Methods of cultivating algae

Abstract

This invention relates to a heterotrophic method for cultivating algae, particularly primitive red algae, to produce valuable pigments such as phycocyanin. The method relies on high oxygen saturation and controlled quantitative application of alkali to provide improved phycocyanin yield. The invention also relates to algal biomass, compositions comprising said biomass or phycocyanin, their use in various products, and reactors for cultivating algae.

Description

[0001] This invention is a divisional application of Chinese invention patent application 202180034883.6, entitled "Method for Cultivating Algae", filed on May 10, 2021. Technical Field

[0002] This invention relates to a heterotrophic method for cultivating algae, particularly for producing valuable pigments such as phycocyanin. The invention also relates to algal biomass produced by this method, the preparation of phycocyanin from said algal biomass, compositions comprising said biomass or phycocyanin, and their use in various products. Furthermore, the invention relates to an improved reactor for cultivating algae, especially under the conditions of this method. Background Technology

[0003] Phycocyanin is a key deep blue protein pigment used as a dye in various industries and in health supplements. The tertiary structure of phycocyanin is crucial for maintaining the color of its chromophore, phycobilin, and ensuring the stability of its linear porphyrin ring form. This stability is lost if denaturation occurs due to exposure to harsh physical conditions, and phycobilin changes its form, turning orange. This loss of color leads to product defects, limited application areas, and manufacturing difficulties.

[0004] Currently, phycocyanin is produced using *Arthrospira platensis* (Spirulina) under autotrophic conditions. However, this requires large, open culture beds that occupy significant space and require constant light. These culture conditions are highly dependent on seasonal fluctuations, and the cultures are susceptible to contamination by toxin-producing cyanobacteria. Furthermore, Spirulina itself has low biomass productivity and low phycocyanin production. The phycocyanin produced by this organism is also unstable and rapidly degrades into an unusable orange color as described above if not maintained under specific conditions.

[0005] Galdieria sulphuraria, a sulfur-loving primitive red algae, lives in acidic and thermophilic environments (such as hot springs), growing at temperatures of 35-55°C and pH levels between 0.0 and 4.0. Furthermore, it has been found to metabolize various organic carbon substrates under heterotrophic conditions while retaining light-harvesting pigments. These light-harvesting pigments include those with both natural edible and biotechnological importance, such as phycocyanin. Moreover, due to its natural environment, Galdieria sulphuraria produces phycocyanin that is more stable than that produced by Spirulina and can tolerate higher temperatures, acidic pH levels, and harsh chemical treatments. This makes it more useful in industrial production processes.

[0006] A 1995 paper sparked interest in the primitive red algae (Galdieria) by describing a "mutant" that overproduced phycocyanin, isolated from a primitive white strain (074W) observed during long-term conventional solid-medium subculturing (Gross and Schnarrenberger, 1995). Of particular importance was that this mutant retained phycocyanin production during heterotrophic growth in the dark. This was unexpected, as phycocyanin is a light-harvesting pigment that is activated by light. Several subsequent studies focused on high-density heterotrophic growth of this primitive red algae strain in conventional stirred-tank reactors (Sloth et al., 2006; Graverholt and Eriksen, 2007). Results showed that phycocyanin production levels varied considerably depending on the heterotrophic substrate, with glucose production being the highest and glycerol production relatively poor.

[0007] A key step in the biosynthetic pathways of chlorophyll and heme, which ultimately lead to the production of photosynthetic pigments such as phycocyanin, is the catalysis of coproporphyrinogen III (CPO) to protoporphyrinogen IX (PROTOGEN) by coproporphyrinogen III oxidase (CPO) (Sarian et al., 2016). It was later found that glucose stimulates CPO production more effectively than other carbon sources, which is associated with good phycocyanin yields when glucose is used as a growth substrate. In fact, CPO has been shown to have a high molecular oxygen requirement, as anaerobic growth leads to its expulsion from cells, thus halting the phycocyanin synthesis pathway (Sarian et al., 2016). This study partly explains the good phycocyanin yields when primitive red algae are cultured on glucose.

[0008] Therefore, current attempts to produce phycocyanin from primitive red algae using heterotrophic culture have demonstrated that oxygen and glucose are required in the dark to promote good phycocyanin production. The highest reported continuous heterotrophic culture using glucose as a carbon source has produced up to 861 mg / L. -1 .sky -1 The intracellular concentration of phycocyanin was 15.6 mg / g. -1 Cell dry weight.

[0009] These levels are inferior to those of primitive red algae cultivation strategies that induce phycocyanin production in a mesotrophic manner using added light. This type of cultivation still provides much higher levels of phycocyanin production and is therefore suitable for industrial production. For example, WO2017 / 050917 describes the use of red light bursts to stimulate phycocyanin production in primitive red algae growing on several different organic carbon substrates. Production capacity has been reported to be significantly increased compared to heterotrophic cultivation methods, with phycocyanin levels ranging from 29 to 100 mg / g. -1 Cell dry weight.

[0010] These studies conclude that, in order to produce adequate amounts of phycocyanin, primitive red algae must grow on glucose and / or in the presence of at least some light, especially if a non-glucose carbon source is used.

[0011] However, large-scale algal cultivation requiring light is expensive, and the use of glucose is not always economically feasible. It is desirable to produce high levels of phycocyanin from pristine red algal strains in a heterotrophic process that does not require any light, and to use alternative carbon sources if needed.

[0012] In addition, there are problems with the supply of nitrogen sources and the pH required to maintain the continuous production of phycocyanin in heterotrophic algae cultivation, especially when trying to use non-glucose carbon sources.

[0013] In the heterotrophic culture of algae such as sulfur-loving primitive red algae, it is known that the overall acidification of the culture medium occurs with cell growth, which is associated with a decrease in phycocyanin production. This is believed to be due to the expression of the AMT ammonium uptake transporter, which leaves protons outside the algal cells when ammonium is deprotonated to ammonia and absorbed. Furthermore, the production of phycocyanin requires large amounts of nitrogen, typically meaning that large amounts of nitrogen sources (such as ammonium sulfate) must be dissolved in the culture medium. This requirement is difficult to meet using glycerol as a carbon source. Glycerol is hygroscopic, removing all the uncomplexed water required to solubilize large amounts of nitrogen sources from the bulk culture medium, making common nitrogen sources (such as ammonium sulfate) insoluble at high concentrations.

[0014] Therefore, it is also desirable to produce high levels of phycocyanin from the original red algal species in a heterotrophic process that can use alternative carbon sources and maintain high nitrogen levels without negatively impacting pH.

[0015] One or more aspects of the present invention are intended to solve one or more of the above-mentioned problems. Summary of the Invention

[0016] According to a first aspect of the present invention, a method for heterotrophic cultivation of algae is provided, comprising:

[0017] (a) Culture algae in a medium containing a carbon source and a nitrogen source, wherein the majority of the carbon source is glycerol and the oxygen saturation of the medium is above 75%.

[0018] According to another first aspect of the present invention, a method for cultivating algae in a heterotrophic manner is provided, comprising:

[0019] (a) Culture algae in a medium containing carbon and nitrogen sources, wherein the oxygen saturation of the medium is above 75%.

[0020] In one embodiment, the carbon source is selected from glucose, sucrose, fructose, and glycerol, or any combination thereof. In one embodiment, the majority of the carbon source is glycerol. In one embodiment, glycerol is the sole carbon source. Therefore, suitably, the carbon source consists of glycerol.

[0021] According to a second aspect of the present invention, a method for heterotrophic cultivation of algae is provided, comprising:

[0022] (a) Algae are cultured in an acidic medium containing a carbon source and a single nitrogen source, wherein the carbon source is glycerol and the single nitrogen source is a base, which is added to the acidic medium during the culture process and alters the pH of the acidic medium.

[0023] According to another second aspect of the present invention, a method for cultivating algae in a heterotrophic manner is provided, comprising:

[0024] (a) Algae are cultured in an acidic medium containing a carbon source and a single nitrogen source, wherein the single nitrogen source is a base, which is added to the acidic medium during the culture process and alters the pH of the acidic medium.

[0025] In one embodiment, the method includes a prior step of providing an acidic culture medium comprising algae and a carbon source, wherein the acidic culture medium does not contain a nitrogen source. In one embodiment, the method includes a prior step of providing an acidic culture medium comprising algae and a carbon source, wherein the majority of the carbon source is glycerol, and wherein the acidic culture medium does not contain a nitrogen source. In one embodiment, the method further includes a prior step of adding a single nitrogen source to the acidic culture medium.

[0026] In one embodiment, a heterotrophic method for culturing algae is provided, comprising:

[0027] (a) Algae are cultured in an acidic medium containing a carbon source and a single nitrogen source, wherein the majority of the carbon source is glycerol, the oxygen saturation of the acidic medium is greater than 75%, and the single nitrogen source is a base, which is added to the acidic medium during the culture process and alters the pH of the acidic medium.

[0028] In another embodiment, a heterotrophic method for culturing algae is provided, comprising:

[0029] (a) Algae are cultured in an acidic medium containing a carbon source and a single nitrogen source, wherein the oxygen saturation of the acidic medium is greater than 75% and the single nitrogen source is a base, which is added to the acidic medium during the culture process and alters the pH of the acidic medium.

[0030] In one embodiment, the method includes a prior step of providing an acidic culture medium comprising algae and a carbon source, wherein the acidic culture medium has an oxygen saturation greater than 75% and wherein the acidic culture medium does not contain a nitrogen source. In another embodiment, the method includes a prior step of providing an acidic culture medium comprising algae and a carbon source, wherein the majority of the carbon source is glycerol, wherein the acidic culture medium has an oxygen saturation greater than 75%, and wherein the acidic culture medium does not contain a nitrogen source. In one embodiment, the method further includes a prior step of adding a single nitrogen source to the acidic culture medium.

[0031] In one implementation, the addition of alkali to the acidic culture medium is automated. Suitablely, this is automated via a pH control system as described herein.

[0032] According to a third aspect of the present invention, a method for producing phycocyanin is provided, the method comprising culturing algae according to the first or second aspect.

[0033] According to a fourth aspect of the invention, algal biomass is provided having a content of at least 25 mg / g. -1 The average intracellular concentration of phycocyanin per cell dry weight is less than 1 mg / g. -1 The average intracellular concentration of phycocyanin by cell dry weight.

[0034] According to a fifth aspect of the invention, algal biomass produced by the method of the first or second aspect is provided.

[0035] According to a sixth aspect of the present invention, a method for preparing phycocyanin from algal biomass of the fourth or fifth aspect is provided.

[0036] According to a seventh aspect of the present invention, a composition comprising algal biomass of the fourth or fifth aspect is provided.

[0037] According to an eighth aspect of the invention, a composition is provided comprising phycocyanin from algal biomass from the fourth or fifth aspect, or phycocyanin produced by the method of the sixth aspect.

[0038] According to a ninth aspect of the invention, a reactor for culturing algae is provided, comprising a container for containing a culture medium, wherein the container has a total volume and a working volume, wherein the working volume is a portion of the total volume operable for containing the culture medium, and wherein the ratio of the total volume to the working volume of the container is at least 2:1.

[0039] In one embodiment, the reactor includes an automated pH control system.

[0040] In one embodiment, the reactor also includes at least one pair of baffles.

[0041] The inventors have discovered a novel method for culturing algae (such as sulfur-loving primitive red algae) under heterotrophic conditions to produce a large quantity of valuable chemicals, particularly photosynthetic pigments (such as phycocyanin). The inventors have found that glycerol, in addition to traditional carbon sources, can be used as an alternative carbon source for this heterotrophic culture, and still provides high productivity as long as high oxygen saturation is maintained in the culture medium.

[0042] The inventors have discovered that high oxygen saturation in the culture medium solves the problem of insufficient pigment production in heterotrophic cultures when light is lacking.

[0043] Unbound by theory, the inventors believe that an oxygen saturation of at least 75% effectively compensates for the lack of light in heterotrophic cultures by providing algae with an alternative source of reactive oxygen species (ROS). These ROS are typically produced as a byproduct of photosynthesis under light, stimulating algae to produce protective pigments, such as phycocyanin. Without light, photosynthesis does not occur, and pigment production typically declines due to inactivation. However, providing a high oxygen saturation can mimic the stimulation typically provided by light. Furthermore, a high oxygen concentration means that the key enzyme CPO remains within the algal cells and can function effectively to produce these pigments.

