NOVEL METHOD OF PRODUCING AN ALGAE COMPOSITION

Abstract

A system and method for growing microalgae capable of mixotrophic metabolism, preferably Chlorella sp. Microalgae grown in the system using the method are able to survive and grow in dark refrigeration, which allows the algae to be stored and transported for application as a live culture. In addition, the microalgae can be grown in sufficient quantities to be sold commercially for application to crops as a biostimulant.

Claims

1. A method of producing an algae composition that comprises viable algae and a liquid, comprising the step(s) of adding oxygen to the liquid to provide for a hyperoxygenated liquid; culturing the algae n the hyperoxygenated liquid at least temporarily under light exposure to allow propagation of the algae.

2. The method of claim 1 comprising the steps of providing a liquid; adding oxygen to the liquid to provide for a hyperoxygenated liquid; inocculating the hyperoxygenated liquid with algae; culturing the algae in the hyperoxygenated liquid at least temporarily under light exposure to allow propagation of the algae.

3. The method of claim 1 comprising the steps of providing a liquid comprising algae; adding oxygen to the liquid to provide for a hyperoxygenated liquid and/or adding hyperoxygenated liquid; culturing the algae in the hyperoxygenated liquid at least temporarily under light exposure to allow propagation of the algae.

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

5. The method of any one of claims 1 to 4, wherein a 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 which is not hyperoxygenated within the same period of time.

6. The method of anyone 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%, still more preferably at most 1%, most preferably no decay in viable cell count within a time period of (i) between 1 and 24 months, preferably between 1 and 20 months, more between 1 and 18 months, even more preferably between 2 and 15 months, still more preferably between 2 and 12 months, even more preferably between 2 and 10 months, even more preferably between 3 and 9 months, most preferably between 4 and 8 months; or (ii) between 1 and 3D days, preferably 1 and 25 days, more preferably 1 and 15 days, even more preferably 1 and 10 days, most preferably 4 and 6 days; or (iii) at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, 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; when compared to a control culture of the same algae which is not hyperoxygenated.

7. The method of any one of claims 1 to 6, wherein the viably cell count increases 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% within a time period of (i) between 1 and 30 days, preferably 5 and 25 days, more preferably 10 and 25 days, even more preferably 15 and 25 days, most preferably 18 and 22 days, or (ii) of between 1 and 24 months, preferably between 1 and 20 months, more between 1 and 18 months, even more preferably between 2 and 15 months, still more preferably between 3 and 12 months, even more preferably between 3 and 10 months, even more preferably between 3 and 8 months, still more preferably between 3 and 6 months, most preferably between 3 and 4 months, when compared to a control culture of the same algae which is not hyperoxygenated.

8. The method of any one of claims 1 to 7, wherein the viable algae can be stored at a temperature of between 0 C. and 10 C., preferably between 2 C. and 8 C., more preferably between 5 C. and 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 as long as 18 months and/or wherein the viable algae can be stored at room temperature, preferably at a temperature of between 10 C. and 30 C., preferably between 15 C. and 25 C., more preferably between 18 C. and 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 8 weeks, still more preferably at least 7 weeks, most preferably at least 8 weeks, in particular at least 2 months without significant decay in viable cell count compared to the viable cell count at the time of harvesting the algae.

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

10. The method of any one of claims 1 to 9, comprising more than one step of adding oxygen to the liquid to provide for a hyperoxygenated liquid and/or adding hyperoxygenated liquid.

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

12. The method of anyone of claims 1 to 11, wherein the method is carried out in a photobioreactor.

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

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

15. The method of any one of claims 1 to 14, wherein exposing the algae in the liquid to light is carried out at a power of 1 to 20 W, preferably 6 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, most preferably 13 W.

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

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

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

20. The method of anyone of claims 1 to 19, wherein the liquid has an oxygen level of at least 20 ppm, preferably at least 25 ppm, more preferably at least 30 ppm, even more preferably at least 35 ppm, still more preferably at least 40, even more preferably at least 45 ppm, most preferably at least 50 ppm.

