvested and the vegetative growth in the shoot was measured as fresh weight and dry weight. The results are shown in Tables 1-4 and FIGURES 1-9. In summary, the lettuce that was treated with the combination of Chlorella whole biomass/Aurantiochytrium acetophilum HS399 extracted/residual biomass showed better growth than the lettuce treated with Chlorella whole biomass microalgae cells alone, Aurantiochytrium acetophilum HS399 extracted/residual biomass microalgae cells alone, the UTC, and the seaweed commercial reference product.
Table 1: No Stress Conditions Fresh Weight % Difference from Untreated Control - Shoot Fresh Weight Treatment Conc. Trial 2 Trial 3 Trial 4 Average Commercial Reference 0.1% -5.0 -2.0 +0.1 -2.3 Product Oat filler 0.03% -0.5 +7.5 -2.5 +1.5 0.3% +3.2 +9.5 +5.4 +6.0 Chlorella whole 0.03% +8.2 +17.1 +10.5 +11.9 biomass 0.3% +6.6 +9.0 +5.7 +7.1 Aurantiochytrium 0.03% +11.9 +14.6 +6.7 +11.1 acetophilum HS399 0.3% +2.6 +15.1 +5.6 +7.8 residual biomass 50:50 blend of Chlorella 0.03% +6.0 +17.4 +4.9 +9.4 whole biomass and 0.3% +0.9 +23.4 -10.6 +4.6 Aurantiochytrium acetophilum HS399 residual biomass 25:75 blend of Chlorella 0.03% +7.9 +13.5 -9.8 +3.9 whole biomass and 0.3% +20.4 +15.0 +18.2 +17.9 Aurantiochytrium acetophilum HS399 residual biomass 75:25 blend of Chlorella 0.03% 0.0 +20.9 0.0 +7.0 whole biomass and 0.3% +21.7 -1.8 +6.1 +8.7 Aurantiochytrium acetophilum HS399 residual biomass Table 2: Salt Stress Conditions Fresh Weight % Difference from Untreated Control -Shoot Fresh Weight Treatment Conc. Trial 2 Trial 3 Trial 4 Average Seaweed commercial 0.1% -8.0 +16.4 -0.5 +2.6 reference Product Oat filler 0.03% +6.8 -18.1 -3.9 -5.1 0.3% +2.3 +0.4 +5.9 +2.9 Chlorella whole biomass 0.03% +1.0 -12.6 +1.3 -3.4 0.3% -9.9 -1.7 -6.9 -6.2 0.03% +3.4 -14.4 -1.1 -4.0 Aurantiochytrium 0.3% +3.7 -6.8 +25.9 +7.6 acetophilum HS399 residual biomass 50:50 blend of Chlorella 0.03% -1.3 -14.7 +13.7 -0.8 whole biomass and 0.3% +25.0 -9.3 +16.2 +10.6 Aurantiochytrium acetophilum HS399 residual biomass 25:75 blend of Chlorella 0.03% +12.0 -10.3 +6.0 +2.6 whole biomass and 0.3% +25.0 -9.3 +16.2 +10.6 Aurantiochytrium acetophilum HS399 residual biomass 75:25 blend of Chlorella 0.03% +31.5 -10.9 +7.7 +9.4 whole biomass and 0.3% +25.2 -7.1 +7.5 +8.5 Aurantiochytrium acetophilum HS399 residual biomass Table 3: No Stress Conditions Dry Weight % Difference from Untreated Control -Shoot Dry Weight Treatment Conc. Trial 2 Trial 3 Average Seaweed commercial 0.1% -5.4 +3.3 -1.1 reference Product Oat filler 0.03% -5.4 +8.9 +1.8 0.3% -3.6 +9.9 +3.2 Chlorella whole biomass 0.03% +8.6 +17.0 +12.8 0.3% +3.2 +9.7 +6.5 Aurantiochytrium 0.03% +11.4 +13.2 +12.3 acetophilum H5399 residual 0.3% +5.4 +16.0 +10.7 biomass 50:50 blend of Chlorella 0.03% +7.5 +16.9 +12.2 whole biomass and 0.3% -4.2 +19.8 +7.8 Aurantiochytrium acetophilum H5399 residual biomass 25:75 blend of Chlorella 0.03% +6.0 +15.6 +10.8 whole biomass and 0.3% +20.2 +14.3 +17.3 Aurantiochytrium acetophilum H5399 residual biomass 75:25 blend of Chlorella 0.03% -2.1 +16.1 +7.0 whole biomass and 0.3% +21.1 -1.3 +9.9 Aurantiochytrium acetophilum H5399 residual biomass Table 4: Salt Stress Conditions Dry Weight % Difference from Untreated Control -Shoot Dry Weight Treatment Conc. Trial 2 Trial 3 Average Seaweed commercial 0.1% +5.8 -11.9 -3.1 reference Product Oat filler 0.03% +10.7 -18.1 -3.7 0.3% +3.1 +2.0 +2.6 Chlorella whole biomass 0.03% +4.6 -10.7 -3.1 0.3% -7.5 -0.2 -3.9 Aurantiochytrium acetophilum 0.03% +6.5 -12.8 -3.2 H5399 residual biomass 0.3% +5.7 -5.5 +0.1 50:50 blend of Chlorella 0.03% +2.1 -8.8 -3.4 whole biomass and 0.3% +26.8 -3.0 +14.9 Aurantiochytrium acetophilum H5399 residual biomass 25:75 blend of Chlorella 0.03% +9.5 -4.1 +2.7 whole biomass and 0.3% +27.8 -11.0 +8.4 Aurantiochytrium acetophilum H5399 residual biomass 75:25 blend of Chlorella 0.03% +22.5 -10.1 +6.2 whole biomass and 0.3% +18.9 -5.0 +7.0 Aurantiochytrium acetophilum H5399 residual biomass Example 2

[0158] A trial was conducted on romaine lettuce (var. Valley Heart) in a Gilbert, AZ
greenhouse to evaluate effects of the PT-0 Chlorella microalgae composition on growth of romaine transplants. Romaine lettuce seedings were transplanted into individual pots of SUN GRO SUNSHINE Mix #4 growth substrate mix (soil). The PT-0 Chlorella microalgae composition was applied as soil drench at the time of seeding.
Organic fertilizer was applied following the 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 enhanced initial plant establishment and accelerated both shoot and root growth up to at least 35 days after planting when applied once at seeding.

