A novel method for producing an algal composition

Abstract

Systems and methods for growing microalgae capable of mixotrophic metabolism, preferably species of the genus Chlorella. The microalgae grown in the system using the method are capable of surviving and growing during cold dark storage, whereby the algae can be stored and transported for application as a live culture. In addition, the microalgae can be grown in an amount sufficient to be commercially sold for application to crops as a biostimulant.

Description

Technical Field

[0001] Background Art The rapid population growth after World War II raised concerns about food security regarding the limits of the Earth's ability to feed the increasing number of people. Malthusians probably predicted famine until 1990. This did not happen thanks to the Green Revolution, an unexpected increase in agricultural efficiency and yields. One element of this was the development of nitrogen fertilizers. This was also a by-product from ammunition technology. Gunpowder factories became fertilizer factories (from swords to carving knives).

[0002] Synthetic chemical fertilizers grow plants. However, after 70 years of use, unintended consequences have occurred, such as 1) a decline in soil fertility and stagnation of crop yields, 2) an increase in greenhouse gases, and 3) groundwater pollution that causes wild algae blooms. The production of synthetic fertilizers requires the combustion of methane. It is estimated that 12% of greenhouse gases are derived from modern agriculture, and most of this is from the use of chemical fertilizers.

[0003] In nature, plant growth depends on the symbiotic relationship between plants and soil microorganisms. Plants exude sugars (carbon compounds) from their roots to supply the microorganisms present in the soil, especially bacteria. In exchange, soil microorganisms process the nutrients necessary for plant growth. Soil bacteria convert atmospheric nitrogen into ammonium compounds that can be absorbed by plant roots. This natural process is known as nitrogen fixation and is mainly the function of bacteria in close contact with the roots (this area is known as the "rhizosphere") and not the function of plants. Rich soil in the rhizosphere has up to 10 billion bacteria per gram. The composition and activity of microorganisms in the soil define "richness." Crop rotation is carried out using leguminous plants to add nitrogen to the soil, but it is the bacteria in the legume root nodules that fix nitrogen. Other bacteria solubilize phosphorus bound to the soil.

[0004] Chemical fertilizers bypass natural processes. Over time, microbial activity - richness - as well as the nitrogen and carbon levels in the soil decline. Chemical fertilizers add macronutrients, nitrogen, phosphorus, and potassium to the soil in forms that can be directly absorbed by the roots. However, plants only absorb about 30% of the nitrogen applied through chemical fertilization. Most of the rest becomes dissolved and ultimately reaches the groundwater. It is well known that the addition of fertilizers to water bodies, as is known to occur through field runoff into ponds and lakes, promotes the growth of wild algae and causes harmful algal blooms. In addition, some of the nitrogen in fertilizers is converted to nitrous oxide, a potent greenhouse gas. Worldwide, agriculture is responsible for 75% of the nitrous oxide released into the atmosphere.

[0005] Applying composted organic materials such as fertilizers to the soil can provide nitrogen as well as carbon to supply bacteria and microorganisms. However, the use of compost is inconvenient due to its bulk and difficult to apply on a large scale. The treatment of fertilizers to produce organic fertilizers is an industrial process and involves energy costs for transportation.

[0006] Biostimulants offer another way to promote plant growth. Plant biostimulants (PBS), as defined in the 2018 U.S. Farm bill, are compounds or organisms that promote natural processes. Numerous studies have shown that living microalgae, particularly Chlorella vulgaris, are effective biostimulants through both foliar and soil applications. Furthermore, the stimulating effect of microalgae on soil microorganisms is regenerative: the application of microalgae increases organic matter, bacterial biomass, and soil respiration (a measure of bacterial activity). In other words, the application of microalgae improves soil richness. However, until now, practical storage of live microalgae has not been available, and live microalgae had to be produced on-site and used immediately after collection, so live microalgae could not be used on a large scale as biostimulants.

[0007] Summary of the Invention The present invention is a novel approach for inducing heterotrophic metabolic activity in microalgae, which means that microalgae grown using this method can consume organic substances from the medium and grow when placed under conditions where photosynthesis is not possible, i.e., in the absence of light. Embodiments of the present invention are directed to improving methods of using an amount sufficient to be commercially sold for application to crops as a biostimulant after the algae are collected for growing species of the genus Chlorella, preferably Chlorella vulgaris. In particular, microalgae grown using the improved method can withstand cold storage and shipping.

[0008] Microalgae, particularly Chlorella vulgaris, have been grown for decades for commercial use. Known methods of growing microalgae such as C. vulgaris at commercial scale require a photobioreactor (PBR) to maximize growth in limited space.

[0009] Most commercial uses of microalgae do not involve living microalgae. For example, when microalgae are grown for oil (biofuel) or used as animal feed additives, the algae are processed immediately after collection. Commercially available algae-based biostimulants include cell-free algae extracts containing growth signaling compounds (plant hormones) such as auxins and cytokinins that stimulate plant growth and promote soil fertility. Among others, there are growth media derived from algae cultures containing the same growth signaling compounds secreted by algae during their growth. Importantly, none of the commercially available biostimulants contain living green algae cells. Most biostimulants are marketed as supplements to chemical fertilizers (not strong enough to replace chemical fertilizers). Initial university research has shown that the application of living algae (grown in the field and applied at the time of algae collection) provides sufficient boost to achieve yields and richness that can eliminate chemical fertilizers. Living cell algal biostimulants have been found to be more effective than algal derivatives. Therefore, methods for preserving living algae during shipping and storage can lead to a reduction in the need for synthetic chemical fertilizers.

[0010] Known commercially available biostimulants containing live cells use cyanobacteria (the so-called "blue-green algae" which are actually bacteria). Cyanobacteria are effective plant biostimulants and produce growth signaling compounds also produced by green algae. Like other bacteria, cyanobacteria can be dried, packaged, and then reconstituted in water for application as live cells. Unfortunately, however, neurotoxins produced by cyanobacteria (such as β-N-methylamino-L-alanine, BMAA) have been associated with outbreaks of neurodegenerative diseases over the past 20 years. Therefore, there is a need for safer biostimulants based on live cells.

[0011] Microalgae grown by the methods taught herein are bottled from the growth medium into the PBR as an "algal concentrate" and remain viable even when stored refrigerated (6 °C). Refrigeration inhibits the growth of common contaminants such as protozoa and bacteria, keeping the concentrate free of contaminants during storage and transport to the application site. Refrigerated storage is typically dark. This is a problem for microalgae because, like higher plants, microalgae are usually autotrophic, i.e., they maintain their survival rate and grow through the production of nutrients by light-dependent photosynthesis. Algae + light + water + CO2 + inorganic nutrients → glucose (and further algae) + O2

[0012] When microalgae are deprived of light, photosynthesis stops and the number of algal cells rapidly decreases (in our inventors' lab, up to 50% in 5 days, with minimal recovery afterwards, Table 1). Thus, with standard propagation methods, live microalgae have to be produced locally for immediate application as PBS. This is a problem for widespread commercial use because microalgae are difficult to grow. Studies showing that live microalgae, i.e., microalgae propagated locally and applied immediately after collection, are effective PBS have been conducted in northern Africa, Southeast Asia, and eastern Europe, but the method has not been adopted for commercial use because it is difficult to reproduce. Without wishing to be bound by theory, the supposed benefit of live algae, particularly Chlorella vulgaris, is the continuous production of signaling compounds after application to plants or soil. Another beneficial effect of live algae, particularly Chlorella vulgaris cells, is that they were observed to take up soil bacteria, transport them into plant root hairs, and ultimately release them into the apoplast, where they function as beneficial endophytes, presumably via phagocytosis. There was no method of storing live microalgae grown on a scale such that they could be grown in one location and distributed to other distant locations without colony breakdown for application. The essence of the present invention is a method for growing microalgae, particularly Chlorella vulgaris, that remain viable without significant reduction in cell number during cold dark storage due to induction of heterotrophic metabolism. That is, microalgae grown by this method can consume organic matter and oxygen and grow in the absence of light. This is achieved by the hyperoxygenation of the water used in the growth medium within the PBR. In a hyperoxygenated environment, microalgae change their metabolic behavior to become "mixotrophic". This means that they continue to produce food / energy by photosynthesis while light is available (autotrophic metabolism), but are capable of heterotrophic growth where they can consume organic nutrients in the dark. Algae are known to be capable of heterotrophic feeding.This is because it has been observed that the addition of sugars such as glucose to the growth medium is induced, especially when exposed to high concentrations of glucose. This method induces heterotrophic metabolism by hyperoxygenating the growth medium, that is, by saturating the growth medium with oxygen nanobubbles. This has not been reported before. Microalgae that can maintain the number of algal colony cells and survival rate during dark storage have not been reported before. Although the method for growing C. vulgaris is described, this method is used for all species of the genus Chlorella and other microalgae capable of mixotrophic metabolism.

[0013] Currently, PBRs are used to grow microalgae in water supplemented with inorganic nutrients. The microalgae grown in this way can be processed and used immediately after collection, but since microalgae cannot withstand refrigerated storage in the dark, they cannot be transported for applications that require live microalgae cells. Growing algae in a photobioreactor has been a common practice. In addition, a nanobubble generator has been used as part of these systems to generate ozone nanobubbles for sterilizing the algal growth medium and to clean the algal growth facilities (tanks and pipes). The improvement described herein includes an oxygen concentrator equipped with a nanobubble generator (NBG). In other words, the present invention provides for the first time a photobioreactor equipped with means for supplying oxygen in the form of nanobubbles. As shown in FIG. 1, the system of the present invention comprises an NBG arranged to receive sterilized water from a source. The sterilized water is supplied through the NBG to receive nanobubbles of oxygen, increasing the measurable oxygen concentration of the water to a saturated state of about 500%, i.e., a hyperoxygenated state.

[0014] Next, pump the hydrogen peroxide to fill at least one PBR. Add a well-known mixture of inorganic nutrients widely used in algal production to the sterilized water in the PBR to create a growth medium. An example of a typical medium that can be used to grow the algae according to the present invention is commercially available Guillard's / F / 2 medium. Preferably, in the method of the present invention, there is no externally added carbohydrate in the medium. Then, inoculate the PBR with microalgae.

[0015] The PBR used in the system is constructed from a translucent material such that light from outside the PBR can be used by the microalgae cells growing within the PBR for photosynthesis. An artificial light source is mounted outside the PBR to simulate a 24-hour day cycle. Also, since photosynthesis requires carbon dioxide, a standard aquarium airstone is used to introduce filtered ambient air into the growth medium to supply carbon dioxide and to mix the microalgae cells to keep them in suspension. Immediately after collection from the PBR, the microalgae cells are bottled together with the growth medium as an algal concentrate containing at least 10 million algae cells / mL. This concentrate is the final product that can be diluted and applied as a plant and soil biostimulant.

[0016] An important feature of the improvement of this method is that the microalgae cells produced can survive in dark cold storage for as long as 18 months, a length of time sufficient for transportation to the agricultural site for application for over 6 months. The bottled algae concentrate is placed directly in a refrigerator (6 °C) for storage prior to sale for use as a biostimulant. However, since algae can grow over a wide range of temperatures, refrigeration is not essential. However, refrigeration favorably prevents the growth of contaminating bacteria and protozoa if storage is extended beyond 2 months. If the product is used within that time, freezing is not required. Contamination resulting in loss of the algal colony was rare during the first 2 months of storage, presumably because heterotrophic feeding suppressed the growth of contaminants. The composition of the present invention containing live algae can be stored at refrigeration temperature, e.g., 6 °C, for over 8 months, but the recommended shelf life is 6 months from collection. Microalgae grown in a system using this method not only remain viable but can continue to grow during storage, thereby maintaining the cell number required for effective application as a biostimulant (see Table 1). The application rate of live algae is 50,000 cells / square foot and is applied to the soil, leaves, or both. Thus, 1 liter of algae concentrate can be diluted to treat 4.5 acres. 5 ml of the concentrate treats 1,000 square feet.