[0044] The inventors also discovered that the effect of high oxygen saturation persists with alternative carbon sources (such as glycerol) and does not require the use of glucose, although glucose can be used to obtain similar good results.

[0045] The phycocyanin production rate of the method of the present invention is 567 times higher than that of existing autotrophic spirulina culture and at least 20 times higher than that of existing spirulina culture based on a cotrophic reactor.

[0046] The method of this invention further improves upon the closest heterotrophic culture of sulfur-loving primitive red algae in the art. The method of this invention can achieve yields exceeding 1.7 g / L. -1 .sky -1 The phycocyanin production rate was significantly higher than the previously reported highest level of 0.86 g / L using glucose as a carbon source. -1 .sky -1 (Graverholt and Eriksen 2007). Surprisingly, the inventors achieved this level of productivity using glycerol, a carbon source generally considered a inferior substrate compared to glucose. When using glucose, the method of the present invention is able to achieve even higher yields exceeding 1.75 g / L. -1 .sky -1 Phycocyanin production rate.

[0047] The inventors have also discovered novel alternative methods for culturing algae (such as sulfur-loving primitive red algae) under heterotrophic conditions to produce large quantities of valuable chemicals, particularly photosynthetic pigments (such as phycocyanin). The inventors have found that glycerol can be used as an alternative carbon source for this heterotrophic culture while still providing high productivity, provided that the nitrogen source is added to the culture when needed and is not premixed with glycerol before entering the bioreactor.

[0048] The inventors have discovered that quantitatively applying an alkaline nitrogen source to the culture medium during cultivation solves the typical problem of nitrogen substrate insolubility when using high concentrations of glycerol and other carbon sources in the initial culture medium, which are necessary to obtain high biomass concentrations and productivity. The inventors have found that by adding alkali to the culture medium as needed during cultivation, the solubility of the alkali in the medium is improved because the concentration of the carbon source (such as glycerol) decreases due to algal uptake. Furthermore, adding alkali to the culture medium as needed maintains the nitrogen requirements for algal growth and allows for fine-tuning of the nitrogen content in the culture medium. The inventors have further discovered that the same alkaline nitrogen source can also be used to control the pH of the culture medium, as it is added during cultivation, which prevents growth-mediated pH drops from leading to a lack of pigment production. Therefore, adding a single alkaline nitrogen source during cultivation solves two problems simultaneously: providing the algae with the required high concentration of nitrogen in the culture medium while controlling the pH of the medium, without requiring the use of any other nitrogen source in the culture medium.

[0049] The inventors have discovered that the quantitative application of a nitrogen source to the culture medium during cultivation can be linked to the pH of the medium, thereby automatically controlling the quantitative application of the nitrogen source. During algal cultivation, ammonium (aq) is directly converted into ammonia gas by algal cells via the AMT transporter, promoting nitrogen diffusion into the cytoplasm and causing proton release into the culture medium. Therefore, the pH does not decrease uncontrollably during cell growth. By using an automated quantitative application system that detects the pH in the culture medium, when the pH drops too low to maintain a constant pH and the high nitrogen concentration in the medium used for phycocyanin production cannot be maintained, alkali (such as ammonia gas) can be easily quantitatively applied to the culture medium. Furthermore, the inventors have found that using ammonia gas is a more reliable way to add alkaline nitrogen to the culture medium without creating high-pH localities due to poor mixing. Not bound by theory, the inventors believe that the large surface area of ​​ammonia gas bubbles allows for better mixing compared to adding liquid nitrogen sources. Moreover, the addition of the gas does not significantly change the volume of the aqueous phase of the culture medium, and therefore does not alter the concentration of other components in the medium.

[0050] The inventors have demonstrated herein a method for heterotrophic culture of sulfur-loving primitive red algae, and that this method, when using ammonia, can maintain stable pH and nitrogen concentration in the culture medium while producing up to 45 mg / g of ammonia.-1 Phycocyanin in cell dry weight.

[0051] Advantageously, the high oxygen saturation solution in the culture medium and the addition of a single alkaline nitrogen source during cultivation can be used together to provide a greatly improved method for heterotrophic cultivation of algae to produce valuable chemicals.

[0052] In summary, the method of the present invention is an improved heterotrophic method for producing valuable chemicals (such as phycocyanin) from algae, which is cheaper and more efficient and can be carried out on an industrial scale.

[0053] The features and implementation schemes of the foregoing aspects are described in the heading section below. Any feature or implementation scheme in any section may be combined with any aspect of the foregoing in any feasible combination.

[0054] The term “a” or “an” as used in this article refers to the singular or plural of a feature.

[0055] The term "comprising" as used herein means that the implementation includes at least the listed features, but may also include other features.

[0056] Unless otherwise stated, the term "about" as used herein may refer to + / -10% of the value, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, or + / -1% of the value.

[0057] illustrate

[0058] oxygen saturation

[0059] This invention relates to a method for culturing algae to produce the valuable chemical phycocyanin, using a culture medium with a high oxygen saturation level. Suitably, the oxygen saturation of the medium is above 75%, above 80%, above 85%, above 90%, or above 95%. A typical method for measuring oxygen saturation is to use a dissolved oxygen probe. Suitably, this would be a polarographic probe that does not interact with the acidic culture medium, as conventional current-based dissolved oxygen probes degrade in highly acidic media. Suitable probes could be, for example, the METTLER TOLEDO polarographic DO sensor InPro series probes or the Hanna Instruments HI-76407 / 2 polarographic dissolved oxygen probe.

[0060] Therefore, the oxygen saturation of the culture medium can be suitable at 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and 95%.

[0061] In one embodiment, the oxygen saturation of the culture medium is approximately 85%.

[0062] Suitablely, oxygen is dissolved in the culture medium. Suitablely, oxygen is dissolved in the culture medium by aeration (suitably with pure oxygen). Suitablely, aeration of the culture medium can be achieved by bubbling air or pure oxygen through the culture medium. Suitablely, aeration of the culture medium can be achieved by bubbling a mixture of air and pure oxygen through the culture medium. Suitablely, the ratio of air to pure oxygen can be adjusted. Suitablely, the mixture of air and pure oxygen can contain between 0-20% pure oxygen, suitablely between 0-15% pure oxygen, suitablely between 0-10% pure oxygen, suitablely between 0-5% pure oxygen. In one embodiment, aeration is achieved by bubbling only air through the culture medium.

[0063] Suitable, oxygen or air is bubbled through the culture medium at a flow rate of 0.1-5 VVM, suitable 0.2-4 VVM, suitable 0.3-3 VVM, suitable 0.4-2 VVM, suitable 0.5-1.5 VVM.

[0064] Suitablely, the flow rate of oxygen or air through the culture medium can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 VVM.

[0065] In one embodiment, oxygen or air is bubbled through the culture medium at a flow rate of 0.5-1.5 VVM.

[0066] Suitable for a bioreactor with a working volume of 3 L, oxygen or air is supplied at a rate of 1-5 L / min. -1 Appropriately, at 2-4 L / min -1 Appropriately, at approximately 3 L / min -1 The flow rate is bubbling through the culture medium.

[0067] Suitablely, oxygen or air is bubbled through the culture medium at a pressure between 0.5 and 5 bar, suitablely between 1 and 3 bar, or suitablely between 1 and 1.5 bar. Suitablely, oxygen or air is bubbled through the culture medium at a pressure of about 1.5 bar.

[0068] carbon source

[0069] This invention relates to a method for culturing algae in a culture medium containing a carbon source.

[0070] Appropriately, the carbon source is used by algae for growth. Therefore, appropriately, the carbon source can be called a carbon substrate. Appropriately, it serves as a carbon substrate that promotes algal growth.

[0071] Suitablely, the carbon source can be selected from any suitable carbon source capable of enabling algae growth. Suitablely, the carbon source can be selected from sugars, carbohydrates, or polyols. Suitable sugars can include monosaccharides, disaccharides, oligosaccharides, or polysaccharides. Suitable monosaccharides can include glucose, mannose, ribose, xylose, fructose, and galactose. Suitable disaccharides can include sucrose, lactose, isomaltose, trehalose, and maltose. Suitable oligosaccharides can include maltotriose, raffinose, stachyose, verbascose, maltodextrose, and cellulose glucose. Suitable polyols can include glycerol, erythritol, inositol, lactitol, mannitol, sorbitol, and xylitol. Suitable carbohydrates can include isomaltitol, cellulose, hemicellulose, pectin, starch, glycogen, chitin, chitosan, guar gum, β-glucan, alginate, gum arabic, β-mannan, inulin, tara gum, xanthan gum, carrageenan, polydextrose, and glucomannan, or any combination thereof.

[0072] Suitablely, the carbon source may include, or be composed of, any of the sugars, carbohydrates or polyols listed above in any combination.

[0073] In one embodiment, the carbon source comprises a sugar. In one embodiment, the carbon source is a sugar. In one embodiment, the carbon source comprises glucose, or fructose, or sucrose, or any combination thereof. In one embodiment, the carbon source is selected from glucose, fructose, sucrose, or any combination thereof. In another embodiment, the carbon source comprises a polyol. In another embodiment, the carbon source is a polyol. In one embodiment, the carbon source comprises glycerol. In one embodiment, the carbon source is glycerol, as described below. In one embodiment, the carbon source comprises glucose, fructose, sucrose, or glycerol, or any combination thereof.

[0074] Suitablely, one or more carbon sources may be used in the method of the present invention. In some embodiments, a mixture of carbon sources may be used. In some embodiments, a single carbon source is used. Suitablely, the term "carbon source" as used herein may be understood to mean all possible carbon sources.

[0075] For example, in one embodiment, a mixture of sugars can be used as a carbon source. In other words, the carbon source can comprise a mixture of sugars. For example, a mixture of glucose and fructose can be used as a carbon source. For example, a mixture of glucose and sucrose can be used as a carbon source. For example, a mixture of fructose and sucrose can be used as a carbon source. For example, a mixture of glucose, fructose, and sucrose can be used as a carbon source. Alternatively, a mixture of polyols and sugars can be used as a carbon source. In other words, the carbon source can comprise a mixture of polyols and sugars. Suitably, a mixture of glycerol and sugars can be used as a carbon source. For example, a mixture of glycerol and glucose can be used as a carbon source. For example, a mixture of glycerol and fructose can be used as a carbon source. For example, a mixture of glycerol and sucrose can be used as a carbon source.

[0076] In embodiments using a carbon source mixture, suitably, the mixture may contain multiple carbon sources, such as a first carbon source, a second carbon source, a third carbon source, etc. Suitably, the carbon source mixture used may be customized according to a specific algae, or may utilize existing resources. Suitably, in some embodiments, the carbon source is obtained from waste. Suitably, "waste" refers to a byproduct or unused product of another process. Suitably, such waste may contain a mixture of multiple carbon sources. Suitably, waste may contain multiple different carbon sources present in varying amounts. Suitably, the carbon sources contained in the waste and their amounts may vary depending on the source of the waste.

[0077] Suitablely, the carbon source mixture may include any combination of carbon sources. For example, in one embodiment, the carbon source mixture may include 50% of a first carbon source and 50% of a second carbon source. Other ratios are contemplated, such as a first carbon source:second carbon source ratio of 10:90, 25:75, 33:66, 75:25, 66:33, 90:10, or any ratio between these ratios. In one embodiment, the carbon source mixture may contain 33% of a first carbon source, 33% of a second carbon source, and 33% of a third carbon source. Other ratios are also contemplated.

[0078] In one implementation, a single carbon source is used. Suitably, in such an implementation, the single carbon source is glycerol.

[0079] Suitablely, the carbon source, or each carbon source, may be impure or pure; suitablely, the purity of the carbon source may be at least 60%, suitablely at least 65%, suitablely at least 70%, suitablely at least 75%, suitablely at least 80%, suitablely at least 85%, suitablely at least 90%, suitablely at least 95%, suitablely at least 99% pure. Suitablely, the total concentration of the carbon source in the culture medium is 10-1000 g / L. -1 Suitable, the concentration is 10 g / L -1 20 gL -1 30 gL -1 40 gL -1 50 gL -1 60 gL -1 70g.L -1 80 gL -1 90 gL -1 100 gL -1 110 gL -1 120 gL -1 130 gL -1 140 gL -1 150 gL -1160 gL -1 170 gL -1 180 gL -1 190 gL -1 200 gL -1 210 gL -1 220 gL -1 230 gL -1 240 gL -1 250 gL -1 300 gL -1 350 gL -1 400 gL -1 450 gL -1 500 gL -1 550 gL -1 600 gL -1 650 gL -1 700 gL -1 750 gL -1 800 gL -1 850 gL -1 900 gL -1 950 gL -1 1000 gL -1 .