STUDY PARAMETERS
Crop Romaine Lettuce, var. Valley Heart Location Heliae research greenhouse, Gilbert, AZ
Seeding Date August 22, 2017 Final Assessment September 19, 2017 Plant density 192 cell trays Irrigation Twice a day overheard with Gilbert, AZ city water Fertilizer solution PHYTAMIN All Purpose Fertilizer (2.7-3.7-2.7);
California Organic Fertilizers Every 3-5 days, diluted 1:320 in city water starting with first true leaf emergence (116ppm N, 84ppm P, 116ppm K) Soil type SUN GRO SUNSHINE Mix #4 Seeding Seeded in 192 cell flats. 1/4" overlay of 40% perlite/60%
vermiculite was added after seeding Replication 80 plants per treatment (five 16 plant replicates) Product applied Overhead as drench at seeding

[0159] FIGURE
10 shows the average leaf canopy area (cm2) 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.
As detailed in FIGURE 10, the increased leaf canopy area (cm2) of the romaine lettuce transplants shows that the microalgae composition enhances early plant establishment.
Table 5: Average Leaf Area at 14 Days After Seeding Treatment Average Leaf Area (cm2) UTC 1.49 0.5% PT-0 composition 1.50 1% PT-0 composition 1.69 2% PT-0 composition 1.84 3% PT-0 composition 2.19 5% PT-0 composition 2.54

[0160] 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-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. As detailed in FIGURE 11, when 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.
Table 6: Shoot Dry Weight Gain as Percent of UTC at 35 Days After Seeding Treatments Average Shoot Dry % UTC Average Shoot Weight (mg) Dry Weight (mg) UTC 136.28 100.00 0.5% PT-0 composition 152.16 111.66 1% PT-0 composition 148.54 109.00 2% PT-0 composition 159.52 117.06 3% PT-0 composition 175.60 128.86 5% PT-0 composition 170.92 125.44

[0161] FIGURE 12 shows the root area (cm2) 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 (cm2), and the % UTC
average root area (cm2) 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. As detailed in 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).
7: Root Area as Percent of Control at 35 Days After Seeding Treatments Average Shoot Dry % UTC Average Shoot Weight (mg) Dry Weight (mg) UTC 16.06 100.00 0.5% PT-0 composition 18.60 115.92 1% PT-0 composition 17.56 109.60 2% PT-0 composition 20.20 125.96 3% PT-0 composition 18.36 114.36 5% PT-0 composition 20.04 125.00

[0162] 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.
Example 3

[0163] A trial was conducted on tomato in Gilbert, AZ greenhouse to evaluate effects of the PT-0 Chlorella microalgae composition and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition on the growth of roma tomato plant establishment and leaf canopy growth.

[0164] 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.
STUDY PARAMETERS
Crop Tomato (var. Keithly Williams K2700) Location Heliae research greenhouse, Gilbert, AZ
Seeding Date August 22, 2017 Final Assessment September 19, 2017 Plant density 192 cell trays Irrigation Twice a day overheard with Gilbert, AZ city water Fertilizer solution PHYTAMIN All Purpose Fertilizer (2.7-3.7-2.7);
California Organic Fertilizers Every 3-5 days, diluted 1:320 in city water starting with first true leaf emergence (116ppm N, 84ppm P, 116ppm K) Soil type SUN GRO SUNSHINE Mix #4 Seeding Seeded in 192 cell flats. 1/4" overlay of 40% perlite/60%

vermiculite was added after seeding Replication 80 plants per treatment (five 16 plant replicates) Product applied Overhead as drench at seeding

[0165] FIGURE 13 shows the leaf canopy area (cm2) 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 (cm2) as a percent of UTC, and the resulting canopy cover per plant (cm2) for each microalgae composition treatment. As detailed in FIGURE 13, it is clear that 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.
Table 8: Leaf Canopy Area at 21 Days After Seeding Treatments % UTC Canopy Canopy Cover Per Plant Cover (cm2) UTC 100 2.1 2.5% PT-0 composition 76 2.6 5% PT-0 composition 269 5.5 0.01% HS399 EB 83 1.6 composition 0.1 HS399 EB composition 83 1.7 1% HS399 EB composition 99 2.1 2.5% HS399 EB composition 154 3.0 5% HS399 EB composition 175 3.6

[0166] A one-time soil application of the PT-0 Chlorella microalgae composition 5% at the time of seeding significantly enhanced shoot development of tomato by greater than 100%. This overall acceleration in plant growth by one-time application of the Chlorella microalgae composition treatment may decrease required growing time from seeding to delivery-ready tomato transplants.
Example 4

[0167] A trial was conducted in a Gilbert, AZ greenhouse to evaluate effects of PT-0 Chlorella microalgae composition on transplants establishment and growth of cauliflower canopy. 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 accelerated initial plant establishment and increased shoot growth through the first 16 days after seeding.
STUDY PARAMETERS
Crop Cauliflower (var. minuteman) Location Heliae research greenhouse, Gilbert, AZ
Seeding Date October 3, 2017 Final Assessment October 19, 2017 Plant density 16 cell trays Irrigation Twice a day overheard with Gilbert, AZ city water Fertilizer solution PHYTAMIN All Purpose Fertilizer (2.7-3.7-2.7);
California Organic Fertilizers Every 3-5 days, diluted 1:320 in city water starting with first true leaf emergence (116ppm N, 84ppm P, 116ppm K) Soil type SUN GRO SUNSHINE Mix #4 Seeding Seeded in 24 cell flats. 1/4" overlay of 40% perlite/60%
vermiculite was added after seeding Replication 80 plants per treatment (five 16 plant replicates) Product applied Overhead as drench at seeding

[0168] FIGURE 14 shows the leaf canopy area (cm2) 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 (cm2) as a percent of UTC, and the resulting canopy cover per plant (cm2) for each microalgae composition treatment. As detailed in FIGURE 14, it is clear that the 3% and 5% concentrations of the PT-Chlorella microalgae composition accelerated cauliflower transplant establishment.
Table 9: %UTC Canopy Area at 16 Days After Seeding Treatments % UTC Canopy Canopy Cover Per Plant Cover (cm2) UTC 100 2.0 3% PT-0 composition 162 3.6 5% PT-0 composition 217 3.9

[0169] A one-time soil application of the PT-0 Chlorella microalgae composition at 5%
at the time of seeding significantly enhanced shoot development of cauliflower by 117%.
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.
Example 5

[0170] A trial was conducted on bell pepper in Camarillo, CA to evaluate effects of the PT-0 Chlorella microalgae composition on growth and yield. The trial was transplanted in late April 2016 and harvested in August 2016. At harvest, marketable peppers were collected from 4 plants in each plot and sorted by size (medium, large, extra-large and jumbo), weighed, and counted. All plots were managed according to local standard practice (see Study Parameters below).
STUDY PARAMETERS
Crop Bell pepper (var. Baron) Location Camarillo, CA
Transplanting Date April 25, 2016 Harvests evaluated August 30, 2016 Bed dimensions 40" centers, 2 seedlines, plants 12" apart Planting density 17,424 plants/A
Fertilizer Before bedding 10 gal/A 10-34-0, 100 lbs N as UAN 32 4 times per seasons Pesticide No pre-plant fumigation Treatments for Phytophthora, weevil and powdery mildew as needed Soil type Clay loam Plot size 200' Lx 3.3' W
Replication 8 Product applied At planting then every 3 weeks via drip

[0171] Relative to local standard practice alone, applications of the PT-0 Chlorella microalgae composition at 0.5-1 gal/A every 3 weeks improved marketable yield of bell peppers. This increase in marketable yield was based on higher yield of United States Department of Agriculture (USDA) grade large and extra-large peppers.

[0172] As shown in FIGURE 15, overall marketable yield was standardized to 1,400 25-lb cartons per acre for local standard practice (UTC) and compared to yield in plots receiving the PT-0 Chlorella microalgae composition. Estimated marketable yield was more than 30% higher for these plots compared to UTC at both 0.5 and 1 gal/A
(P < 0.05).

[0173] Referring to FIGURE 16, marketable yield of large and extra-large fruit was increased by 50% in plots that received the PT-0 Chlorella microalgae composition (P <
0.1). This was observed for both yield by weight and by count. No differences were observed for other size classes.