21. The method of anyone of claims 1 to 20, wherein the liquid has an oxygen saturation of at least 100%, preferably of 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 hyperoxygenated liquid which is 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 of anyone of claims 1 to 22, wherein the liquid is an aqueous solution, preferably selected from water, fresh water, sea water, sterilized water, culture medium and/or buffer.

24. The method of any one of claims 1 to 17, wherein the liquid is a culture medium, preferably the culture medium comprising (a) water, preferably sterilized water; (b) nitrate, preferably an alkalimetal salt thereof, more preferably sodium nitrate; (c) dihydrogenphosphate preferably an alkalimetal salt thereof, more preferably sodium dihydrogenphosphate; (d) silicate, preferably an alkalimetal salt thereof, more preferably sodium silicate; (e) one or more trace metals, preferably inorganic salts thereof, more preferably the trace metals are selected from cobalt, copper, iron, manganese, molybdenmum and/or zink; and/or (f) one or more vitamins, preferably selected from vitamin B.sub.12, biotin, and/or thiamine.

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

26. The method of anyone of claims 1 to 25, wherein no additional carbohydrate is added to the liquid to induce heterotrophic metabolism in the algae.

27. The method 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 of anyone of claims 1 to 27, wherein the algae are capable of mixotrophic metabolism, preferably wherein the algae are capable of both autotrophic and heterotrophic metabolism, even more preferably the algae have autotrophic metabolism when grown under light exposure and are capable of heterotrophic metabolism in the absence of light.

29. The method of any one of claims 1 to 28, wherein the algae are obligate mixotroph, obligate autotroph and facultative heterotroph, facultative autotroph and obligate heterotroph, and/or facultative mixotroph.

30. The method of anyone of claims 1 to 29, wherein the algae are selected from the group of unicellular algae, preferably green algae, more preferably the algae are Chlorella, even more preferably the algae are Chlorella vulgaris.

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

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

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

34. The composition of anyone of claims 31 to 33, wherein the liquid has an oxygen level of at least 20 ppm, preferably at least 25 ppm, more preferably at least 30 ppm, even more preferably at least 35 ppm, still more preferably at least 40, even more preferably at least 45 ppm, most preferably at least 50 ppm.

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

37. The composition of any one of claims 31 to 36, wherein the liquid is a culture medium comprising (a) water, preferably sterilized water; (b) nitrate, preferably an alkalimetal salt thereof, more preferably sodium nitrate; (c) dihydrogenphosphate preferably an alkalimetal salt thereof, more preferably sodium dihydrogenphosphate; (d) silicate, preferably an alkalimetal salt thereof, more preferably sodium silicate; (e) one or more trace metals, preferably inorganic salts thereof, more preferably the trace metals are selected from cobalt, copper, iron, manganese, molybdenmum and/or zink; and/or (f) one or more vitamins, preferably selected from vitamin B.sub.12, biotin, and/or thiamine.

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

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

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

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

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

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

44. The composition of any one of claims 31 to 43, wherein a 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 which is not hyperoxygenated in the same period of time.

45. The composition of any one of claims 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%, most preferably no decay in viable cell count within a time period of (i) between 1 and 24 months, preferably between 1 and 20 months, more between 1 and 18 months, even more preferably between 2 and 15 months, still more preferably between 2 and 12 months, even more preferably between 2 and 10 months, even more preferably between 3 and 6 months, most preferably between 3 and 5 months; or (ii) between 1 and 30 days, preferably 1 and 25 days, more preferably 1 and 15 days, even more preferably 1 and 10 days, most preferably 4 and 6 days; or (iii) at least 1 month, preferably at least 2 months, more preferably at least 3 months, even more preferably at least 4 months, 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; when compared to a control culture of the same algae which is not hyperoxygenated.