[0080] Suitable for continuous culture, the total concentration of carbon source in the culture medium for algae is close to zero (0-5 g / L). -1 This is to prevent the inhibition of pigment production.

[0081] However, appropriately, the carbon source is 50-300 g / L. -1 The total concentration present in the fed medium is, appropriately, approximately 50 g / L. -1 60 gL -1 70 gL -1 80 gL -1 90 gL -1 100 gL -1 110 gL -1 120 gL -1 130g.L -1 140 gL -1 150 gL -1 160 gL -1 170 gL -1 180 gL -1 190 gL-1 200 gL -1 210g.L -1 220 gL -1 230 gL -1 240 gL -1 250 gL -1 260 gL -1 270 gL -1 280 gL -1 290g.L -1 300 gL -1 In one implementation, the carbon source is approximately 200 g / L. -1 The total concentration of [the substance] is present in the fed culture medium. Suitably, in this embodiment, the method of the invention is a continuous process.

[0082] Appropriately, the carbon source is added to the culture medium at a rate proportional to the cell concentration, growth rate, and feed concentration.

[0083] In some implementations, the carbon source is 10-30 g / L. -1 The total concentration of carbon source is present in the culture medium. In one embodiment, the carbon source is at a concentration of 20 g / L. -1 The total concentration of [the substance] is present in the culture medium. Suitablely, in this embodiment, the method of the invention is a batch process.

[0084] In other embodiments, the carbon source is 400-750 g / L. -1 The total concentration of carbon source is present in the fed culture medium. In one embodiment, the carbon source is at a concentration of 500 g / L. -1 The total concentration is present in the fed culture medium. Suitably, in this embodiment, the method of the present invention is a semi-continuous process.

[0085] glycerin

[0086] The present invention relates to a method for culturing algae in a culture medium containing a carbon source, wherein in some embodiments the majority of the carbon source is glycerol, and in some embodiments the sole carbon source is glycerol.

[0087] Suitablely, the glycerol may be laboratory-grade glycerol or waste glycerol. Suitablely, the glycerol may be biodiesel-grade glycerol. Suitablely, the glycerol may be a byproduct of an industrial process.

[0088] Suitablely, glycerol may be impure or pure, and suitablely, the purity of glycerol may be at least 60%, suitablely at least 65%, suitablely at least 70%, suitablely at least 75%, suitablely at least 80%, suitablely at least 85%, suitablely at least 90%, suitablely at least 95%, suitablely at least 99% pure.

[0089] In one implementation, the glycerol is waste glycerol. Suitablely, in such an implementation, the purity of the glycerol is between approximately 65-90%.

[0090] In one embodiment, the glycerol is laboratory grade. Suitably, in such an embodiment, the purity of the glycerol is at least 95%, suitably at least 99%.

[0091] Suitablely, the carbon source is formed from glycerol and other carbon-containing contaminants. Suitablely, the carbon source does not contain any other growth substrate besides glycerol, which may contain sugars such as glucose. Suitablely, the carbon source does not contain glucose.

[0092] Suitablely, a majority of the carbon source means that the majority of the carbon in the culture medium is provided by glycerol. Suitablely, a small portion of the carbon source may be provided by contaminants, suitable organic contaminants, or suitable carbon-containing contaminants. Suitablely, the majority of the carbon source is glycerol, and a small portion of the carbon source is provided by contaminants. Suitablely, more than 50% of the carbon source is glycerol. Suitablely, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of the carbon source is glycerol. Suitablely, less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the carbon source is contaminants. "Carbon source" means all organic matter containing carbon in a solution in a culture medium. In one embodiment, at least 90% of the carbon source is glycerol, and at most 10% of the carbon source may be contaminants. In one embodiment, at least 95% of the carbon source is glycerol, and at most 5% of the carbon source may be contaminants. In one embodiment, the carbon source is substantially composed of glycerol.

[0093] Suitablely, the carbon source is composed of glycerol. Therefore, suitablely, the only carbon source is glycerol.

[0094] Suitable, glycerin is used in doses of 10 to 1000 g / L. -1 The concentration present in the culture medium is, appropriately, 10 g / L. -1 20 gL -1 30 gL -1 40 gL -1 50 gL -1 60 gL -1 70 gL -1 80 gL-1 90 gL -1 100 gL -1 110 gL -1 120 gL -1 130 gL -1 140 gL -1 150 gL -1 160 gL -1 170 gL -1 180 gL -1 190 gL -1 200 gL -1 210 gL -1 220 gL -1 230 gL -1 240 gL -1 250 gL -1 300 gL -1 350 gL -1 400 gL -1 450 gL -1 500 gL -1 550 gL -1 600 gL -1 650 gL -1 700 gL -1 750 gL -1 800 gL -1 850 gL -1 900 gL -1 950 gL -1 1000 gL -1 .

[0095] Suitable for continuous culture, the concentration of glycerol in the culture medium for algae is close to zero (0-5 g / L). -1 This is to prevent the inhibition of pigment production.

[0096] However, glycerol is suitably used in doses of 10 to 1000 g / L. -1 The concentration present in the fed culture medium is, appropriately, 10 g / L. -1 20 gL -1 30 gL -1 40 gL -1 50 gL -1 60 gL -1 70 gL -1 80 gL-1 90 gL -1 100 gL -1 110 gL -1 120 gL -1 130 gL -1 140 gL -1 150 gL -1 160 gL -1 170 gL -1 180 gL -1 190 gL -1 200 gL -1 210 gL -1 220 gL -1 230 gL -1 240 gL -1 250 gL -1 300 gL -1 350 gL -1 400 gL -1 450 gL -1 500 gL -1 550 gL -1 600 gL -1 650 gL -1 700 gL -1 750 gL -1 800 gL -1 850 gL -1 900 gL -1 950 gL -1 1000 gL -1 .

[0097] In one implementation, glycerol is used at 10-30 gL. -1 The concentration of glycerol present in the culture medium is [specifically, 20 g / L]. In one embodiment, glycerol is [specifically, 20 g / L]. -1 The concentration of [the substance] is present in the culture medium. Suitablely, in this embodiment, the method of the invention is a batch process.

[0098] In one implementation, glycerol is used at 50-300 g / L. -1 The concentration of glycerol is present in the fed culture medium. In one embodiment, glycerol is present at 100 g / L. -1 The concentration of [the substance] is present in the fed culture medium. Suitablely, in this embodiment, the method of the present invention is a continuous process.

[0099] In one implementation, glycerol is used at a concentration of 400-750 g / L. -1 The concentration of glycerol is present in the fed culture medium. In one embodiment, glycerol is present at 500 g / L. -1 The concentration of [the substance] is present in the fed culture medium. Suitablely, in this embodiment, the method of the present invention is a semi-continuous process.

[0100] algae

[0101] This invention relates to a method for cultivating algae to produce valuable chemicals such as phycocyanin.

[0102] Suitablely, the algae are red algae. Suitablely, the algae are red algae of the class Cyanidiophyceae, suitablely red algae of the order Cyanidiales, suitablely red algae of the family Cyanidiaceae or Galdieriaceae, suitablely red algae of the genera Cyanidioschyzon, Cyanidium, or Galdieria, suitablely red algae of species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, or sulfur-loving Galdieria species.

[0103] Appropriately, the algae are sulfur-loving primitive red algae.

[0104] Suitable algae are sulfur-loving primitive red algae strains selected from ACUF141, SAG 108.79, 074G, 074G-G1, 074G-G2, CCMEE5587.1, SAG 108.71 and UTEX#2919.

[0105] In one implementation, the sulfur-loving primitive red algae strain is ACUF141 (Algal Collection University Federico II).

[0106] Suitable algae are rich in valuable chemicals. Suitable algae are rich in pigments. Suitable algae are rich in phycobiliproteins and / or carotenoids. Suitable phycobiliproteins and / or carotenoids are described below. Suitable algae are rich in phycocyanin.

[0107] Suitable, the algae can be wild-type algae or mutant algae. Suitable, mutant algae can be spontaneously generated or genetically modified.

[0108] Appropriately, algae are spontaneously generated mutants.

[0109] Suitablely, the algae are highly colored. Suitablely, the highly colored algae can be wild-type algae, or may contain genetic changes or mutations. In some embodiments, the algae may contain epigenetic changes, suitablely resulting in their high coloration, suitablely through an overproduction of phycocyanin. Suitablely, an overproduction of phycocyanin may mean that the algae produces more phycocyanin compared to wild-type algae of the same strain not cultured under the conditions of the method of the present invention.

[0110] Suitablely, the mutant algae possess mutations or genetic changes that favor the production of valuable chemicals. Suitablely, the mutant algae possess mutations or genetic changes that lead to an overproduction of valuable chemicals. Suitablely, the algae possess epigenetic changes that favor the production of valuable chemicals. Suitablely, the algae possess epigenetic changes that lead to an overproduction of valuable chemicals. Therefore, suitablely, the algae that can be mutant algae are overproducers of phycocyanin.

[0111] Suitable algae can be a single species of algae or a mixture of different species of algae.

[0112] Suitable algae can be a single species of algae or a mixture of different species of algae.

[0113] Suitable methods may be used to cultivate a single species of a given genus, several species of a single given genus, or several species of different given genera (at least two species of two different genera).

[0114] Suitable methods may be used to cultivate a single plant of a given species, several plants of a single given species, or several plants of different given species (at least two plants of two different species).

[0115] In one embodiment, the method of the present invention uses a single strain of algae, suitably the sulfur-loving primitive red algae strain ACUF141.

[0116] culture medium

[0117] This invention relates to a method for heterotrophic cultivation of algae, wherein the algae grow in a culture medium.

[0118] Appropriately, the initial culture medium used at the start of cultivation is the starting medium. Appropriately, the starting medium may contain different components compared to the culture medium used during cultivation. Appropriately, cultivation begins once all the components required for algal growth have been added to the starting medium. Appropriately, the culture medium used during cultivation can be simply referred to as "culture medium".

[0119] Suitably, in some embodiments of the invention, a starting culture medium that does not contain a nitrogen source may be provided. Suitably, a nitrogen source is added to the starting culture medium to begin culturing, and a further nitrogen source may be added during culturing as needed.

[0120] Therefore, suitably, algae are included in the culture medium during cultivation. Suitably, the culture medium contains nutrients, minerals, etc., necessary for algal growth. Suitably, the culture medium contains at least one nitrogen source and a carbon source, as defined in the method of the present invention and elsewhere herein. Suitably, the culture medium also contains a phosphorus source as described elsewhere herein.

[0121] Suitablely, the culture medium is a heterotrophic medium. Suitablely, the culture medium can be any suitable heterotrophic medium known to those skilled in the art, such as the medium described in Minoda et al. 2004.

[0122] Suitably, the culture medium can be an aqueous solution. Suitably, the culture medium may further include the addition of salts and minerals, such as magnesium salts, calcium salts, iron salts, boron salts, manganese salts, zinc salts, sodium salts, cobalt salts, and copper salts. Suitably, the culture medium may further include the addition of the following salts and minerals: for example, MgSO4, CaCl2, FeCl3, H3BO3, MnCl2, ZnCl2, Na2MoO2, CoCl2, and CuCl2.

[0123] Suitable, the concentration of the magnesium salt can be (for 10 g of carbon): at 0.3 g / L. -1 Up to 0.7 gL -1 Between, at 0.4gL -1 Up to 0.6 gL -1 Between, approximately 0.5 gL -1 .

[0124] Suitable, the concentration of the calcium salt can be (for 10 g of carbon): at 0.03 g / L. -1 Up to 0.07 gL -1 Between 0.04 gL -1 Up to 0.06 gL -1 Between, approximately 0.05 gL -1 .

[0125] Suitable, the concentration of the iron salt can be (for 10 g of carbon): at 0.01 g / L. -1 and 0.04 gL -1 Between 0.015 g / L -1 and 0.03 gL -1 Between 0.02 gL -1 and 0.03 gL -1 Between, approximately 0.025 g / L -1 .