[0174] In summary, for this Example, the PT-0 Chlorella microalgae composition improved marketable yield of bell peppers by both weight and count. Estimated marketable yield increased by 30%. Additional testing is underway in California on both fumigated conventional fields in 2017.

Example 6

[0175] A trial was conducted on snap beans in Paynesville, MN to evaluate the PHYCOTERRA Chlorella microalgae composition. The trial was transplanted in late June 2016 and was harvested in early September 2016 (75 days). All plots were managed according to local standard practice (see Study Parameters below). Relative to standard practice alone, bi-weekly additions of 1 gal/acre increased vegetative growth and marketable bean yield.
STUDY PARAMETERS
Crop Snap bean (var. Provider) Location Crow River Research Farm, Paynesville MN
Transplanting Date June 25, 2016 Harvests evaluated September 8, 2016 Bed dimensions 8,800 plants/A
Planting density Via pivot as needed Fertilizer At planting application: 80 lbs/A Urea 30 lbs/A K, 30 lbs/A P, 1 lb/A Zn, 1 lb/A B
Soil type Estherville sandy loam, silt loam Plot size 5' W x 20' L
Replication 8 plots per treatment, RCB design Product applied Via temporary drip at planting then every 2 wks (6 total)

[0176] As shown in FIGURE 17, fresh shoot biomass was increased by 2% when the PT-0 Chlorella microalgae composition was applied bi-weekly at 1-2 gal/A
compared to standard practice alone (UTC).

[0177] 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.

[0178] The results show that in the field trial in Minnesota, the PT-0 Chlorella microalgae composition applied bi-weekly at 1 gal/acre (for a total of 6 applications) showed an increase in shoot growth in snap beans by an increased % yield of 7%
over the UTC and a 1,419 lb/acre increase over the UTC, and thus showing a significantly increased marketable yield for an estimated favorable ROI of over S600/acre.

Example 7

[0179] 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.
STUDY PARAMETERS
Crop Snap pea (var. Sugar Sprint) Location Crow River Research Farm, Paynesville, MN
Transplanting Date June 23, 2016 Harvests evaluated August 25, 2016 Bed dimensions 8,600 plants/A
Planting density Via pivot as needed Fertilizer At planting application: 80 lbs/A Urea 30 lbs/A K, 30 lbs/A P, 1 lb/A Zn, 1 lb/A B
Soil type Estherville sandy loam, silt loam Plot size 5' W x 20' L
Replication 8 plots per treatment, RCB design Product applied Via temporary drip at planting then every 2 wks (5 total)

[0180] 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.

[0181] As shown in FIGURES 20-21, 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.

[0182] The results show that in the field trial in MN, the PT-0 Chlorella microalgae composition applied bi-weekly at 2-4 gal/A resulted in higher marketable yield that was in part due to increased number of pods produced per plant.

IMPROVED SOIL HEALTH
Example 8

[0183] Field trials were conducted on sweet corn, snap peas, and snap beans to evaluate the effect of the PT-0 Chlorella microalgae composition on soil health. Sweet corn, snap beans and snap peas were transplanted in adjacent fields in Paynesville, MN
during the summer of 2016 and compared to plots within each trial that just received water at the time of application. All plots were managed according to standard local practice (see Study Parameters below). Soil was collected from the root zone of each plot during harvest and evaluated for bacterial community structure using a next-generation ILLUMINA MISEQ sequencing system.
STUDY PARAMETERS
Crop Sweet corn (var. Snap bean (var. Snap pea (var.
Temptation) Provider) Sprint) Location Crow River Research Farm, Paynesville MN
Transplanting June 24, 2016 June 25, 2016 June 23, Date Harvests September 11, September 8, 2016 August 25, 2016 evaluated 2016 Planting density 34,800 plants/A 8,800 plants/A 8,600 plants/A
Irrigation Via pivot as needed Fertilizer at 120 lbs/A Urea 80 lbs/A Urea Planting 30 lbs/A K, 30 lbs/A P, 1 lb/A Zn, 1 lb/A B
Soil type Estherville sandy loam, silt loam Plot size 5' W x 20' L
Replication 8 plots per treatment, RCB design Product Applied Then every 2 weeks (6 total) Then every 2 weeks Via Temporary (5 total) Drip at Planting

[0184] As shown in FIGURE 22, 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).

[0185] Referring to FIGURE 23, 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.
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.

[0186] In summary, for the three crops planted in the same field in Minnesota, the PT-0 Chlorella microalgae composition had a significant impact on standardizing the bacterial community. Abundances of three soil bacteria increased two-fold, two of which are known to be beneficial to plant performance.
Example 9

[0187] Greenhouse trials were conducted to evaluate the effect of PT-0 Chlorella microalgae composition on soil health. Soil was collected from a field planted with alfalfa from Gilbert, AZ classified as Antho sandy loam and diluted by 40% with a peat based soil mix and perlite to allow drainage. Quart pots were filled with soil mixed with the PT-0 Chlorella microalgae composition (0.3-3% solution v/v in city water), a seaweed commercial reference product, or city water alone (UTC). Pots were kept moist by watering every 2 days with city water. Soil samples were collected every 5 days for 2 weeks 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) dry soil aggregate size distribution ¨
indicator of soil quality (e.g. porosity, erosion, resistance, and root penetration).

[0188] As shown in FIGURES 24-25, 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<0.1) 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<0.1). As shown in FIGURE 25, 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.

[0189] As shown in FIGURE 26, 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.

[0190] In summary, for a greenhouse study using native Arizona soil, significant improvement in biological soil health based on the accumulation of organic carbon and nitrogen and physical soil quality were observed within 2 weeks of a single application of the PT-0 Chlorella microalgae composition. Additional studies will target the effect of repeated applications representing the schedule followed during a crop season.
Example 10

[0191]
Greenhouse trials were conducted to evaluate the effect of the PT-0 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.

[0192] As shown in 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. As shown in FIGURE 28, soil protein was "Very Low' in the initial soil but increased to "medium" over 30 days compared to the UTC.

[0193] As shown in FIGURE 29, 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).

[0194] In summary, for a greenhouse study, two applications of PT-0 Chlorella microalgae composition significantly improved the biological health of native Arizona field soil by promoting the accumulation of organic carbon and nitrogen and improving water holding capacity.
IMPROVED SHELF-LIFE QUALITY AND POST-STORAGE MARKETABILITY

[0195] Applying a microalgae composition to plants increases the shelf-life quality and consumer taste preference of harvested fruits. 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. Generally, 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.

[0196] 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. For testing firmness, a penetrometer is used to measure the force that it takes to penetrate the fruit surface. A firmer fruit indicates longer shelf-life. With respect to 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.

Example 11

[0197] A trial was conducted on strawberry (var. Camarosa) in Winter Garden, FL to evaluate performance of the PHYCOTERRA Chlorella microalgae composition on berry quality after storage and on consumer preference. The trial was transplanted in late October 2016 and harvested through early March 2017. All plots were managed according to the local standard practice (see Study Parameters below). Raw data is included in the table shown in FIGURE 30.
STUDY PARAMETERS
Crop Strawberry (var. Camarosa) Location Winter Garden, FL
Transplanting Date October 24, 2016 Pick Frequency Weekly culls when not assessed Bed dimensions 60" W x 6" H, 2 rows Planting density 17,424 plants/A
Drip irrigation 6" emitters, 0.3" applied daily Fertilizer 20-100 lbs 20-20-20 NPK monthly via drip Pesticide Ridomil, Sevin, Dipel and Captec as needed Soil Type Sandy, non-fumigated Plot Size 12 ft sections of 100 ft bed Replication 4 Product applied 0.5 gal/A via drip at planting then every 2 wks

[0198] Berries from one harvest (day 113) were shipped cold overnight to a University lab in New York, stored for 3 days and then assessed for post-storage quality, particularly appearance. Berries from an additional harvest (day 128) were shipped to the Sensory Evaluation Center at a University in New York for consumer testing.
Relative to standard practice alone (UTC), bi-weekly additions of the microalgae composition at 0.5 gal/A improved berry appearance after shipping and cold storage. Consumers consistently preferred berries that had come from plots treated with the microalgae composition over standard practice.

