Novel method for producing algae composition

Abstract

Systems and methods for growing microalgae, preferably Chlorella genus (Chlorella sp.), capable of metabolism of mixed nutrition. Microalgae grown in a system using the method can survive and grow in dark refrigeration, which enables the algae to be stored and transported for application as live cultures. Furthermore, the microalgae can grow in sufficient amounts for commercial sales for application as a biostimulant to crops.

Description

Background Art

[0001] Rapid population growth after World War II led to concerns about food security (the limits of the planet's ability to feed a growing population). Malthusians predicted possible famine in the 1990s. This did not happen, thanks to the Green Revolution, an unexpected increase in agricultural efficiency and yields. One factor was the development of nitrogen fertilizers, a byproduct of munitions technology. Gunpowder factories became fertilizer factories (turning swords into plowshares).

[0002] Synthetic chemical fertilizers make plants grow. However, after seven decades of use, unintended consequences have emerged, such as: 1) declining soil fertility and stagnant crop yields, 2) increased greenhouse gases, and 3) contamination of groundwater leading to wild algae blooms. The production of synthetic fertilizers requires the burning of methane; an estimated 12% of greenhouse gases originate from modern agriculture, much of which comes from the use of chemical fertilizers.

[0003] In nature, plant growth depends on a symbiotic relationship between the plant and soil microorganisms. Plants exude sugars (carbon compounds) from their roots, which feed microorganisms present in the soil, especially bacteria. In turn, soil microorganisms process the nutrients needed for plant growth. Soil bacteria convert atmospheric nitrogen into ammonium compounds that can be taken up by plant roots. This natural process is called nitrogen fixation and is primarily the work of bacteria that live in close contact with the roots (this area is called the "rhizosphere"), not the work of the plant. Fertile soil in the rhizosphere has up to 10 billion bacteria per gram. The microbial composition and activity in the soil defines "fertility." Crop rotation adds nitrogen to the soil with legumes, but it is bacteria in the legume root nodules that fix the nitrogen. Other bacteria dissolve soil-bound phosphorus.

[0004] Chemical fertilizers bypass natural processes. Over time, microbial activity (fertility) as well as the soil's nitrogen and carbon levels decline. Chemical fertilizers add large amounts of nutrients, nitrogen, phosphorus and potassium, to the soil in a form that can be directly absorbed by the roots. However, plants absorb only 30% of the nitrogen applied through chemical fertilization. Most of the rest goes into solution and eventually reaches the groundwater. It is well known that the addition of fertilizers to water bodies, for example, is known to occur through runoff from agricultural soil into ponds and lakes, promoting the growth of wild algae, leading to harmful algal blooms. In addition, some of the nitrogen in fertilizers is converted into nitrous oxide, a potent greenhouse gas. Globally, agriculture accounts for 75% of nitrous oxide emissions into the atmosphere.

[0005] Composted organic matter such as manure can be applied to the soil to provide nitrogen as well as bacteria and carbon, which feeds the microorganisms. However, the use of compost is inconvenient due to its large volume and is difficult to apply on a large scale. Processing manure to make organic fertilizer is an industrial process and there are energy costs in transportation.

[0006] Biostimulants provide another way to promote plant growth. Defined in the 2018 U.S. Farm Bill, plant biostimulants (plant biostimulant, PBS) are compounds or organisms that promote natural processes. A large number of studies have shown that live microalgae, especially Chlorella vulgaris, are effective biostimulants applied through both foliar and soil. In addition, the stimulation of microalgae to soil microorganisms is regenerative: applying microalgae increases organic matter, bacterial mass and soil respiration (a measure of bacterial activity). In other words, applying microalgae improves soil fertility. However, in the past, it was impossible to use live microalgae as biostimulants on a large scale because the actual storage of live microalgae was unavailable, and the live microalgae had to be produced on site and used immediately after harvest. Summary of the invention

[0007] The present invention is a novel method for inducing heterotrophic metabolic activity in microalgae, which means that microalgae cultivated using the method can consume organic matter from the culture medium to grow if placed under conditions where photosynthesis is not possible (i.e., without light). Embodiments of the present invention are directed to improving methods for cultivating sufficient quantities of Chlorella sp., preferably Chlorella vulgaris, which are commercially sold as biostimulants for application to crops after harvesting the algae. In particular, microalgae cultivated using the improved method are able to survive dark refrigerated storage and transportation.

[0008] Microalgae, and in particular Chlorella vulgaris, have been cultivated for commercial use for decades. Known methods for cultivating microalgae such as C. vulgaris in commercial volumes involve photobioreactors (PBRs) to maximize growth within a limited space.

[0009] Most commercial uses of microalgae do not involve live microalgae. For example, when microalgae are cultivated for oil content (biofuel) or used as animal feed additives, algae are processed immediately after harvest. Commercially available algae-based biostimulants include acellular algae extracts, which contain growth signal compounds (plant hormones) that stimulate plant growth and also promote soil fertility, such as auxins and cytokinins. Others are growth media from algae culture, containing the same growth signal compounds secreted by algae during its growth. Importantly, no commercially available biostimulants contain live green algae cells. Most biostimulants are sold as supplements to chemical fertilizers; they are not enough to replace chemical fertilizers. Early university studies have shown that applying live algae (cultivated on site and applied when algae are harvested) provides enough promotion to yield and fertility, thereby eliminating chemical fertilizers. Live cell algae biostimulants have been shown to be more effective than algae derivatives. For this reason, methods for preserving live algae during transportation and storage can achieve a reduction in the demand for synthetic chemical fertilizers.

[0010] Known commercially available biostimulants containing living cells use cyanobacteria ("blue-green algae", which are actually bacteria). Cyanobacteria are effective plant biostimulants and produce growth signal compounds, which are also produced by green algae. Like other bacteria, cyanobacteria can be dried, packaged, then reconstituted in water, and applied as living cells. However, disadvantageously, in the past two decades, neurotoxins produced by cyanobacteria (including β-N-methylamino-L-alanine, BMAA) have been associated with outbreaks of neurodegenerative diseases. For this reason, safer biostimulants based on living cells are needed.

[0011] Microalgae cultivated by the methods taught herein remain viable even when bottled as "algae concentrate" in growth medium from a PBR and stored under refrigeration (6°C). Refrigeration keeps the concentrate free of contaminants during storage and transportation to the application site because it inhibits the growth of common contaminants, including protozoa and bacteria. Refrigerated storage is typically dark, which is problematic for microalgae because, like higher plants, microalgae are typically autotrophic, meaning they produce nutrients via photosynthesis that relies on light to stay alive and grow:

[0012] Algae + light + water + CO2 + inorganic nutrients → glucose (& more algae) + O2

[0013] When microalgae lose light, photosynthesis stops, and algae cell counts drop rapidly (in our laboratory, drop up to 50% in five days, then recover very little, Table 1). Therefore, with standard propagation methods, live microalgae must be produced locally, so as to be used as PBS immediately. Because microalgae are not easy to grow, this is problematic for a wide range of commercial uses. Studies showing that live microalgae, i.e., local cultivation and microalgae used immediately after harvesting are effective PBSs have been conducted in North Africa, Southeast Asia, and Eastern Europe, but the method has not yet been adopted for commercial use, because it is difficult to replicate. Without wishing to be bound by theory, the supposed benefit of live algae, particularly Chlorella vulgaris, is to continuously produce signal compounds after being applied to plants or soil. Another beneficial effect of live algae (particularly Chlorella vulgaris cells) is that it is observed that it ingests soil bacteria (possibly by phagocytosis) to transport them to plant root hairs, and finally releases them into interstitial spaces, where they play a role as beneficial endophytes. There is no method for large-scale cultivation and storage of live microalgae, so that it can grow in one place, and be distributed to other distant places to apply, without colony degradation. The essence of the present invention is a method for cultivating microalgae, and in particular Chlorella vulgaris, which remains viable during refrigerated, dark storage due to the induction of heterotrophic metabolism without a significant drop in cell count; that is, the microalgae cultivated by this method are able to consume organic matter and oxygen and grow in the absence of light. This is achieved by high oxygenation of the water used in the growth medium of the PBR. In a highly oxygenated environment, the microalgae change metabolic behavior and become "mixotrophic", which means that although they continue to produce food / energy (autotrophic metabolism) by photosynthesis in the presence of light, they become able to grow heterotrophically in the dark and consume organic nutrients. It is known that algae are able to feed heterotrophically because it has been observed that it is induced by adding sugars such as glucose to the growth medium, especially when exposed to high concentrations of glucose. The present method induces heterotrophic metabolism by high oxygenation of the growth medium, i.e., by saturating the growth medium with oxygen nanobubbles. This has not been reported before. There have also been no reports of microalgae that can maintain algae colony cell counts and viability during dark storage. Although the method is described for the cultivation of C. vulgaris, it is applicable to all Chlorella species as well as other microalgae capable of mixotrophic metabolism.

[0014] Currently, PBRs are used to cultivate microalgae in water supplemented with inorganic nutrients. Microalgae cultivated in this manner can be processed and used immediately after harvesting, but cannot be transported for any use requiring living microalgae cells because the microalgae cannot survive dark, refrigerated storage. It is common practice to cultivate algae in photobioreactors. In addition, the use of nanobubble generators as part of these systems has been used to produce ozone nanobubbles for sterilization of algae growth media and cleaning of algae growth equipment (tanks and pipes). The improvements described herein include an oxygen concentrator with an attached nanobubble generator (NBG). In other words, the present invention provides for the first time a photobioreactor equipped with a device for supplying oxygen in the form of nanobubbles. As Figure 1 As shown, the system of the present invention includes a NBG positioned to receive sterile water from a source. The sterile water is fed through the NBG to receive nanobubbles of oxygen, thereby increasing the measurable oxygen concentration in the water to about 500% saturation, i.e., a hyperoxygenated state.

[0015] The highly oxygenated water is then pumped to fill at least one PBR. A mixture of well-known inorganic nutrients widely used in algae production is added to the sterile water of the PBR to produce a growth medium. An example of a typical culture medium that can be used to cultivate algae according to the present invention is the commercially available Guillard's / F / 2 culture medium. Preferably, in the method of the present invention, no externally added carbohydrates are present in the culture medium. The PBR is then inoculated with microalgae.

[0016] The PBR used in the system is made of a translucent material so that light from outside the PBR can be used by the microalgae cells growing inside the PBR for photosynthesis. Artificial light sources are mounted outside the PBR and simulate a 24-hour day cycle. Since photosynthesis also requires carbon dioxide, a standard aquarium stone bubbler is used to introduce filtered ambient air into the growth medium, thereby providing carbon dioxide and keeping the microalgae cells mixed and suspended. After harvesting from the PBR, the microalgae cells are bottled with their growth medium as an algae concentrate containing at least 10 million algae cells per mL; the concentrate is the final product, which can be diluted and used as a plant and soil biostimulant.