[0126] Suitable, the culture medium may include the addition of trace elements, such as boron salts, manganese salts, zinc salts, sodium salts, cobalt salts, and copper salts. Suitablely, the concentration of each trace element (for 10 g of carbon) is 0.01 mg / L. -1 and 6 mg.L -1 between.

[0127] The culture medium may further contain a chelating agent, such as EDTA. Suitably, the concentration of EDTA may be (for 10 g of carbon): at 0.005 g / L. -1 and 0.025 gL -1 Between 0.01gL -1 and 0.02gL -1 Between, approximately 0.015 g / L -1 .

[0128] In one implementation, the concentration of this salt and mineral can be as follows (for 10 g of carbon (g / L)). -1 The trace elements are: MgSO4·7H2O 0.5, CaCl2·2H2O 0.056, FeCl3 0.028, and EDTA·Na2 0.016. In one embodiment, the concentration of the trace elements is (mg·L). -1 ): H3BO3 5.72, MnCl2.4H2O 3.64, ZnCl2 0.21, Na2MoO2.2H2O 0.78, CoCl2.6H2O 0.08, CuCl2 0.086.

[0129] The culture medium may further contain an antifoaming agent, such as antifoaming agent A (Sigma-Aldrich).

[0130] Suitablely, during the culture process, the culture medium may be replaced or added; suitablely, fresh culture medium may be used. Suitablely, this is carried out in a continuous process. Suitablely, this culture medium may be called a "feed" medium. Suitablely, the feed medium may contain any of the same components as the culture medium described above at the same concentration. However, in some cases, the feed medium may be different. For example, suitablely, as mentioned above, the feed medium may contain a higher concentration of carbon source than the culture medium.

[0131] Cultivation methods

[0132] This invention relates to a method for cultivating algae.

[0133] Appropriately, this method is heterotrophic. Therefore, appropriately, this method is performed in darkness. Appropriately, this method is performed entirely in darkness. Appropriately, this method is neither autotrophic nor polytrophic.

[0134] Suitable of the method, it can be batch, semi-continuous, or continuous. In one implementation, the method is continuous.

[0135] Suitablely, in the case of batch processing, each batch is carried out for 7 to 21 days, suitablely 10 to 19 days, and suitablely 14 to 16 days. Suitablely, the method is batched and carried out for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 days. In one implementation, the method is batched, with each batch carried out for 8 days.

[0136] Suitablely, in addition to cultivating algae, the method of the present invention may include further steps. Suitablely, these steps may be performed before or after the cultivation step.

[0137] Suitablely, the method of the present invention may include one or more prior steps.

[0138] Suitably, the method of the present invention may include the following prior steps: (a) providing an acidic culture medium comprising algae and a carbon source, wherein the acidic culture medium does not contain a nitrogen source. Optionally, the oxygen saturation of the culture medium is at least 75%.

[0139] Suitably, the method of the present invention may include the following prior steps: (a) providing an acidic culture medium comprising algae and a carbon source, wherein the majority of the carbon source is glycerol, and wherein the acidic culture medium does not contain a nitrogen source. Optionally, the oxygen saturation of the culture medium is at least 75%.

[0140] Suitablely, in such a step, the acidic culture medium can be regarded as the starting culture medium.

[0141] Suitably, the method may further include the step of adding a single nitrogen source to the acidic culture medium. Suitably, it is added to an acidic starting medium. Suitably, such a step initiates the culture by providing the algae with a nitrogen source for growth. Suitably, during the culture process, the single nitrogen source is then periodically added to the culture medium as needed.

[0142] Suitablely, the method of the present invention may further include one or more subsequent steps.

[0143] Suitably, the method of the present invention may include the step of collecting algal biomass. Suitably, the step of collecting algal biomass occurs after the culture step or each culture step. Suitably, algal biomass is collected from the culture medium. Suitably, algal biomass is collected from the culture medium by any suitable technique (such as gravity or low-pressure filtration, decantation, or sedimentation).

[0144] Suitable algal biomass contains high concentrations of one or more valuable chemicals, such as phycocyanin, as described below.

[0145] Suitablely, the algal biomass can be used as is, or valuable chemicals can be further extracted. Suitablely, the method of the present invention may further include the step of extracting valuable chemicals from the algal biomass. Suitablely, the method of the present invention may further include the step of extracting phycocyanin from the algal biomass. Suitable extraction techniques are known in the art, such as cell lysis. Suitable cell lysis techniques may include, for example, freeze-thaw lysis, high-pressure homogenization, or sonication. Suitablely, cell lysis releases lysates from the algal cells. Suitablely, the lysates contain valuable chemicals, such as phycocyanin.

[0146] Suitably, the method of the present invention may further include a purification step, wherein valuable chemicals are purified. Suitably, this purification step occurs after the extraction step, thus suitably purifying valuable chemicals from algal cell lysates. Therefore, suitably, phycocyanin is purified from algal cell lysates. Suitable purification techniques are known in the art, such as those commonly used in the preparation of phycocyanin derived from spirulina. A suitable purification technique may be ammonium sulfate precipitation.

[0147] Suitablely, the method may therefore include the steps of: (i) lysing algal cells and (ii) purifying valuable chemicals from the lysate.

[0148] Suitablely, the method may therefore include the steps of: (i) lysing algal cells by freeze-thaw lysis, high-pressure homogenization or sonication, and (ii) purifying valuable chemicals from the lysate by ammonium sulfate precipitation.

[0149] In one implementation scheme, the valuable chemical is phycocyanin.

[0150] Alternatively or additionally, the method of the present invention may include the step of drying algal biomass.

[0151] Additionally, the method of the present invention may include the step of reducing the particle size of the dried algal biomass, suitably by grinding the dried algal biomass. Suitably, this can produce a powder. The characteristics and uses of such powder are described elsewhere herein.

[0152] In one embodiment, the method of the present invention includes the following steps:

[0153] (b) Collect algal biomass from the culture medium.

[0154] In one embodiment, the method of the present invention includes the following steps:

[0155] (b) Collecting algal biomass from the culture medium; and

[0156] (c) Extract one or more valuable chemicals from algal biomass.

[0157] Suitablely, the extraction step may include one of the techniques described above. Suitablely, the method may further include a purification step as described above.

[0158] In one embodiment, the method of the present invention includes the following steps:

[0159] (b) Collecting algal biomass from the culture medium; and

[0160] (c) Dry algal biomass.

[0161] In one embodiment, the method of the present invention includes the following steps:

[0162] (b) Collecting algal biomass from the culture medium;

[0163] (c) dried algal biomass; and

[0164] (d) Grind the dried algal biomass into powder.

[0165] Nitrogen source and pH

[0166] This invention relates to a method for culturing algae in a culture medium containing a nitrogen source.

[0167] Suitably, the nitrogen source may be present in the initial culture medium. Alternatively or additionally, the nitrogen source may be added to the culture medium during cultivation. In any case, the nitrogen source is essential for algal growth. Suitably, in a first aspect of the invention, the nitrogen source is present in the initial culture medium, and optionally, an additional nitrogen source may be added during cultivation. Suitably, in a second aspect of the invention, the nitrogen source is not present in the initial culture medium, but is added to the culture medium during cultivation.

[0168] Appropriately, adding a nitrogen source to a culture medium can be referred to as applying a nitrogen source quantitatively to the culture medium.

[0169] For example, in one embodiment of the second aspect, a heterotrophic method for culturing algae is provided, comprising the following steps:

[0170] (a) Provide an acidic culture medium containing algae and a carbon source, wherein the majority of the carbon source is glycerol, and wherein the acidic culture medium does not contain a nitrogen source;

[0171] (b) Add a nitrogen source to the acidic medium to begin culturing;

[0172] (c) If necessary, add a nitrogen source to the acidic culture medium during the culture process;

[0173] The nitrogen source is an alkali, and the alkali is the only nitrogen source for algae and the only nitrogen source used to control the pH of the culture medium.

[0174] For example, in one embodiment of the second aspect, a heterotrophic method for culturing algae is provided, comprising the following steps:

[0175] (a) Provide an acidic culture medium containing algae and a carbon source, wherein the acidic culture medium does not contain a nitrogen source;

[0176] (b) Add a nitrogen source to the acidic medium to begin culturing;

[0177] (c) If necessary, add a nitrogen source to the acidic culture medium during the culture process;

[0178] The nitrogen source is an alkali, and the alkali is the only nitrogen source for algae and the only nitrogen source used to control the pH of the culture medium.

[0179] Appropriately, cultivation can only begin after the addition of a nitrogen source, as algal growth requires nitrogen. Therefore, appropriately, the addition of a nitrogen source allows algal growth and controls the initiation of cultivation.

[0180] Suitablely, step (b) is carried out in a reactor. Therefore, suitablely, a nitrogen source is added to the reactor containing an acidic culture medium. Therefore, suitablely, step (a) may include providing an acidic culture medium in the reactor.

[0181] Suitablely, the nitrogen source is alkaline. Suitablely, the nitrogen source is a base. Suitablely, the nitrogen source includes ammonia. Suitablely, the nitrogen source is selected from ammonia, ammonium hydroxide, ammonium sulfate, and ammonium phosphate. Suitablely, the nitrogen source can be a gas, liquid, or solid.

[0182] Suitablely, a nitrogen source is added to the culture medium during the culturing process. Suitablely, a nitrogen source is added to the culture medium when needed. Suitablely, if a gaseous nitrogen source is used, it is added to the culture medium by spraying. Suitablely, an automated pH control system is used to add the nitrogen source to the culture medium. Suitablely, the automated system is described elsewhere in this document.

[0183] Suitably, a nitrogen source is added to the culture medium when the pH decreases. Suitably, the pH of the culture medium drops below the optimal value for algal growth. Suitably, this optimal value is specific to the algae being cultured. In embodiments where the algae are or contain sulfur-loving proto-red algae, the optimal pH is approximately 2. In such embodiments, suitably, a nitrogen source is added to the culture medium when the pH drops below pH 2. Suitably, as described above, an automated pH control system is configured to measure the pH of the culture medium and add a nitrogen source to the medium as needed. Suitable automated pH control systems will be further described below.

[0184] In some embodiments, suitably, the starting medium does not contain a nitrogen source. Suitably, in such embodiments, as described below, an acid is used instead of a nitrogen source in the starting medium. Suitably, in such embodiments, the starting medium is acidic. Suitably, this is an embodiment of the second aspect of the invention.

[0185] If a gaseous nitrogen source is used, it is added to the culture medium at a flow rate between 0.1 and 1 L / min, preferably about 0.5 L / min.

[0186] If a gaseous nitrogen source is used, it should be added to the culture medium at a pressure difference of 0.5 to 2 bar, preferably about 1 bar, from the air or oxygen supply.

[0187] In one embodiment, the nitrogen source is ammonia.

[0188] Suitablely, the carbon to nitrogen ratio in the culture medium is controlled during the culture process. Suitablely, during the culture process, the carbon to nitrogen ratio is between 5:1 and 15:1, preferably between 10:1 and 15:1. In one embodiment, during the culture process, the carbon to nitrogen ratio is maintained at approximately 10:1.

[0189] Suitable, the nitrogen concentration in the culture medium is 0.5-5 g / L. -1 Between. Suitable, the concentration is approximately 0.5 g / L. -1 1g.L-1 1.5 gL -1 2 gL -1 2.5 gL -1 3 gL -1 3.5 gL -1 4 gL -1 4.5 gL -1 5 gL -1 .

[0190] In one embodiment, the nitrogen concentration in the culture medium is 0.5-1.5 g / L. -1 Suitablely, in such an implementation, the method is semi-continuous, and suitablely, the nitrogen source is ammonium hydroxide.

[0191] In one embodiment, the nitrogen concentration in the culture medium is 0.6-1.4 g / L. -1 Suitablely, in such an implementation, the method is semi-continuous, and suitablely, the nitrogen source is ammonia.

[0192] In one embodiment, the nitrogen concentration in the culture medium is approximately 5 g / L. -1 Suitablely, in such an implementation, the method is continuous, and suitablely, the nitrogen source is ammonium hydroxide.

[0193] In one embodiment, the nitrogen concentration in the culture medium is 1 g / L. -1 Suitablely, in such an implementation, the method is continuous, and suitablely, the nitrogen source is ammonia.