[0199] As shown in FIGURE 31, 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. Referring to FIGURE 32, 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.

[0200] In summary, for the Florida trial, 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).
Example 12 A trial was conducted on strawberry (var. Camarosa) in Winter Garden, FL to evaluate performance of each of the PHYCOTERRA Chlorella microalgae composition and the Aurantiochytrium acetophilum HS399 extracted biomass (EB) microalgae composition on strawberry shelf-life and quality post-harvest. All plots received the standard fertilization regimen used by the grower for these crops excluding biostimulants. The Chlorella microalgae composition and the Aurantiochytriurn acetophilum HS399 extracted biomass (EB) microalgae composition were added in addition to standard fertilization. Strawberry plants were transplanted to the field. First application of the microalgae compositions each occurred at time of transplanting and then every 14 days afterward until harvest via drip irrigation. The UTC received the same amount of carrier water as other treatments at the time of each application. The 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).
STUDY PARAMETERS
Crop Strawberry (var. Camarosa) Location Winter Garden, FL
Conventional Row Spacing 2 rows/60" bed with plants every 12"
Plot size minimum 12 ft section after first 2 ft along 100 ft drip line Treatment Treatment rows were randomized across field and untreated buffer rows were planted on both ends to minimize edge effects One 100 ft row, randomized in order throughout field Observations From multiple subsamples per 12 ft section Replication 14 treatments x 8 replicates = 112 treatment plots (12 ft sections) Local Standard Production Fertility, weed, insect management Standard Management Fungicide application. Record disease management Practice measures

[0201]
Application rates of the PHYCOTERRA Chlorella microalgae composition and the Aurantiochytrium acetophilum H5399 extracted biomass (EB) microalgae composition were as detailed in Table 10 below.
Table 10: Treatments Application Treatment Rate Number Product gallon/acre Ti Standard Practice (Untreated) N/A
T2 PHYCOTERRA composition 0.4 T3 PHYCOTERRA composition 0.5 T4 PHYCOTERRA composition 1 T5 PHYCOTERRA composition 2 T6 H5399 Extracted Biomass (EB) 0.4 T7 H5399 Extracted Biomass (EB) 0.5 T8 H5399 Extracted Biomass (EB) 1 T9 H5399 Extracted Biomass (EB) 2 T10 Green Water Polyculture 0.4 T11 Green Water Polyculture 0.5 T12 Green Water Polyculture 1 T13 Green Water Polyculture 2 T14 Seaweed-based Commercial Reference 0.5

[0202]
Marketable berries from all replicates were harvested. Replicate 2 lb clamshells were kept for an on-site storage assessment or shipped overnight to a university lab in NY for an additional storage assessment. On-site, clamshells were placed at 38 F for 4-5 days, followed by 24 hrs at ambient temperature. Berry quality was assessed after the storage period, using sub-sampling of individual berries when appropriate for the following characteristics: sweetness (% brix), firmness, post-harvest disease & decay. Once the second batch of replicates was received by the University, they were stored for 4 days at 34 F and then assessed for post-storage quality.

[0203] 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.

[0204] As shown in 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 H5399 extracted biomass (EB) microalgae composition showed the most advantage.

[0205] Nineteen weeks after planting, strawberries were harvested and shipped overnight to a university sensory lab where they were kept at ambient conditions. The next day they were assessed by 74 respondents for various hedonics attributes.
All treatments were tested at the 1/2 gallon/A rate. Referring to FIGURE 35, for every attribute, the PHYCOTERRA Chlorella microalgae composition and the seaweed commercial reference were preferred over standard practice. The PHYCOTERRA
Chlorella microalgae composition was especially preferred over most treatments for aroma and texture liking. The Aurantiochytrium acetophilum H5399 extracted biomass (EB) microalgae composition was especially preferred for flavor liking.
Greenwater Polyculture (GWP) tracked with other treatments in terms of advantages over standard practice.

Example 13

[0206] A trial was conducted on strawberry (var. Portola) in Guadalupe Valley, CA to evaluate performance of various microalgae compositions on strawberry growth, yield, and post-harvest berry quality; specifically, the PHYCOTERRA Chlorella microalgae composition, PT-090 Chlorella microalgae composition, the Aura ntiochytrium acetophilum HS399 whole biomass (WB) microalgae composition, the Aurantiochytrium ace tophilum 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. Each of the 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.
STUDY PARAMETERS
Crop Strawberry (var. Portola) Location Guadalupe Valley, CA
Conventional Row Spacing 40" furrow spacing with 24" wide bed spacing, and plants on plant lines 12" apart and plant lines 12" apart Harvest Schedule As frequently as standard local grower practice with estimated 12-16 picks Fumigation Schedule Early May, 32 gal/a PicChlor60 Plot size minimum 1 double-line bed 45 ft length per plot with 80+
plants per plot Trial Design Randomized Complete Block Observations Taken from 70 plants inside 3 ft buffer zone of each plot end Replication 6 replicate plots for each treatment Local Standard Production Fertility, weed, insect management, etc.
Standard Management Fungicide application. Record disease management Practice measures Fungicides will be applied weekly when flowers and fruit are present j Advantage over %Advantage oyez %Advantage over Standar standar standar ...............
.....................
Holding Rate RWOR
d Comm. MNiMME d Comm. MMOINM d Comm.
Test Treatment lgaliA) Practice Ref. Practice Ref, ii;.4tikittia4 Practice Ref.
1 Standa; es: Practice 2.13 128 1.63 Comm. Ref. 0.50 1.88 -12% 1.22 -5% 1.40 -14%
PhycoTerra 0.25 1.87 -12% -1% 0.93 -27% -23% 1.43 -12% 2%
NwcoTerra 0.50 1.77 -17% -5% 1.10 -14% -10%
1.47 5%
Terrehe90 0.25 1.78. -16% -5% 102 -219 -16% 1.43 -12% 2%
Terrene.90 0.50 1.5 -13% -2% 1.12 -13% -8%
1.48 -9% 5%, FES355WB 0.25 1.90 -11% 1% 1.07 -17% -12% 1.35 -17% -4%
FS99W 0.50 1.11 -11% -6% G. -23% -19% 1.42 -n% 1%
FES399 V16 washed 0.25 1.83 -14% -3% 117 -9% -4% 1.45 11% 4%
liS399 W8 washed 0.50 1.78 -15% _5% 095 -25% -22% 1.25 -23% -11%
Combo 399WB 0.25 1.78 -16% -S% 0.93 -27% -.23% 1.30 -20% -7%
Combo 399W13 0.50 1.83 -14% -3% 1.15 -10% -5%
1.53 -6% 10%
Table 11: Raw Data