[0017] An important feature of the improved method is that the microalgae cells produced by it can survive for more than six months, even up to eighteen months, under dark, refrigerated storage, which is enough to allow it to be transported to agricultural sites for application. Bottled algae concentrates are placed in refrigeration (6°C) for storage directly before being sold as biostimulants. However, refrigeration is not necessary because algae can grow in a wide range of temperatures. However, if the storage is extended for more than two months, refrigeration advantageously prevents the growth of bacteria and protozoa contaminants. If the product is used during this period, refrigeration is unnecessary. During the initial two months of storage, contamination leads to algae colony loss rarely, which may be because heterotrophic feeding inhibits the growth of contaminants. The composition of the present invention comprising live algae can be stored at refrigerator temperature, for example, at 6°C for more than eight months, but the recommended use date is six months from harvest. The microalgae cultivated in the system using this method not only remain alive, but also continue to grow during storage, which maintains the number of cells necessary for the application of effective biostimulants (see Table 1). The live algae is applied at a rate of 50,000 cells per square foot, to the soil, foliage, or both; thus, one liter of algae concentrate can be diluted to treat 4.5 acres; 5 ml of concentrate treats 1000 square feet.

[0018] Another feature of the improved method is that it enables microalgae to grow on a large scale at low cost for agricultural purposes, requires minimal engineering, and provides high yields of microalgae in a small space. In particular, the present invention enables algae to grow in a liter photobioreactor tank up to 750 liters of volume. Previously, there was a problem that the increase in tank diameter caused the algae in the center of the tank to be shaded, and therefore hindered growth due to the low efficiency of light exposure. However, the inventors unexpectedly found that the algae cell count in the larger tank rose as fast as the count in the 300-liter PBR with its smaller diameter. One explanation for this is that algae is constantly mixed with an air bubbler. This prevents algae cells from gathering in the middle of the tank away from the light source. Another explanation is that algae are mixed nutrients, that is, they do not rely on photosynthesis, so they continue to grow even in more shaded or even dark areas in the reactor. The use of larger PBR tanks has caused increased efficiency and productivity.

[0019] In the context of the present invention, at least the following embodiments E1 to E72 are described:

[0020] E1. According to a first embodiment, the present invention relates to a method for producing an algae composition comprising living algae and a liquid, comprising the following steps:

[0021] - adding oxygen to the liquid to provide a highly oxygenated liquid;

[0022] - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

[0023] E2. The method of embodiment 1 comprises the following steps:

[0024] - provide fluids;

[0025] - adding oxygen to the liquid to provide a highly oxygenated liquid;

[0026] - inoculating the highly oxygenated liquid with algae;

[0027] - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

[0028] E3. The method of embodiment 1 comprises the following steps:

[0029] - providing a liquid comprising algae;

[0030] - adding oxygen to the liquid to provide a highly oxygenated liquid and / or adding a highly oxygenated liquid;

[0031] - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

[0032] E4. The method of any one of embodiments 1 to 3, wherein the viable cell count in the liquid is 1 million cells / mL to 20 million cells / mL, preferably 2 million cells / mL to 18 million cells / mL, more preferably 5 million cells / mL to 15 million cells / mL, even more preferably 7 million cells / mL to 14 million cells / mL, still more preferably 10 million cells / mL to 13 million cells / mL, and most preferably 11 million cells / mL to 13 million cells / mL.

[0033] E5. The method of any one of embodiments 1 to 4, wherein the decay in viable cell count is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, still more preferably at most 50%, still more preferably at most 40%, still more preferably at most 30%, still more preferably at most 20%, still more preferably at most 10%, still more preferably at most 5%, of the decay in viable cell count of a control culture of the same algae over the same time period that is not hyperoxygenated.

[0034] E6. The method of any one of embodiments 1 to 5, wherein there is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, still more preferably at most 2%, still more preferably at most 1% decrease in viable cell count, and most preferably no decrease in viable cell count, when compared to a control culture of the same algae that is not hyperoxygenated within the following time period,

[0035] (i) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 2 months to 12 months, even more preferably 2 months to 10 months, even more preferably 3 months to 9 months, most preferably 4 months to 8 months; or

[0036] (ii) 1 day to 30 days, preferably 1 day to 25 days, more preferably 1 day to 15 days, even more preferably 1 day to 10 days, most preferably 4 days to 6 days; or

[0037] (iii) at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, still more preferably at least 5 months, even more preferably at least 6 months, still more preferably at least 7 months, and most preferably at least 8 months.

[0038] E7. The method of any one of embodiments 1 to 6, wherein the viable cell count is increased by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, still more preferably at least 30%, most preferably at least 40% over the following time period when compared to a control culture of the same algae that is not hyperoxygenated:

[0039] (i) 1 day to 30 days, preferably 5 days to 25 days, more preferably 10 days to 25 days, even more preferably 15 days to 25 days, most preferably 18 days to 22 days, or

[0040] (ii) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 3 months to 12 months, even more preferably 3 months to 10 months, even more preferably 3 months to 8 months, still more preferably 3 months to 6 months, most preferably 3 months to 4 months.

[0041] E8. The method of any one of embodiments 1 to 7, wherein the live 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, still more preferably at least 6 months, even more preferably at least 7 months, even more preferably at least 8 months, most preferably up to 18 months, and / or wherein the live 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, still more preferably at least 5 weeks, even more preferably at least 6 weeks, still more preferably at least 7 weeks, most preferably at least 8 weeks, in particular at least 2 months, without a significant decrease in the viable cell count compared to the viable cell count when the algae is harvested.

[0042] E9. The method of any one of embodiments 1 to 8, wherein oxygen is added only once.

[0043] E10. The method of any one of embodiments 1 to 9, comprising adding oxygen to the liquid to provide a highly oxygenated liquid and / or more than one step of adding a highly oxygenated liquid.

[0044] E11. The method of any one of embodiments 1 to 8, wherein oxygen is continuously added to the liquid.

[0045] E12. The method of any one of embodiments 1 to 11, wherein the method is performed in a photobioreactor.

[0046] E13. The method of any one of embodiments 1 to 12, wherein exposing the algae in the liquid to light is performed at intervals of 1 to 12 hours for at least 1 to 24 hours, preferably at intervals of 8 hours for at least 16 hours.

[0047] 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 200nm to 800nm, preferably 250nm to 650nm, more preferably 300nm to 550nm, even more preferably 400nm to 500nm, and most preferably 440nm.

[0048] E15. The method of any one of embodiments 1 to 14, wherein exposing the algae in the liquid to light is carried out at a power of 1 W to 20 W, preferably 5 W to 20 W, more preferably 10 W to 20 W, even more preferably 10 W to 15 W, most preferably 13 W.

[0049] E16. The method of any one of embodiments 1 to 15, wherein exposing the algae in the liquid to light is performed at a light intensity of 1,000 lux to 20,000 lux, preferably 5,000 lux to 15,000 lux, more preferably 8,000 lux to 12,000 lux.

[0050] E17. The method of any one of embodiments 1 to 16, wherein the method further comprises:

[0051] (i) monitoring viable cell counts; and / or

[0052] (ii) harvesting the algae; and / or

[0053] (iii) concentrating the harvested algae; and / or

[0054] (iv) storing the algae.

[0055] E18. The method of any one of embodiments 1 to 17, wherein the living algae is stored:

[0056] (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

[0057] (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

[0058] (iii) In darkness.

[0059] E19. The method of any one of embodiments 1 to 18, wherein oxygen is added to the liquid by supplying oxygen nanobubbles to the liquid.

[0060] E20. The method of any one of embodiments 1 to 19, 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, still more preferably at least 40, even more preferably at least 45 ppm, and most preferably at least 50 ppm.

[0061] E21. The method of any one of embodiments 1 to 20, wherein 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%.

[0062] E22. The method of any one of embodiments 2 to 21, wherein the highly oxygenated liquid inoculated with the algae has an oxygen level of at least 50 ppm and / or an oxygen saturation of at least 500%.

[0063] E23. The method of any one of embodiments 1 to 22, wherein the liquid is an aqueous solution, preferably selected from water, fresh water, sea water, sterile water, culture medium and / or buffer.

[0064] E24. The method of any one of embodiments 1 to 17, wherein the liquid is a culture medium, preferably the culture medium comprises:

[0065] a) water, preferably sterile water;

[0066] b) nitrates, preferably alkali metal salts thereof, more preferably sodium nitrate;

[0067] c) dihydrogen phosphate, preferably an alkali metal salt thereof, more preferably sodium dihydrogen phosphate;

[0068] d) silicates, preferably alkali metal salts thereof, more preferably sodium silicate;

[0069] e) one or more trace metals, preferably inorganic salts thereof, more preferably said trace metals are selected from cobalt, copper, iron, manganese, molybdenum and / or zinc; and / or

[0070] f) one or more vitamins, preferably selected from vitamin B 12 , biotin and / or thiamine.

[0071] E25. The method of embodiments 1 to 24, wherein no externally added carbohydrates are present in the liquid.

[0072] E26. The method of any one of embodiments 1 to 25, wherein no additional carbohydrates are added to the liquid to induce heterotrophic metabolism in the algae.

[0073] E27. The method of any one of embodiments 1 to 26, operating in batch mode, fed-batch mode, semi-continuous mode or continuous mode.

[0074] E28. The method of any one of embodiments 1 to 27, wherein the algae is capable of mixed trophic metabolism, preferably wherein the algae is capable of both autotrophic and heterotrophic metabolism, even more preferably wherein the algae has autotrophic metabolism when grown under light exposure and is capable of heterotrophic metabolism in the absence of light.

[0075] E29. The method of any one of embodiments 1 to 28, wherein the algae is an obligate mixotroph, an obligate autotroph and a facultative heterotroph, a facultative autotroph and an obligate heterotroph, and / or a facultative mixotroph.

[0076] E30. The method of any one of embodiments 1 to 29, wherein the algae is selected from the group of unicellular algae, preferably green algae, more preferably the algae is of the genus Chlorella, even more preferably the algae is Chlorella vulgaris.

[0077] E31. A composition comprising living algae and a liquid, wherein said composition is obtainable by the method according to any one of embodiments 1 to 30.

[0078] E32. A composition comprising living algae and a liquid, wherein the liquid is highly oxygenated.

[0079] E33. The composition of embodiment 31 or 32, wherein the liquid comprises oxygen nanobubbles, preferably wherein the liquid is saturated with oxygen nanobubbles.

[0080] E34. The composition of any of embodiments 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, still more preferably at least 40, even more preferably at least 45 ppm, and most preferably at least 50 ppm.

[0081] E35. The composition of any one of embodiments 31 to 34, wherein the oxygen saturation of the liquid is at least 200%, preferably at least 300%, more preferably at least 400%, most preferably at least 500%.

[0082] E36. The composition of any one of embodiments 31 to 35, wherein the liquid is selected from an aqueous solution, preferably selected from water, fresh water, sea water, sterile water, culture medium and / or buffer.

[0083] E37. The composition of any one of embodiments 31 to 36, wherein the liquid is a culture medium comprising:

[0084] a) water, preferably sterile water;

[0085] b) nitrates, preferably alkali metal salts thereof, more preferably sodium nitrate;

[0086] c) dihydrogen phosphate, preferably an alkali metal salt thereof, more preferably sodium dihydrogen phosphate;

[0087] d) silicates, preferably alkali metal salts thereof, more preferably sodium silicate;

[0088] e) one or more trace metals, preferably inorganic salts thereof, more preferably said trace metals are selected from cobalt, copper, iron, manganese, molybdenum and / or zinc; and / or

[0089] f) one or more vitamins, preferably selected from vitamin B 12, biotin and / or thiamine.