[0194] Properly, the culture medium is kept at a constant pH.

[0195] Suitablely, the pH of the culture medium is acidic. Suitablely, the pH of the culture medium is between 0 and 4. Suitablely, the pH can be about 0, 1, 2, 3, or 4. In one embodiment, the pH of the culture medium is 2. Suitablely, the pH of the acidic starting medium is about pH 2.

[0196] Suitable nitrogen sources can also be used to control the pH of the culture medium. Suitable nitrogen sources can be added to acidic culture media during the culture process to change the pH of the acidic medium, in addition to providing nitrogen for algal growth.

[0197] Suitablely, the nitrogen source is a base. Suitablely, the base is added to the acidic medium during the culture process and changes the pH of the acidic medium.

[0198] Suitably, the method of the present invention includes a single nitrogen source. Suitably, the single nitrogen source may be an alkali. Suitably, the single nitrogen source serves two functions: providing nitrogen for algal growth and controlling the pH of the culture medium.

[0199] Suitablely, the sole nitrogen source is a base. Therefore, suitablely, the base is the sole nitrogen source for algae and the sole nitrogen source for controlling the pH of the culture medium.

[0200] Suitablely, the method of the present invention does not contain any other nitrogen source.

[0201] In one embodiment, the sole nitrogen source is ammonia. Suitably, ammonia is the sole nitrogen source for algae and also the sole nitrogen source used to control the pH of the culture medium.

[0202] Suitablely, the concentration of the base can be between 1 M and 10 M, and suitablely, the concentration of the base is 10 M.

[0203] Suitablely, acid can be added to the culture medium to control the pH. Suitablely, any acid can be used. Suitablely, the acid is soluble in the culture medium; suitablely, the acid is soluble in glycerol. Suitablely, the acid is sulfuric acid.

[0204] Suitablely, the concentration of the acid can be between 1 M and 10 M, and suitablely, the concentration of the acid is about 5 M.

[0205] In some embodiments, acid is added to the culture medium at the start of the culture. Suitably, the acid is added to the starting culture medium. Suitably, the acid can be added instead of a nitrogen source. Suitably, this embodiment occurs if a nitrogen source is added as needed throughout the culture process, but is not present in the starting culture medium at the start of the culture. Suitably, this occurs in a second aspect of the invention. Suitably, in such an embodiment, the process is continuous.

[0206] Suitable, the concentration of acid in the initial culture medium is proportional to the concentration of carbon in the culture medium, and the ratio is comparable to the ratio of acid to nitrogen (i.e., 1:5, 1:10, 1:15 acid:carbon).

[0207] Suitably, in one embodiment, when the method is continuous, the acid is added at a molar ratio of 1:2 to the nitrogen source. Suitably, when the nitrogen source is ammonia or ammonium hydroxide, the acid is added at a molar ratio of 1:2 to the nitrogen source.

[0208] Suitablely, in implementation schemes where the nitrogen source is ammonium sulfate, no acid is added to the starting culture medium.

[0209] Phosphorus source

[0210] The present invention relates to a method for culturing algae in a culture medium, wherein suitably, the culture medium may further contain a phosphorus source.

[0211] Suitable for use as an absorbable phosphorus source.

[0212] Suitable phosphorus sources are selected from: phosphoric acid or phosphate salts. Suitable phosphate salts may be selected from: sodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), potassium dihydrogen phosphate (KH2PO4), potassium hydrogen phosphate (KHPO4), or mixtures thereof in any proportion.

[0213] In one implementation, the phosphorus source is potassium dihydrogen phosphate (KH2PO4).

[0214] Suitable, the phosphorus source is approximately 0.3 to 0.7 gL per 10 g of carbon. -1 Appropriately, approximately 0.4 to 0.6 gL per 10 g of carbon. -1 Appropriately, approximately 0.5 gL per 10 g of carbon. -1 The phosphorus source is present in the culture medium at a concentration of 0.54 g / L per 10 g of carbon. In one embodiment, the phosphorus source is present at a concentration of 0.54 g / L. -1 The concentration of is present in the culture medium.

[0215] temperature

[0216] This invention relates to a method for heterotrophic culture of algae in a culture medium.

[0217] Appropriately, the culture medium is maintained at a constant temperature. Appropriately, the culture medium is maintained at the optimal temperature for culturing the selected algae.

[0218] Suitablely, the temperature of the culture medium is warm. Suitablely, the temperature of the culture medium is between 35 and 55°C. Suitablely, the temperature can be approximately 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55°C. In one embodiment, the temperature of the culture medium is 42°C.

[0219] Phycocyanin

[0220] This invention relates to a method for cultivating algae to produce valuable chemicals. This invention also relates to a method for producing phycocyanin by cultivating algae.

[0221] Suitablely, the method of the first or second aspect can be used to produce any valuable chemical. Suitablely, it can be used to produce any valuable chemical derived from algae.

[0222] Suitablely, algae can produce proteins, lipids, and / or carbohydrates. Suitablely, algae can produce pigments. Suitablely, algae can produce pigments selected from phycobiliproteins and / or carotenoids.

[0223] Phycobiliproteins are water-soluble pigments found in phycobilisomes, which are photosynthetic complexes present in cyanobacteria and some microalgae. There are three types of phycobiliproteins: phycocyanin, phycoerythrin, and isophycocyanin.

[0224] Suitable algae can produce phycobiliproteins selected from phycocyanin, phycoerythrin, isophycocyanin, or any combination thereof.

[0225] The term carotenoids includes carotene (α, β, ε, γ, δ, and ζ-carotene, lycopene, and phytohexenoside) and lutein (astaxanthin, antheroxin, hesperidin, cryptoxanthin, canthaxanthin, diatomoxanthin, diatomoxanthin, ranunculusoxanthin, fucoxanthin, lutein, neoxanthin, pinocembrin, zeaxanthin, tubuloxanthin, zeaxanthin, and zeaxanthin). Carotenoids are fat-soluble pigments, and suitably, they are orange and yellow.

[0226] Suitablely, algae can produce carotenoids selected from carotene, lutein, or combinations thereof. Suitablely, algae can produce carotene selected from α, β, ε, γ, δ, ζ-carotene, lycopene, and phytohexenochrome, or any combination thereof. Suitablely, algae can produce lutein selected from astaxanthin, antheroxin, hesperidin, cryptoxanthin, canthaxanthin, diatomoxanthin, diatomoxanthin, ranunculusoxanthin, fucoxanthin, lutein, neoxanthin, pinocemaxanthin, zeaxanthin, tubuloxanthin, azadirachtin, and zeaxanthin, or any combination thereof.

[0227] Suitablely, algae can produce one valuable chemical, or more than one type of valuable chemical. Suitablely, algae can produce a mixture of valuable chemicals. Suitablely, algae can produce phycocyanin, fucoidin, and glycogen.

[0228] In one implementation, algae produce phycocyanin.

[0229] In one embodiment, the method of the present invention is a method for culturing algae to produce phycocyanin.

[0230] If appropriate, fucosides and / or glycogen can also be produced.

[0231] use

[0232] This invention relates to the cultivation of algae to produce valuable chemicals such as phycocyanin. Suitablely, these chemicals have a variety of uses, and suitablely, the algal biomass itself has a variety of uses.

[0233] Suitablely, algal biomass can be used as food or ingredient for humans or animals. Suitablely, algal biomass can be used as a nutritional supplement. Suitablely, algal biomass can be used as a protein or fiber supplement. Suitablely, algal biomass can also be used in cosmetics or pharmaceuticals.

[0234] On the other hand, the use of the algal biomass of the present invention as food, cosmetic or pharmaceutical is provided.

[0235] Suitable algae biomass for use in the food, cosmetics or pharmaceutical industries can be provided in liquid or powder form.

[0236] Suitablely, algal biomass can be processed. Suitablely, algal biomass can be dried. Suitablely, algal biomass can be dried and ground to form powder. Suitablely, the powder can have a particle size between 10 and 100 μm.

[0237] In another aspect of the invention, a powder formed from algal biomass of the fourth or fifth aspect is provided. Suitably, the powder is formed by drying and grinding the algal biomass.

[0238] Suitablely, the powder can be used as a food or ingredient for humans or animals. Suitablely, the powder can be used as a nutritional supplement. Suitablely, as a protein or fiber supplement.

[0239] Suitablely, algal biomass can also be digested. Suitablely, algal biomass can be enzymatically digested. Suitablely, algal proteins bound to polysaccharides are released. Suitablely, algal biomass can be enzymatically digested using one or more glycoenzymes. Suitable mixtures of glycoenzymes are available in the art, such as Viscozyme L.

[0240] In another aspect of the invention, an algal protein composition derived from algal biomass of the fourth or fifth aspect is provided. Suitably, the protein composition is obtained by enzymatic digestion of the algal biomass.

[0241] Algal protein can be used appropriately as a food or ingredient for humans or animals. It can also be used appropriately as a nutritional supplement.

[0242] Suitablely, phycocyanin produced by the method of the present invention can be used as a food or ingredient for humans or animals. Suitablely, phycocyanin can be used as a nutritional supplement. Suitablely, phycocyanin can also be used in cosmetics or pharmaceuticals.

[0243] On the other hand, the use of the phycocyanin of the present invention as food, cosmetic or pharmaceutical is provided.

[0244] Phycocyanin for use in the food, cosmetic, or pharmaceutical industries may be provided in liquid, powder, or capsule form.

[0245] In another aspect of the invention, a capsule comprising phycocyanin produced by the method of the invention is provided.

[0246] Suitablely, the capsule can be hard or soft. Suitablely, the capsule is formed from a digestible material. Any known digestible material can be used, such as: starch, cellulose, gelatin, carrageenan, collagen, hydroxypropyl methylcellulose, amylopectin.

[0247] Suitably, phycocyanin produced by the method of the present invention can be used as a dye. Suitably, phycocyanin can be used as a blue dye. Suitably, phycocyanin can be used as a natural blue dye. Suitably, it can be used as a dye in food or ingredients. Suitably, phycocyanin produced by the method of the present invention can be used as an edible dye. Alternatively, it can be used as a dye in cosmetics or pharmaceuticals.

[0248] According to another aspect of the invention, the use of algal biomass or phycocyanin produced by the method of the invention as food, food ingredient, nutritional supplement, cosmetic or pharmaceutical is provided.

[0249] According to another aspect of the invention, the use of phycocyanin produced by the method of the invention as a dye is provided.

[0250] Suitably, according to the seventh and eighth aspects of the invention, the algal biomass of the invention or the phycocyanin produced by the invention may be included in the composition. Suitably, the composition may also have a variety of uses, and suitably, the uses described herein also apply to the composition.

[0251] Suitablely, in addition to algal biomass or phycocyanin, the composition may contain other components and ingredients. Suitablely, these other components and ingredients are those typically included in compositions intended for the relevant purpose (e.g., as food, cosmetic, or pharmaceutical).

[0252] algal biomass

[0253] This invention relates to algal biomass with high intracellular phycocyanin concentrations.

[0254] Suitablely, algal biomass is produced by culturing algae. Suitablely, algae are cultured as described above. Suitablely, algae are cultured using the method of the present invention.

[0255] Suitable, algal biomass has a content of at least 25 mg / g. -1 The average intracellular concentration of phycocyanin by cell dry weight. Suitable, algal biomass contains 25-50 mg / g. -1The average intracellular concentration of phycocyanin per cell dry weight. Suitable, the algal biomass contains 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg.g -1 The average intracellular concentration of phycocyanin per gram of cell dry weight. Appropriately, the amount of phycocyanin per gram of cell dry weight may be referred to as the "phycocyanin concentration".

[0256] Suitablely, in one embodiment, the algal biomass has approximately 28 mg / g. -1 The average intracellular concentration of phycocyanin by cell dry weight. Suitablely, this algal biomass is produced through continuous culture.

[0257] Suitablely, in one embodiment, the algal biomass has 45 mg / g -1 The average intracellular concentration of phycocyanin by cell dry weight. Suitablely, this algal biomass is produced through batch or semi-continuous culture.

[0258] Suitablely, this algal biomass exhibits a reduced concentration of isophycocyanin compared to algal biomass produced by autotrophic or co-trophic methods. Isophycocyanin is a pigment considered an impurity in industrially produced phycocyanin due to its slightly different color. Food-grade phycocyanin obtained from Spirulina is commonly contaminated with isophycocyanin, causing the originally blue product to have a slightly greenish tint. Separating the two proteins, which have similar size and physical properties to phycocyanin, is challenging and typically requires expensive chromatographic techniques. Advantageously, the algal biomass produced by the heterotrophic cultivation method of this invention has very low levels of isophycocyanin.