46. The composition of any one of claims 31 to 44, wherein the viably cell count increases 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% within a time period of (i) between 1 and 30 days, preferably 5 and 25 days, more preferably 10 and 25 days, even more preferably 15 and 25 days, most preferably 18 and 22 days, or (ii) between 1 and 24 months, preferably between 1 and 20 months, more between 1 and 18 months, even more preferably between 2 and 15 months, still more preferably between 3 and 12 months, even more preferably between 3 and 10 months, even more preferably between 3 and $ months, still more preferably between 3 and 8 months, most preferably between 3 and 4 months, when compared to a control culture of the same algae which is not hyperoxygenated.

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

48. The use of claim 47, wherein the plants are selected from fruits, vegetables and/or crops, preferably the crops are agricultural plants grown for food or fiber.

49. The use of claim 48, wherein the fruits are selected from fruit-bearing trees, berry bushes, and/or pineapples.

50. The use of claim 48, wherein the vegetables are selected from garden vegetables, preferably tomatoes, potatoes, cucumbers, pepper, carrots, squash, and/or pumpkin.

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

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

53. The method of claim 52, wherein the soil is agricultural land, pastureland, a playing field, a golf course, and/or a civic green space.

54. The method of claim 52 or 53, comprising the step of diluting the composition of 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 between 10 and 100,000 cells per sq ft, preferably between 100 and 90,000 cells per sq ft, preferably between 1,000 and 80,000 cells per sq ft, more preferably between 10,000 and 70,000 cells per sq ft, still more preferably between 20,000 and 60,000 cells per sq ft, even more preferably between 30,000 and 55,000 cells per sq ft, still more preferably between 35,000 and 55,000 cells per sq ft, still more preferably between 40,000 and 55,000 cells per sq ft, even more preferably between 45,000 and 55,000 cells per sq ft, most preferably in an amount of 50,000 cells per sq ft.

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 comprise 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 are operably connected to the means for generating nanobubbles.

59. The photobioreactor of any one of claims 56 to 58, comprising one or more of (a) one or more reaction vessels, preferably made from fiberglass; (b) one or more reservoirs; (c) one or more light sources, preferably LED light sources, more preferably a tubular LED grow light and/or LED bulbs; (d) one or more means for supplying dissolved gas to the photobioreactor; (e) an oxygen concentrator; (f) piping; and/or (g) one or more valves.

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

61. The photobioreactor of any one of claims 56 to 60, comprising a light source, preferably a tubular LED grow light and/or a 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 to 550 nm, even more preferably 400 to 500 nm, most preferably 440 nm; and/or (b) a light intensity of between 1,000 and 20,000 lux, preferably 5,000 and 15,000 lux, more preferably of between 8,000 and 12,000 lux; and/or (c) a power of 1 to 20 W, preferably 5 to 20 W, more preferably 10 to 20 W, even more preferably 10 to 15 W, most preferably 13 W; and/or (d) the light source being positioned vertically and equidistant around the one or more reaction vessels, preferably the light source being positioned at a distance of 0.5 to 50 cm, preferably 1 to 10 cm, more preferably 2 to 8 cm, even more preferably 5 to 6 cm, most preferably 5 cm from the one or more reaction vessels; and/or (e) the light source comprising a timer operable to switch on and off the light source, preferably the timer is set to cycle said light source on for at least 1 to 24 hours and off for at least 1 to 12 hours, preferably on for 16 hours and off for 8 hours.

62. The photobioreactor of any one of claims 56-61, comprising one or more means for supplying dissolved gas to the one or more reaction vessels which is not the 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 dissolved gas into the one or more reaction vessels, tubing for the dissolved gas to pass through, 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.

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

64. The photobioreactor of any one of claims 56 to 63 comprising piping, preferably wherein the piping is adapted to provide for fluid connection of the of the photobioreactor's components.

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 in fluid connection.

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 valves, wherein the valves are operable to allow separation of said at least one photobioreactor from other photobioreactors by opening or closing said valves.