[0090] E38. The composition of any one of embodiments 31 to 37, wherein no externally added carbohydrates are present in the liquid.

[0091] E39. The composition of any one of embodiments 31 to 38, wherein no additional carbohydrates are added to the liquid.

[0092] E40. The composition of any one of embodiments 31 to 3, wherein the algae is capable of mixed trophic metabolism, preferably wherein the algae is capable of both autotrophic and heterotrophic metabolism, even more preferably the algae has autotrophic metabolism when grown under light exposure and is capable of heterotrophic metabolism in the absence of light.

[0093] E41. The composition of any one of embodiments 31 to 40, wherein the algae is an obligate mixotroph, an obligate autotroph and a facultative heterotroph, a facultative autotroph and an obligate heterotroph, and / or a facultative mixotroph.

[0094] E42. The composition of any one of embodiments 31 to 41, wherein the algae is selected from the group of unicellular algae, preferably green algae, more preferably the algae is of the genus Chlorella, even more preferably the algae is Chlorella vulgaris.

[0095] E43. The composition of any one of embodiments 31 to 42, wherein the viable cell count in the liquid is 1 million cells / mL to 20 million cells / mL, preferably 2 million cells / mL to 18 million cells / mL, more preferably 5 million cells / mL to 15 million cells / mL, even more preferably 7 million cells / mL to 14 million cells / mL, still more preferably 10 million cells / mL to 13 million cells / mL, and most preferably 11 million cells / mL to 13 million cells / mL.

[0096] E44. The composition of any of embodiments 31 to 43, wherein the decay in viable cell count is at most 90%, preferably at most 80, more preferably at most 70%, even more preferably at most 60%, still more preferably at most 50%, still more preferably at most 40%, still more preferably at most 30%, still more preferably at most 20%, still more preferably at most 10%, still more preferably at most 5%, when compared to the decay in viable cell count of a control culture of the same algae over the same time period that is not hyperoxygenated.

[0097] E45. The composition of any one of embodiments 31 to 44, wherein there is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, still more preferably at most 2%, still more preferably at most 1% decrease in viable cell count, most preferably no decrease in viable cell count, when compared to a control culture of the same algae that is not hyperoxygenated within the following time period:

[0098] (i) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 2 months to 12 months, even more preferably 2 months to 10 months, even more preferably 3 months to 6 months, most preferably 3 months to 5 months; or

[0099] (ii) 1 day to 30 days, preferably 1 day to 25 days, more preferably 1 day to 15 days, even more preferably 1 day to 10 days, most preferably 4 days to 6 days; or

[0100] (iii) at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, still more preferably at least 5 months, even more preferably at least 6 months, still more preferably at least 7 months, and most preferably at least 8 months.

[0101] E46. The composition of any one of embodiments 31 to 44, wherein the viable cell count is increased by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, still more preferably at least 30%, most preferably at least 40% over the following time period when compared to a control culture of the same algae that is not hyperoxygenated:

[0102] (i) 1 day to 30 days, preferably 5 days to 25 days, more preferably 10 days to 25 days, even more preferably 15 days to 25 days, most preferably 18 days to 22 days, or

[0103] (ii) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 3 months to 12 months, even more preferably 3 months to 10 months, even more preferably 3 months to 8 months, still more preferably 3 months to 6 months, most preferably 3 months to 4 months.

[0104] E47. Use of the composition of any one of embodiments 31 to 46 for improving plant growth.

[0105] E48. The use according to embodiment 47, wherein the plant is selected from fruits, vegetables and / or crops, preferably the crops are agricultural plants grown for food or fiber.

[0106] E49. The use according to embodiment 48, wherein the fruit is selected from fruiting trees, berry bushes and / or pineapples.

[0107] E50. The use according to embodiment 48, wherein the vegetable is selected from garden vegetables, preferably tomatoes, potatoes, cucumbers, peppers, carrots, winter squash and / or pumpkins.

[0108] E51. The use according to embodiment 48, wherein the crop is selected from vegetable crops, sugar beets, corn, beans, hay, peanuts, cotton, hemp and / or tobacco.

[0109] E52. A method for maintaining or improving soil fertility and / or improving plant growth, comprising the step of applying the composition of any one of embodiments 31 to 46 to soil and / or plants.

[0110] E53. The method described in embodiment 52, wherein the soil is agricultural land, pasture, sports field, golf course and / or urban green space.

[0111] E54. The method of embodiment 52 or 53, comprising the step of diluting the composition of any one of embodiments 25 to 37 before applying the composition to the soil and / or plants.

[0112] E55. The method of any one of embodiments 52 to 54, wherein the composition is applied in an amount of 10 to 100,000 cells per square foot, preferably 100 to 90,000 cells per square foot, preferably 1,000 to 80,000 cells per square foot, more preferably 10,000 to 70,000 cells per square foot, still more preferably 20,000 to 60,000 cells per square foot, even more preferably 30,000 to 55,000 cells per square foot, still more preferably 35,000 to 55,000 cells per square foot, still more preferably 40,000 to 55,000 cells per square foot, even more preferably 45,000 to 55,000 cells per square foot, and most preferably 50,000 cells per square foot.

[0113] E56. A photobioreactor comprising means for supplying oxygen in the form of nanobubbles.

[0114] E57. The photobioreactor of embodiment 56, wherein the means for supplying oxygen in the form of nanobubbles comprises means for providing oxygen, preferably an oxygen concentrator, and means for generating nanobubbles, preferably a nanobubble generator.

[0115] E58. The photobioreactor of embodiment 57, wherein the means for providing oxygen is operably connected to the means for generating nanobubbles.

[0116] E59. The photobioreactor of any one of embodiments 56 to 58, comprising one or more of the following:

[0117] a) one or more reaction vessels, preferably made of fiberglass;

[0118] b) one or more storage devices;

[0119] c) one or more light sources, preferably LED light sources, more preferably tubular LED grow lights and / or LED bulbs;

[0120] d) one or more means for supplying dissolved gas to the photobioreactor;

[0121] e) oxygen concentrator;

[0122] f) pipelines; and / or

[0123] g) One or more valves.

[0124] E60. The photobioreactor of any one of embodiments 56 to 59, comprising a reaction vessel, preferably wherein the reaction vessel is a vessel characterized by one or more of the following:

[0125] (a) the container is liquid-impermeable;

[0126] (b) the container is cylindrical;

[0127] (c) the container has fixed side walls and a bottom;

[0128] (d) the container has a removable lid; and / or

[0129] (e) The container is made of a translucent material, preferably a translucent material containing glass fibers.

[0130] E61. The photobioreactor of any one of embodiments 56 to 60, comprising a light source, preferably a tubular LED grow light and / or an LED bulb, more preferably wherein the light source is characterized by one or more of the following:

[0131] 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

[0132] b) a light intensity of 1,000 lux to 20,000 lux, preferably 5,000 lux to 15,000 lux, more preferably 8,000 lux to 12,000 lux; and / or

[0133] c) a power of 1 W to 20 W, preferably 5 W to 20 W, more preferably 10 W to 20 W, even more preferably 10 W to 15 W, most preferably 13 W; and / or

[0134] d) the light source is positioned vertically and equidistantly around the one or more reaction vessels, preferably the light source is positioned at a distance of 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 from the one or more reaction vessels; and / or

[0135] e) The light source comprises a timer operable to turn the light source on and off, preferably, the timer is set to cycle the light source on for at least 1 hour to 24 hours and off for at least 1 hour to 12 hours, preferably on for 16 hours and off for 8 hours.

[0136] E62. The photobioreactor described in any one of embodiments 56 to 61, comprising one or more devices for supplying dissolved gas to the one or more reaction containers, the one or more devices being not devices for supplying oxygen in the form of nanobubbles, preferably, wherein the one or more devices for supplying dissolved gas to the one or more reaction containers comprise a pump operable to push the dissolved gas into the one or more reaction containers, a pipe for the dissolved gas to pass through, and a check valve, more preferably, wherein the one or more devices for supplying dissolved gas to the one or more reaction containers are aquarium stone bubblers.

[0137] E63. The photobioreactor of any one of embodiments 56 to 62, comprising a conduit, preferably wherein the conduit is made of a polymer, preferably polyethylene, and / or stainless steel, preferably wherein the conduit is made of a combination of polyethylene and stainless steel.

[0138] E64. The photobioreactor of any one of embodiments 56 to 63 comprises a conduit, preferably wherein the conduit is suitable for providing fluid connections of components of the photobioreactor.

[0139] E65. A system comprising one or more photobioreactors according to any one of embodiments 56 to 64.

[0140] E66. The system of embodiment 65, comprising at least two photobioreactors, preferably wherein the photobioreactors are fluidically connected.

[0141] E67. The system of embodiment 66, wherein the photobioreactors are arranged in a photobioreactor array.

[0142] E68. The system of embodiment 66 or 67, wherein the photobioreactors are connected in parallel.

[0143] E69. The system of any one of embodiments 65 to 68, comprising a valve, wherein the valve is operable to allow the at least one photobioreactor to be separated from the other photobioreactors by opening or closing the valve.

[0144] E70. The system of any one of embodiments 65 to 69, wherein the one or more photobioreactors are fluidically connected to a nanobubble generator.

[0145] E71. The system according to any one of claims 65 to 70, comprising a reservoir fluidically connected to the one or more photobioreactors.

[0146] E72. The system according to any of claims 65 to 71, comprising a drain line operably connected to the one or more photobioreactors for removing liquid from the one or more photobioreactors, preferably the drain line is made of polyethylene.

[0147] These and other features and advantages thereof will be apparent to those skilled in the art of propagating microalgae using photobioreactors from a careful reading of the detailed description of the invention and the illustrative embodiments taken in conjunction with the accompanying drawings. DETAILED DESCRIPTION

[0148] According to a first aspect, the present invention relates to a method for producing an algae composition comprising living algae and a liquid, comprising the following steps:

[0149] - adding oxygen to the liquid to provide a highly oxygenated liquid;

[0150] - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

[0151] The term "live" algae refers to live algae or living algae. Cultivating algae in a highly oxygenated liquid at least temporarily under light exposure so that the algae can reproduce refers to exposing the algae to light for a certain period of time, such as exposing the algae to light for a certain period of time by irradiating the reaction vessel in a photobioreactor. However, exposure to light does not need to be continuous, and there may be a temporary stage without exposure to light. Algae has an autotrophic metabolism when grown under light exposure, and is capable of heterotrophic metabolism in the absence of light. In particular, exposure to light should not be continuous, because algae grows more efficiently under circadian exposure, typically 16 hours in the case of light and 8 hours in the absence of light. The dark phase is when heterotrophic metabolism occurs. Algal reproduction refers to the cell growth and division of algae cells, thereby providing an increase in cell counts, but may also include a stable state of dividing cells and / or living cells as well as dead cells. Algae can be cultivated using typical conditions known to the skilled person for cultivating algae, such as those described in Chapter 2.3 "Algae production" in FAO Fisheries Technical Paper 361, Rome 1996, ISBN 92-5-103934-8, 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.