[0207]
Application rates of the PHYCOTERRA Chlorella microalgae composition, PT-090 Chlorella microalgae composition, the Aurantiochytrium acetophilum H5399 whole biomass (WB) microalgae composition, the Aurantiochytrium acetophilum H5399 washed whole biomass (WB washed) microalgae composition, and the combination 25% Chlorella: 75% Aurantiochytrium acetophilum H5399 whole biomass (WB) microalgae composition were as detailed in Table 12 below.
Table 12: Treatments Application Treatment Rate Number Product gallon/acre Ti Untreated control (UTC/standard practice) N/A
T2 Seaweed Commercial Reference 0.5 T3 PHYCOTERRA Chlorella composition 0.25 T4 PHYCOTERRA Chlorella composition 0.5 T5 H5399 Whole Biomass (WB) washed 0.25 T6 H5399 Whole Biomass (WB) washed 0.5 T7 H5399 Whole Biomass (WB) 0.25 T8 HS399 Whole Biomass (WB) 0.5 T9 PT-090 0.25 T10 PT-090 0.5 T11 25% Chlorella: 75% HS399 WB 0.25 T12 25% Chlorella: 75% HS399 WB 0.5

[0208] Fifteen weeks after transplanting, berries were harvested and stored in cold storage for 5 days and weights before and after storage were compared. Percent weight (water) loss is shown in FIGURE 36. Water loss was fairly low for all water-holding capacity tests across treatments (<3% of original weight) but advantages were observed across the board. Berries from microalgae-treated plants lost 11-17% less water than the plants grown with standard practice and 2-6% less water than plants grown with standard practice and the seaweed commercial reference.

[0209] Fifteen weeks after transplanting, berries were harvested and stored in cold storage for 5 or 7 days and bruising severity on the stored berries was compared between treatments. As shown in FIGURE 37, berries from microalgae-treated plants had a lower degree of bruising (5-27% reduction) than from plants grown using standard practice.
Berries assessed 5 days after storage showed an advantage for microalgae compositions compared to the seaweed commercial reference (5-23% reduction), but the same pattern was not consistent 7 days after storage (see FIGURE 38).
Example 14

[0210] For the treatments referred to in this Example as Commercial Reference +
PT-065, 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.

[0211] For the treatments referred to in this Example as Commercial Reference +
PT-090, the commercial reference was applied first to the soil at a rate of 20 gal/acre.
The PT-090 microalgae composition was then added on top via drip irrigation.
The commercial reference was only applied 3 times per season, whereas the PT-090 microalgae composition was applied every 14 days until harvest.

[0212] A trial was conducted on strawberry (var. Portola - Organic) in Santa Maria, CA to evaluate performance of various microalgae compositions on strawberry growth, yield, and post-harvest berry quality, particularly PT-065 microalgae composition, PT-090 microalgae composition, the combination PT-065 microalgae composition: microbial based commercial reference product microalgae composition, and the combination PT-090 microalgae composition: microbial based commercial reference product microalgae composition. All plots received standard local fertigation practice, including NEPTUNE'S HARVEST fertilizer and NFORCE fertilizer. A control was added with standard local fertigation practice plus 4 applications of a microbial-based commercial reference product that is standard to this location. Treatments included two versions of an OMRI certified Chlorella microalgae composition that differ by pasteurization temperature (PT-065 microalgae composition and PT-090 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. Berries were harvested according to local commercial schedule (twice per week during fruiting season). The timing of the commercial reference applications was once at the time of planting (6/20), once 14-21 days after planting (7/5), once in late July/early August (7/31) and the last in early September (9/11).
All plots were managed according to the local standard practice (see Study Parameters below).
STUDY PARAMETERS
Crop Strawberry (var. Portola) Location Santa Maria, CA
Conventional Row Spacing Wide 4-row beds, 64-inches center-to-center; plants spaced 14 inches apart in each of the four rows Harvest Schedule As frequently as standard local grower practice with estimated 32 picks Fumigation Schedule None (Organic) Plot size minimum 1 four-row bed 25-30 ft length per plot with 80+
plants per plot. Plots will be located away from any field edges with 1-2 commercial buffer beds in between Trial Design Randomized complete block Observations Yield data taken from 40 inside plants, outside 40 combined with inside 40 for post-harvest assessments Replication 6 replicate plots for each treatment and untreated control Local Standard Production Fertility, weed, insect management, etc.
Standard Management Standard management practices for organic production.
Practice Record disease management measures

[0213] Application rates of PT-065 microalgae composition, PT-090 microalgae composition treatment, the combination Commercial Reference + PT-065 microalgae composition treatment, and the combination Commercial Reference + PT-090 microalgae composition treatment were as detailed in Table 13 below. Raw data is included in the table shown in FIGURE 39.
Table 13: Treatments Application Treatment Product Rate Number gallon/acre Ti Standard practice only (UTC) Water T2 Commercial reference (No PT-0) 20 T3 Commercial Reference + PT-065 0.5 T4 Commercial Reference + PT-090 0.5 T5 PT-065C 0.25 T6 PT-090 0.25 T7 PT-065 0.5 T8 PT-090 0.5

[0214] At 11, 14 and 18 weeks after transplanting, berries were harvested and stored in cold storage for 6 days and weights before and after storage were compared. As shown in FIGURE 40, percent weight (water) loss is shown for all 3 assessments. Across all 3 assessments, berries from plants treated with a combination of Commercial Reference + PT-090 microalgae composition lost 8-38% less water than those treated with standard practice. Berries from plants treated with PT-090 microalgae composition lost 6-11% less water than standard practice for the first two assessments.
Almost all treatments had a positive effect over standard practice for the 2nd assessment. For the last two assessments, almost all treatments had an advantage over the commercial reference alone (3-70%). Overall, PT-065 microalgae composition and PT-090 microalgae composition at 0.5 gal/A alone and combined with the commercial reference showed the most advantage in Santa Maria.

[0215] At 11, 14 and 18 weeks after transplanting, berries were harvested and stored in cold storage for 6 days and berry skin firmness was assessed using a penetrometer. The force needed to pierce the skin (g force) is shown below for all 3 assessments performed on Santa Maria berries. Referring to FIGURE 41, across all 3 assessments, berries from plants treated with a combination of the microbial-based Commercial Reference + PT-090 microalgae composition were 4-9% firmer after storage than those treated with standard practice and 2-10% firmer than those receiving just the commercial reference. PT-090 microalgae composition (1/4 gal/A) showed an advantage over standard practice (3-11%) and the commercial reference (4-14%) in 2 of the 3 assessments.

[0216] At 11, 14 and 18 weeks after transplanting, berries were harvested and stored in cold storage for 6 days and bruising was assessed at the end of this period.
Referring to FIGURE 42, marketability (% of berries with mild to moderate bruising) is shown for all 3 assessments performed on Santa Maria berries. In all 3 assessments, PT-090 microalgae composition (1/2 gal/A) showed an increase in marketability due to reduced bruising compared to standard practice (5-9%). The microbial-based Commercial Reference + PT-065micr0a1gae composition (1/2 gal/A; 11-40%), PT-microalgae composition alone (1/4 gal/A; 4-8%), and PT-090 microalgae composition alone (1/4 gal/A, 2-9%) all showed an advantage over standard practice in 2 of assessments. Compared to the commercial reference alone, PT-065 microalgae composition and PT-090 microalgae composition showed an advantage increasing marketability in 2 of 3 assessments (5-30%).