[0017] Another feature of the improvement of this method is to enable the large-scale growth of microalgae for agricultural use at low cost, which requires only minimal operation and achieves a high yield of microalgae in a small space. In particular, the present invention enables the growth of algae in a liter photobioreactor tank with a maximum capacity of 750 liters. Heretofore, when the diameter of the tank was increased, there was a concern that light shielding of the algae occurred in the center of the tank, resulting in growth being hindered due to inefficient exposure to light. However, the inventors have surprisingly found that the number of algal cells in a larger tank increases almost as fast as that in a 300-liter PBR with a smaller diameter. One explanation for this is to constantly mix the algae with air bubbles. This prevents the algal cells from gathering in the center of the tank and from gathering from the light source. Another explanation is that the algae are mixotrophic. That is, the algae do not depend on photosynthesis, so they continue to grow even in a region of the reactor that is more shaded or even in the dark. The larger the PBR tank used, the greater the improvement in efficiency and productivity.

[0018] In the context of the present invention, at least the following embodiments E1 to E72 are described. E1. According to a first embodiment, the present invention is a method for producing an algal composition comprising viable algae and a liquid, comprising: - adding oxygen to the liquid to prepare a peroxidized liquid; and - culturing the algae in the peroxidized liquid at least temporarily under light exposure to enable the growth of the algae. The method relates to a method comprising. E2. - preparing a liquid; - adding oxygen to the liquid to prepare a peroxidized liquid; - planting the algae in the peroxidized liquid; - culturing the algae in the peroxidized liquid at least temporarily under light exposure to enable the growth of the algae. The method of embodiment 1 comprising. E3. - Preparing a liquid containing algae; - Adding oxygen to the liquid to prepare a peroxidized liquid and / or adding a peroxidized liquid; - Culturing the algae in the peroxidized liquid, at least temporarily under light exposure, to enable growth of the algae The method of Embodiment 1, comprising the above steps. E4. The number of viable cells in the liquid is 1 million to 20 million cells / mL, preferably 2 million to 18 million cells / mL, more preferably 5 million to 15 million cells / mL, even more preferably 7 million to 14 million cells / mL, even more preferably 10 million to 13 million cells / mL, and most preferably 11 million to 13 million cells / mL. The method according to any one of Embodiments 1 to 3. E5. The attenuation of the number of viable cells is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, even more preferably at most 50%, even more preferably at most 40%, even more preferably at most 30%, even more preferably at most 20%, even more preferably at most 10%, even more preferably at most 5% of the attenuation of the number of viable cells in a control culture of the same algae that has not been peroxidized, within the same period. The method according to any one of Embodiments 1 to 4. E6. When compared with a control culture of the same algae that has not been peroxidized, (i) between 1 and 24 months, preferably between 1 and 20 months, more preferably between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 2 and 12 months, even more preferably between 2 and 10 months, even more preferably between 3 and 9 months, and most preferably between 4 and 8 months; or (ii) between 1 and 30 days, preferably between 1 and 25 days, more preferably between 1 and 15 days, even more preferably between 1 and 10 days, and most preferably between 4 and 6 days; or (iii) for at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, and most preferably at least 8 months A method according to any one of Embodiments 1 to 5, wherein within the period, the decay of viable cell count is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, even more preferably at most 2%, even more preferably at most 1%, and most preferably there is no decay of viable cell count. When compared with a control culture of the same algae that has not been peroxidized, (i) between 1 and 30 days, preferably between 5 and 25 days, more preferably between 10 and 25 days, even more preferably between 15 and 25 days, most preferably between 18 and 22 days, or (ii) between 1 and 24 months, preferably between 1 and 20 months, more preferably between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 3 and 12 months, even more preferably between 3 and 10 months, even more preferably between 3 and 8 months, even more preferably between 3 and 6 months, and most preferably between 3 and 4 months A method according to any one of Embodiments 1 to 6, wherein within the period, the viable cell count increases by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, even more preferably at least 30%, and most preferably at least 40%. The viable algae can be stored for a period of at least 1 month, preferably at least 2 months, more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, even more preferably at least 8 months, and most preferably 18 months at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, compared to the number of viable cells at the time of algae collection, and / or the viable algae can be stored at room temperature, preferably at a temperature of 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C, for at least 1 week, preferably at least 2 weeks, more preferably at least 3 weeks, even more preferably at least 4 weeks, more preferably at least 5 weeks, even more preferably at least 6 weeks, even more preferably at least 7 weeks, most preferably at least 8 weeks, especially at least 2 months, without significant decay in the number of viable cells, according to the method of any one of Embodiments 1 to 7. E9. The method of any one of Embodiments 1 to 8, wherein oxygen is added only once. E10. The method of any one of Embodiments 1 to 9, including steps of adding oxygen to a liquid to prepare a peroxygenated liquid and / or adding the peroxygenated liquid multiple times. E11. The method of any one of Embodiments 1 to 8, wherein oxygen is continuously added to the liquid. E12. The method of any one of Embodiments 1 to 11, which is carried out in a photobioreactor. E13. The method of any one of Embodiments 1 to 12, wherein exposing the algae in the liquid to light is carried out for at least 1 hour to 24 hours at intervals of 1 hour to 12 hours, preferably for at least 16 hours at intervals of 8 hours. E14. The method of any one of Embodiments 1 to 13, wherein exposing the algae in the liquid to light is carried out at a wavelength of 200 nm to 800 nm, preferably 250 nm to 650 nm, more preferably 300 to 550 nm, even more preferably 400 to 500 nm, and most preferably 440 nm. Exposing the algae in the liquid to light is carried out at a power of 1 to 20 W, preferably 5 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, and most preferably 13 W, according to any one of the methods of Embodiments 1 to 14. Exposing the algae in the liquid to light is carried out at a light intensity between 1,000 and 20,000 lux, preferably between 5,000 and 15,000 lux, more preferably between 8,000 and 12,000 lux, according to any one of the methods of Embodiments 1 to 15. E17. (i) Monitoring the number of viable cells; and / or (ii) Collecting the algae; and / or (iii) Concentrating the collected algae; and / or (iv) Storing the algae which is further included in any one of the methods of Embodiments 1 to 16. E18. The viable algae are: (i) at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C; and / or (ii) at a temperature of 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C; and / or (iii) in the dark stored, according to any one of the methods of Embodiments 1 to 17. E19. Oxygen is added to the liquid by supplying oxygen nanobubbles to the liquid, according to any one of the methods of Embodiments 1 to 18. The liquid has an oxygen level of at least 20 ppm, preferably at least 25 ppm, more preferably at least 30 ppm, even more preferably at least 35 ppm, even more preferably at least 40 ppm, even more preferably at least 45 ppm, and most preferably at least 50 ppm, according to any one of the methods of Embodiments 1 to 19. E21. The liquid is in accordance with any one of the methods of Embodiments 1 to 20, having an oxygen saturation of at least 100%, preferably at least 200%, more preferably at least 300%, even more preferably at least 400%, and most preferably at least 500%. E22. The peroxidized liquid for cultivating algae is in accordance with any one of the methods of Embodiments 2 to 21, having an oxygen level of at least 50 ppm and / or an oxygen saturation of at least 500%. E23. The liquid is an aqueous solution, preferably selected from water, pure water, seawater, sterilized water, culture medium, and / or buffer, in accordance with any one of the methods of Embodiments 1 to 22. E24. The liquid is a culture medium. Preferably, the culture medium is: a) water, preferably sterilized water; b) nitrate, preferably its alkali metal salt, more preferably sodium nitrate; c) dihydrogen phosphate, preferably its alkali metal salt, more preferably sodium dihydrogen phosphate; d) silicate, preferably its alkali metal salt, more preferably sodium silicate; e) one or more trace metals, preferably their inorganic salts, more preferably trace metals selected from cobalt, copper, iron, manganese, molybdenum, and / or zinc; and / or f) preferably vitamin B 12 , one or more vitamins selected from biotin and / or thiamine and is included in accordance with any one of the methods of Embodiments 1 to 17. E25. The method of Embodiments 1 to 24, wherein no externally added carbohydrate is present in the liquid. E26. The method of any one of Embodiments 1 to 25, wherein no additional carbohydrate for inducing heterotrophic metabolism in the algae is added to the liquid. E27. The method according to any one of Embodiments 1 to 26, which is operated in batch mode, fed-batch mode, semi-continuous mode, or continuous mode. E28. Algae are capable of mixotrophic metabolism. Preferably, algae are capable of both autotrophic and heterotrophic metabolism. More preferably, in the case of growing under light exposure, algae carry out autotrophic metabolism, and are capable of heterotrophic metabolism in the absence of light, according to any one of Methods 1 to 27. E29. Algae are obligate mixotrophs, obligate autotrophs and facultative heterotrophs, facultative autotrophs and obligate heterotrophs, and / or facultative mixotrophs, according to any one of Methods 1 to 28. E30. Algae are selected from the group of unicellular algae, preferably green algae. More preferably, algae belong to the genus Chlorella. Even more preferably, algae are Chlorella vulgaris, according to any one of Methods 1 to 29. E31. A composition comprising viable algae and a liquid, which can be obtained by a method according to any one of Embodiments 1 to 30. E32. A composition comprising viable algae and a liquid, wherein the liquid is peroxidized. E33. The liquid of the composition according to Embodiment 31 or 32 contains oxygen nanobubbles, preferably the liquid is saturated with oxygen nanobubbles. E34. The liquid of the composition according to any one of Embodiments 31 to 33 has an oxygen level of at least 20 ppm, preferably at least 25 ppm, more preferably at least 30 ppm, even more preferably at least 35 ppm, even more preferably at least 40 ppm, even more preferably at least 45 ppm, and most preferably at least 50 ppm. E35. The liquid of the composition according to any one of Embodiments 31 to 34 has an oxygen saturation of at least 200%, preferably at least 300%, more preferably at least 400%, and most preferably at least 500%. E36. The liquid is selected from aqueous solutions, preferably selected from water, pure water, seawater, sterilized water, culture medium, and / or buffer, of the composition according to any one of Embodiments 31 to 35. E37. The liquid is: (a) water, preferably sterilized water; (b) Nitrate, preferably its alkali metal salt, more preferably sodium nitrate; (c) Dihydrogen phosphate, preferably its alkali metal salt, more preferably sodium dihydrogen phosphate; (d) Silicate, preferably its alkali metal salt, more preferably sodium silicate; (e) One or more trace metals, preferably their inorganic salts, more preferably trace metals selected from cobalt, copper, iron, manganese, molybdenum, and / or zinc; and / or (f) Preferably vitamin B 12 , one or more vitamins selected from biotin and / or thiamine A composition according to any one of embodiments 31 to 36, which is a culture medium containing E38. A composition according to embodiments 31 to 37, wherein the carbohydrate added externally is not present in the liquid. E39. A composition according to any one of embodiments 31 to 38, wherein no additional carbohydrate is added to the liquid. E40. The algae are capable of mixotrophic metabolism. Preferably, the algae are capable of both autotrophic and heterotrophic metabolism. More preferably, when the algae grow under light, they undergo autotrophic metabolism, and when light is absent, they are capable of heterotrophic metabolism. A composition according to any one of embodiments 31 to 3. E41. The algae are obligate mixotrophs, obligate autotrophs and facultative heterotrophs, facultative autotrophs and obligate heterotrophs, and / or facultative mixotrophs. A composition according to any one of embodiments 31 to 40. E42. The algae are unicellular algae, preferably selected from the group of green algae. More preferably, the algae are of the genus Chlorella. Even more preferably, the algae are Chlorella vulgaris. A composition according to any one of embodiments 31 to 41. The number of viable cells in the liquid is 1 million to 20 million cells / mL, preferably 2 million to 18 million cells / mL, more preferably 5 million to 15 million cells / mL, even more preferably 7 million to 14 million cells / mL, even more preferably 10 million to 13 million cells / mL, and most preferably 11 million to 13 million cells / mL, for any one composition of Embodiments 31 to 42. E44. The attenuation of the number of viable cells is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, even more preferably at most 50%, even more preferably at most 40%, even more preferably at most 30%, even more preferably at most 20%, even more preferably at most 10%, even more preferably at most 5%, when compared with the attenuation of the number of viable cells in a control culture of the same algae that has not been peroxidized, for any one composition of Embodiments 31 to 43. E45. When compared with a control culture of the same algae that has not been peroxidized (i) between 1 and 24 months, preferably between 1 and 20 months, more preferably between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 2 and 12 months, even more preferably between 2 and 10 months, even more preferably between 3 and 6 months, and most preferably between 3 and 5 months; or (ii) between 1 and 30 days, preferably between 1 and 25 days, more preferably between 1 and 15 days, even more preferably between 1 and 10 days, and most preferably between 4 and 6 days; or (iii) at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, and most preferably at least 8 months During the period, the attenuation of the viable cell count is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, even more preferably at most 2%, even more preferably at most 1%, and most preferably there is no attenuation of the viable cell count, for any one of the compositions of Embodiments 31 to 44. E46. When compared to a control culture of the same non-peroxygenated algae, (i) between 1 and 30 days, preferably between 5 and 25 days, more preferably between 10 and 25 days, even more preferably between 15 and 25 days, most preferably between 18 and 22 days, or (ii) between 1 and 24 months, preferably between 1 and 20 months, more preferably between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 3 and 12 months, even more preferably between 3 and 10 months, even more preferably between 3 and 8 months, even more preferably between 3 and 6 months, most preferably between 3 and 4 months During the period, the viable cell count increases by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, even more preferably at least 30%, and most preferably at least 40%, for any one of the compositions of Embodiments 31 to 44. E47. Use of any one of the compositions of Embodiments 31 to 46 for improving plant growth. E48. The plant is selected from fruits, vegetables, and / or crops, and preferably the crop is an agricultural crop grown for food or fiber, for the use of Embodiment 47. E49. The fruit is selected from fruit trees, berry thickets, and / or pineapples, for the use of Embodiment 48. E50. The vegetable is a horticultural vegetable, preferably selected from tomatoes, potatoes, cucumbers, peppers, carrots, pumpkins and / or squashes, for the use of Embodiment 48. E51. The crop is selected from leafy vegetables, sugar beets, corns, beans, hay, peanuts, cottons, flax, and / or tobaccos, for the use of Embodiment 48. A method for maintaining or improving soil fertility and / or improving plant growth, comprising the step of applying a composition according to any one of Embodiments 31 to 46 to the soil and / or plants. E53. The method according to Embodiment 52, wherein the soil is agricultural land, pasture land, a sports field, a golf course, and / or urban green space. E54. The method according to Embodiment 52 or 53, comprising the step of diluting a composition according to any one of Embodiments 25 to 37 before applying the composition to the soil and / or plants. E55. The composition is applied in an amount of 10 to 100,000 cells / square foot, preferably 100 to 90,000 cells / square foot, preferably 1,000 to 80,000 cells / square foot, more preferably 10,000 to 70,000 cells / square foot, even more preferably 20,000 to 60,000 cells / square foot, even more preferably 30,000 to 55,000 cells / square foot, even more preferably 35,000 to 55,000 cells / square foot, even more preferably 40,000 to 55,000 cells / square foot, even more preferably 45,000 to 55,000 cells / square foot, and most preferably 50,000 cells / square foot, according to any one of Embodiments 52 to 54. E56. A photobioreactor comprising means for supplying oxygen in the form of nanobubbles. E57. The photobioreactor according to Embodiment 56, wherein the means for supplying oxygen in the form of nanobubbles comprises means for supplying oxygen, preferably an oxygen concentrator, and means for generating nanobubbles, preferably a nanobubble generator. E58. The photobioreactor according to Embodiment 57, wherein the means for supplying oxygen is operably connected to the means for generating nanobubbles. E59. a) One or more reaction vessels, preferably made of glass fiber; b) One or more reservoirs; c) One or more light sources, preferably an LED light source, more preferably a tubular LED growth light, and / or an LED bulb; d) One or more means for supplying dissolved gas to the photobioreactor; e) Oxygen concentrator; f) Piping system; and / or g) One or more valves The photobioreactor of any one of Embodiments 56 to 58, comprising one or more of the above. E60. A reaction vessel is provided, preferably the reaction vessel is as follows: a) Being liquid-impermeable; b) Being cylindrical in shape; c) Having a fixed side wall and bottom; d) Having a removable lid; and / or e) Preferably being made of a translucent material containing glass fibers The container characterized by one or more of the above, which is the photobioreactor of any one of Embodiments 56 to 59. E61. A light source, preferably a tubular LED growth light and / or an LED bulb, more preferably the light source is as follows: a) A wavelength of 200 nm to 800 nm, preferably 250 nm to 650 nm, more preferably 300 nm to 550 nm, even more preferably 400 nm to 500 nm, most preferably 440 nm; and / or b) An illuminance between 1,000 and 20,000 lux, preferably between 5,000 and 15,000 lux, more preferably between 8,000 and 12,000 lux; and / or c) A power of 1 to 20 W, preferably 5 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, most preferably 13 W; and / or d) A light source arranged vertically and equidistantly around one or more reaction vessels, preferably a light source arranged 0.5 cm to 50 cm, preferably 1 cm to 10 cm, more preferably 2 cm to 8 cm, even more preferably 5 cm to 6 cm, most preferably 5 cm away from one or more reaction vessels; and / or e) A light source comprising a timer operable to switch the light source on and off, preferably the timer is set to cycle the light source on for at least 1 to 24 hours and off for at least 1 to 12 hours, preferably 16 hours on and 8 hours off One or more of characterize any one of embodiments 56 - 60 of a photobioreactor E62. One or more means for supplying dissolved gas to one or more reaction vessels, which are not means for supplying oxygen in the form of nanobubbles, preferably the one or more means for supplying dissolved gas to one or more reaction vessels comprise a pump operable to push dissolved gas into one or more reaction vessels, a piping system through which the dissolved gas passes, and a check valve, more preferably the one or more means for supplying dissolved gas to one or more reaction vessels is a water tank stone bubbler, of any one of embodiments 56 - 61 of a photobioreactor E63. Comprising a piping system, preferably the piping system is made of a polymer, preferably polyethylene and / or stainless steel, preferably the piping system is made of a combination of polyethylene and stainless steel, of any one of embodiments 56 - 62 of a photobioreactor E64. Comprising a piping system, preferably the piping system is adapted to provide fluid communication between components of the photobioreactor, of any one of embodiments 56 - 63 of a photobioreactor E65. A system comprising one or more photobioreactors according to any one of embodiments 56 - 64 E66. A system according to embodiment 65, comprising at least two photobioreactors, preferably the photobioreactors are in fluid communication E67. A system according to embodiment 66, wherein the photobioreactor is arranged within a photobioreactor array E68. A system according to embodiment 66 or 67, wherein the photobioreactors are connected in parallel Embodiment 65-68, a system comprising a valve, the valve being operable to separate the at least one photobioreactor from other photobioreactors by opening and closing the valve. Embodiment 65-69, a system wherein one or more photobioreactors are in fluid communication with a nanobubble generator. Embodiment 65-70, a system comprising a reservoir in fluid communication with one or more photobioreactors. Embodiment 65-71, a system comprising a discharge line operably connected to one or more photobioreactors for removing liquid from the one or more photobioreactors, preferably the discharge line is made of polyethylene. These and other features, as well as their advantages, will become apparent to those skilled in the art of growing microalgae using photobioreactors by carefully reading the detailed description of the invention and the exemplary embodiments accompanied by the drawings.