[0259] Suitablely, the algal biomass of the present invention has a mean intracellular concentration of isophycocyanin lower than the mean intracellular concentration of phycocyanin. Suitablely, the algal biomass has a mean intracellular concentration of isophycocyanin lower than 1 mg / g. -1 The average intracellular concentration of phycocyanin by cell dry weight. Suitablely, algal biomass may have an undetectable average intracellular concentration of heterophycocyanin.

[0260] Unbound by theory, the inventors believe that since phycocyanin and heterophycocyanin are usually light-induced, the heterotrophic culture of this invention, in which phycocyanin is induced by high oxygen concentration, provides a purer phycocyanin product after conventional extraction techniques.

[0261] Suitablely, the method of the present invention produces algal biomass. Suitablely, the method of the present invention produces at least 35 g / L. -1 .sky -1 Algal biomass. Suitablely, the method of the present invention produces 35 to 45 g / L of algal biomass. -1.sky -1 Algal biomass. Suitably, the method of the present invention produces 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 g / L. -1 .sky -1 The amount of algal biomass produced daily can be referred to as "biomass productivity".

[0262] Suitablely, in one embodiment, the method of the present invention produces approximately 42 gL. -1 .sky -1 Algal biomass.

[0263] Suitably, the method of the present invention produces phycocyanin. Suitably, the method of the present invention produces at least 1 g / L. -1 .sky -1 Phycocyanin. Suitablely, the method of the present invention produces 1-2.5 g / L. -1 .sky -1 Phycocyanin. Suitably, the method of the present invention produces 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 g / L. -1 .sky -1 Phycocyanin. Appropriately, the amount of phycocyanin produced daily can be referred to as "phycocyanin productivity".

[0264] Suitablely, in one embodiment, the method of the present invention produces at least 1.7 gL. -1 .sky -1 Phycocyanin. Suitably, in one embodiment, when glucose is used as the carbon source, the method of the present invention produces 1.75 g / L. -1 .sky -1 Phycocyanin. Suitablely, even when using more challenging carbon sources such as glycerol, the method of the present invention produces at least 1.5 g / L. -1 .sky -1 .

[0265] Suitablely, the phycocyanin produced by the method of the present invention and contained in algal biomass is thermally stable. Suitablely, the phycocyanin is thermally stable at temperatures up to 65°C.

[0266] Suitably, the phycocyanin produced by the method of the present invention and contained in algal biomass is pH stable. Suitably, the phycocyanin is pH stable between pH 2 and pH 8.

[0267] Suitablely, algal biomass can further have a high concentration of protein. Suitablely, algal biomass can contain 26-32% protein. Suitablely, this protein can be used as food.

[0268] Suitable algal biomass can further have a high concentration of insoluble carbohydrates. Suitable algal biomass can contain 63-78% carbohydrates. When algal biomass is used as food, these insoluble carbohydrates can provide fiber.

[0269] reactor

[0270] This invention relates to a reactor for cultivating algae.

[0271] Suitablely, the reactor is used to cultivate algae to produce valuable chemicals, suitablely phycocyanin. Suitablely, the reactor is used to cultivate algae as defined above.

[0272] Suitably, the reactor is used to perform the method of the invention. Suitably, the reactor is used in the method for culturing algae in the first or second aspect, or in the method for producing phycocyanin in the third aspect.

[0273] Suitablely, the method of the present invention may include culturing algae in any suitable bioreactor (e.g., flask, fermenter, canal, open pond, etc.). However, suitablely, the method of the present invention is carried out in the reactor of the present invention (suitably, as defined in the ninth aspect).

[0274] Suitablely, the reactor is an airlift reactor.

[0275] Suitably, the reactor includes a container. Suitably, the container has a total volume. Suitably, the total volume of the container can be up to 200 L. Suitably, the total volume of the container can be 1 L, 2.5 L, 4 L, 5 L, 10 L, 100 L, 200 L, or any suitable industrial volume.

[0276] Suitablely, the container is made of plastic (suitably, acid-resistant plastic). Suitable plastics may include HDPE, acrylic, PTFE, PEEK, PVC, and PP.

[0277] Suitably, the container also has a working volume. Suitably, the working volume is a portion of the total volume. Suitably, the working volume is operable to contain culture medium. Suitably, the working volume is between 1 / 2 and 1 / 3 of the total container volume. Suitably, the working volume is approximately 1 / 3 of the total container volume.

[0278] Suitably, the ratio of the total volume of the container to the working volume is at least 2:1. Suitably, the ratio of the total volume of the container to the working volume is at least 3:1 or at least 4:1.

[0279] In one implementation, the ratio of total volume to working volume is 3:1.

[0280] Appropriately, the container volume, which is the total volume minus the working volume, can be defined as "headspace".

[0281] Ideally, the headroom should be between 3 / 4 and 1 / 2 of the container's total volume. Ideally, the headroom should be approximately 2 / 3 of the container's total volume.

[0282] Suitable for this purpose, the top space is operable to accommodate algal foam. Algal foam is generated during algal cultivation and typically resides on top of the culture medium. Algal foam is considered important for the stability of algal cultivation and needs to be protected. However, currently available reactor designs do not allow for the containment of this foam. The inventors discovered that adjusting the reactor design to have a large volume available for foam resulted in better algal biomass productivity, particularly for cultivating phycocyanin-producing species.

[0283] Suitably, the container includes width and depth. Suitably, the width and / or depth of the container can vary depending on the total volume required by the container. Suitably, the width and / or depth, but not the height, is varied to avoid altering the hydrodynamics of the culture medium.

[0284] Appropriately, the depth and / or width can be varied as follows to obtain different volumes:

[0285]

[0286] Suitably, the reactor further includes an aerator. Suitably, the aerator is operable to deliver air bubbles into the culture medium within the container. Suitably, the aerator can deliver oxygen into the culture medium and / or deliver a nitrogen source into the culture medium. Suitably, the reactor may include a first aerator for delivering oxygen into the culture medium and a second aerator for delivering a nitrogen source into the culture medium.

[0287] Suitably, the container also includes one or more baffles, suitably at least one pair of baffles. Suitably, the reactor includes two or four baffles. Suitably, the baffles are operable to guide the flow of the culture medium. Suitably, the baffles are located near the bottom of the container, suitably such that they are submerged in the culture medium during use. Suitably, the baffles span the entire width of the container.

[0288] Suitably, the baffle has a triangular or hemispherical profile. Suitably, the baffle is configured to guide the flow of culture medium. Suitably, the baffle is configured to guide the flow of air bubbles from the aerator or each aerator. Suitably, the baffle is configured to guide the flow of culture medium generated by the flow of air bubbles from the aerator or each aerator.

[0289] Currently available reactor designs generate significant shear forces within the culture medium via mixing baffles. The inventors discovered that by further refining the reactor design, particularly the baffle design, algal biomass productivity increased.

[0290] Ideally, the height of the baffle should be in a 1:1 ratio with the bottom of the container.

[0291] Suitablely, the reactor includes a pH control system as described below.

[0292] Automated pH control

[0293] The reactor of the present invention may further include an automated system operable to control the pH of the culture medium.

[0294] Suitablely, the pH control system is automated. Suitablely, the pH control system is operable to quantitatively apply acid or base (suitably, at suitable time intervals) to the culture medium. Suitablely, the pH control system can quantitatively apply a nitrogen source to the culture medium, suitablely, the nitrogen source is a base as described above. Suitable nitrogen sources are as described above.

[0295] Suitablely, the addition of a nitrogen source to the culture medium is automated. Suitablely, this is done through a pH control system. Therefore, suitablely, in the second aspect of the method, the addition of the nitrogen source is performed through a pH control system. Suitablely, the quantitative application of alkali mentioned herein can be considered as the quantitative application of a nitrogen source to the culture medium.

[0296] Suitable, the control system can be operable to quantitatively apply liquid alkali or gaseous alkali or both to the culture medium.

[0297] Suitably, when the base is a liquid (e.g., ammonium hydroxide), the control system includes a pump. Suitably, the pump is operable to apply the base quantitatively into the culture medium. Suitably, the pump is a peristaltic pump.

[0298] Suitably, when the alkali is a gas (e.g., ammonia), the control system includes a valve. Suitably, the valve is operable to apply the alkali quantitatively to the culture medium. Suitably, the valve is a solenoid valve.

[0299] Suitablely, the automated system includes an alkali reservoir. Suitablely, the alkali reservoir is operable to store alkali. Suitablely, the alkali reservoir is connected to the reactor via piping. Suitablely, a valve or pump for metering the application of alkali is installed between the respective reservoir and the reactor.

[0300] Suitably, the automated system includes a computer operable to control the quantitative application of alkali to the culture medium. Suitably, the computer is operable to control the valve or pump, or each valve or pump. Suitably, the computer is operable to open or close the valve. Suitably, the computer is operable to turn the pump, or each pump, on or off.

[0301] Suitably, the automated system includes a pH probe operable to sense the pH of the culture medium and transmit the measured pH value to a computer. Suitably, the pH probe is operable to be placed in the culture medium. Suitably, the pH probe is operable to measure the pH of the culture medium at intervals. Suitably, the intervals are every 10 seconds, every 5 seconds, every 2 seconds, or every second.

[0302] Ideally, the computer can preset the optimal pH of the culture medium, such as pH 2. Ideally, the computer can preset the maximum and minimum pH thresholds.

[0303] If the probe measures a pH value higher than the optimum, the computer triggers a quantitative application of acid to the culture medium. Alternatively, this may occur if the pH value is higher than the optimum but not higher than the maximum threshold.

[0304] Appropriately, if the pH measured by the probe is below the optimal value, the computer triggers the quantitative application of alkali to the culture medium. Appropriately, this will occur if the pH is below the optimal value but not below the minimum threshold.

[0305] Appropriately, the computer calculates the appropriate dosage of alkali to maintain the optimal pH of the culture medium.

[0306] Appropriately, the computer is operable to open valves or start pumps to quantitatively apply acid or alkali to the culture medium.

[0307] Suitablely, the valve or pump is opened or actuated for 100 ms to 1000 ms, suitablely 200 ms to 800 ms, suitablely 400 ms to 600 ms. Suitablely, the valve or pump is opened or actuated for approximately 500 ms. Suitablely, when the valve is opened or the pump is actuated, a certain dose of acid or base is released into the culture medium.

[0308] Appropriately, once a certain dose of acid or base is delivered to the culture medium, there is a pause period to allow the medium to reach equilibrium. Appropriately, during the pause, the pH probe does not take any measurements. Appropriately, the pause period lasts between 15 and 45 seconds, appropriately between 20 and 40 seconds, appropriately between 25 and 35 seconds, and appropriately approximately 30 seconds.

[0309] Suitable for use, the computer can lock the system if the measured pH value is higher or lower than a threshold. For example, for an optimal pH of 2, the threshold for the maximum pH value can be set around pH 2.2, and the threshold for the minimum pH value can be set around pH 1.8. Suitable for use, the computer can lock the automated system for up to 90 minutes, or appropriately for about 60 minutes.

[0310] A suitable control system could be the Minifors control system, which can be used in conjunction with an Arduino pH controller (Biochemfluidics, UK).

[0311] In another aspect of the invention, a system is provided comprising a reactor according to the ninth aspect of the invention and an automated pH control system, wherein the automated pH control system is operable to control the pH of a culture medium within the reactor. Attached Figure Description

[0312] The invention will now be described with reference to the following figures, in which:

[0313] Figure 1 It showed that in 0.6 days -1 Sulfophilic primitive red algae grown under continuous flow conditions, of which 20 g / L -1 Glycerol was used as a growth-limiting substrate. Batch growth was initiated at 10 g / L prior to the start of continuous flow on day 4. -1 Glucose support to provide a high starting point for intracellular phycocyanin (circle) The oxygen saturation level is automatically controlled (linearly) by the reactor's computer system at 50% or 35%. During continuous flow, the cell dry weight remains stable at approximately 10 g / L throughout the culture process. -1 (square ).