[0152] According to a specific embodiment, the method of the first aspect comprises the following steps:

[0153] - provide fluids;

[0154] - adding oxygen to the liquid to provide a highly oxygenated liquid;

[0155] - inoculating the highly oxygenated liquid with algae;

[0156] - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

[0157] In this way, fresh algae cultures can be provided.

[0158] According to another specific embodiment, the method of the first aspect comprises the following steps:

[0159] - providing a liquid comprising algae;

[0160] - adding oxygen to the liquid to provide a highly oxygenated liquid and / or adding a highly oxygenated liquid;

[0161] - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

[0162] In a specific embodiment of the method of the first aspect, one or more of the following applies:

[0163] a) the liquid has a viable cell count, wherein the viable cell count is from 1 million cells / mL to 20 million cells / mL, preferably from 2 million cells / mL to 18 million cells / mL, more preferably from 5 million cells / mL to 15 million cells / mL, even more preferably from 7 million cells / mL to 15 million cells / mL, still more preferably from 10 million cells / mL to 15 million cells / mL, and most preferably from 12 million cells / mL to 14 million cells / mL;

[0164] b) the decrease in viable cell count is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, still more preferably at most 50%, still more preferably at most 40%, still more preferably at most 30%, still more preferably at most 20%, still more preferably at most 10%, still more preferably at most 5% of the decrease in viable cell count of a control culture of the same algae over the same period of time that is not hyperoxygenated;

[0165] c) there is at most a 10% decrease in viable cell count, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, still more preferably at most 2%, yet more preferably at most 1%, and most preferably substantially no decrease in viable cell count when compared to a control culture of the same algae that is not hyperoxygenated within the following time period:

[0166] 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 2 months to 12 months, even more preferably 2 months to 10 months, even more preferably 3 months to 6 months, most preferably 3 months to 5 months,

[0167] or

[0168] 1 day to 30 days, preferably 1 day to 25 days, more preferably 1 day to 15 days, even more preferably 1 day to 10 days, most preferably 4 days to 6 days,

[0169] or

[0170] At least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, still more preferably at least 5 months, even more preferably at least 6 months, still more preferably at least 7 months, most preferably at least 8 months; and / or

[0171] d) the viable cell count is increased by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, still more preferably at least 30%, most preferably at least 40% when compared to a control culture of the same algae that is not hyperoxygenated, during the following time period:

[0172] 1 day to 30 days, preferably 5 days to 25 days, more preferably 10 days to 25 days, even more preferably 15 days to 25 days, most preferably 18 days to 22 days,

[0173] or

[0174] 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 3 months to 12 months, even more preferably 3 months to 10 months, even more preferably 3 months to 8 months, still more preferably 3 months to 6 months, most preferably 3 months to 4 months.

[0175] It is contemplated that in the context of the present invention, when comparing viable cell counts between a highly oxygenated liquid containing live algae and a non-highly oxygenated liquid containing live algae, the culture conditions are identical, including but not limited to culture temperature, salinity, pH, culture ingredients, light / dark cycles, etc. In a preferred embodiment, the viable cell count of the liquid is between 12 million cells / mL and 14 million cells / mL, particularly when the algae is harvested. In the same or further preferred embodiments, within the same time period, preferably within 5 days after harvest, there is a decay in viable cell count of at most 5% of the decay in viable cell count of a control culture of the same algae that is not highly oxygenated. In the same or further preferred embodiments, within a time period of 4 to 6 days, there is a decay in viable cell count of at most 4%. In the same or further preferred embodiments, there is substantially no decay in viable cell count over a time period of at least 2 months, most preferably 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, over a period of at least 2 months, there is substantially no decay in the viable cell count. Further preferably, when refrigerated, in particular at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, over a period of at least 8 months, there is substantially no decay in the viable cell count. Typically, at harvest, a small drop in cell count is observed, which is attributed to the shock caused by the sudden change in environment, but the heterotrophic algae are able to quickly start to recover and even reproduce. For example, within a period of 18 to 22 days, in particular, for example within 19 days, the viable cell count increases by at least 5%. In another example, within a period of 3 to 4 months, such as in particular 119 days, the viable cell count increases by at least 40%.

[0176] The skilled artisan is aware of methods for determining viable cell counts, such as using a hemacytometer or plate counting method (e.g., according to ASTM D5465) or its automated equivalent, such as the electric induction zone method of counting and sizing a single cell suspension according to ASTM F2149-16. Electric induction zone instruments are commonly referred to as Coulter counters. Other methods known to the skilled artisan include dye exclusion assays, colorimetric assays, fluorometric assays, photometric assays, and flow cytometric assays.

[0177] In a specific embodiment of the method according to the first aspect, oxygen is initially added only once. In an alternative, the method comprises one or more further steps of adding oxygen to the liquid to provide a highly oxygenated liquid and / or adding a highly oxygenated liquid. In another alternative, oxygen is added continuously to the liquid.

[0178] In a particular embodiment of the method according to the first aspect, the method is performed in a photobioreactor.

[0179] In a particular embodiment of the method according to the first aspect, the algae in the liquid are exposed to light:

[0180] a) light exposure at intervals of 1 to 12 hours for at least 1 to 24 hours, preferably at intervals of 8 hours for at least 16 hours;

[0181] b) at 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;

[0182] c) at a power of 1 W to 20 W, preferably 5 W to 20 W, more preferably 10 W to 20 W, even more preferably 10 W to 15 W, most preferably 13 W; and / or

[0183] d) is carried out under a light intensity of 1,000 lux to 20,000 lux, preferably 5,000 lux to 15,000 lux, more preferably 8,000 lux to 12,000 lux.

[0184] In this way, the algae are effectively illuminated with light to support autotrophic metabolism by photosynthesis. In addition, algae have / need a circadian light cycle for mixed nutrition, in particular switching from an autotrophic mode to a heterotrophic mode. Therefore, the light exposure scheme according to the present invention promotes switching between autotrophic mode and heterotrophic mode.

[0185] In a specific embodiment of the method according to the first aspect, the method further comprises:

[0186] a) monitoring viable cell counts; and / or

[0187] b) harvesting algae; and / or

[0188] c) concentrating the harvested algae; and / or

[0189] d) Storage of algae.

[0190] In a specific embodiment of the method according to any one of the first to fifth aspects, the method is operated in batch mode, fed-batch mode, semi-continuous mode or in continuous mode. This provides flexible handling of the growing algae, simple adaptation to expansion and different reactor types. The batch mode of operation allows the entire algae concentrate to be harvested from the photobioreactor. However, the inventors have found that it is more effective to perform a partial harvest, i.e. operate in a semi-continuous mode, and remove 40% to up to 70% of the tank and add highly oxygenated culture medium to refill the tank. The unharvested algae is then re-exposed to the highly oxygenated liquid (particularly culture medium and / or buffer) as it continues to grow. Cell growth is fast enough to allow harvesting at intervals of 4 to 5 days. The partial harvest process is effective because cleaning between partial harvests is not required for longer operating times. After 4 to 5 months, the rate of increase in cell counts from the tank decreases; at this point, the "tired" tank is taken offline, drained, cleaned and restarted.

[0191] According to a second aspect, the present invention relates to a composition comprising living algae and a liquid, wherein the composition is obtainable by a method according to any one of the first to fifth aspects.

[0192] According to a third aspect, the present invention relates to a composition comprising living algae and a liquid, wherein the liquid is highly oxygenated.

[0193] In a specific embodiment of any of the methods according to the first aspect or any of the compositions according to the second and third aspects, the living algae may be stored:

[0194] 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

[0195] 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

[0196] c) in the dark.

[0197] The living algae can be stored at a temperature of 0°C to 10°C and / or in the dark. The expression "stored in the dark" means that the living algae is kept in the dark, i.e. in the absence of light and / or in the absolute or substantially complete exclusion of light. However, refrigeration is not necessary, since the algae can grow over a wide temperature range. However, refrigeration advantageously prevents the growth of bacterial and protozoan contaminants. On the other hand, the algae can be stored at room temperature for up to two months, which advantageously simplifies storage conditions and transportation. For more extended storage, i.e. storage for a period of time longer than two months from harvest, refrigeration is recommended, i.e. it is recommended to keep 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.

[0198] In specific embodiments of any of the methods according to the first aspect or any of the compositions according to the second and third aspects, the live 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, still more preferably at least 6 months, even more preferably at least 7 months, still more preferably at least 8 months, most preferably up to 18 months without a significant decrease in the viable cell count compared to the viable cell count when the algae is harvested. In other or the same specific 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 live 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, still more preferably at least 5 weeks, even more preferably at least 6 weeks, still more preferably at least 7 weeks, most preferably at least 8 weeks, in particular at least 2 months, without a significant decay in the live cell count compared to the live cell count at the time of harvesting the algae. Thus, the present invention enables simplified and easy filling, storage, transportation and use of live algae, and thus facilitates their large-scale use as biostimulants.

[0199] In a specific embodiment 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 live algae and liquid or the liquid comprising live algae, respectively, is characterized by a viable cell count, wherein:

[0200] a) a viable cell count of 1 million cells / mL to 20 million cells / mL, preferably 2 million cells / mL to 18 million cells / mL, more preferably 5 million cells / mL to 15 million cells / mL, even more preferably 7 million cells / mL to 14 million cells / mL, still more preferably 10 million cells / mL to 13 million cells / mL, most preferably 11 million cells / mL to 13 million cells / mL;

[0201] b) a reduction in viable cell count of at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, still more preferably at most 50%, still more preferably at most 40%, still more preferably at most 30%, still more preferably at most 20%, still more preferably at most 10%, still more preferably at most 5% of the reduction in viable cell count of a control culture of the same algae that is not hyperoxygenated;

[0202] c) there is at most a 10% decrease in viable cell count, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, still more preferably at most 2%, yet more preferably at most 1%, and most preferably no decrease in viable cell count when compared to a control culture of the same algae that is not hyperoxygenated within the following time period:

[0203] 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 2 months to 12 months, even more preferably 2 months to 10 months, even more preferably 3 months to 9 months, most preferably 4 months to 8 months,

[0204] or

[0205] 1 day to 30 days, preferably 1 day to 25 days, more preferably 1 day to 15 days, even more preferably 1 day to 10 days, most preferably 4 days to 6 days,

[0206] or

[0207] At least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, still more preferably at least 5 months, even more preferably at least 6 months, still more preferably at least 7 months, most preferably at least 8 months; and / or

[0208] d) the viable cell count is increased by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, still more preferably at least 30%, most preferably at least 40% when compared to a control culture of the same algae that is not hyperoxygenated, during the following time period:

[0209] 1 day to 30 days, preferably 5 days to 25 days, more preferably 10 days to 25 days, even more preferably 15 days to 25 days, most preferably 18 days to 22 days,

[0210] or

[0211] 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 3 months to 12 months, even more preferably 3 months to 10 months, even more preferably 3 months to 8 months, most preferably 3 months to 6 months, still more preferably 3 months to 6 months, most preferably 3 months to 4 months.