[0019] Mode for Carrying Out the Invention According to a first aspect, the present invention is a method for producing an algal composition containing viable algae and a liquid, comprising: - adding oxygen to the liquid to prepare a peroxidized liquid; - culturing the algae in the peroxidized liquid, at least temporarily under light exposure, to allow growth of the algae and a method related thereto. The term "living" algae refers to algae that are alive. Culturing the algae in a peroxidized liquid under light exposure, at least temporarily, to enable growth of the algae refers to exposing the algae to light for a certain period of time, for example, by irradiating a reaction vessel in a photobioreactor. However, the exposure to light need not be continuous, and there may be a temporary stage without exposure to light. When the algae grow under light exposure, they undergo autotrophic metabolism and are capable of heterotrophic metabolism in the absence of light. In particular, since the algae typically grow more efficiently under a diurnal exposure of 16 hours with light and 8 hours without light, the exposure to light need not be continuous. The dark period is when heterotrophic metabolism occurs. The growth of the algae refers to cell growth and cell division of the algal cells that result in an increase in the number of cells, but may also include a steady state of dividing cells and / or living cells, as well as dead cells. The algae can be cultured using typical conditions known to those skilled in the art for culturing algae, such as those described in Chapter 2.3, "Algal production" of the Manual on the Production and Use of Live Food for Aquaculture, Food and Agricultural Organization of the United Nations Fisheries Technical Paper 361, Rome 1996, ISBN 92-5-103934-8.

[0020] According to a particular embodiment, the method of the first aspect - a step of preparing a liquid; - a step of adding oxygen to the liquid to prepare a peroxidized liquid; - a step of inoculating the peroxidized liquid with the algae; - a step of culturing the algae in the peroxidized liquid under light exposure, at least temporarily, to enable growth of the algae comprises. In this way, a fresh algal culture can be provided.

[0021] According to another particular embodiment, the method of the first aspect - a step of preparing a liquid containing the algae; - adding oxygen to a liquid to prepare a peroxidized liquid and / or adding a peroxidized liquid; - culturing algae in the peroxidized liquid, at least temporarily under light exposure, to allow growth of the algae and comprising.