[0314] Figure 2 (A) shows that the growth rate of sulfur-loving primitive red algae decreases with increasing impeller speed (circles). Growth rate (days) -1 ),(square Biomass concentration (gL) -1 ) and (triangle (A) shows oxygen saturation (%), and (B) shows that the growth rate decreases at impeller speeds above 500 rpm; growth rate (black circle) The relationship between the glycerol concentration and the impeller velocity was obtained from several stirred tank bioreactors operating at 42°C, with glycerol concentrations ranging from 5 to 15 g / L. -1 The line represents the regression average with 95% confidence intervals on both sides.

[0315] Figure 3 This shows sulfur-loving primitive red algae cultured in a fed-batch manner in a 4 L airlift bioreactor, with a feed concentration of 20 g / L. -1 Glycerin was used as the initial substrate, starting on day 3.5 at a dose of 500 g / L. -1 Glycerol-fed solution. Despite rapid growth and high carbon concentration, oxygen (measured rhomboid shape) is low. Trace information from the bioreactor remained above 85% throughout the study. The decrease in O2 around day 6 was caused by biofilm buildup on the dissolved oxygen probe, which recovered to >85% after cleaning. Aside from a period of pump blockage on day 6, cell dry weight (square) remained relatively stable. The growth rate is exponential. Intracellular phycocyanin (circles) After the batching stage, it reached 42 mg / g. -1 At the end of the culture, it reached 45 mg / g. -1 Glycerin (triangular) During the replenishment and batching phase, the level remains close to zero.

[0316] Figure 4 Continuous incubation on glycerol at different temperatures in the dark is shown.

[0317] Figure 5A The reactor for culturing algae according to the invention, used in the cultivation process, is shown.

[0318] Figure 5B The reactor design according to the present invention is shown. The reactor 100 includes a container 101 with a lid 103 and two triangular baffles 102 disposed inside the container 101 at the lower end of the container 101. The total volume is represented by arrow A, the working volume is represented by arrow B, and arrow C represents the non-working volume or top space of the container 101.

[0319] Figure 6 This shows how the increase in hydrogen ions in the culture medium is related to the concentration of phycocyanin in cells, (A) Δ[H] after 167 hours of flask culture. + [ ], with different initial culture pH, (B) intracellular phycocyanin concentration after 167 hours.

[0320] Figure 7 This shows how hydrogen ion release is correlated with NH4 uptake from the growth medium. (A) 20 g / L of medium grown in a 42°C shaking water bath. -1 Growth curves of glucose flask cultures. Culture variation depends on initial pH. (Circles) This is a pH 2.0 control culture medium, square in shape. The control is glycyl-glycine buffer, triangle. It has a pH of 2.6 and an inverted triangle shape. It has a pH of 3.1 and is an orange diamond shape. It is pH 3.6. (B) The concentration of hydroxyl groups in the culture medium, measured by a pH probe, indicates a decrease in pH and H+ in the growth medium. + Increase. (C) Total N concentration in the culture medium as determined by the Berthelot reaction.

[0321] Figure 8 The study demonstrated the detection of nitrogen uptake by algal cell membranes and an automated pH control system; NH4 + Extracellular deprotonation of the AMT transporter facilitates the diffusion of NH3(g) into the cell. Monitoring the extracellular proton concentration with a pH probe leads to the automatic addition of alkaline ammonium hydroxide / ammonia to balance the NH4+ in the culture medium. + concentration.

[0322] Figure 9 Semi-continuous culture based on ammonium hydroxide is shown, (A) starting from day 2.3 in an MK 3 airlift bioreactor with a growth rate of 1.0 day. -1 The expected growth rate, through 500 gL -1 Glycerol supplementation was used to culture the original red algae for more than 6 days. Cell dry weight (g / L) -1 ) ; Glycerol concentration (gL) -1 ) Oxygen saturation of growth medium (%) Specific phycocyanin concentration (mg.g) -1 ) (B) By pH The nitrogen concentration in the culture medium was controlled to maintain a constant level. Throughout the study, 150 mL of 10 M NH4OH was automatically added. Nitrogen should be maintained at 0.5-1.5 g / L. -1 The decreasing trend in culture medium nitrogen can be explained by the difference between the feed solution (pH 2.0) and the reactor (pH 1.9).

[0323] Figure 10An automated pH control system based on ammonia, with a capacity of 3 L, is shown. The system includes a reactor and an automated pH control system with a pH probe, an ammonia reservoir, and a solenoid valve.

[0324] Figure 11 The feedback loop of the automated pH control system is shown; pH is continuously monitored by the system and recorded at 1-second intervals. A setpoint is selected, in this case pH 2.0, and either side is locked as a safety feature. If the pH is detected to be below the setpoint but above the low lockout point, the valve will open, creating a 30-second pause to allow the pH to reach equilibrium. If the pH is detected to be above or below the lockout value, a 60-minute lockout will occur. This is to prevent the accidental addition of NH3 in the event of valve failure, probe failure, or a disconnection between the reactor and the NH3 supply.

[0325] Figure 12 Semi-continuous culture of primitive red algae using automated NH3 gas as the nitrogen source is shown, (A) starting from day 3.5 with a 1.0-day incubation period. -1 The expected growth rate was achieved in the MK 4 airlift bioreactor using 500 g / L -1 Glycerol was added to the culture feed for more than 7 days to culture red algae. Cell dry weight (g / L) -1 ) ; Glycerol concentration (gL) -1 ) Oxygen saturation of growth medium (%) Specific phycocyanin concentration (mg.g) -1 ) Biofilm accumulation on the dissolved oxygen probe led to a decrease in oxygen saturation measured around day 6. (B) The nitrogen concentration in the culture medium was maintained by pH control. Throughout the study, although a total of 43 g of NH3 was added, nitrogen levels remained between 0.5 and 1.5 g / L. -1 Between. Phycocyanin volumetric productivity (mg.L) -1 ) It increases exponentially with cell growth.

[0326] Figure 13 The initial continuous glycerol culture using an automated nitrogen source of ammonium hydroxide is shown; after a semi-continuous phase from day 3 to day 6, the culture was carried out in an MK 2 airlift bioreactor at a dilution of 0.5 times per day and 75 g / L. -1 Glycerol was cultured in a continuous flow environment. Throughout the study, nitrogen was automatically added to maintain the pH at 1.7. Cell dry weight (g / L) -1 ) ; Glycerol concentration (gL) -1 ) Oxygen saturation of growth medium (%) Nitrogen concentration × (g / L) -1 ).

[0327] Figure 14 This demonstrates how continuous culture using nitrogen was maintained in an MK 4 airlift bioreactor via automated pH control; continuous flow culture was conducted in the MK 4 airlift bioreactor at a dilution rate of 1.0 per day, using 50 g / L... -1 Glycerol was used as a growth-limiting substrate. From day 4 to day 9, oxygen was maintained at approximately 100%, at which point it was reduced to 50% by decreasing the airflow rate. Reduced mixing and high dilution created a flushing effect, resulting in a sharp increase in glycerol concentration in the reactor. Cell dry weight (g / L) -1 ) ; Glycerol concentration (gL) -1 ) Oxygen saturation of growth medium (%) Specific phycocyanin concentration (mg.g) -1 ) Nitrogen concentration × (g / L) -1 pH .

[0328] Figure 15 The purified cell extract of the primitive red algae (B) grown heterotrophically on glycerol showed a lack of phycocyanin (APC) pigment compared to autotrophic spirulina (A).

[0329] Figure 16 This is another graph showing that the growth rate of sulfur-loving primitive red algae decreases as the impeller speed increases. (Using 15 g / L...) -1 Glycerol as the starting growth substrate and 300 g / L -1 Glycerol is fed in a batch culture process within a stirred tank reactor. The system automatically sets the parameters to allow for the use of 4.0 L / min. -1 The air was maintained at 100% oxygen saturation. The natural logarithm of the biomass concentration was used to calculate the growth rate (square) for three sample intervals at increasing impeller speed. 400-500 rpm, triangle 500-700 rpm, diamond shape 700-800 rpm).

[0330] Figure 17 Showing more details with Figure 4 Same data. Using refined glycerin (800 g / L) -1Primitive red algae were cultured in a MK 4 bioreactor at 4.0 L scale at 42°C using a feed-batch, 120 g / L continuous feed method. Under semi-continuous conditions, biomass (square...) was cultured until day 8. When the sample reaches 45 g / L, it is then fed at a 0.7-day interval. -1 The dilution rate was initially fed continuously for 12 days. Automatic addition of 10 M NH4OH maintained the pH at 2.0 and provided the primary nitrogen source for the culture. Phycocyanin (circle) Maintained at 35 mg / g throughout the continuous phase -1 The above corresponds to a high oxygen saturation (inverted triangle) in the reactor throughout the same period. ).

[0331] Figure 18 and 19 More images of the reactor for culturing algae according to the present invention, used in the cultivation process, are shown. Figure 19 Showing with Figure 18 The same reactor, with the panels removed, reveals the original red algae culture inside.

[0332] Figure 20 It shows D-glucose, fructose and sucrose (500 g / L) -1 Feeding should be done in batches, 150-200 g / L -1 (Continuous feed) was used to culture primitive red algae at a scale of 4.0 L in an MK 4 bioreactor at 42 °C. During the semi-continuous phase (day 0), 500 g / L was used. -1 Glucose growth monitoring began, with a growth rate of 0.6 days. -1 Under semi-continuous conditions, up to day 6, biomass (square) )Grow to 25 gL -1 From this point on, use 150 gL -1 D-glucose at 0.6 days -1 The dilution rate was then increased to allow for continuous feeding. Once the culture reached a stable biomass concentration (~65 g / L)... -1 The glucose concentration in the supplemental feed was increased to 200 g / L on day 11. -1 Stable production was maintained for 5 days, during which time carbon was converted to fructose. Some representative data points were lost. Pump blockage on day 19 caused a halt to continuous flow, and sucrose (200 g / L) was resumed 2 days later. -1 The phycocyanin (circled) was used as a carbon source to restart. The stable state lasted for another 5 days until the experiment was stopped. Except for the feeding on day 19, when it turned into fructose, phycocyanin (circled) remained a carbon source throughout the continuous phase. Maintain at 35 mg / g -1 The above corresponds to a high oxygen saturation (inverted triangle). Cell dry weight measurements were performed in triplicate, with error bars only displayed when the value was greater than the specified value (±SEM).

[0333] Some aspects and embodiments of the invention will now be illustrated by the following non-limiting examples. Detailed Implementation

[0334] Example

[0335] Materials and methods

[0336] A. culture medium

[0337] The heterotrophic medium is an optimization based on Minoda, containing a suitable 10 g / L... -1 The following concentrations of inorganic components limit the growth of carbon substrates (Minoda et al., 2004). These elements are scaled proportionally according to the final carbon concentration of the medium, typically 20 g / L for batch media. -1 Carbon, semi-continuous culture medium 500-750 g / L -1 Carbon, continuous culture medium 50-300 g / L -1 Carbon. The flask studies described use glucose as the primary carbon substrate, while the bioreactor-scale studies use glycerol, unless otherwise stated.

[0338] For 10 g of carbon, the final inorganic composition and concentration are as follows (g / L). -1 The final concentrations of trace elements are: (NH₄)₂SO₄ 2.62, KH₂PO₄ 0.54, MgSO₄·7H₂O 0.5, CaCl₂·2H₂O 0.056, FeCl₃ 0.028, EDTA·Na₂ 0.016; -1 The concentrations of the culture medium and agar were as follows: H3BO3 5.72, MnCl2·4H2O 3.64, ZnCl2 0.21, Na2MoO2·2H2O 0.78, CoCl2·6H2O 0.08, CuCl2 0.086. For plate preparation, the double-concentrated solutions of culture medium and agar were sterilized separately by autoclaving and then mixed in a 1:1 ratio after brief cooling.

[0339] Unless otherwise specified, adjust the pH of the culture medium to 2.0 with 5 M H2SO4. For continuous cultures using glycerol, replace (NH4)2SO4 with H2SO4 at a molar ratio of 1:2, see Table 1. For feed solutions for semi-continuous cultures, do not add (NH4)2SO4 and adjust the pH to 2.0.