70. The system of any one of claims 65 to 69, wherein the one or more photobioreactors are in fluid connection with 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 discharge line operably connected to the one or more photobioreactors to remove liquid from the one or more photobioreactors, preferably the discharge line is made from polyethylene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLE

[0283] FIG. 1 is a schematic representation of the growing system illustrating the improvement of the system used to grow microalgae using a PBR growing system comprising addition of the NBG and attached oxygen concentrator.

[0284] FIG. 2 illustrates a preferred arrangement for eight PBRs (10) plumbed in parallel with valves (25) separating each PBR from others to prevent contamination for a growing system. The valves (25) are also operated to allow filling and harvest of microalgae from individual PBRs from piping at the base of the PBR (45).

[0285] FIG. 3 is a detailed drawing of a preferred embodiment of a growing system (90) improved by addition of a nanobubble generator (NBG) (50) with an attached oxygen concentrator (55), such that an improved method can be used for growth of microalgae. As shown, the growing system (90) uses two holding tanks (30), each having a capacity of 500 gallons (approximately 2000 liters). The holding tanks (30) are positioned higher than the PBRs (10) to allow gravity-feeding of the sterile water to the PBRs (10), after passing it through the nanobubble generator (NBG) (50) with an attached oxygen concentrator (55), to saturate the water with oxygen nanobubbles. Each PBR (10) has a close-fitting, removable lid (15) that can be opened to access the inside of the PBR (40), which allows for the addition of inorganic nutrient solution to the water as well as the algae inoculant once each PBR used in the system is filled. The PBRs (10) in FIG. 3 are shaded to show growth medium (40) containing microalgae, some of which are at a lower level reflecting recent harvest before replenishment of sterilized, hyperoxygenated water and inorganic nutrient solution and reinoculation.

DETAILED DESCRIPTION OF THE FIGURES AND ILLUSTRATIVE EMBODIMENTS

[0286] Referring to the drawings, FIG. 1 is a schematic representation of a preferred embodiment of a growing system improved by the addition of an NBG with an attached oxygen concentrator. Preparation of growth medium used to fill each PBR for growing microalgae using the improved method begins with sterilization of water drawn from a municipal source or a well and is not distilled water. The water is sterilized for use in the growing system, which is done by the introduction of ozone to the holding tank, as sometimes used in the commercial production of microalgae.

[0287] Sterilized water from the holding tank is gravity fed through a nanobubble generator. An oxygen concentrator is attached to the nanobubble generator to supply oxygen for the creation of oxygen nanobubbles to saturate the sterilized water. At sea level and room temperature, the oxygen content of water in the holding tank is 7 ppm. After addition of oxygen nanobubbles, the oxygen content of water reaching the PBR is at least 50 ppm, with an oxygen saturation of approximately 500%. Following this hyperoxygenation process, the water is then pumped to at least one PBR, a cylindrical container constructed of translucent fiberglass.

[0288] FIG. 2 is detailed depiction of eight PBRs (10) plumbed in parallel for use in a growing system. The growing system in depicted in FIG. 3. The PBRs (10) as illustrated each have a capacity of 80 gallons (300 liters). Each PBR (10) carries a fitted, removable lid (15) constructed of the same translucent material, preferably fiberglass, which prevents contamination of the contents by airborne particles or dust, but which can also be removed for access into the PBR.

[0289] FIG. 3 is a detailed drawing of a preferred embodiment of a growing system (90) improved by the addition of a NBG (50) with an attached oxygen concentrator (55). FIG. 3 does not depict the plumbing system described in FIG. 2. In this embodiment tap water fills at least one holding tank (30) placed at a higher elevation than the top of the PBRs (10) used in the growing system (90). As described in FIG. 1, sterilized water is gravity-fed from the holding tank (30) through a valve (25) carried on the tank and connected to pipe (35) that is attached to a nanobubble generator or NBG (50). An oxygen concentrator (55) is attached (60) to the NBG (50) to add oxygen to generate oxygen-filled nanobubbles that are injected into the water as it passes through the NBG (50). After receiving the oxygen nanobubbles, the hyperoxygenated water is pumped through tubing (60) connected to the plumbing system illustrated in FIG. 2. By opening and closing the valves the plumbing can be used to fill or drain individual PBRs (10) comprising the production system (90). The plumbing in the growing system (90) consists of polyethylene and stainless steel tubing. As shown in FIG. 2, the plumbing can be arranged to transport the hyperoxygenated water from the nanobubble generator to multiple PBRs plumbed in parallel and separable by valves (25) installed onto the piping that can be opened and closed to fill or drain any individual PBR (10). Both FIGS. 2 and 3 depict PBRs (10) filled with growth medium at different levels of harvest.