[0022] In certain embodiments of the method of the first aspect, one or more of the following apply: a) The liquid has a viable cell count of from 1 million to 20 million cells / mL, preferably from 2 million to 18 million cells / mL, more preferably from 5 million to 15 million cells / mL, even more preferably from 7 million to 15 million cells / mL, even more preferably from 10 million to 15 million cells / mL, most preferably from 12 million to 14 million cells / mL; b) The decay of the viable cell count is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, even more preferably at most 50%, even more preferably at most 40%, even more preferably at most 30%, even more preferably at most 20%, even more preferably at most 10%, even more preferably at most 5% of the decay of the viable cell count of a control culture of the same algae that has not been peroxidized during the same period; c) When compared to a control culture of the same algae that has not been peroxidized, for between 1 and 24 months, preferably between 1 and 20 months, more preferably still between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 2 and 12 months, even more preferably between 2 and 10 months, even more preferably between 3 and 6 months, most preferably between 3 and 5 months, or for between 1 and 30 days, preferably between 1 and 25 days, more preferably between 1 and 15 days, even more preferably between 1 and 10 days, most preferably between 4 and 6 days, or For at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, and most preferably at least 8 months within which period, the decay of viable cell count is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, even more preferably at most 2%, even more preferably at most 1%, and most preferably essentially none; and / or when compared to a control culture of the same non-peroxidized algae the viable cell count is, between 1 and 30 days, preferably between 5 and 25 days, more preferably between 10 and 25 days, even more preferably between 15 and 25 days, and most preferably between 18 and 22 days, or between 1 and 24 months, preferably between 1 and 20 months, more preferably between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 3 and 12 months, even more preferably between 3 and 10 months, even more preferably between 3 and 8 months, even more preferably between 3 and 6 months, and most preferably between 3 and 4 months in which period, it increases by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, even more preferably at least 30%, and most preferably at least 40%. In the context of the present invention comparing the number of viable cells between a peroxidized liquid containing viable algae and a non-peroxidized liquid containing viable algae, culture conditions such as culture temperature, salt concentration, pH, culture components, light-dark cycle, etc., although not limited thereto, are intended to be the same. In a preferred embodiment, the liquid has a viable cell count of 12 million to 14 million cells / mL, particularly at the time of algae collection. In the same or even more preferred embodiments, within the same period, preferably within 5 days after collection, there is at most a 5% decay in the viable cell count of a control culture of the same non-peroxidized algae. In the same or even more preferred embodiments, within a period of 4 to 6 days, there is at most a 4% decay in the viable cell count. In the same or further preferred embodiments, there is essentially no decay in the viable cell count within at least 2 months, most preferably within at least 8 months. Preferably, at room temperature, preferably at a temperature of 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C, there is essentially no decay in the viable cell count within at least 2 months. Even more preferably, especially when refrigerated at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, there is essentially no decay in the viable cell count within at least 8 months. Typically, at the time of collection, a slight decrease in the cell count is observed due to shock from a sudden change in the environment, but heterotrophic algae can quickly initiate recovery and even grow. For example, the viable cell count increases by at least 5% within a period of 18 to 22 days, particularly within 19 days or less. In another example, the viable cell count increases by at least 40% within a period of 3 to 4 months, particularly within 119 days or less. One skilled in the art knows methods for determining the number of viable cells, for example, using a hemocytometer or a plate count method (e.g., in accordance with ASTM D5465) or an automated equivalent thereof, such as the Electrical Sensing Zone Method of Enumerating and Sizing Single Cell Suspensions in accordance with ASTM F2149-16. Electrical Sensing Zone instrumentation is generally referred to as a Coulter counter. Other methods known to those skilled in the art include dye exclusion assays, colorimetric assays, fluorescence assays, luminometry assays, and flow cytometry assays.

[0023] In certain embodiments of the method according to the first aspect, oxygen is added only once initially. In an alternative form, the method includes one or more additional steps of adding oxygen to a liquid to prepare a peroxidized liquid and / or adding the peroxidized liquid. In a further alternative form, oxygen is continuously added to the liquid.

[0024] In certain embodiments of the method according to the first aspect, the method is carried out in a photobioreactor.

[0025] In certain embodiments of the method according to the first aspect, exposing the algae in the liquid to light is: a) for at least 1 hour to 24 hours with an exposure interval of 1 hour to 12 hours, preferably for at least 16 hours with an exposure interval of 8 hours; b) at a wavelength of 200 nm to 800 nm, preferably 250 nm to 650 nm, more preferably 300 to 550 nm, even more preferably 400 to 500 nm, and most preferably 440 nm; c) at a power of 1 to 20 W, preferably 5 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, and most preferably 13 W, and / or d) at a light intensity of 1,000 to 20,000 lux, preferably 5,000 to 15,000 lux, and more preferably 8,000 to 12,000 lux is carried out. In this way, the algae are efficiently irradiated with light to support autotrophic metabolism using photosynthesis. Also, because the algae are mixotrophic, they have / require a circadian photoperiod, especially to switch from the autotrophic mode to the heterotrophic mode. Therefore, the exposure protocol according to the present invention facilitates the switching between the autotrophic mode and the heterotrophic mode.

[0026] In a particular embodiment of the method according to the first aspect, the method further: a) monitoring the viable cell count; and / or b) collecting the algae; and / or c) concentrating the collected algae; and / or d) storing the algae is included.

[0027] In a particular embodiment of the method according to any one of the first to fifth aspects, the method is operated in a batch mode, a fed-batch mode, a semi-continuous mode, or a continuous mode. This enables flexible handling of the grown algae, scale-up, and easy adaptation to different reactor types. The batch operation mode allows the collection of the entire algae concentrate from the photobioreactor. However, the inventors have found that it is more efficient to collect partially, i.e., operate in a semi-continuous mode, collect from 40% to a maximum of 70% of the tank, and refill the tank by adding a peroxidized medium. Then, the algae that were not collected continue to grow and are thus re-exposed to the peroxidized liquid (especially the culture medium and / or buffer). The cells grow fast enough to allow collection at 4- to 5-day intervals. The partial collection process is efficient because the washing between partial collections does not require a longer operation time. After 4 to 5 months, the rate of increase in the cell count from the tank decreases; at that point, the "tired" tank is taken offline, drained, washed, and restarted.

[0028] According to a second aspect, the present invention relates to a composition comprising viable algae and a liquid, which can be obtained by a method according to any one of the first to fifth aspects.

[0029] According to a third aspect, the present invention relates to a composition comprising viable algae and a liquid, the liquid being peroxidized.

[0030] In certain embodiments, in any of the methods according to the first aspect, or in any of the compositions according to the second and third aspects, the viable algae are: a) at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C; and / or b) at a temperature of 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C; and / or c) in the dark and can be stored. The viable algae can be stored at a temperature of 0°C to 10°C and / or in the dark. The expression "storage in the dark" refers to maintaining the viable algae in the dark, i.e., in the absence of light and / or under absolute or substantially complete exclusion of light. However, since algae can grow over a wide range of temperatures, refrigeration is not essential. However, refrigeration advantageously prevents the growth of bacterial and protozoal contaminants. On the other hand, the algae can be stored at room temperature for as long as two months, which advantageously simplifies storage conditions and transportation. For longer-term storage, i.e., storage for a period longer than two months from collection, refrigeration is recommended, i.e., it is recommended to maintain the algae at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, most preferably 6°C.

[0031] In certain embodiments of any of the methods according to the first aspect, or any of the compositions according to the second and third aspects, the viable algae can be stored at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, for at least 1 month, preferably at least 2 months, more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, even more preferably at least 8 months, and most preferably for up to 18 months, without significantly decreasing the viable cell count as compared to the viable cell count at the time of algae collection. In other or the same certain embodiments, in any of the methods according to the first aspect, or any of the compositions according to the second and third aspects, the viable algae can be stored at room temperature, preferably 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C, for at least 1 week, preferably at least 2 weeks, more preferably at least 3 weeks, even more preferably at least 4 weeks, more preferably at least 5 weeks, even more preferably at least 6 weeks, even more preferably at least 7 weeks, most preferably at least 8 weeks, particularly at least 2 months, without significantly decreasing the viable cell count as compared to the viable cell count at the time of algae collection. Thus, the present invention enables easy and simple filling, storage, transportation, and use of live algae, facilitating large-scale use as a biostimulant.

[0032] In certain embodiments of any of the methods according to the first aspect, or any of the compositions according to the second and third aspects, the composition comprising viable algae and a liquid, or the liquid comprising viable algae, is each characterized by the viable cell count, a) the viable cell count is 1 million to 20 million cells / mL, preferably 2 million to 18 million cells / mL, more preferably 5 million to 15 million cells / mL, even more preferably 7 million to 14 million cells / mL, even more preferably 10 million to 13 million cells / mL, and most preferably 11 million to 13 million cells / mL; b) the decay of the viable cell count is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, even more preferably at most 50%, even more preferably at most 40%, even more preferably at most 30%, even more preferably at most 20%, even more preferably at most 10%, even more preferably at most 5% of the decay of the viable cell count of the same non-peroxygenated algal control culture; c) when compared to the same non-peroxygenated algal control culture, during 1 to 24 months, preferably during 1 to 20 months, more preferably during 1 to 18 months, even more preferably during 2 to 15 months, even more preferably during 2 to 12 months, even more preferably during 2 to 10 months, even more preferably during 3 to 9 months, most preferably during 4 to 8 months, or during 1 to 30 days, preferably during 1 to 25 days, more preferably during 1 to 15 days, even more preferably during 1 to 10 days, most preferably during 4 to 6 days or for at least 1 month, preferably for at least 2 months, more preferably for at least 3 months, even more preferably for at least 4 months, even more preferably for at least 5 months, even more preferably for at least 6 months, even more preferably for at least 7 months, most preferably for at least 8 months within this period, the decay of the viable cell count is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, even more preferably at most 2%, even more preferably at most 1%, and most preferably not; and / or d) the viable cell count, when compared to the same non-peroxygenated algal control culture, during 1 to 30 days, preferably during 5 to 25 days, more preferably during 10 to 25 days, even more preferably during 15 to 25 days, most preferably during 18 to 22 days, or Between 1 and 24 months, preferably between 1 and 20 months, more preferably between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 3 and 12 months, even more preferably between 3 and 10 months, even more preferably between 3 and 8 months, most preferably between 3 and 6 months, even more preferably between 3 and 6 months, most preferably between 3 and 4 months Within this period, it increases by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, even more preferably at least 30%, most preferably at least 40%. In the context of the present invention comparing the number of viable cells between a peroxidized liquid containing viable algae and a non-peroxidized liquid containing viable algae, culture conditions such as culture temperature, salt concentration, pH, culture components, light-dark cycle, etc., although not limited thereto, are intended to be the same. In a preferred embodiment, the liquid has a viable cell count of 12 million to 14 million cells / mL, particularly at the time of algae collection. In the same or a further preferred embodiment, within the same period, particularly within 5 days after collection, there is at most a 5% decay in the viable cell count of a control culture of the same non-peroxidized algae. In the same or a further preferred embodiment, within a period of 4 to 6 days, there is at most a 4% decay in the viable cell count. In the same or a further preferred embodiment, within a period of at least 2 months, most preferably at least 8 months, there is essentially no decay in the viable cell count. Preferably, at room temperature, preferably at a temperature of 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C, within a period of at least 2 months, there is essentially no decay in the viable cell count. More preferably, when refrigerated particularly at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, within a period of at least 8 months, there is essentially no decay in the viable cell count. Typically, at the time of collection, a slight decrease in the cell count is observed due to shock caused by a sudden change in the environment, but heterotrophic algae can quickly initiate recovery and even grow. For example, the viable cell count increases by at least 5% within a period of 18 to 22 days, particularly within 19 days or the like. In another example, the viable cell count increases by at least 40% within a period of 3 to 4 months, particularly within 119 days or the like. One skilled in the art knows methods for determining the number of viable cells, for example, using a hemocytometer or plate count method (e.g., in accordance with ASTM D5465) or an automated equivalent thereof, such as the Electrical Sensing Zone Method of Enumerating and Sizing Single Cell Suspensions in accordance with ASTM F2149-16. Electrical Sensing Zone instrumentation is commonly referred to as a Coulter counter. Other methods known to those skilled in the art include dye exclusion assays, colorimetric assays, fluorescence assays, luminometry assays, and flow cytometry assays. Also, cell viability is verified by assays such as microscopy: viable algal cells have a distinctive appearance with green protoplasts, while non-viable cells appear like cell fragments.