[0217] The percent of berries with severe bruising is also shown in FIGURE 43.
Compared to standard practice, PT-065 microalgae composition (1/4 gal/A) and the combination of the Commercial Reference + PT-090 microalgae composition reduced severe bruising (10-30%) in 2 of 3 assessments. Compared to the commercial reference, multiple treatments showed a benefit.

[0218] For the consumer preference testing, one harvest was performed for each trial. The first occurred on or about October 11, 2017. Marketable berries from 6 replicates each of 6 treatments were harvested and packaged into clamshells.
These were delivered to the University sensory lab the same day. The second harvest occurred on or about November 2, 2017. Marketable berries from 6 replicates each of 5 treatments were harvested and packaged into clamshells. Once shipment was received, a consumer taste panel was conducted. Berries were washed and prepared into halves for approximately 100 consumer volunteers who received one half of 5-6 berries (UTC and 4-5 treatments).
Berries were scored for appearance and flavor. It should be noted that for the consumer sensory panel testing, the treatments were as shown below in Table 14.
Table 14: Treatments Application Treatment Product Rate Number gallon/acre Ti Standard practice only (UTC) Water T2 Commercial reference (No PT-0) 20 T3 Commercial Reference + PT-065 0.5 T5 PT-065 0.25 T7 PT-065 0.5

[0219] Twelve weeks after transplanting, berries were harvested and transported to a University sensory lab where they were kept overnight at 4 C. The next day they were assessed by 102 respondents for various liking attributes. Referring to FIGURE
44, 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.
Example 15

[0220] A trial was conducted on strawberry (var. Portola) in Oxnard, CA to evaluate performance of various microalgae compositions on strawberry growth, yield, and post-harvest berry quality; particularly, the PHYCOTERRA Chlorella microalgae composition, PT-065 microalgae composition, the Aurantiochytrium acetophilum whole biomass (WB) microalgae composition, the Aurantiochytrium acetophilum extracted biomass (EB) microalgae composition, and the combination 25%
Chlorella:
75% H5399 whole biomass (WB) microalgae composition. All plots receive standard local fertilization regimen used by the grower for this crop, excluding biostimulants. 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).

[0221] Application rates of the PHYCOTERRA Chlorella microalgae composition, the Aurantiochytrium acetophilum H5399 extracted biomass (EB) microalgae composition, the Aurantiochytrium acetophilum H5399 whole biomass (WB) microalgae composition, the PT-065 microalgae composition, and the combination 25% Chlorella: 75%

whole biomass (WB) microalgae composition were as detailed in Table 15 below.
Raw data is included in the table shown in FIGURES 45-46.
STUDY PARAMETERS
Crop Strawberry (var. Portola) Location Oxnard, CA
Conventional Row Wide 4-row beds, 64-inches center-to-center; plants Spacing spaced 14 inches apart in each of the four rows Harvest Schedule As frequently as standard local grower practice with estimated 24 picks Fumigation Schedule Local practice (recorded) ¨ timing will be in June Plot size minimum 1 four-row bed 25 ft length per plot with 80+
plants per plot. Plots will be located away from any field edges with 1-2 commercial buffer beds in between Trial Design Randomized complete block Observations Yield data taken from 40 inside plants, outside 40 combined with inside 40 for post-harvest assessments Replication 6 replicate plots for each treatment and untreated control Local Standard Production Fertility, weed, insect management, etc Standard Management Standard management practices, including fungicide Practice application. Record disease management measures.
Fungicides will be applied as necessary (by grower) when flowers and fruit are present Table 15: Treatments Application Treatment Rate Number Product gallon/acre Ti Untreated control (UTC/standard practice) .. Water T2 Seaweed Commercial Reference 0.5 T3 PHYCOTERRA Chlorella composition 0.25 T4 PHYCOTERRA Chlorella composition .. 0.5 T5 H5399 Extracted Biomass (EB) 0.25 T6 H5399 Extracted Biomass (EB) 0.5 T7 H5399 Whole Biomass (WB) 0.25 T8 H5399 Whole Biomass (WB) 0.5 T9 PT-065 0.25 T10 PT-065 0.5 T11 25% Chlorella: 75% H5399 WB 0.25 T12 25% Chlorella: 75% H5399 WB 0.5

[0222] At 11, 15, 19, 21, and 26 weeks after transplanting, berries were harvested and stored in cold storage for 6 to 10 days and weights before and after storage were compared. As shown in FIGURE 47, water loss was fairly low for all holding tests across treatments (< 13% of original weight) but some advantages were observed.
Compared to standard practice, the PT-065 microalgae composition (1/4 gal/A) and Aurantiochytrium acetophilum H5399 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 H5399 extracted biomass (EB) microalgae composition showed an advantage in 3 of 5 assessments. Compared to the commercial reference, only the Aurantiochytrium acetophilum H5399 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).

[0223] At 11, 15, 19, 21 and 26 weeks after transplanting, berries were harvested and stored in cold storage for 6 to 10 days and berry skin firmness was assessed using a penetrometer. The force needed to pierce the skin (g force) is shown in FIGURE
48 for all 5 assessments performed on the Oxnard strawberries. In 2 of 5 assessments, the PT-065 microalgae composition, Aurantiochytrium acetophilum H5399 extracted biomass (EB) microalgae composition (1/4 gal/A), Aurantiochytrium acetophilum H5399 whole biomass (WB) microalgae composition, and the combination 25% Chlorella: 75%

whole biomass (WB) microalgae composition all showed an advantage in skin firmness (4-20%) compared to standard practice. These advantages occurred during the extreme ends of the harvest season (September and January) when berries may be off-peak and increasing shelf-life advantage would be most desirable. Compared to the seaweed-based commercial reference, the PHYCOTERRA Chlorella microalgae composition and the Aurantiochytrium acetophilum H5399 extracted biomass (EB) microalgae composition showed an advantage in 3 of 5 assessments (4-14%).

[0224] At 11, 15, 19, 21 and 26 weeks after transplanting, berries were harvested and stored in cold storage for 6-10 days and bruising was assessed at the end of this period.
Marketability (% of berries with mild to moderate bruising) is shown in FIGURE
49 for all 4 assessments performed on Oxnard strawberries. Compared to standard practice, improvements were only observed for the final two assessments (late season).
The PT-065 microalgae composition (6-18%) and the Aurantiochytrium acetophilum H5399 extracted biomass (EB) microalgae composition (3-20%) showed increased marketability due to bruising reduction in both assessments. Improvements over the seaweed commercial reference were only observed in the first and last assessment (early and late season). All 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 H5399 extracted biomass (EB) microalgae composition (6-11%). As shown in FIGURE
50, the percent of berries with severe bruising was especially reduced during late season by multiple products (10-60%).

[0225] For the consumer preference testing, one harvest was performed for each trial.
The first occurred on or about October 11, 2017. Marketable berries from 6 replicates each of 6 treatments were harvested and packaged into clamshells. These were delivered to the University sensory lab the same day. The second harvest occurred on or about November 2, 2017. Marketable berries from 6 replicates each of 5 treatments were harvested and packaged into clamshells. Once shipment was received, a consumer taste panel was conducted. Berries were washed and prepared into halves for approximately 100 consumer volunteers who received one half of 5-6 berries (UTC and 4-5 treatments).
Berries were scored for appearance and flavor. It should be noted that for the consumer sensory panel testing, the treatments were as shown below in Table 16.