[0212] It is contemplated that in the context of the present invention, in the case of comparing viable cell counts between a highly oxygenated liquid containing live algae and a non-highly oxygenated liquid containing live algae, the culture conditions are the same, including but not limited to culture temperature, salinity, pH, culture ingredients, light / dark cycle, etc. In a preferred embodiment, the viable cell count of the liquid is 12 million cells / mL to 14 million cells / mL, especially when the algae is harvested. In the same or further preferred embodiments, within the same time period, especially within 5 days after harvesting, there is a decay in viable cell count of up to 5% of the decay in viable cell count of a control culture of the same algae that is not highly oxygenated. In the same or further preferred embodiments, within a time period of 4 to 6 days, there is a decay of up to 4% of the viable cell count. In the same or further preferred embodiments, there is substantially no decay in viable cell count for a period of at least 2 months, most preferably for a period of 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, for a period of at least 2 months, there is substantially no decay in viable cell count. Further preferably, when refrigerated, in particular at a temperature of 0°C to 10°C, preferably 2°C to 8°C, more preferably 5°C to 7°C, there is substantially no decay in the viable cell count over a period of at least 8 months. Typically, at harvest, a small drop in cell count is observed, which is attributed to the shock caused by the sudden change in environment, but the heterotrophic algae are able to quickly start to recover and even reproduce. For example, within a period of 18 to 22 days, in particular such as within 19 days, the viable cell count increases by at least 5%. In another example, within a period of 3 to 4 months, such as in particular 119 days, the viable cell count increases by at least 40%.

[0213] The skilled person is aware of methods for determining viable cell counts, such as the electric induction zone method of counting and sizing single cell suspensions using a hemacytometer or plate counting (e.g., according to ASTM D5465) or its automated equivalent, such as the electric induction zone method according to ASTM F2149-16. Electric induction zone instruments are often referred to as Coulter counters. Other methods known to the skilled person include dye exclusion assays, colorimetric assays, fluorometric assays, photometric assays, and flow cytometric assays. In addition, cell viability is verified by inspections such as microscopic inspection: viable algal cells have a distinctive appearance of having green protoplasts, while non-viable cells appear as cell debris.

[0214] In a specific embodiment of any of the methods according to the first aspect or any of the compositions according to the second and third aspects, the liquid is highly oxygenated. The term "highly oxygenated" means more oxygenated than usual. Molecular oxygen has poor water solubility. In its aqueous solution, oxygen is present in (H2O) 20Inclusion complexes. The solubility of oxygen in water is temperature-dependent and pressure-dependent. At 25 ° C and 101.3 kPa, fresh water contains about 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 about 4.95 mL / L of molecular oxygen. In the context of the present invention, a highly oxygenated liquid refers to a liquid with a particularly high content of dissolved oxygen, preferably with a chemically and physically possible maximum value of dissolved oxygen. In other words, a highly oxygenated liquid is supersaturated with dissolved oxygen. It is expected that the technician understands that the highly oxygenated state of water changes with environmental conditions. In the context of the present invention, if not otherwise specified, the highly oxygenated 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 high oxygenation induces heterotrophic metabolism in algae capable of mixed nutrition. Advantageously, high oxygenation allows induction of heterotrophic metabolism in algae even in the absence of any externally added carbohydrates. Preferably, there are no externally added carbohydrates in the liquid. Preferably, the amount of carbohydrate in the liquid is less than 10 weight percent, preferably less than 8 weight percent, more preferably less than 6 weight percent, even more preferably less than 4 weight percent, still more preferably less than 2 weight percent, and most preferably less than 1 weight percent, based on the gross weight of the total culture medium. For example, when utilizing high oxygenation to induce heterotrophic metabolism in algae, it is not necessary to add additional carbohydrates to induce heterotrophic metabolism in algae. The liquid can be continuously maintained in a high oxygenation state, or the oxygen content of the liquid can decay over time. For example, in the context of the present invention, the oxygen content of fresh high oxygenation liquid is at least 50ppm. After 48 hours in a photobioreactor, the oxygen content of the liquid is at least 30ppm, and when harvesting at about 4 days, the oxygen content of the liquid is about 20ppm.

[0215] In any one of the methods according to the first aspect or in a specific embodiment of any one of the compositions according to the second aspect and the third aspect, in order to add oxygen to the liquid, oxygen nanobubbles are supplied to the liquid, and the oxygen can be present in the liquid as oxygen nanobubbles. The term "nanobubble" refers to bubbles with a diameter in the nanometer range, for example, in the range of 1nm to 1,000nm. Nanobubbles are generally characterized by 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), especially according to DLS of ASTM E3247-20, known to technicians. The size of the bubbles in the nanometer range provides unique physical, chemical and biological properties, such as high total bubble surface area, strong surface negative charge, thereby providing stability of the solution, minimizing degassing and / or agglomeration. Under standard conditions, the oxygen nanobubbles of the present invention can last up to 2 weeks in solution. Therefore, the algae concentrate harvested after 4 to 5 days of growth still has an elevated oxygen content. Nanobubbles promote increasing the concentration of dissolved gases in liquids. Nanobubbles can be formed using any gas and injected into any liquid. In the context of the present invention, nanobubbles are preferably formed by injecting oxygen into aqueous liquids such as sterile water, culture media or buffers. The use of oxygen nanobubbles allows the provision of high levels of dissolved oxygen (DO), which cannot be achieved by other conventional aeration methods that do not utilize oxygen nanobubbles. Therefore, oxygen nanobubbles provide a more efficient use of oxygen. Nanobubbles can be produced using a nanobubble generator.

[0216] In a specific embodiment of any of the methods according to the first aspect or any of the compositions according to the second and third aspects, 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, still more preferably at least 40, even more preferably at least 45 ppm, most preferably at least 50 ppm, and / or the oxygen saturation of the liquid is at least 200%, preferably at least 300%, more preferably at least 400%, most preferably at least 500%. In a preferred embodiment of the method according to the first aspect of the invention, in the step of inoculating the highly oxygenated liquid with algae, the liquid is highly oxygenated and has an oxygen level of at least 50 ppm and / or an oxygen saturation of at least 500%, preferably the liquid is highly oxygenated using oxygen nanobubbles. The oxygen level or oxygen saturation of the liquid, respectively, can be measured using any standard analytical method known to those skilled in the art, such as titration techniques, electrochemical analysis, for example using a Clark electrode or photochemical analysis. It is contemplated that corresponding sensors for determining the oxygen level in the liquid can be used. Without wishing to be bound by theory, the inventors believe that high oxygenation or oxygen levels, e.g., of at least 50 ppm and / or oxygen saturation, e.g., of about 500%, in the liquid induce heterotrophic metabolism in the algae. In this way, the induction of heterotrophic metabolism in the algae can be advantageously achieved even in the absence of any externally added carbohydrates in the liquid, preferably the culture medium and / or the buffer. For example, when high oxygenation is utilized to induce heterotrophic metabolism in the algae, no additional carbohydrate source needs to be added to the liquid.

[0217] In a specific embodiment of any one of the methods according to the first aspect or any one of the compositions according to the second and third aspects, the liquid is an aqueous solution, preferably selected from water, fresh water, sea water, sterile water, culture medium and / or buffer. In this way, the algae are provided with an optimal supply for growth and / or maintenance of viability.

[0218] In a specific embodiment of any of the methods according to the first aspect or any of the compositions according to the second and third aspects, the liquid is a culture medium, preferably, the culture medium comprises:

[0219] a) water, preferably sterile water;

[0220] b) nitrates, preferably alkali metal salts thereof, more preferably sodium nitrate;

[0221] c) dihydrogen phosphate, preferably an alkali metal salt thereof, more preferably sodium dihydrogen phosphate;

[0222] d) silicates, preferably alkali metal salts thereof, more preferably sodium silicate;

[0223] e) one or more trace metals, preferably inorganic salts thereof, more preferably the trace metals are selected from cobalt, copper, iron, manganese, molybdenum and / or zinc; and / or

[0224] f) one or more vitamins, preferably selected from vitamin B 12 , biotin and / or thiamine.

[0225] In some embodiments, no additional or external carbohydrates are added to the liquid to induce heterotrophic metabolism in the algae. As used herein, the term "carbohydrate" includes monosaccharides, such as glucose, fructose and / or galactose; disaccharides, such as sucrose, maltose, trehalose and / or lactose; oligosaccharides, such as raffinose and / or maltodextrin. In the context of the present invention, the expression "no additional or external carbohydrates are added" or "externally added carbohydrates" means that no carbohydrates are added to the liquid, which have not yet formed part of the liquid, in particular the culture medium, while it is not excluded that carbohydrates such as glucose may be formed during the culture, for example by autotrophic algae that are alive or dead and released into the liquid. According to the present invention, an example of a typical culture medium that can be used to grow algae is the commercially available Guillard's / F / 2 medium.

[0226] In the specific embodiment of any one of the methods according to the first aspect or in the composition according to the second aspect and the third aspect, algae can carry out mixed nutrition metabolism, preferably wherein algae can carry out both autotrophic metabolism and heterotrophic metabolism. In a specific embodiment, algae has autotrophic metabolism when growing under light exposure, and can carry out heterotrophic metabolism in the absence of light. In a specific embodiment, algae is a dedicated mixed nutrition organism, a dedicated autotrophic organism and a facultative heterotrophic organism, a facultative autotrophic organism and a dedicated heterotrophic organism, and / or a facultative mixed nutrition organism. In this way, algae can switch from autotrophy to heterotrophy, and vice versa. Mixed nutrition algae can adapt its metabolism to environmental conditions. In the context of the present invention, for example, when stored in the dark, algae can switch from autotrophy to heterotrophy during the growth or cultivation phase in a photobioreactor. Particularly useful algae in the context of the present invention are selected from the group of unicellular algae, preferably green algae, more preferably algae is Chlorella, and even more preferably algae is Chlorella vulgaris.

[0227] In one illustrative embodiment, the method according to the first aspect comprises:

[0228] - forming a growth medium by feeding sterile water through a nanobubble generator to add oxygen nanobubbles to the water to achieve hyperoxygenation or an oxygen level of at least 50 ppm and about 500% oxygen saturation;

[0229] - pumping the sterile highly oxygenated water through a pipe connected at one end to the nanobubble generator and at a second end to a stainless steel pipe running vertically into at least one photobioreactor to fill the photobioreactor with growth medium;

[0230] - adding an inorganic nutrient solution formulated to support the growth of microalgae to the at least one photobioreactor comprising sterile highly oxygenated water to form a growth medium, and inoculating the growth medium with a substantially homogeneous single culture of microalgae (preferably Chlorella) capable of mixotrophic metabolism at a concentration of 6 million cells / mL to 8 million cells / mL;

[0231] - Expose the inoculated growth medium to light for 16 hours at 8 hour intervals;

[0232] - continuous delivery of ambient air via an aquatic stone bubbler with a filter placed inside the PBR near the bottom;

[0233] - monitoring the growth rate of the microalgae by draining a volume of the growth medium from the PBR and counting the cells; and

[0234] - Harvesting the microalgae by draining the growth medium comprising the microalgae grown to a concentration of 12 million cells / mL into a container and storing the container at 6°C, the container preferably consisting of polyethylene.

[0235] According to a fourth aspect, the present invention relates to the use of the composition according to the second aspect or the third aspect for improving plant growth.