[0033] In certain embodiments, in any of the methods according to the first aspect, or in any of the compositions according to the second and third aspects, the liquid is peroxidized. The term "peroxidized" means being oxygenated more than normal. Molecular oxygen is poorly water-soluble. In an aqueous solution, oxygen is (H2O) 20It exists within the class rating. The solubility of oxygen in water depends on temperature and pressure. At 25 °C and 101.3 kPa, fresh water contains approximately 6.04 mL / L of oxygen. Mineral salts in water reduce the solubility of oxygen. For example, at 25 °C and 101.3 kPa, seawater contains approximately 4.95 mL / L of molecular oxygen. In the context of the present invention, a hyperoxygenated liquid preferably refers to a liquid having a particularly high dissolved oxygen content in the maximum amount possible chemically and physically. In other words, the hyperoxygenated liquid is supersaturated with dissolved oxygen. It is believed that those skilled in the art understand that the hyperoxygenated state of water changes depending on ambient conditions. In the context of the present invention, unless otherwise specified, the hyperoxygenated state refers to standard conditions, i.e., a temperature of 20 °C and a pressure of 1013 hPa. Without wishing to be bound by theory, the inventors believe that hyperoxygenation induces heterotrophic metabolism in mixotrophic algae. Advantageously, hyperoxygenation enables the induction of heterotrophic metabolism in algae even without any externally added carbohydrates. It is preferred that no externally added carbohydrates are present in the liquid. Preferably, the amount of carbohydrates in the liquid is less than 10% by weight, preferably less than 8% by weight, more preferably less than 6% by weight, even more preferably less than 4% by weight, even more preferably less than 2% by weight, and most preferably less than 1% by weight, based on the total weight of the total culture medium. For example, when hyperoxygenation is used to induce heterotrophic metabolism in algae, no additional carbohydrates are required to induce heterotrophic metabolism in the algae. The liquid may remain in a hyperoxygenated state continuously, or the oxygen content of the liquid may decay over time. For example, freshly hyperoxygenated liquid in the context of the present invention has an oxygen content of at least 50 ppm. After 48 hours in a photobioreactor, the liquid has an oxygen content of at least 30 ppm, and at the time of collection on about the fourth day, the liquid has an oxygen content of about 20 ppm.

[0034] In certain embodiments, for any of the methods according to the first aspect or any of the compositions according to the second and third aspects, to add oxygen to a liquid, oxygen nanobubbles are supplied to the liquid such that oxygen can be present in the liquid as oxygen nanobubbles. The term "nanobubble" refers to bubbles with diameters in the nanometer range, for example, in the range of 1 to 1,000 nm. Nanobubbles are typically characterized by a size distribution. Nanobubbles can be characterized by high-speed camera image analysis, electrical signals (Coulter counter), resonance mass, particle trajectories (Nanoparticle Tracking Analysis (NTA)), laser diffraction, and dynamic light scattering (DLS), known to those skilled in the art, particularly DLS according to ASTM E3247-20. The dimensions of bubbles within the nanometer range achieve unique physical, chemical, and biological properties that minimize off-gas generation and / or aggregation in solution, such as a high total bubble surface area and a strong negative surface charge. The oxygen nanobubbles of the present invention can persist in solution for up to two weeks under standard conditions. Thus, the algal concentrate collected after 4 to 5 days of growth still has a high oxygen content. Nanobubbles facilitate an increase in the concentration of dissolved gas in a liquid. Nanobubbles can be formed using any gas and injected into any liquid. In the context of the present invention, nanobubbles are preferably formed from oxygen injected into an aqueous liquid, such as sterile water, culture medium, or buffer. The use of oxygen nanobubbles can achieve high levels of dissolved oxygen (DO) that cannot be achieved by other conventional aeration methods that do not utilize oxygen nanobubbles. Thus, oxygen nanobubbles enable more efficient use of oxygen. Nanobubbles can be generated using a nanobubble generator.

[0035] In certain embodiments, for any of the methods according to the first aspect or for any of the compositions according to the second and third aspects, the liquid has an oxygen level of at least 20 ppm, preferably at least 25 ppm, more preferably at least 30 ppm, even more preferably at least 35 ppm, even more preferably at least 40 ppm, even more preferably at least 45 ppm, and most preferably at least 50 ppm, and / or the liquid has an oxygen saturation of at least 200%, preferably at least 300%, more preferably at least 400%, and most preferably at least 500%. In a preferred embodiment of the method according to the first aspect of the present invention, in the step of inoculating the hyperoxygenated liquid with algae, the liquid is hyperoxygenated, has an oxygen level of at least 50 ppm, and / or has an oxygen saturation of at least 500%, and preferably the liquid is hyperoxygenated using oxygen nanobubbles. The oxygen level or oxygen saturation of the liquid can be measured using any standard analytical method known to those skilled in the art, such as titration techniques, such as electrochemical analysis using a Clark electrode, or photochemical analysis. It is contemplated that a corresponding sensor for determining the oxygen level in the liquid can be used. Without wishing to be bound by theory, the inventors believe that hyperoxygenation or an oxygen level of at least 50 ppm, for example, and / or an oxygen saturation of, for example, about 500% in the liquid induces heterotrophic metabolism in algae. In this way, the induction of heterotrophic metabolism in algae can be advantageously achieved even in the absence of externally added carbohydrates in the liquid, preferably the culture medium and / or buffer. For example, when hyperoxygenation is used to induce heterotrophic metabolism in algae, there is no need to add an additional carbohydrate source to the liquid.

[0036] In certain embodiments, for any of the methods according to the first aspect or for any of the compositions according to the second and third aspects, the liquid is preferably an aqueous solution selected from water, fresh water, seawater, sterilized water, culture medium, and / or buffer. In this way, an optimal supply for the growth and / or maintenance of viability of the algae is provided.

[0037] In certain embodiments, for any of the methods according to the first aspect, or for any of the compositions according to the second and third aspects, the liquid is a culture medium, and preferably the culture medium is: a) water, preferably sterilized water; b) nitrates, preferably alkali metal salts thereof, more preferably sodium nitrate; c) dihydrogen phosphate, preferably alkali metal salts thereof, more preferably sodium dihydrogen phosphate; d) silicates, preferably alkali metal salts thereof, more preferably sodium silicate; e) one or more trace metals, preferably inorganic salts thereof, more preferably trace metals selected from cobalt, copper, iron, manganese, molybdenum, and / or zinc; and / or f) preferably vitamin B 12 , one or more vitamins selected from biotin, and / or thiamine is included. In some particular embodiments, no additional or external carbohydrates are added to the liquid for inducing heterotrophic metabolism in algae. As used herein, the term "carbohydrates" includes monosaccharides such as glucose, fructose, and / or galactose, disaccharides such as sucrose, maltose, trehalose, and / or lactose, and oligosaccharides such as raffinose and / or maltodextrin. The expression "no additional or external carbohydrates are added" or "externally added carbohydrates" in the context of the present invention means that no carbohydrates are added to the liquid, which is a liquid that has not yet formed part of it, particularly the culture medium. At the same time, it does not exclude the possibility that carbohydrates such as glucose can be formed during culture by, for example, living or dead autotrophic algae, or released into the liquid. An example of a typical medium that can be used to grow algae according to the present invention is commercially available Guillard / F / 2 medium.

[0038] In certain embodiments, for any of the methods according to the first aspect, or for any of the compositions according to the second and third aspects, the algae are capable of mixotrophic metabolism, preferably the algae are capable of both autotrophic and heterotrophic metabolism. In certain embodiments, the algae exhibit autotrophic metabolism when growing under light exposure and are capable of heterotrophic metabolism in the absence of light. In certain embodiments, the algae are obligate mixotrophs, obligate autotrophs and facultative heterotrophs, facultative autotrophs and obligate heterotrophs, and / or facultative mixotrophs. In this way, the algae can switch from autotrophy to heterotrophy and vice versa. Mixotrophic algae can adapt their metabolism to ambient conditions. In the context of the present invention, for example, the algae can switch from autotrophy to heterotrophy during growth in a photobioreactor or in a culture phase when stored in the dark. Algae particularly useful in the context of the present invention are selected from the group of unicellular algae, preferably green algae, more preferably the algae are of the genus Chlorella, and even more preferably the algae are Chlorella vulgaris.

[0039] In an exemplary embodiment, the method according to the first aspect is: - creating a growth medium by adding oxygen nanobubbles to sterilized water by means of peroxidation or by feeding water through a nanobubble generator to reach an oxygen level of at least 50 ppm with an oxygen saturation of about 500%; - pumping the sterilized peroxidized water through a piping system connected to the nanobubble generator at one end and to a stainless-steel piping system piped to at least one photobioreactor at a second end to fill the photobioreactor with the growth medium; - An inorganic nutrient solution formulated to support the growth of microalgae is added to at least one photobioreactor containing sterilized hydrogen peroxide to create a growth medium, and a substantially homogeneous single culture of mixotrophic microalgae, preferably a species of the genus Chlorella, having a concentration of 6 to 8 million cells / mL is inoculated into the growth medium; - Exposing the inoculated growth medium to light for 16 hours at 8-hour intervals; - Continuously delivering ambient air through an underwater stone bubbler having a filter disposed within the PBR near the base; - Monitoring the growth rate of the microalgae by draining a specific volume of the growth medium from the PBR and counting the cells; - Collecting the microalgae by draining the growth medium containing the microalgae grown to a concentration of 12 million cells / mL into a container, preferably composed of polyethylene, and storing the container at 6°C comprising.

[0040] According to a fourth aspect, the present invention relates to the use of a composition according to the second or third aspect for improving the growth of plants.

[0041] In a preferred embodiment of the use according to the fourth aspect, the composition stimulates the plants, preferably the plants are selected from fruits, vegetables, and / or crops, and preferably the crops are agricultural plants grown for food or fiber. Examples of fruits can include, but are not limited to, fruit trees, berry thickets, and pineapples. Examples of vegetables can include, but are not limited to, garden vegetables, preferably tomatoes, potatoes, cucumbers, peppers, carrots, pumpkins, and / or squashes. Examples of crops can include, but are not limited to, sugar beets, sugar cane, corn, beans, hay, peanuts, tobacco, cotton, flax, and / or hemp.

[0042] According to a fifth aspect, the present invention is a method for maintaining or improving the fertility of soil and / or improving the growth of plants, - applying a composition according to the second or third aspect to soil and / or plants relates to a method comprising: In certain embodiments of the method according to the ninth aspect, the soil can be agricultural land, pasture land, sports fields, golf courses, and / or urban green spaces. Thus, the compositions of the present invention can be applied to a wide range of lands. In certain further embodiments of the method according to the ninth aspect, the method comprises diluting the composition according to any one of claims 25 to 37 before applying it to the soil and / or plants. In particular, the composition is from 10 to 100,000 cells per square foot (108 to 1,080,000 cells / m 2 ), preferably from 100 to 90,000 cells per square foot (1,080 to 970,000 cells / m 2 ), preferably from 1,000 to 80,000 cells per square foot (10,800 to 860,000 cells / m 2 ), more preferably from 10,000 to 70,000 cells per square foot (108,000 to 750,000 cells / m 2 ), even more preferably from 20,000 to 60,000 cells per square foot (215,000 to 645,000 cells / m 2 ), even more preferably from 30,000 to 55,000 cells per square foot (323,000 to 592,000 cells / m 2 ), even more preferably from 35,000 to 55,000 cells per square foot (378,000 to 592,000 cells / m 2 ), even more preferably from 40,000 to 55,000 cells per square foot (431,000 to 592,000 cells / m 2 ), even more preferably from 45,000 to 55,000 cells per square foot (484,000 to 592,000 cells / m 2 ), most preferably 50,000 cells per square foot (538,000 cells / m 2 ) and applied in an amount. Thus, the compositions and methods of the present invention enable efficient maintenance or improvement of soil fertility and / or plant growth.

[0043] According to a sixth aspect, the present invention relates to a photobioreactor comprising means for supplying oxygen in the form of nanobubbles.