[0290] As illustrated in FIG. 2, the hyperoxygenated water is introduced through piping connected near the base of each PBR (45). After filling the PBR, inorganic nutrient solution, known in the industry as f/2 or F/2, is added to the sterilized, hyperoxygenated water from the top of the PBR tank by removing the lid (15). At this point the hyperoxygenated growth medium is complete and ready for introduction of algae inoculant.

[0291] Immediately after creating the growth medium (40) an algae inoculant, 5 gallons (20 liters) of Chlorella vulgaris grown to a cell count of 6-8 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.

[0292] While not shown in the figures, tubular LED grow lights having a wave length of 440 nM are positioned vertically around each PBR a 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 introduction of ambient air through an aquarium stone bubbler positioned inside the PBR at the base (not shown in the figures). The stone bubbler is attached by tubing to an external pump mounted outside of the PBR that contains a filter and an air dryer (not shown in the figures). The delivery of ambient air also provides carbon dioxide needed for photosynthesis.

[0293] In reference to FIG. 3, growth of the microalgae is monitored over time by drawing samples from the selected PBR (10) from the discharge line (70) attached to the piping (35) connected to each PBR (10) comprising the system (90). Specifically, the valve (25) carried on piping (45) connected to the base of the PBR (10) is opened so that the growth medium and microalgae (40) can be drained from the PBR through the piping (35) for sampling and for harvest. Cells are counted with a hemocytometer or automatic cell counter. Once the cell count in the growth medium (40) exceeds 12 million cells/mL, the culture is a finished algae concentrate ready for harvesting, again by opening the valve (25) to drain the desired volume of the through the piping at the base (45) of the PBR (10). A discharge line (70) is connected to the piping to be used for filling polyethylene containers that are placed into refrigerated storage at 6 C. The containers can be any size.

[0294] All of the algae concentrate can be harvested from a single PBR tank, or the harvest may be partial, typically drawing 10%-20% of the volume from the PBR. After partial harvest, the volume is replaced with new growth medium (i.e., hyperoxygenated, sterile water to which additional inorganic nutrients may be added). Partial harvest does not require re-inoculation with additional algae culture since the algae remaining in the PBR continue to grow. Typically, the cell count in the PBR recovers to the pre-harvest level in 4-5 days. Thus, with partial harvest, as much as 20% of the PBR's volume can be taken at 5-day intervals. In this case, a PBR can remain in active service for as long as 5 months.

[0295] The size of PBRs (10) can vary. The preferred embodiment of the system (90) includes PBRs constructed to 6 feet tall with a capacity of 360 gallons (1350 liters). FIGS. 2 and 3 show the preferred assembly of multiple PBR tanks (10), with FIG. 3 illustrating a photobioreactor array (55) involving eight PBRs (10) held on a rack (100). The rack (100) is constructed of metal, preferably extruded aluminum. The number of individual PBRs that may be used in the PBR array is limited only by available space of the building housing the production system (90). The number of holding tanks can be expanded as well, again depending upon available space of the building.

[0296] It is intended that the scope of the present invention include all modifications that incorporate its principal design features, and that the scope and limitation of the present invention are to be determined by the scope of the appended claims and their equivalents. It also should be understood, therefore, that the inventive concepts herein described are interchangeable and/or they can be used together in still other permutations of the present invention, and that other modifications and substitutions will be apparent to those skilled in the art of propagation of microalgae using photobioreactors from the foregoing description of the preferred embodiments without departing from the spirit or scope of the present invention.