[0236] In a preferred embodiment of the use according to the fourth aspect, the composition stimulates plants, preferably plants selected from fruits, vegetables and / or crops, preferably crops are agricultural plants grown for food or fiber. Examples of fruits can be fruiting trees, berry bushes, pineapples without limitation. Examples of vegetables can be garden vegetables without limitation, preferably tomatoes, potatoes, cucumbers, peppers, carrots, winter squash and / or pumpkins. Examples of crops can be beets, sugar cane, corn, beans, hay, peanuts, tobacco, cotton, hemp and / or flax without limitation.

[0237] According to a fifth aspect, the present invention relates to a method for maintaining or improving soil fertility and / or for improving plant growth, comprising the following steps:

[0238] - applying the composition according to the second aspect or the third aspect to the soil and / or plants.

[0239] In a specific embodiment of the method according to the ninth aspect, the soil can be agricultural land, pasture, sports field, golf course and / or urban green space. Therefore, the composition of the present invention can be applied to a wide range of land. In other specific embodiments of the method according to the ninth aspect, the method includes a step of diluting the composition before applying the composition of any one of claims 25 to 37 to soil and / or plants. In particular, the composition is applied in the following amounts: 10 to 100,000 cells per square foot (per m 2 108 to 1,080,000 cells per square foot), preferably 100 to 90,000 cells per square foot (per m 2 1,080 to 970,000 cells per square foot), preferably 1,000 to 80,000 cells per square foot (per m 2 10,800 to 860,000 cells per square foot), more preferably 10,000 to 70,000 cells per square foot (per m 2 108,000 to 750,000 cells per square foot), and more preferably 20,000 to 60,000 cells per square foot (per m 2 215,000 to 645,000 cells per square foot), even more preferably 30,000 to 55,000 cells per square foot (per m 2 323,000 to 592,000 cells per square foot), and more preferably 35,000 to 55,000 cells per square foot (per m 2 378,000 to 592,000 cells per square foot), and more preferably 40,000 to 55,000 cells per square foot (per m 2 431,000 to 592,000 cells per square foot), even more preferably 45,000 to 55,000 cells per square foot (per m 2 484,000 to 592,000 cells per square foot (50,000 to 592,000 cells per square foot) is preferred. 2 Thus, the compositions and methods of the present invention allow effective maintenance or improvement of soil fertility and / or plant growth.

[0240] According to a sixth aspect, the present invention relates to a photobioreactor comprising means for supplying oxygen in the form of nanobubbles.

[0241] In a specific embodiment of the photobioreactor according to the sixth aspect, the means for supplying oxygen in the form of nanobubbles comprises a means for providing oxygen, preferably an oxygen concentrator, and a means for generating nanobubbles, preferably a nanobubble generator. The nanobubble generator allows efficient and uniform generation of nanobubbles, i.e. nanobubbles with a specific size distribution.

[0242] In a specific embodiment of the photobioreactor according to the sixth aspect, the photobioreactor comprises one or more of the following:

[0243] a) one or more reaction vessels, preferably made of fiberglass;

[0244] b) one or more storage devices;

[0245] c) one or more light sources, preferably LED light sources, more preferably tubular LED grow lights and / or

[0246] or LED bulbs;

[0247] d) one or more means for supplying dissolved gases to the photobioreactor;

[0248] e) oxygen concentrator;

[0249] f) pipelines; and / or

[0250] g) One or more valves.

[0251] In particular embodiments, the photobioreactor comprises a reaction vessel, preferably wherein the reaction vessel is a vessel characterized by one or more of the following:

[0252] a) the container is liquid-impermeable;

[0253] b) the container is cylindrical;

[0254] c) the container has fixed side walls and a bottom;

[0255] d) the container has a removable lid; and / or

[0256] e) The container is made of a translucent material, preferably a translucent material containing glass fibers.

[0257] In this way, a suitable reaction vessel for photobiosynthesis is provided. The reaction vessel of the photobioreactor according to the present invention can have any volume. The preferred volume is up to 750 liters. There is a problem that the increase in tank diameter causes the algae in the center of the tank to be shaded and slows down the growth. However, the inventors of the present invention unexpectedly found that the algae cell counts in the larger tanks rose as fast as the counts in the 350 liter photobioreactor with its smaller diameter (under comparable conditions). One explanation for this is that the algae is constantly mixed with the air bubbler. This prevents the algae cells from gathering in the middle of the tank away from the light source. Another explanation is that the algae are mixed nutrients, so they continue to grow in the dark. They do not rely solely on photosynthesis. The use of larger reaction vessels improves efficiency and productivity.

[0258] In particular embodiments, the photobioreactor preferably comprises a tubular LED grow light, more preferably wherein the light source is characterized by one or more of the following:

[0259] 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

[0260] Those

[0261] b) a light intensity of 1,000 lux to 20,000 lux, preferably 5,000 lux to 15,000 lux, more preferably 8,000 lux to 12,000 lux; and / or

[0262] c) a power of 1 W to 20 W, preferably 5 W to 20 W, more preferably 10 W to 20 W, even more preferably 10 W to 15 W, most preferably 13 W; and / or

[0263] d) the light source is positioned vertically and equidistantly around the one or more reaction vessels, preferably, the light source is positioned at a distance of 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 from the one or more reaction vessels; and / or

[0264] e) The light source comprises a timer operable to turn the light source on and off, preferably the timer is set to cycle the light source on for at least 1 hour to 24 hours and off for at least 1 hour to 12 hours, preferably on for 16 hours and off for 8 hours.

[0265] In this way, the algae can be efficiently exposed to light during growth in the photobioreactor.

[0266] In a specific embodiment, the photobioreactor comprises one or more means for supplying dissolved gas to the one or more reaction vessels, which is not a means for supplying oxygen in the form of nanobubbles, preferably, wherein the one or more means for supplying dissolved gas to the one or more reaction vessels comprises a pump operable to push the dissolved gas into the one or more reaction vessels, a conduit for passing the dissolved gas, and a check valve, more preferably, wherein the one or more means for supplying dissolved gas to the one or more reaction vessels is an aquarium stone bubbler. In this way, for example, ambient air or any other gas mixture can be effectively supplied to the liquid in the photobioreactor, in particular to the reaction vessels of the photobioreactor.

[0267] In a preferred embodiment, the photobioreactor comprises a conduit, preferably wherein the conduit is made of a polymer, preferably polyethylene, and / or stainless steel, preferably the conduit is made of a combination of polyethylene and stainless steel. In this way, liquids can pass through the photobioreactor. Stainless steel allows for safety, long equipment life, and easy sterilization of the equipment during maintenance, as corrosion is minimized. Preferably, the conduit is adapted to provide fluid connections of components of the photobioreactor, such as a fluid connection of the following devices: a device for supplying oxygen in the form of nanobubbles, preferably a nanobubble generator; one or more reaction vessels; one or more reservoirs; one or more devices for supplying dissolved gas to the photobioreactor; an oxygen concentrator; and / or one or more valves. For example, the conduit may provide a fluid connection of the reservoir with the reaction vessel and the device for supplying oxygen in the form of nanobubbles, and the conduit may be provided with one or more valves and ultimately allow the photobioreactor to be filled with sterile highly oxygenated water or the growth medium containing algae to be discharged from the photobioreactor.

[0268] According to a seventh aspect, the present invention relates to a system comprising one or more photobioreactors according to the sixth aspect.

[0269] In a specific embodiment, the system comprises at least two photobioreactors, preferably wherein the photobioreactors are fluidically connected and / or wherein the photobioreactors are arranged in a photobioreactor array. Furthermore, the bioreactors are preferably connected in parallel. In this way, the growth of algae can be easily scaled up, since a large number of algae can be cultivated in parallel.

[0270] In a specific embodiment, the system comprises a valve, wherein the valve is operable to allow the at least one photobioreactor to be separated from the other photobioreactors by opening or closing the valve. This allows individual filling and / or draining of the bioreactors in the system. The valve also allows control of access to and from individual photobioreactors and access between individual photobioreactors.

[0271] In a specific embodiment, one or more photobioreactors are fluidly connected to a nanobubble generator. In this way, oxygen nanobubbles can be effectively supplied to one or more bioreactors. Each of the one or more photobioreactors can have a nanobubble generator. In an alternative, a plurality of photobioreactors can have a nanobubble generator together. In this way, the supply of oxygen nanobubbles is centralized and the system is simplified. This achieves lower cost and easy maintenance of the system.

[0272] In a preferred embodiment, the system comprises a reservoir connected to one or more photobioreactor fluids. The reservoir may contain a liquid supplied to one or more photobioreactors, such as an aqueous solution, preferably selected from water, fresh water, seawater, sterile water, culture medium and / or an aqueous solution in a buffer. A reservoir may feed a single photobioreactor or all photobioreactors of the system. The reservoir may be, for example, a storage tank. Preferably, the reservoir is positioned in a manner that allows gravity feeding of the photobioreactor.

[0273] In a specific embodiment, the system includes a discharge line operably connected to one or more photobioreactors to remove liquid from one or more photobioreactors, preferably, the discharge line is made of polyethylene. The discharge line facilitates harvesting of algae and allows continuous algae production.

[0274] In an illustrative embodiment, in a system according to the seventh aspect, the system comprises at least one reservoir, preferably a storage tank; a photobioreactor; a nanobubble generator; a light source; one or more devices for supplying dissolved gas to one or more reaction vessels, preferably an aquarium rock bubbler,

[0275] - wherein the storage tank is connected via a valve carried on the tank to a first end of a pipe, the pipe preferably comprising stainless steel, the second end of the pipe being connected to the nanobubble generator, preferably the pipe being connected to a photobioreactor near the bottom of the photobioreactor, the pipe having a first valve positioned above the connection to the photobioreactor and a second valve positioned below the connection to the photobioreactor, for filling the photobioreactor with sterile highly oxygenated water or draining the growth medium containing microalgae from the photobioreactor via a section of the pipe connected at a second end to a drain line, the drain line preferably consisting of a polyethylene tube;

[0276] - wherein the system comprises more than one photobioreactor arranged in an array of photobioreactors, the photobioreactors being vertically connected in parallel such that the pipe connected at the first end to the nanobubble generator and at the second end to the exhaust line carries an additional length of pipe connected to the additional photobioreactor near the bottom of each of the additional photobioreactors, the additional pipe carrying a first valve positioned above the connection to the additional photobioreactor and a second valve positioned below the connection to the additional photobioreactor, the valves allowing the at least one photobioreactor to be separated from the other photobioreactors by opening or closing the valves.

[0277] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa.

[0278] In the claims and / or description, the use of the word "a / kind" when used in conjunction with the terms "comprising," "including," "having," or "containing," or any variations of these terms, may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0279] 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, although indicating specific embodiments of the present invention, are given by way of illustration only. In addition, it is expected that changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on this detailed description.

[0280] Example

[0281] To illustrate the advantages of the methods and compositions of the present invention, serial cell counts observed after cold storage of microalgae cultures propagated using a system with oxygen nanobubbles ("treated") and without oxygen nanobubbles ("untreated") were compared. Three representative experiments, two with paired controls (A and B), and a third without an untreated control (C) are described in Table 1 below. For the experiments, algae from a monoculture of Chlorella vulgaris was used. The monoculture of Chlorella vulgaris was purchased from the University of Texas Phycology Laboratory and had been propagated under standard conditions for growing algae. As culture medium, commercially available Guillard's / F / 2 medium was used.