[0044] In a particular embodiment of a photobioreactor according to the sixth aspect, the means for supplying oxygen in the form of nanobubbles comprises means for supplying oxygen, preferably an oxygen concentrator, and means for generating nanobubbles, preferably a nanobubble generator. The nanobubble generator enables the efficient and uniform generation of nanobubbles, i.e., nanobubbles with a specific size distribution.

[0045] In a particular embodiment of a photobioreactor according to the sixth aspect, the photobioreactor comprises: a) one or more reaction vessels, preferably made of glass fiber; b) one or more reservoirs; c) one or more light sources, preferably an LED light source, more preferably a tubular LED growth light, and / or an LED bulb; d) one or more means for supplying dissolved gas to the photobioreactor; e) an oxygen concentrator; f) a piping system; and / or g) one or more valves and comprises one or more of them.

[0046] In a particular embodiment, the photobioreactor comprises a reaction vessel, and preferably the reaction vessel is characterized by one or more of the following: a) being liquid-impermeable; b) being cylindrical in shape; c) having fixed side walls and a bottom; d) having a removable lid; and / or e) being preferably made of a translucent material containing glass fiber and is a container characterized by one or more of them. In this way, a reaction vessel suitable for photosynthesis is provided. The reaction vessel of the photobioreactor according to the present invention can have any volume. A preferred volume is up to 750 liters. When the diameter of the tank increases, there is a concern that the algae at the center of the tank will be shaded and the growth will slow down. However, the inventors of the present invention have surprisingly found that the number of algal cells in a larger tank increases as rapidly as that in a 350-liter photobioreactor with a smaller diameter (under the same conditions). One explanation for this is that the algae are constantly mixed with air bubbles. This prevents the algal cells from gathering at the center of the tank and from gathering from the light source. Another explanation is that since the algae are mixotrophic, they continue to grow in the dark. Algae do not simply rely on photosynthesis. The larger the reaction vessel used, the greater the improvement in efficiency and productivity.

[0047] In certain embodiments, the photobioreactor preferably comprises tubular LED growth lights, and more preferably the light source is characterized by one or more of the following: a) a wavelength of 200 nm to 800 nm, preferably 250 nm to 650 nm, more preferably 300 nm to 550 nm, even more preferably 400 nm to 500 nm, and most preferably 440 nm; and / or b) a light intensity between 1,000 and 20,000 lux, preferably between 5,000 and 15,000 lux, more preferably between 8,000 and 12,000 lux; and / or c) a power of 1 to 20 W, preferably 5 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, and most preferably 13 W; and / or d) a light source arranged vertically and equidistantly around one or more reaction vessels, preferably a light source arranged 0.5 cm to 50 cm, preferably 1 cm to 10 cm, more preferably 2 cm to 8 cm, even more preferably 5 cm to 6 cm, and most preferably 5 cm away from one or more reaction vessels; and / or e) A light source equipped with a timer operable to switch the light source on and off, preferably the timer is set to cycle the light source on for at least 1 to 24 hours and off for at least 1 to 12 hours, preferably on for 16 hours and off for 8 hours. In this way, the algae can be efficiently exposed to light during growth in the photobioreactor.

[0048] In certain embodiments, the photobioreactor comprises one or more means for supplying dissolved gas to one or more reaction vessels, which are not means for supplying oxygen in the form of nanobubbles, preferably the one or more means for supplying dissolved gas to one or more reaction vessels comprise a pump operable to push the dissolved gas into one or more reaction vessels, a piping system through which the dissolved gas passes, and a check valve, more preferably the one or more means for supplying dissolved gas to one or more reaction vessels is a water tank stone bubbler. In this way, for example, ambient air or any other gas mixture can be efficiently supplied to the liquid in the photobioreactor, particularly to the reaction vessels of the photobioreactor.

[0049] In a preferred embodiment, the photobioreactor comprises a piping system, preferably the piping system is made of a polymer, preferably polyethylene and / or stainless steel, and preferably the piping system is made of a combination of polyethylene and stainless steel. In this way, liquid can be passed through the photobioreactor. Stainless steel enables safety and a long equipment life because erosion is minimized and it is easy to sterilize the equipment during maintenance. Preferably, the piping system is adapted to effect fluid communication of the components of the photobioreactor, for example, means for supplying oxygen in the form of nanobubbles, preferably a nanobubble generator, one or more reaction vessels, one or more reservoirs, one or more means for supplying dissolved gas to the photobioreactor, an oxygen concentrator, and / or fluid communication of one or more valves. For example, the piping system can effect fluid communication between a reservoir and a reaction vessel and means for supplying oxygen in the form of nanobubbles, the piping system can have one or more valves, and ultimately enable filling the photobioreactor with sterilized hydrogen peroxide solution or draining a growth medium containing algae from the photobioreactor.

[0050] According to a seventh aspect, the present invention relates to a system comprising one or more photobioreactors according to the sixth aspect.

[0051] In certain embodiments, the system comprises at least two photobioreactors, preferably the photobioreactors are in fluid communication and / or the photobioreactors are arranged within a photobioreactor array. Also, the bioreactors are preferably connected in parallel. In this way, a large amount of algae can be cultured in parallel, so that the growth of algae can be easily scaled up.

[0052] In certain embodiments, the system comprises a valve, which is operable to separate the at least one photobioreactor from other photobioreactors by opening and closing the valve. The valve enables individual filling and / or draining of the bioreactors within the system. The valve also enables control of passage into and out of individual photobioreactors, as well as passage between individual photobioreactors.

[0053] In certain embodiments, one or more photobioreactors are in fluid communication with a nanobubble generator. In this way, oxygen nanobubbles can be efficiently supplied to one or more bioreactors. There may be one nanobubble generator per one or more photobioreactors. Alternatively, there may be one nanobubble generator for a plurality of photobioreactors together. In this way, the supply of oxygen nanobubbles is centralized and the system is simplified. This enables cost reduction and easier maintenance of the system.

[0054] In a preferred embodiment, the system comprises a reservoir in fluid communication with one or more photobioreactors. The reservoir can contain a liquid, preferably an aqueous solution selected from water, fresh water, seawater, sterilized water, culture medium, and / or buffer, which is supplied to the one or more photobioreactors. One reservoir can feed individual photobioreactors or all of the photobioreactors of the system. The reservoir may be, for example, a holding tank. Preferably, the reservoir is arranged to enable gravity feed of the photobioreactors.

[0055] In certain embodiments, the system comprises a discharge line operably connected to one or more photobioreactors for removing liquid from the one or more photobioreactors, preferably the discharge line is manufactured from polyethylene. The discharge line facilitates the collection of algae and enables continuous algae production.

[0056] In an exemplary embodiment, in a system according to the seventh aspect, the system comprises at least one reservoir, preferably a holding tank, a photobioreactor, a nanobubble generator, a light source, and one or more means for supplying dissolved gas to one or more reaction vessels, preferably an aquarium stone bubbler. - The holding tank is connected to a first end of a piping system via a valve provided on the tank. The piping system preferably includes stainless steel. The second end of the piping system is connected to the nanobubble generator. Preferably, the piping system is connected to the photobioreactor near the base of the photobioreactor. The piping system is connected at its second end to a discharge line, preferably including a polyethylene tube, via the length of the piping system connected at the second end, to fill the photobioreactor with sterilized hydrogen peroxide solution or to drain a growth medium containing microalgae from the photobioreactor. The piping system has a first valve disposed above the connection to the photobioreactor and a second valve disposed below the connection to the photobioreactor. - The system includes a plurality of photobioreactors disposed within a photobioreactor array. The photobioreactors are connected to the nanobubble generator at the first end, and the piping system connected to the discharge line at the second end is piped in parallel such that it has an additional length of piping system. The additional piping system is connected to each additional photobioreactor near the base of the additional photobioreactor. The additional piping system has a first valve disposed above the connection to the additional photobioreactor and a second valve disposed below the connection to the additional photobioreactor. The valves make it possible to separate the at least one photobioreactor from other photobioreactors by opening and closing the valves.

[0057] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the present invention and vice versa.

[0058] In the claims and / or in this specification, the use of the word "a" or "an" when used in combination with the terms "comprising", "including", "having", "containing", or any variations of these terms may mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or plural".

[0059] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and examples, while indicating specific embodiments of the present invention, are given by way of illustration only. In addition, changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description.

[0060] Examples To illustrate the advantages of the methods and compositions of the present invention, the continuous cell counts observed after refrigerated storage of microalgae cultures grown using a system with oxygen nanobubbles ("treated") and a system without oxygen nanobubbles ("untreated") were compared. Three representative experiments are described in Table 1 below, two of which used paired controls (A and B), and the third did not use an untreated control (C). For the experiments, algae from a single culture of Chlorella vulgaris were used. A single culture of Chlorella vulgaris was purchased from the Phycology Laboratory at the University of Texas. Since then, the algae have been grown under standard conditions for growing algae. As the medium, commercially available Guillard's F / 2 medium was used. [Table 1]

[0061] As can be derived from Table 1, the methods and compositions according to the present invention enable an equal or increased number of cells at the time of collection, a less attenuation of the number of cells 5 days after collection, and even an increase in the number of cells 19 days and 119 days after collection, respectively. This is due to mixotrophic algae that can switch from autotrophic to heterotrophic. Mixotrophy is not induced in untreated samples. Mixotrophic algae can survive in the dark, for example when stored, since they can switch from autotrophic to heterotrophic. The controls of the experiments according to A. and B. of Table 1 show a nearly 50% decrease in the number of cells. However, the algal compositions of the present invention advantageously decrease only to a much lesser extent and even show growth when the number of cells in the untreated control continues to decrease. The experiments according to B. and C. of Table 1 show that the algae even continue to grow, indicating that the algae are living healthily.

Brief Description of the Drawings

[0062]

Figure 1

Figure 2

Figure 3

[0063] Detailed Description of the Drawings and Embodiments Served as Examples Referring to the drawings, FIG. 1 is a schematic view of a preferred embodiment of a growth system improved by the addition of an NBG with an oxygen concentrator attached thereto. The preparation of the growth medium used to fill each PBR for growing microalgae using the improved method begins with the sterilization of water drawn from a municipal source or well rather than distilled water. The water is sterilized for use in the growth system, and the sterilization is performed by introducing ozone into the holding tanks, as is sometimes used in the commercial production of microalgae.

[0064] Sterilized water from the holding tank is gravity-fed through a nanobubble generator. An oxygen concentrator is attached to the nanobubble generator to supply oxygen to create oxygen nanobubbles and saturate the sterilized water. At sea surface and room temperature, the oxygen content of the water in the holding tank is 7 ppm. After the addition of oxygen nanobubbles, the oxygen content of the water reaching the PBR is at least 50 ppm, and the oxygen saturation is approximately 500%. Following this hyperoxygenation process, the water is pumped into a cylindrical container constructed of translucent glass fiber, which is at least one PBR.

[0065] Figure 2 is a detailed view of eight PBRs (10) piped in parallel used in the growth system. The growth system is shown in Figure 3. Each of the illustrated PBRs (10) has a capacity of 80 gallons (300 liters). Each PBR (10) has a fitted removable lid (15) constructed of the same translucent material, preferably glass fiber, which prevents contamination of the contents by airborne particles or dust, but can also be removed to access the PBR.