Table 16: Treatments Application Treatment Rate Number Product gallon/acre Ti Standard practice only (UTC) Water T2 Seaweed Commercial Reference 0.5 T4 PHYCOTERRA Chlorella composition 0.5 T6 H5399 Extracted Biomass (EB) 0.5 T8 H5399 Whole Biomass (WB) 0.5 T10 PT-065 0.5

[0226] Sixteen weeks after transplanting, berries were harvested and transported to a university sensory lab where they were kept overnight at 4 C. The next day they were assessed by 100 respondents for various liking attributes. All treatments were tested at the 1/2 gallon/A rate. As shown in FIGURE 51, the Aurantiochytrium acetophilum whole biomass (WB) microalgae composition was preferred over standard practice and the seaweed-based commercial reference for all attributes. This treatment particularly stood out for overall liking, aroma and flavor. The PHYCOTERRA Chlorella composition was also preferred over standard practice for all attributes but not to the same degree as the Aurantiochytrium acetophilum H5399 whole biomass (WB) microalgae composition. The PT-065 microalgae composition was only preferred over standard practice for appearance and was preferred less for overall liking, aroma and flavor.
Example 16

[0227] A trial was conducted in a greenhouse to test the effects of the PT-0 microalgae composition, the Aurantiochytrium acetophilum H5399 extracted (EB) microalgae composition, and the combination 25% Chlorella: 75% H5399 extracted biomass (EB) microalgae composition on Espresso tomato, a brown/red cocktail variety that normally is grown in hydroponic systems. The objective was to test the effect of these formulations on the shelf life of the tomato fruits after harvest. Plants were grown in a greenhouse environment in rockwool cubes and then transplanted into 3-gallon coco coir blocks (EARTHSCAPE CUT' N GRO 6-gal bags). 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 mm, each for a 1 mm 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). On 12/06/17, the first fruit harvest occurred and continued weekly until experiment termination on 02/14/2018; a total of 32 harvests. Initial weight was measured of each fruit harvested. Espresso tomato fruits were placed in ambient conditions to observe shelf life overtime. Weight measurements were taken throughout the storage period (among 1 and 20 days post-harvest). Raw data is included in the table shown in FIGURE 52.
Table 17: Vegetative and Fruiting Fertilizer Solutions Vegetative Fruiting mg/L
183.81 167.19 47.11 47.11 205.67 293.89 Ca 207.00 207.00 Mg 67.05 67.05 98.39 152.77 Fe 1.98 1.98 Zn 0.33 0.33 Mn 0.91 0.91 0.30 0.30 Cu 0.05 0.05 Mo 0.06 0.06

[0228] Water retention measurements were calculated from total weight of fruit after each harvest; fruit water-holding capacity is an indicator of shelf-life. As shown in FIGURE
53 tomatoes grown in coco bag with the PT-0 microalgae composition showed better water retention capacity in comparison to UTC, with a 22% increase of water retention than UTC across all harvests and storage times. Referring to FIGURES 54-55, this pattern held up even for longer storage periods of 10 and 18 days. After 18 days post-harvest, the effect of water retention on fruit is was visually ascertainable.
The UTC
showed a negative effect on fruit because the tomatoes lost more water in comparison with the other treatments. The PT-0 microalgae composition, among all the microalgae treatments, showed better capacity to retain water, whereas the UTC did not present the same effects.

[0229] As detailed in Table 18 below, overall, 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%

extracted biomass (EB) microalgae composition). At day 10 post-harvest, tomato fruits which were grown in coco blocks with the PT-0 microalgae composition showed 22.2%
advantage of water retention compare with UTC. 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. At day 18 post-harvest, 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%

extracted biomass (EB) microalgae composition showed an increase of water holding capacity compared to UTC with 6.95% and 10%, respectively.
Table 18: Average Overall Advantage Over UTC for Fruit Water Holding Capacity Overall % Advantage in Fruit Water-Treatment Holding Capacity Over UTC
PT-0 18.9 HS399EB 5.4 PT-0:HS399EB 11.9 days PT-0 22.2 HS399EB 8.2 PT-0:HS399EB 18.2 18 days PT-0 19.0 HS399EB 6.95 PT-0:HS399EB 10.9 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.

[0231] Sixteen Douglass Fir trees (6'-7') were purchased from a local retail garden center within one hour of delivery from wholesaler. The 16 trees were divided into two groups of eight, a "fresh tree" group and "stored tree" group. The fresh tree group was used to populate one half of the experiment on the same day they were purchased.
The stored tree group were kept in a warehouse for two weeks, to simulate unfavorable real-world storage conditions found in retail centers. After the storage period, the stored trees were set up alongside the fresh tree group and treated. Within both of the two groups four unique treatments were applied to two replicates.

[0232] 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).

[0233] The PT-0 microalgae composition was applied at 0.1%, 1.0% and 5.0%
(vol/vol). A commercial reference (CR) product was also applied at 1.0%
vol/vol, as listed on the product label. Reapplication of each treatment solution occurred when the untreated control trees had consumed nearly all water in the tree stand. The volume of each solution required to top-off the reservoir was quantified and tracked over time.

[0234] Fallen needles were collected from the floor under each tree periodically.
Needles were placed in paper bags (separate for each replicate) and dried in a dehydrator at 160 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.

[0235] The cumulative needle drop weight for each treatment was compared in three ways: raw weight values, normalized by height and normalized by circumference are shown below in Table 19. Normalization by height and circumference did not affect the patterns between treatments. Needle drop weight per treatment was also examined as a function of time (see Table 20 below).
Table 19: Average Cumulative Needle Dry Weight Block Treatment Ave. Needle Ave. Ave. Height- Ave. Ave.
Drop Heigh corrected Circumf Circumferenc Dry Weight t (ft) cumulative erence e-corrected (cumulative, needle weight (ft) cumulative (g/ft) needle weight (g/ft) Fresh Untreated 51.705 6.58 7.89 0.81 62.79 trees Fresh Commercial 23.99 7.23 3.28 0.72 32.89 trees Reference 1.0%
Fresh PT-0 1.0% 31.435 7.13 4.46 0.88 37.88 trees Fresh PT-0 5.0% 14.89 6.77 2.23 0.65 23.75 trees Stored Untreated 10.3 6.92 1.49 0.76 14.18 trees Stored Commercial 12.965 6.54 1.98 0.70 18.55 trees Reference 1.0%
Stored PT-0 0.1% 14.2 6.75 2.10 0.80 17.62 trees Stored PT-0 1.0% 26.02 6.60 3.94 0.96 27.24 trees Table 20: Average Needle Drop Dry Weight (g) by Treatment Over Time Date Block Treatment Average Cumulative Needle Needle Dry Weight (g) Dry Weight (g) 12/7/2017 Fresh Untreated 17.265 51.7 12/7/2017 Fresh Commercial 12.825 Reference 1.0% 24.0 12/7/2017 Fresh PT-0 1.0% 13.285 31.4 12/7/2017 Fresh PT-0 5.0% 7.935 14.9 12/13/2017 Fresh Untreated 9.15 12/13/2017 Fresh Commercial 4.7 Reference 1.0%
12/13/2017 Fresh PT-0 1.0% 5.685 12/13/2017 Fresh PT-0 5.0% 2.27 1/5/2018 Fresh Untreated 25.29 1/5/2018 Fresh Commercial 6.465 Reference 1.0%
1/5/2018 Fresh PT-0 1.0% 12.465 1/5/2018 Fresh PT-0 5.0% 4.685 12/13/2017 Stored Untreated 6.025 10.8 12/13/2017 Stored Commercial 5.98 12.88 Reference 1.0%
12/13/2017 Stored PT-0 0.1% 9.39 14.2 12/13/2017 Stored PT-0 1.0% 14.99 26.02 1/5/2018 Stored Untreated 4.275 1/5/2018 Stored Commercial 6.985 Reference 1.0%
1/5/2018 Stored PT-O0.1% 4.81 1/5/2018 Stored PT-0 1.0% 11.03

[0236] As shown in FIGURE 56, within the fresh tree group, both the PT-0 microalgae composition and the commercial reference reduced the total weight of fallen needles by 70% across 3 collection dates. However, this same pattern did not hold true for trees in the stored tree block.