[0282] Table 1. Algal cell counts were taken at harvest and later after a period of dark refrigerated storage. This compares algae grown using standard commercial methods and EnSoil algae grown using a proprietary technique that allows for continued algal growth while in storage.

[0283]

[0284] As can be seen from Table 1, the methods and compositions according to the invention allow for comparable or increased cell counts at harvest, less decay in cell counts 5 days after harvest, and even increased cell counts 19 and 119 days after harvest, respectively. This is attributed to the mixotrophic algae that are able to switch from autotrophy to heterotrophy. Mixotrophy was not induced in the untreated samples. Mixotrophic algae are able to switch from autotrophy to heterotrophy and are therefore able to survive in the dark, for example, when stored. The controls in the experiments according to A. and B. of Table 1 showed a drop in cell counts of almost 50%. However, while the untreated controls continued to drop in cell counts, the algae compositions of the invention advantageously dropped to a much smaller extent and even showed growth. The experiments according to B. and C. of Table 1 showed that the algae even continued to reproduce, indicating that they are healthy and alive. BRIEF DESCRIPTION OF THE DRAWINGS

[0285] 1. Figure 1 is a schematic diagram of a growing system showing the modification of a system for growing microalgae using a PBR growing system, including the addition of NBG and an attached oxygen concentrator.

[0286] 2. Figure 2 A preferred arrangement of eight parallel vertical PBRs 10 is shown, wherein valves 25 separate each PBR from the rest to prevent contamination of the growing system. Valve 25 is also operated to allow microalgae to be filled and harvested from a single PBR from a pipe at the bottom 45 of the PBR.

[0287] 3. Figure 3 Detailed diagram of a preferred embodiment of a growing system 90, which is improved by adding a nanobubble generator (NBG) 50 with an attached oxygen concentrator 55 so that an improved method can be used for the growth of microalgae. As shown, the growing system 90 uses two storage tanks 30, each with a capacity of 500 gallons (about 2000 liters). The storage tanks 30 are positioned higher than the PBR 10 to allow sterile water to be gravity fed to the PBR 10 after passing it through the nanobubble generator (NBG) 50 with an attached oxygen concentrator 55, thereby saturating the water with aerobic nanobubbles. Each PBR 10 has a tight removable lid 15 that can be opened to access the interior 40 of the PBR, which allows the addition of inorganic nutrient solutions and algae inoculants to the water after each PBR used in the system is filled. Figure 3 The PBR 10 in FIG. 1 is shaded to show growth medium 40 containing microalgae, some of which is at a lower level, reflecting the most recent harvest prior to replenishment with sterile highly oxygenated water and inorganic nutrient solution and re-inoculation.

[0288] Embodiments of the invention

[0289] With reference to the accompanying drawings, Figure 1 is a schematic diagram of a preferred embodiment of a growing system modified by the addition of a NBG with an attached oxygen concentrator. Preparation of growth medium for filling each PBR for growing microalgae using the modified method begins with the sterilization of water that is drawn from a municipal source or well and is not distilled water. The water is sterilized for use in the growing system by introducing ozone into the storage tank, as is sometimes used in commercial production of microalgae.

[0290] The sterile water gravity feed from the storage tank is passed through the nanobubble generator. An oxygen concentrator is attached to the nanobubble generator to supply oxygen for producing oxygen nanobubbles, thereby saturating the sterile water. At sea level and room temperature, the oxygen content of the water in the storage tank is 7ppm. After adding oxygen nanobubbles, the oxygen content of the water arriving at the PBR is at least 50ppm, and the oxygen saturation is about 500%. After this high oxygenation process, the water is then pumped to at least one PBR (a cylindrical container consisting of translucent fiberglass).

[0291] Figure 2 A detailed description of eight parallel vertical PBRs 10 used in a growth system is shown in FIG. Figure 3 As shown, the PBRs 10 each have a capacity of 80 gallons (300 liters). Each PBR 10 is provided with a suitable, removable cover 15 made of the same translucent material, preferably fiberglass, which protects the contents from contamination by airborne particles or dust, but which can also be removed to gain access to the PBR.

[0292] Figure 3 is a detailed view of a preferred embodiment of a growing system 90 modified by the addition of a NBG 50 with an attached oxygen concentrator 55. Figure 3 Not depicted Figure 2 In this embodiment, tap water fills at least one storage tank 30, which is set at a higher elevation than the top of the PBR 10 used in the growing system 90. Figure 1 As described in , sterile water is gravity fed from a storage tank 30 via a valve 25 carried on the tank and connected to a pipe 35 attached to a nanobubble generator or NBG 50. An oxygen concentrator 55 is attached 60 to the NBG 50 to add oxygen to produce oxygenated nanobubbles that are injected into the water as it passes through the NBG 50. After receiving the oxygen nanobubbles, the highly oxygenated water is pumped through a tube connected to the NBG 50. Figure 260 of the piping system shown in FIG. By opening and closing valves, the piping can be used to fill or empty a single PBR 10 that makes up the production system 90. The piping in the growth system 90 is composed of polyethylene and stainless steel pipes. Figure 2 As shown in , the piping can be arranged to deliver highly oxygenated water from the nanobubble generator to multiple PBRs that are vertically connected in parallel and can be separated by valves 25 installed on the pipes, which can be opened and closed to fill or empty any single PBR 10. Figure 2 and Figure 3 Both depict a PBR 10 filled with growth medium at different harvest levels.

[0293] like Figure 2 As shown in , highly oxygenated water is introduced via a pipe connected near the bottom 45 of each PBR. After filling the PBR, an inorganic nutrient solution (referred to in the industry as f / 2 or F / 2) is added to the sterile highly oxygenated water from the top of the PBR tank by removing the cap 15. At this point, the highly oxygenated growth medium is complete and ready to introduce the algae inoculum.

[0294] Immediately after forming the growth medium 40, the algal inoculum, i.e., 5 gallons (20 liters) of Chlorella vulgaris grown to a cell count of 6 million cells / mL to 8 million cells / mL, is poured into the PBR by removing the lid 15. The Chlorella vulgaris strain currently used by the authors was originally purchased from the Phycology Laboratory at the University of Texas and has been propagated using standard methods.

[0295] Although not shown in the figure, tubular LED growth lights with a wavelength of 440nM are vertically positioned around each PBR at regular intervals to provide light for photosynthesis. This lighting is set to cycle on for 16 hours and off for 8 hours to simulate a 24-hour day. The inoculated growth medium 40 in the PBR 10 is continuously mixed by introducing ambient air through an aquarium stone bubbler (not shown) positioned inside the bottom of the PBR. The stone bubbler is attached to an external pump installed on the outside of the PBR by a pipe, and the external pump includes a filter and an air dryer (not shown). The delivery of ambient air also provides the carbon dioxide required for photosynthesis.

[0296] Reference Figure 3, the growth of microalgae is monitored over time by extracting samples from the selected PBR 10 from the discharge line 70 attached to the pipe 35 connected to each PBR 10 that constitutes the system 90. Specifically, the valve 25 carried on the pipe 45 connected to the bottom of the PBR 10 is opened so that the growth medium and microalgae 40 can be discharged from the PBR via the pipe 35 for sampling and for harvesting. Count the cells with a hemocytometer or an automatic cell counter. After the cell count in the growth medium 40 exceeds 12 million cells / mL, the culture is a finished product "algae concentrate" ready for harvest, and the desired volume is discharged again through the pipe at the bottom 45 of the PBR 10 by opening the valve 25. The discharge line 70 is connected to a pipe for filling a polyethylene container, which is placed in a refrigerated storage at 6°C. The container can be of any size.

[0297] All algae concentrate can be harvested from a single PBR tank, or the harvest can be partial, typically withdrawing 10% to 20% of the volume from the PBR. After partial harvesting, the volume is replaced with new growth medium (i.e., highly oxygenated sterile water to which additional inorganic nutrients can be added). Since the algae remaining in the PBR continues to grow, partial harvesting does not require re-inoculation with additional algae culture. Typically, the cell count in the PBR returns to the level before harvesting within 4 to 5 days. Therefore, in the case of partial harvesting, up to 20% of the volume of the PBR can be taken at intervals of 5 days. In this case, the PBR can remain in active service for up to 5 months.

[0298] The dimensions of the PBR 10 may vary. A preferred embodiment of the system 90 includes a PBR configured to be 6 feet tall and having a capacity of 360 gallons (1350 liters). Figure 2 and Figure 3 A preferred assembly of multiple PBR tanks (10) is shown, wherein Figure 3 A photobioreactor array 55 is shown comprising eight PBRs 10 held on a rack 100. The rack 100 is constructed of metal, preferably extruded aluminum. The number of individual PBRs that can be used in a PBR array is limited only by the available space in the building housing the production system 90. The number of storage tanks can also be increased, again depending on the available space in the building.

[0299] The scope of the present invention is intended to include all modifications incorporating its main design features, and the scope and limitation of the present invention will be determined by the scope of the appended claims and their equivalents. Therefore, it should also be understood that the inventive concepts described herein are interchangeable and / or they can be used together in yet other arrangements of the present invention, and for those skilled in the art of using photobioreactors to propagate microalgae, other modifications and alternatives will be apparent from the description of the foregoing preferred embodiments without departing from the spirit or scope of the present invention.

Claims

1. A method for producing an algae composition comprising living algae and a liquid, comprising the steps of: - adding oxygen to the liquid to provide a highly oxygenated liquid; - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

2. The method according to claim 1, comprising the steps of: - provide fluids; - adding oxygen to the liquid to provide a highly oxygenated liquid; - inoculating the highly oxygenated liquid with algae; - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

3. The method according to claim 1, comprising the steps of: - providing a liquid comprising algae; - adding oxygen to the liquid to provide a highly oxygenated liquid and / or adding a highly oxygenated liquid; - cultivating said algae in said highly oxygenated liquid at least temporarily under light exposure to enable said algae to reproduce.

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

5. The method according to any one of claims 1 to 4, wherein the decay in viable cell count is at most 90%, preferably at most 80%, more preferably at most 70%, even more preferably at most 60%, still more preferably at most 50%, still more preferably at most 40%, still more preferably at most 30%, still more preferably at most 20%, still more preferably at most 10%, still more preferably at most 5% of the decay in viable cell count of a control culture of the same algae over the same period of time that is not hyperoxygenated.

6. The method according to any one of claims 1 to 5, wherein there is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, still more preferably at most 2%, yet more preferably at most 1% decrease in viable cell count, most preferably no decrease in viable cell count when compared to a control culture of the same algae that is not hyperoxygenated within the following time period: (i) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 2 months to 12 months, even more preferably 2 months to 10 months, even more preferably 3 months to 9 months, most preferably 4 months to 8 months; or (ii) 1 day to 30 days, preferably 1 day to 25 days, more preferably 1 day to 15 days, even more preferably 1 day to 10 days, most preferably 4 days to 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, still more preferably at least 5 months, even more preferably at least 6 months, still more preferably at least 7 months, and most preferably at least 8 months.