[0066] Figure 3 is a detailed view of a preferred embodiment of a growth system (90) improved by the addition of an NBG (50) to which an oxygen concentrator (55) is attached. Figure 3 does not show the piping system described in Figure 2. In this embodiment, tap water fills at least one holding tank (30) located at a height higher than the upper part of the PBR (10) used in the growth system (90). As shown in Figure 1, sterilized water is gravity-fed from the holding tank (30) through a valve provided on the holding tank (30) and connected to a pipe (35) attached to a nanobubble generator or NBG (50). An oxygen concentrator (55) is attached to the NBG (50) (60) to add oxygen to generate oxygen-filled nanobubbles, and the oxygen-filled nanobubbles are injected into the water as they pass through the NBG (50). After receiving the oxygen nanobubbles, the hydrogen peroxide water is pumped through a piping system (60) connected to the piping system illustrated in Figure 2. By opening and closing the valves, the individual PBRs (10) that make up the production system (90) can be filled or drained using the piping. The piping in the growth system (90) consists of a polyethylene and stainless steel piping system. As shown in Figure 2, the piping is arranged in parallel and separably to transport hydrogen peroxide water from the nanobubble generator to a plurality of PBRs installed on a piping system that can be opened and closed to fill or drain any individual PBR (10). Both Figure 2 and Figure 3 show PBRs (10) filled with growth medium at different collection levels.

[0067] As shown in Figure 2, hydrogen peroxide water is introduced through a piping system connected near the base of each PBR (45). After filling the PBR, an inorganic nutrient solution known in the industry as f / 2 or F / 2 is added to the sterilized hydrogen peroxide water from the top of the PBR tank by removing the lid (15). At this point, the hydrogen peroxide growth medium is complete and ready for the introduction of the algal inoculum.

[0068] Immediately after preparing the growth medium (40), 5 gallons (20 liters) of Chlorella vulgaris grown to a cell density of 6 to 8 million cells / mL, which is the algal inoculum, is poured into the PBR by removing the lid (15). The Chlorella vulgaris strain currently used by the authors was originally purchased from the algal research laboratory at the University of Texas and grown using standard methods.

[0069] Although not shown, tubular LED growth lights having a wavelength of 440 nM are vertically arranged at regular intervals around each PBR to provide light for photosynthesis. This lighting is set to repeat 16 hours on and 8 hours off to simulate 24 hours a day. The planted growth medium (40) within the PBR (10) is continuously mixed by introducing ambient air through an aquarium stone bubbler located inside the PBR at the base (not shown). The stone bubbler is attached by a piping system to an external pump mounted outside the PBR that contains a filter and an air dryer (not shown). Also, the delivery of ambient air provides the carbon dioxide necessary for photosynthesis.

[0070] Referring to FIG. 3, the growth of microalgae is monitored over time by withdrawing samples from a selected PBR (10) from a discharge line (70) attached to a piping system (35) connected to each PBR (10) constituting the system (90). Specifically, by opening a valve (25) provided on a piping system (45) connected to the base of the PBR (10), the growth medium and microalgae (40) can be drained from the PBR through a piping system (35) for sampling and collection. Cells are counted using a hemocytometer or an automated cell counter. When the cell count in the growth medium (40) exceeds 12 million cells / mL, the valve (25) is opened again and a desired volume is drained through the piping system at the base (45) of the PBR (10), and the culture becomes an "algal concentrate" ready to be collected. A discharge line (70) is connected to a piping system that will be used to fill a polyethylene container placed in a 6°C refrigerator. The container can be of any size.

[0071] All of the algal concentrate can also be collected from a single PBR tank, or the collection can be partial, typically withdrawing 10% - 20% of the volume from the PBR. After partial collection, the volume is exchanged with fresh growth medium (i.e., peroxygen-sterilized water to which additional inorganic nutrients can be added). Since the algae remaining in the PBR continue to grow, partial collection does not require replanting of additional algal cultures. Typically, the cell count in the PBR recovers to pre-collection levels in 4 - 5 days. Thus, in partial collection, an amount of up to 20% of the PBR volume can be taken at 5-day intervals. In this case, the PBR can remain in active operation for up to 5 months.

[0072] The size of the PBR (10) can be various. A preferred embodiment of the system (90) comprises a PBR constructed at a height of 6 feet with a capacity of 360 gallons (1350 liters). FIGS. 2 and 3 show a preferred assembly of a plurality of PBR tanks (10), and FIG. 3 illustrates a photobioreactor array (55) comprising eight PBRs (10) held in a rack (100). The rack (100) is constructed of metal, preferably extruded aluminum. The number of individual PBRs that can be used in the PBR array is limited only by the available space in the building that houses the manufacturing system (90). Similarly, the number of holding tanks can also be expanded here depending on the available space in the building.

[0073] It is intended that the scope of the present invention include all modifications incorporating its main design features, and that the scope and limitations of the present invention should be determined by the appended claims and their equivalents. Accordingly, the concepts of the present invention described herein are interchangeable and / or can be used together in still other alternative forms of the present invention, and other modifications and substitutions will be apparent to those skilled in the art of growing microalgae using photobioreactors from the foregoing description of the preferred embodiments without departing from the spirit or scope of the present invention.

Claims

1. A method for producing an algal composition comprising viable algae and a liquid, comprising: - a step of adding oxygen to the liquid to prepare a peroxidized liquid; and - a step of culturing the algae in the peroxidized liquid at least temporarily under light exposure to enable growth of the algae. A method comprising the above steps.

2. - a step of preparing a liquid; - a step of adding oxygen to the liquid to prepare a peroxidized liquid; - a step of inoculating the peroxidized liquid with algae; - a step of culturing the algae in the peroxidized liquid at least temporarily under light exposure to enable growth of the algae. The method according to claim 1, comprising the above steps.

3. - a step of preparing a liquid containing algae; - a step of adding oxygen to the liquid to prepare a peroxidized liquid and / or adding a peroxidized liquid; - a step of culturing the algae in the peroxidized liquid at least temporarily under light exposure to enable growth of the algae. The method according to claim 1, comprising the above steps.

4. The number of viable cells in the liquid is 1 million to 20 million cells / mL, preferably 2 million to 18 million cells / mL, more preferably 5 million to 15 million cells / mL, even more preferably 7 million to 14 million cells / mL, even more preferably 10 million to 13 million cells / mL, and most preferably 11 million to 13 million cells / mL. The method according to any one of claims 1 to 3.

5. The attenuation of the number of viable cells is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, even more preferably at most 50%, even more preferably at most 40%, even more preferably at most 30%, even more preferably at most 20%, even more preferably at most 10%, and even more preferably at most 5% of the attenuation of the number of viable cells in a control culture of the same algae that has not been peroxidized during the same period. The method according to any one of claims 1 to 4.

6. When compared with a control culture of the same algae that has not been peroxidized, (i) between 1 and 24 months, preferably between 1 and 20 months, more preferably between 1 and 18 months, even more preferably between 2 and 15 months, even more preferably between 2 and 12 months, even more preferably between 2 and 10 months, even more preferably between 3 and 9 months, and most preferably between 4 and 8 months; or during a period of 1 to 30 days, preferably 1 to 25 days, more preferably 1 to 15 days, even more preferably 1 to 10 days, most preferably 4 to 6 days; or during a period of at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, most preferably at least 8 months the method according to any one of claims 1 to 5, wherein the decay of the viable cell count is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, even more preferably at most 2%, even more preferably at most 1%, and most preferably there is no decay of the viable cell count.

7. when compared with a control culture of the same non-peroxygenated algae during a period of 1 to 30 days, preferably 5 to 25 days, more preferably 10 to 25 days, even more preferably 15 to 25 days, most preferably 18 to 22 days, or during a period of 1 to 24 months, preferably 1 to 20 months, more preferably 1 to 18 months, even more preferably 2 to 15 months, even more preferably 3 to 12 months, even more preferably 3 to 10 months, even more preferably 3 to 8 months, even more preferably 3 to 6 months, most preferably 3 to 4 months the method according to any one of claims 1 to 6, wherein the viable cell count increases by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, even more preferably at least 30%, most preferably at least 40%.

8. The viable algae can be stored at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, without significant attenuation of the viable cell count compared to the viable cell count at the time when the algae were collected, for at least 1 month, preferably at least 2 months, more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, even more preferably at least 8 months, and most preferably for a period as long as 18 months, and / or at room temperature, preferably 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C, for at least 1 week, preferably at least 2 weeks, more preferably at least 3 weeks, even more preferably at least 4 weeks, more preferably at least 5 weeks, even more preferably at least 6 weeks, even more preferably at least 7 weeks, most preferably at least 8 weeks, and particularly for at least 2 months, according to the method of any one of claims 1 to 7.

9. The method according to any one of claims 1 to 8, wherein oxygen is added only once.

10. The method according to any one of claims 1 to 9, comprising steps of adding oxygen to the liquid to prepare a peroxidized liquid and / or adding the peroxidized liquid a plurality of times.

11. The method according to any one of claims 1 to 8, wherein oxygen is continuously added to the liquid.

12. The method according to any one of claims 1 to 11, which is carried out in a photobioreactor.

13. The method according to any one of claims 1 to 12, wherein exposing the algae in the liquid to light is carried out for at least 1 hour to 24 hours at intervals of 1 hour to 12 hours, preferably for at least 16 hours at intervals of 8 hours.

14. The method according to any one of claims 1 to 13, wherein exposing the algae in the liquid to light is carried out at a wavelength of 200 nm to 800 nm, preferably 250 nm to 650 nm, more preferably 300 to 550 nm, even more preferably 400 to 500 nm, and most preferably 440 nm.

15. Exposing the algae in the liquid to light is carried out at a power of 1 to 20 W, preferably 5 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, and most preferably 13 W, according to the method of any one of claims 1 to 14.

16. Exposing the algae in the liquid to light is carried out at a light intensity between 1,000 and 20,000 lux, preferably between 5,000 and 15,000 lux, more preferably between 8,000 and 12,000 lux, according to the method of any one of claims 1 to 15.

17. (i) Monitoring the number of viable cells; and / or (ii) Collecting the algae; and / or (iii) Concentrating the collected algae; and / or (iv) Storing the algae The method according to any one of claims 1 to 16, further comprising.

18. The viable algae are: (i) At a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C; and / or (ii) At a temperature of 10°C to 30°C, preferably 15°C to 25°C, more preferably 18°C to 22°C; and / or (iii) Stored in the dark The method according to any one of claims 1 to 17.

19. By supplying oxygen nanobubbles to the liquid, oxygen is added to the liquid, according to the method of any one of claims 1 to 18.

20. The oxygen level of the liquid is at least 20 ppm, preferably at least 25 ppm, more preferably at least 30 ppm, even more preferably at least 35 ppm, even more preferably at least 40 ppm, even more preferably at least 45 ppm, and most preferably at least 50 ppm, according to the method of any one of claims 1 to 19.

21. The oxygen saturation of the liquid is at least 100%, preferably at least 200%, more preferably at least 300%, even more preferably at least 400%, and most preferably at least 500%, according to the method of any one of claims 1 to 20.

22. The oxygen level of the peroxygenated liquid for planting the algae is at least 50 ppm and / or the oxygen saturation is at least 500%, according to the method of any one of claims 2 to 21.

23. The method according to any one of claims 1 to 22, wherein the liquid is an aqueous solution, preferably selected from water, pure water, seawater, sterilized water, culture medium, and / or buffer.

24. The liquid is a culture medium, preferably the culture medium is: a) water, preferably sterilized water; b) nitrate, preferably an alkali metal salt thereof, more preferably sodium nitrate; c) dihydrogen phosphate, preferably an alkali metal salt thereof, more preferably sodium dihydrogen phosphate; d) silicate, preferably an alkali metal salt thereof, more preferably sodium silicate; e) one or more trace metals, preferably inorganic salts thereof, more preferably trace metals selected from cobalt, copper, iron, manganese, molybdenum, and / or zinc; and / or f) Preferably vitamin B １２ , one or more vitamins selected from biotin and / or thiamine The method according to any one of claims 1 to 17, comprising the above.