[0237] The volume of each solution required to top-off the reservoir of each tree varied between treatments and the actual volumes applied were recorded (see Table 21 below). The average volume across all top-off events are represented graphically in FIGURE 57. Only data for the fresh trees block was recorded. For 4 of 6 different top-off events, the trees treated with the PT-0 microalgae composition at 5%
required 11-60% less water than untreated control. Results were more variable compared to the commercial reference but ranged from 5-40% less water in 3 of 6 top off events.
Table 21: Volumes of Treatment Solution (mL) to Replenish Individual Tree-Watering Reservoirs Date Block Treatment Average Top-off Volume (mL) 11/27/2017 Fresh Untreated 1500 11/27/2017 Fresh Commercial Reference 1.0% 1500 11/27/2017 Fresh PT-0 1.0% 1500 11/27/2017 Fresh PT-O5.0% 1500 11/29/2017 Fresh Untreated 1200 11/29/2017 Fresh Commercial Reference 1.0% 650 11/29/2017 Fresh PT-0 1.0% 1150 11/29/2017 Fresh PT-O5.0% 950 12/3/2017 Fresh Untreated 900 12/3/2017 Fresh Commercial Reference 1.0% 800 12/3/2017 Fresh PT-0 1.0% 1100 12/3/2017 Fresh PT-0 5.0% 800 12/7/2017 Fresh Untreated 650 12/7/2017 Fresh Commercial Reference 1.0% 450 12/7/2017 Fresh PT-0 1.0% 475 12/7/2017 Fresh PT-O5.0% 375 12/13/2017 Fresh Untreated 1100 12/13/2017 Fresh Commercial Reference 1.0% 750 12/13/2017 Fresh PT-0 1.0% 600 12/13/2017 Fresh PT-O5.0% 450 12/20/2017 Fresh Untreated 950 12/20/2017 Fresh Commercial Reference 1.0% 1100 12/20/2017 Fresh PT-0 1.0% 800 12/20/2017 Fresh PT-0 5.0% 1050

[0238] Overall, the trees treated with the PT-0 microalgae composition at 5% lost fewer needles and required less water during the month after trees were cut and set up in tree stands.

[0239] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

[0240] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0241] Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). All provided ranges of values are intended to include the end points of the ranges, as well as values between the end points.

[0242] The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of', "consists essentially of', or "substantially comprises"
that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0243] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0244] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0245] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

[0246] This invention includes all modifications and equivalents of the subject matter recited in the claims and/or aspects appended hereto as permitted by applicable law.

Claims (23)

78What is claimed is:

1. A method of enhancing a plant comprising the step of administering to the plant, seedling, or seed a liquid composition treatment comprising a culture of microalgae, the microalgae comprising at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum H5399 cells in an amount effective to enhance at least one characteristic of a plant compared to a substantially identical untreated plant, wherein the characteristic is selected from improved shelf life, increased water retention, and diminished needle-drop.

2. The method of Claim 1 wherein administering comprises contacting soil in the immediate vicinity of the plant, seedling, or seed with an effective amount of the liquid composition treatment.

3. The method of Claim 2 wherein the liquid composition is administered at a rate in the range of .25-2 gallons/acre.

4. The method of Claim 3 wherein the liquid composition comprises between 100g-800g per acre of at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells.

5. The method of Claim 2 wherein the contacting comprises a drip irrigation system and/or process.

6. The method of Claim 1 wherein the liquid composition treatment further comprises phosphoric acid and potassium sorbate.

7. The method of Claim 1 wherein the liquid composition treatment further comprises citric acid.

8. The method of Claim 1 wherein 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.

9. The method of Claim 1 wherein the at least one of pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells are pasteurized for between 90-150 minutes.

10. The method of Claim 1 wherein the microalgae comprises Chlorella cells and Aurantiochytrium acetophilum HS399 cells in an amount effective amount to improve shelf-life of the plant compared to a substantially identical untreated plant.

11. The method of Claim 1 wherein the microalgae comprises Aurantiochytrium cells and the liquid composition is applied in an amount effective to increase water retention of the plant by at least 5% compared to a substantially identical untreated plant.

12. The method of Claim 1 wherein the microalgae comprises only Chlorella cells and the liquid composition is applied in an effective amount to reduce needle-drop of the plant by at least 5% compared to a substantially identical untreated plant.

13. The method of Claim 10 wherein the liquid composition comprises pasteurized Chlorella cells and pasteurized Aurantiochytrium acetophilum HS399 cells in a ratio of 25:75.

14. The method of Claim 1 wherein the Aurantiochytrium acetophilum HS399 cells have been subjected to an extraction process to remove oils from the Aurantiochytrium acetophilum HS399 cells.

15. A composition for enhancing at least one plant characteristic comprising a microalgae biomass comprising at least two species of microalgae, and wherein the composition causes synergistic enhancement of at least one plant characteristic selected from improved shelf life, increased water retention, and diminished needle-drop.

16. The composition of claim 15, wherein the microalgae species is selected from the group consisting of Botryococcus, Chlorella, Chlamydomonas, Scenedesmus, Pavlova, Phaeodactylum, Nannochloropsis, Aurantiochytrium. Spirulina, Galdieria, Haematococcus, Isochrysis, Porphyridium, Schizochytrium, and Tetraselmis.

17. The composition of claim 15, wherein the microalgae biomass comprises whole biomass and/or residual biomass.

18. The composition of claim 15, wherein the composition comprises a first species of microalgae and a second species of microalgae, wherein the ratio of the first species of microalgae and the second species of microalgae is between 1:20 and 1:1.

19. The composition of claim 15, wherein the composition comprises a first species of microalgae and a second species of microalgae, wherein the ratio of the first species of microalgae and the second species of microalgae is between 1:4 and 1:1.

20. The composition of claim 18, wherein the first species of microalgae is Chlorella and the second species of microalgae is Aurantiochytrium.

21. The composition of claim 20, wherein ratio of Chlorella and Aurantiochytrium is 25:75, 50:50 or 75:25

22. The composition of claim 20, wherein the Chlorella is whole biomass and Aurantiochytrium is residual biomass or wherein Chlorella is residual biomass and Aurantiochytrium is whole biomass.

23. A method of plant enhancement comprising administering to a plant, seedling, or seed the composition treatment of claim 15, wherein the composition treatment enhances at least one plant characteristic synergistically, wherein the characteristic is selected from wherein the characteristic is selected from improved shelf life, increased water retention, and diminished needle-drop.