7. The method according to any one of claims 1 to 6, wherein the viable cell count is increased by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, still more preferably at least 30%, most preferably at least 40% over the following time period when compared to a control culture of the same algae that is not hyperoxygenated: (i) 1 day to 30 days, preferably 5 days to 25 days, more preferably 10 days to 25 days, even more preferably 15 days to 25 days, most preferably 18 days to 22 days, or (ii) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 3 months to 12 months, even more preferably 3 months to 10 months, even more preferably 3 months to 8 months, still more preferably 3 months to 6 months, most preferably 3 months to 4 months.

8. The method according to any one of claims 1 to 7, wherein the live 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, still more preferably at least 6 months, even more preferably at least 7 months, even more preferably at least 8 months, most preferably up to 18 months, and / or wherein the live 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, yet more preferably at least 5 weeks, even more preferably at least 6 weeks, yet more preferably at least 7 weeks, most preferably at least 8 weeks, in particular at least 2 months, without a significant decrease in the viable cell count compared to the viable cell count when the algae is harvested.

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

10. A method according to any one of claims 1 to 9, comprising more than one step of adding oxygen to the liquid to provide a highly oxygenated liquid and / or adding a highly oxygenated liquid.

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

12. The method according to any one of claims 1 to 11, wherein the method is performed 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 performed at intervals of 1 to 12 hours for at least 1 to 24 hours, preferably at intervals of 8 hours for at least 16 hours.

14. The method according to any one of claims 1 to 13, wherein exposing the algae in the liquid to light is performed at 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.

15. The method according to any one of claims 1 to 14, wherein exposing the algae in the liquid to light is performed at a power of 1 W to 20 W, preferably 5 W to 20 W, more preferably 10 W to 20 W, even more preferably 10 W to 15 W, most preferably 13 W.

16. The method according to any one of claims 1 to 15, wherein exposing the algae in the liquid to light is performed at a light intensity of 1,000 lux to 20,000 lux, preferably 5,000 lux to 15,000 lux, more preferably 8,000 lux to 12,000 lux.

17. The method according to any one of claims 1 to 16, wherein the method further comprises (i) monitoring the viable cell count; and / or (ii) harvesting the algae; and / or (iii) concentrating the harvested algae; and / or (iv) storing the algae.

18. The method according to any one of claims 1 to 17, wherein the living algae is stored (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 darkness.

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

20. The method according to any one of claims 1 to 19, 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, still more preferably at least 40, even more preferably at least 45 ppm, most preferably at least 50 ppm.

21. The method according to any one of claims 1 to 20, wherein 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%, most preferably at least 500%.

22. The method of any one of claims 2 to 21, wherein the highly oxygenated liquid inoculated with the algae has an oxygen level of at least 50 ppm and / or an oxygen saturation of at least 500%.

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

24. The method according to any one of claims 1 to 17, wherein the liquid is a culture medium, preferably the culture medium comprises (a) water, preferably sterile water; (b) nitrates, preferably alkali metal salts thereof, more preferably sodium nitrate; (c) dihydrogen phosphate, preferably an alkali metal salt 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 said trace metals are selected from cobalt, copper, iron, manganese, molybdenum and / or zinc; and / or (f) one or more vitamins, preferably selected from vitamin B 12 , biotin and / or thiamine.

25. The method of any one of claims 1 to 24, wherein no externally added carbohydrates are present in the liquid.

26. The method of any one of claims 1 to 25, wherein no additional carbohydrates are added to the liquid to induce heterotrophic metabolism in the algae.

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

28. The method according to any one of claims 1 to 27, wherein the algae is capable of mixotrophic metabolism, preferably wherein the algae is capable of both autotrophic and heterotrophic metabolism, even more preferably the algae has an autotrophic metabolism when grown under light exposure and is capable of heterotrophic metabolism in the absence of light.

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

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

31. A composition comprising living algae and a liquid, wherein the composition is obtainable by the method according to any one of claims 1 to 30.

32. A composition comprising living algae and a liquid, wherein the liquid is highly oxygenated.

33. The composition of claim 31 or 32, wherein the liquid comprises oxygen nanobubbles, preferably wherein the liquid is saturated with oxygen nanobubbles.

34. A 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, still more preferably at least 40, even more preferably at least 45 ppm, most preferably at least 50 ppm.

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

36. A composition according to any one of claims 31 to 35, wherein the liquid is selected from an aqueous solution, preferably selected from water, fresh water, sea water, sterile water, culture medium and / or buffer.

37. A composition according to any one of claims 31 to 36, wherein the liquid is a culture medium comprising: (a) water, preferably sterile water; (b) nitrates, preferably alkali metal salts thereof, more preferably sodium nitrate; (c) dihydrogen phosphate, preferably an alkali metal salt 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 said trace metals are selected from cobalt, copper, iron, manganese, molybdenum and / or zinc; and / or (f) one or more vitamins, preferably selected from vitamin B 12 , biotin and / or thiamine.

38. A composition according to any one of claims 31 to 37, wherein no externally added carbohydrates are present in the liquid.

39. A composition according to any one of claims 31 to 38, wherein no further carbohydrates are added to the liquid.

40. The composition according to any one of claims 31 to 39, wherein the algae is capable of mixotrophic metabolism, preferably wherein the algae is capable of both autotrophic and heterotrophic metabolism, even more preferably the algae has an autotrophic metabolism when grown under light exposure and is capable of heterotrophic metabolism in the absence of light.

41. The composition of any one of claims 31 to 40, wherein the algae is an obligate mixotroph, an obligate autotroph and a facultative heterotroph, a facultative autotroph and an obligate heterotroph, and / or a facultative mixotroph.

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

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

44. The composition of any one of claims 31 to 43, wherein the decay in viable cell count is at most 90%, preferably at most 80, more preferably at most 70%, even more preferably at most 60%, still more preferably at most 50%, still more preferably at most 40%, still more preferably at most 30%, still more preferably at most 20%, still more preferably at most 10%, still more preferably at most 5%, when compared to the decay in viable cell count of a control culture of the same algae over the same period of time that is not hyperoxygenated.

45. The composition of any one of claims 31 to 44, wherein there is at most a 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 4%, still more preferably at most 2%, yet more preferably at most 1% decrease in viable cell count, most preferably no decrease in viable cell count, when compared to a control culture of the same algae that is not hyperoxygenated within the following time period: (i) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 2 months to 12 months, even more preferably 2 months to 10 months, even more preferably 3 months to 6 months, most preferably 3 months to 5 months; or (ii) 1 day to 30 days, preferably 1 day to 25 days, more preferably 1 day to 15 days, even more preferably 1 day to 10 days, most preferably 4 days to 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, still more preferably at least 5 months, even more preferably at least 6 months, still more preferably at least 7 months, and most preferably at least 8 months.

46. ​​The composition of any one of claims 31 to 44, wherein the viable cell count is increased by at least 1%, preferably at least 5%, more preferably at least 10%, even more preferably at least 20%, still more preferably at least 30%, most preferably at least 40% over the following time period when compared to a control culture of the same algae that is not hyperoxygenated: (i) 1 day to 30 days, preferably 5 days to 25 days, more preferably 10 days to 25 days, even more preferably 15 days to 25 days, most preferably 18 days to 22 days, or (ii) 1 month to 24 months, preferably 1 month to 20 months, further 1 month to 18 months, even more preferably 2 months to 15 months, still more preferably 3 months to 12 months, even more preferably 3 months to 10 months, even more preferably 3 months to 8 months, still more preferably 3 months to 6 months, most preferably 3 months to 4 months.

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

48. Use according to claim 47, wherein the plant is selected from fruits, vegetables and / or crops, preferably the crops are agricultural plants grown for food or fiber.

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

50. Use according to claim 48, wherein the vegetables are selected from garden vegetables, preferably tomatoes, potatoes, cucumbers, peppers, carrots, winter squash and / or pumpkins.

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

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

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

54. A 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 soil and / or plants.

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

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

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

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

59. The photobioreactor of any one of claims 56 to 58, comprising one or more of the following: (a) one or more reaction vessels, preferably the one or more reaction vessels are made of fiberglass; (b) one or more storage devices; (c) one or more light sources, preferably LED light sources, more preferably tubular LED grow lights and / or LED bulbs; (d) one or more means for supplying dissolved gas to the photobioreactor; (e) oxygen concentrators; (f) pipelines; and / or (g) One or more valves.

60. The photobioreactor according to any one of claims 56 to 59, comprising a reaction vessel, preferably wherein the reaction vessel is a vessel characterized by one or more of the following: (a) the container is liquid-impermeable; (b) the container is cylindrical; (c) the container has fixed side walls and a bottom; (d) the container has a removable lid; and / or (e) The container is made of a translucent material, preferably a translucent material containing glass fiber.

61. The photobioreactor according to any one of claims 56 to 60, comprising a light source, preferably a tubular LED grow light and / or an LED bulb, more preferably wherein 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, most preferably 440 nm; and / or (b) a light intensity of 1,000 lux to 20,000 lux, preferably 5,000 lux to 15,000 lux, more preferably 8,000 lux to 12,000 lux; and / or (c) a power of 1 W to 20 W, preferably 5 W to 20 W, more preferably 10 W to 20 W, even more preferably 10 W to 15 W, most preferably 13 W; and / or (d) the light sources are positioned vertically and equidistantly around the one or more reaction vessels, preferably the light sources are positioned at a distance of 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 from the one or more reaction vessels; and / or (e) The light source comprises a timer operable to turn the light source on and off, preferably the timer is set to cycle the light source on for at least 1 hour to 24 hours and off for at least 1 hour to 12 hours, preferably on for 16 hours and off for 8 hours.

62. The photobioreactor according to any one of claims 56 to 61, comprising one or more devices for supplying dissolved gas to the one or more reaction vessels, the one or more devices being not devices for supplying oxygen in the form of nanobubbles, preferably, wherein the one or more devices for supplying dissolved gas to the one or more reaction vessels comprise a pump operable to push dissolved gas into the one or more reaction vessels, a pipe for passing dissolved gas, and a check valve, more preferably, wherein the one or more devices for supplying dissolved gas to the one or more reaction vessels are aquarium stone bubblers.

63. The photobioreactor according to any one of claims 56 to 62, comprising a conduit, preferably wherein the conduit is made of a polymer, preferably polyethylene, and / or stainless steel, preferably the conduit is made of a combination of polyethylene and stainless steel.

64. The photobioreactor according to any one of claims 56 to 63, comprising a conduit, preferably wherein the conduit is adapted to provide fluid connections of components of the photobioreactor.

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

66. The system of claim 65, comprising at least two photobioreactors, preferably wherein the photobioreactors are fluidically connected.

67. The system of claim 66, wherein the photobioreactors are arranged in a photobioreactor array.

68. The system of claim 66 or 67, wherein the photobioreactors are connected in parallel.

69. The system of any one of claims 65 to 68, comprising a valve, wherein the valve is operable to allow the at least one photobioreactor to be separated from other photobioreactors by opening or closing the valve.

70. The system of any one of claims 65 to 69, wherein the one or more photobioreactors are fluidly connected to a nanobubble generator.

71. The system of any one of claims 65 to 70, comprising a reservoir in fluid connection with the one or more photobioreactors.

72. The system of any one of claims 65 to 71, comprising a drain line operably connected to the one or more photobioreactors to remove liquid from the one or more photobioreactors, preferably the drain line is made of polyethylene.

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