25. The method according to any one of claims 1 to 24, wherein no carbohydrate added from outside is present in the liquid.

26. The method according to any one of claims 1 to 25, wherein no additional carbohydrate for inducing heterotrophic metabolism in the algae is added to the liquid.

27. The method according to any one of claims 1 to 26, which is operated in batch mode, fed-batch mode, semi-continuous mode, or continuous mode.

28. The algae are capable of mixotrophic metabolism, preferably the algae are capable of both autotrophic metabolism and heterotrophic metabolism, and even more preferably, when the algae grow under light, they carry out autotrophic metabolism and are capable of heterotrophic metabolism in the absence of light. The method according to any one of claims 1 to 27.

29. The algae are obligate mixotrophs, obligate autotrophs and facultative heterotrophs, facultative autotrophs and obligate heterotrophs, and / or facultative mixotrophs. The method according to any one of claims 1 to 28.

30. The algae are unicellular algae, preferably selected from the group of green algae, more preferably the algae belong to the genus Chlorella, and even more preferably the algae are Chlorella vulgaris. The method according to any one of claims 1 to 29.

31. A composition comprising viable algae and a liquid, which can be obtained by the method according to any one of claims 1 to 30.

32. A composition comprising viable algae and a liquid, wherein the liquid is peroxidized.

33. The composition according to claim 31 or 32, wherein the liquid contains oxygen nanobubbles, preferably the liquid is saturated with oxygen nanobubbles.

34. The composition according to any one of claims 31 to 33, wherein the oxygen level of the liquid is at least 20 ppm, preferably at least 25 ppm, more preferably at least 30 ppm, even more preferably at least 35 ppm, even more preferably at least 40 ppm, even more preferably at least 45 ppm, and most preferably at least 50 ppm.

35. The composition according to any one of claims 31 to 34, wherein the oxygen saturation of the liquid is at least 200%, preferably at least 300%, more preferably at least 400%, and most preferably at least 500%.

36. The composition according to any one of claims 31 to 35, wherein the liquid is selected from aqueous solutions, preferably selected from water, pure water, seawater, sterilized water, culture media, and / or buffers.

37. The liquid is: (a) water, preferably sterilized water; (b) nitrate, preferably an alkali metal salt thereof, more preferably sodium nitrate; (c) dihydrogen phosphate, preferably an alkali metal salt thereof, more preferably sodium dihydrogen phosphate; (d) silicate, preferably an alkali metal salt thereof, more preferably sodium silicate; (e) one or more trace metals, preferably inorganic salts thereof, more preferably trace metals selected from cobalt, copper, iron, manganese, molybdenum, and / or zinc; and / or (f) Preferably vitamin B １２ One or more vitamins selected from biotin and / or thiamine a culture medium containing the same. The composition according to any one of claims 31 to 36.

38. The composition according to any one of claims 31 to 37, wherein no externally added carbohydrate is present in the liquid.

39. The composition according to any one of claims 31 to 38, wherein no additional carbohydrate is added to the liquid.

40. The algae are capable of mixotrophic metabolism, preferably the algae are capable of both autotrophic and heterotrophic metabolism, and even more preferably the algae exhibit autotrophic metabolism when growing under light exposure and are capable of heterotrophic metabolism in the absence of light. The composition according to any one of claims 31 to 39.

41. The composition according to any one of claims 31 to 40, wherein the algae are obligate mixotrophs, obligate autotrophs and facultative heterotrophs, facultative autotrophs and obligate heterotrophs, and / or facultative mixotrophs.

42. The composition according to any one of claims 31 to 41, wherein the algae are selected from the group of unicellular algae, preferably green algae, more preferably the algae are of the genus Chlorella, and even more preferably the algae are Chlorella vulgaris.

43. The composition according to any one of claims 31 to 42, wherein the number of viable cells in the liquid is 1 million to 20 million cells / mL, preferably 2 million to 18 million cells / mL, more preferably 5 million to 15 million cells / mL, even more preferably 7 million to 14 million cells / mL, even more preferably 10 million to 13 million cells / mL, and most preferably 11 million to 13 million cells / mL.

44. The composition according to any one of claims 31 to 43, wherein the decay of the number of viable cells is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, even more preferably at most 50%, even more preferably at most 40%, even more preferably at most 30%, even more preferably at most 20%, even more preferably at most 10%, even more preferably at most 5% when compared with the decay of the number of viable cells of a control culture of the same algae that has not been peroxidized during the same period.

45. When compared with a control culture of the same algae that has not been peroxidized, (i) for 1 to 24 months, preferably for 1 to 20 months, more preferably for 1 to 18 months, even more preferably for 2 to 15 months, even more preferably for 2 to 12 months, even more preferably for 2 to 10 months, even more preferably for 3 to 6 months, and most preferably for 3 to 5 months; or (ii) for 1 to 30 days, preferably for 1 to 25 days, more preferably for 1 to 15 days, even more preferably for 1 to 10 days, and most preferably for 4 to 6 days; or (iii) within a period of at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, even more preferably at least 5 months, even more preferably at least 6 months, even more preferably at least 7 months, most preferably at least 8 months the composition according to any one of claims 31 to 44, wherein the attenuation of viable cell count is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, even more preferably at most 2%, even more preferably at most 1%, and most preferably there is no attenuation of viable cell count.

46. when compared with a control culture of the same non-peroxygenated algae (i) during the period of 1 to 30 days, preferably 5 to 25 days, more preferably 10 to 25 days, even more preferably 15 to 25 days, most preferably 18 to 22 days, or (ii) during the period of 1 to 24 months, preferably 1 to 20 months, more preferably 1 to 18 months, even more preferably 2 to 15 months, even more preferably 3 to 12 months, even more preferably 3 to 10 months, even more preferably 3 to 8 months, even more preferably 3 to 6 months, most preferably 3 to 4 months the composition according to any one of claims 31 to 44, wherein the viable cell count increases by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, even more preferably at least 30%, most preferably at least 40%.

47. Use of the composition according to any one of claims 31 to 46 for improving plant growth.

48. The use according to claim 47, wherein the plant is selected from fruits, vegetables, and / or crops, and preferably the crop is an agricultural plant grown for food or fiber.

49. The use according to claim 48, wherein the fruit is selected from fruit trees, berry bushes, and / or pineapples.

50. The use according to claim 48, wherein the vegetable is a horticultural vegetable, preferably selected from tomatoes, potatoes, cucumbers, peppers, carrots, pumpkins and / or squashes.

51. The use according to claim 48, wherein the crop is selected from vegetables, sugar beets, corn, beans, hay, peanuts, cotton, flax, and / or tobacco.

52. A method for maintaining or improving soil fertility and / or improving plant growth, comprising the step of applying the composition according to any one of claims 31 to 46 to the soil and / or plants.

53. The method according to claim 52, wherein the soil is agricultural land, pasture land, a sports field, a golf course, and / or urban green space.

54. The method according to claim 52 or 53, comprising the step of diluting the composition according to any one of claims 25 to 37 before applying the composition to the soil and / or plants.

55. The composition is applied in an amount of 10 to 100,000 cells / square foot, preferably 100 to 90,000 cells / square foot, preferably 1,000 to 80,000 cells / square foot, more preferably 10,000 to 70,000 cells / square foot, even more preferably 20,000 to 60,000 cells / square foot, even more preferably 30,000 to 55,000 cells / square foot, even more preferably 35,000 to 55,000 cells / square foot, even more preferably 40,000 to 55,000 cells / square foot, even more preferably 45,000 to 55,000 cells / square foot, and most preferably 50,000 cells / square foot, according to any one of claims 52 to 54.

56. A photobioreactor comprising means for supplying oxygen in the form of nanobubbles.

57. The photobioreactor according to claim 56, wherein the means for supplying oxygen in the form of nanobubbles comprises means for supplying oxygen, preferably an oxygen concentrator, and means for generating nanobubbles, preferably a nanobubble generator.

58. The photobioreactor according to claim 57, wherein the means for supplying oxygen is operably connected to the means for generating nanobubbles.

59. a) one or more reaction vessels, preferably made of glass fiber; b) one or more reservoirs; c) one or more light sources, preferably an LED light source, more preferably a tubular LED growth light, and / or an LED bulb; d) one or more means for supplying dissolved gas to the photobioreactor; e) an oxygen concentrator; f) A piping system; and / or g) One or more valves The photobioreactor according to any one of claims 56 to 58, comprising one or more of the above.

60. Comprising a reaction vessel, preferably the reaction vessel is as follows: a) Being liquid-impermeable; b) Being cylindrical; c) Having a fixed side wall and bottom; d) Having a removable lid; and / or e) Preferably being made of a translucent material containing glass fibers The photobioreactor according to any one of claims 56 to 59, which is a container characterized by one or more of the above.

61. Comprising a light source, preferably a tubular LED growth light and / or an LED bulb, more preferably the light source is as follows: a) A wavelength of 200 nm to 800 nm, preferably 250 nm to 650 nm, more preferably 300 nm to 550 nm, even more preferably 400 nm to 500 nm, most preferably 440 nm; and / or b) An illuminance between 1,000 and 20,000 lux, preferably between 5,000 and 15,000 lux, more preferably between 8,000 and 12,000 lux; and / or c) A power of 1 to 20 W, preferably 5 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, most preferably 13 W; and / or d) A light source arranged vertically and equidistantly around one or more reaction vessels, preferably a light source arranged 0.5 cm to 50 cm, preferably 1 cm to 10 cm, more preferably 2 cm to 8 cm, even more preferably 5 cm to 6 cm, most preferably 5 cm away from one or more reaction vessels; and / or e) Comprising a timer operable to switch the light source on and off, preferably the timer is set to cycle the light source on for at least 1 to 24 hours and off for at least 1 to 12 hours, preferably on for 16 hours and off for 8 hours, a light source The photobioreactor according to any one of claims 56 to 60, characterized by one or more of the above.

62. One or more means for supplying dissolved gas to one or more reaction vessels, which are not means for supplying oxygen in the form of nanobubbles. Preferably, the one or more means for supplying dissolved gas to one or more reaction vessels comprise a pump operable to push the dissolved gas into the one or more reaction vessels, a piping system through which the dissolved gas passes, and a check valve. More preferably, the one or more means for supplying dissolved gas to one or more reaction vessels is a water tank stone bubbler. The photobioreactor according to any one of claims 56 to 61.

63. Comprising a piping system. Preferably, the piping system is made of a polymer, preferably polyethylene and / or stainless steel, and more preferably, the piping system is made of a combination of polyethylene and stainless steel. The photobioreactor according to any one of claims 56 to 62.

64. Comprising a piping system. Preferably, the piping system is adapted to achieve fluid communication of the components of the photobioreactor. The photobioreactor according to any one of claims 56 to 63.

65. A system comprising one or more photobioreactors according to any one of claims 56 to 64.

66. Comprising at least two photobioreactors. Preferably, the at least two photobioreactors are in fluid communication. The system according to claim 65.

67. The photobioreactor is arranged in a photobioreactor array. The system according to claim 66.

68. The photobioreactors are connected in parallel. The system according to claim 66 or 67.

69. Comprising a valve, the valve being operable to separate the at least one photobioreactor from other photobioreactors by opening and closing the valve. The system according to any one of claims 65 to 68.

70. The one or more photobioreactors are in fluid communication with a nanobubble generator. The system according to any one of claims 65 to 69.

71. Comprising a reservoir in fluid communication with the one or more photobioreactors. The system according to any one of claims 65 to 70.

72. A system according to any one of claims 65 to 71, comprising a discharge line operably connected to the one or more photobioreactors for removing liquid from the one or more photobioreactors, preferably wherein the discharge line is manufactured from polyethylene.

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