[0340] Table 1 - Continuous Nitrogen-Free Culture Medium

[0341]

[0342] Table 2 - Semi-continuous nitrogen-free culture medium

[0343]

[0344] B. Cultivation technology

[0345] flask

[0346] Flask cultures were performed in 250 mL Erlenmeyer flasks at a working volume of 100 mL, using an Incu-Shake MIDI incubator (SciQuip, UK) at 42°C and 200 rpm in the dark. Offline pH monitoring was performed using a benchtop pH-temperature probe (Eppendorf, UK).

[0347] Stirred tank bioreactor

[0348] The stirred tank bioreactor experiments used a Minifors 2 (Infors, Switzerland) glass reactor with a working volume of 1.0 - 2.0 L, which contained a 2-bladed Rushton turbine and operated between 400-800 rpm depending on the study.

[0349] airlift bioreactor

[0350] A series of airlift bioreactors with working volumes ranging from 2.5 to 4.0 L were used in this study. All of these were constructed from 0.6–0.8 mm thick acrylic sheets for custom construction. The airflow rate used depended on the working volume and was set between 0.5 and 1.5 VVM. Excess foaming in the reactors was controlled using an antifoaming agent (Sigma, UK).

[0351] Bioreactor Control

[0352] The Minifors control system is used in conjunction with all variant bioreactors and is connected to a computer via the OPC-UA standard to allow for time-dependent parameter control. The exponential pump feeding curve for semi-continuous culture was calculated using Microsoft Excel, with the expected growth rate being 1.0–1.2 days. -1 between.

[0353] A custom Arduino-based pH controller was designed to control the metered application of ammonia via a solenoid valve 100T3MP 12-32 (Biochemfluidics, UK).

[0354] C. monitor

[0355] growth

[0356] Growth was monitored on a time-dependent basis. To prevent any pigment interactions (Gross et al., 1998), the absorbance of the cell suspension was measured at 800 nm using an S-200 spectrophotometer (Boeco, Germany) with a path length of 1 cm. Deionized water was used as the blank. Cells were diluted with deionized water as needed to ensure absorbance measurements were below 0.7 cm⁻¹. -1 .

[0357] Unless otherwise specified, 1–10 mL of cells (or 5–50 mg of stem cells) in triplicate are used for determining cell dry weight (DCW). Centrifuge the cells at 18,000 × g for 30 seconds, discard the supernatant, and resuspend in deionized water. Repeat twice to remove culture medium components. Resuspend the washed cells in 1 mL of deionized water and freeze at -80°C for at least overnight, followed by freeze-drying under vacuum for 24 hours.

[0358] Phycocyanin

[0359] Resuspend 10–50 mg of frozen stem cells in 1.5 mL of 100 mM potassium phosphate buffer at pH 7.2. Mechanically disrupt the cells using cell disruption tubes (2 mL capacity, 0.5 mm zirconium beads, Sigma-Aldrich, USA) and a BeadBug homogenizer (Benchmark Scientific Inc., USA) at 4000 rpm for 5–8 cycles of 60 seconds each, cooling the tubes to 4°C between cycles.

[0360] The cell lysate was centrifuged at 18,000×g for 60 minutes at 4°C, and 500 µL of the blue supernatant was collected and diluted as needed for spectrophotometric analysis as described below.

[0361] Phycocyanin extract was transferred to a 1 cm path length cuvette for analysis. Absorbance was read at 320, 562, 620, and 652 nm using a spectrophotometer, and C-PC content was estimated according to the method described in (Kursar and Alberte, 1983), expressed as Equation 1 below.

[0362]

[0363] glycerin

[0364] When glycerol is added to the culture medium, monitor the glycerol concentration in the medium. Use a glycerol-free reagent (Sigma-Aldrich) according to the manufacturer's instructions; this is an enzymatic assay that leads to the production of green quinone imine from glycerol. Dilute the culture medium sample 100-fold and read the final absorbance at 540 nm.

[0365] nitrogen

[0366] The ammonium concentration in the growth medium was determined using a modified Berlethot reaction (Rhine et al., 1998), in which the ammonium-containing solution was made alkaline, which produces ammonia. The ammonia reacts with 2-phenylphenol to produce a blue indophenol complex. Initial samples were diluted 250–1000 times and incubated in the reaction mixture for 1 hour. The concentrations were then determined using a plate reader (Victor). 2 The absorbance of 1420 (Wallac) was read at 660 nm.

[0367] result

[0368] Oxygen saturation and reactor efficiency in the production of phycocyanin from sulfur-loving primitive red algae

[0369] a) Failed to promote high oxygen saturation in the stirred tank reactor and reduce phycocyanin levels during glycerol growth.

[0370] In a standard Minifors 2 (Infors, Switzerland) dual Rushton turbine mixing glass bioreactor, with a working volume of 2.0 L, at 20 g / L... -1 Glycerol, as a growth-limiting substrate, was observed to be present in 0.6 days. -1 Sulfophilic primitive red algae were grown under continuous flow conditions. Batch growth was initiated on day 4 before the start of continuous flow, starting with 10 g / L of [amount missing]. -1 Glucose support was provided to deliver 32.8 ± 0.8 mg / g. -1 Intracellular phycocyanin at a high starting point of cell dry weight ( Figure 1 Under conditions of 50% oxygen saturation and with glycerol as the carbon source, after four days of continuous culture, intracellular phycocyanin decreased to approximately 10 mg / g. -1 Low cell dry weight. Cell dry weight remained stable throughout the culture (black square). On day 11, 500 rpm and 2.0 L.min -1 The maximum non-destructive setting of the airflow-controlled stirred tank reactor cannot achieve near 100% oxygen saturation, despite glycerol substrate concentrations as low as 20 g / L. -1 The oxygen saturation level remained stable at 70%.

[0371] b) Growth rate decreases when impeller speed exceeds 500 rpm.

[0372] Figure 2 B and Figure 16 The study showed that under heterotrophic conditions, the growth rate of sulfur-loving primitive red algae decreased as the impeller speed of the stirred tank bioreactor increased.

[0373] 15 gL -1 Glycerol was used as the starting carbon substrate to prevent glycerol-mediated growth inhibition, ensuring 1.35 days. -1 High initial growth rate. Semi-continuous culture was started on day 4 to prevent glycerol from dropping to 5 g / L. -1 The following, and for the remainder of the experiment, not exceeding 12 g / L. -1 The impeller speed is automatically controlled by the system, with a minimum setting of 400 rpm to maintain an oxygen saturation of >95%. Above 10 OD 800 (3.25 gL) -1 (Cell dry weight), the system began to increase impeller speed, causing the growth rate to decrease to 1.05 days within 24 hours from 500-700 rpm. -1 The growth rate decreased to 0.54 days at 780-700 rpm from 4.5 to 6 days. -1 A peak impeller velocity was observed at 4.5 days, after which the velocity began to decline. This is likely due to the relationship between the density of oxygen-absorbing cells and the density of cells damaged by the impeller, leading to a balance in oxygen demand.

[0374] In a stirred tank reactor, shear forces are applied to the culture medium to reduce the size of flowing bubbles and decrease mixing time, thereby improving mass transfer in the system. Our observations show that high growth rates and high oxygen saturation are maintained during heterotrophic growth, with cell densities exceeding 10 g / L. -1 The required impeller speed would damage the organism. An alternative strategy is being sought, namely, the use of an airlift system.

[0375] c) Maintain increased oxygen levels in the airlift reactor and increased phycocyanin production in the dark.

[0376] Initial attempts to increase oxygen concentration in conventional stirred-tank bioreactors failed because the shearing of algal cells caused damage (see Sections (a) and (b)). It is impossible to maintain high oxygen concentrations (through mixing) while simultaneously maintaining high productivity of biomass and phycocyanin. To address this problem, we developed a low-shear airlift bioreactor design, particularly suitable for achieving high oxygen concentrations in high-density cultivation of sulfur-loving primitive red algae.

[0377] In airlift bioreactors ( Figure 5A , Figure 5B , Figure 18 and Figure 19 In 10.5 gL -1 Glycerin, pH 2.0, 42°C, and 6.0 L / min -1 Incubation began under airflow and a working volume of 4.0 L. Figure 4 and Figure 17 Batch growth at 1.0 day -1 It was administered and reached 5.6 gL on day 3. -1 The biomass concentration. At this point, at 0.6 days... -1 The rate is 800 g / L -1 Glycerol-feeded semi-continuous growth was initiated. Nitrogen was automatically added under pH feedback control, and the pH in the reactor was maintained at 1.9 using 10 M NH4OH. 500 mL of semi-continuous feed was added until day 7, resulting in a growth rate of 0.54 days. -1 The biomass concentration was 43.4 g / L. -1 On the 8th day, at 0.6 days -1 The dilution rate started at 120 g / L. -1 A continuous flow of nitrogen-free glycerol was used. On day 15, for the remainder of the experiment, the dilution rate and the glycerol concentration were increased to 0.7 days. -1 and 130 gL -1 ( Figure 4 and Figure 17 ).

[0378] At a dilution rate of 0.6 days -1 During continuous flow, for a substrate yield of 0.435, the average biomass productivity was 32.3 ± 0.8 g / L. -1 .sky -1 This is very close to the previously reported data (Y). x / s 0.43) (Graverholt and Eriksen, 2007). Compared with the literature (861 mg / L / day) -1 Compared to the previous culture, this culture produced 1151 ± 61 mg / L. -1 .sky -1 The high phycocyanin production rate indicates that the yield of phycocyanin produced from glycerol is 15.5 mg / g. -1 When Y x / s At a concentration of 0.45, increasing the dilution rate and the amount of glycerol fed in the feed can increase the biomass productivity to 40.5 ± 0.6 g / L. -1 .sky -1 During this period, with 0.6 days -1 Compared to dilutions (85–75% saturation), oxygen levels decreased slightly; however, intracellular phycocyanin continued to increase, reaching 41.0 mg / g on day 20. -1 The values ​​were high, and a very high phycocyanin production rate of 1560 ± 57 mg / L was achieved in the last three days. -1 .sky -1 It is almost twice that of previous literature reports (Graverholt and Eriksen, 2007).

[0379] Table 3 shows the high productivity achieved by our airlift reactor system in the dark. These data include an average intracellular phycocyanin content of 27.95 mg / g over a 10-day process at 42°C. -1 Cell dry weight was better than that reported in existing literature. During this period, the glycerol concentration in the feed ranged from 90 to 230 g / L. -1 The concentration varied between these values, using a concentration of 100 g / L. -1 Laboratory-grade glycerol yielded 1.7 g / L within 2.5 days. -1 .sky -1 High volumetric productivity. These figures are twice the highest productivity observed in the literature using glucose in stirred tank reactors: Graverholt 2007 achieved 15.6 mg / g. -1 Intracellular concentration of cell dry weight and 0.86 g / L -1 .sky -1 Volumetric productivity.

[0380] In fact, the expressed phycocyanin level is more comparable to the level achieved by additionally irradiating cells with a light-emitting diode array to reach 35-40 mg / g within the region. -1 The data from DCW (WO2017 / 050917) are comparable. Other observations by Sloth also indicate that when the growing algae are exposed to glycerol and light (65 μmol photons / m²), the algae respond well to light. -2 .s -1 When grown under these conditions, the phycocyanin content increases significantly, reaching 20 mg / g. -1 DCW (Sloth et al., 2006).

[0381] Table 3: Productivity of continuous culture on different glycerol substrates in the novel reactor

[0382]

[0383] d) Compared with the literature, fed-batch culture also showed improvement.

[0384] Figure 3 This adds further evidence confirming increased phycocyanin production and intracellular expression when using high oxygen saturation in our airlift system. Although the feed pump became clogged near the end of the study, at 20 g / L... -1 Exponential growth on glycerol, initiated with batches of substrate concentrations, lasted for 7 days, with an average growth rate of 0.81 days. -1 Throughout the study, oxygen was maintained at high saturation, which was actually associated with very high intracellular phycocyanin levels, reaching 37.7 ± 2.4 mg / g at the end of the day 3.5 batch phase.-1 The level reached 45.2 ± 3.0 mg / g when supplemental feeding was stopped on day 7. -1 This is favorable compared to our own stirred tank data, which achieved 32.8 ± 0.8 mg / g of glucose during the batching stage. -1 This difference suggests that it was caused by a brief period of decreased oxygen concentration between day 2 and day 3. Figure 1 It is believed that the oxygen saturation level dropping to 50% around day 6 was a measurement error, as inspection revealed a large biofilm clogging the dissolved oxygen probe. Further supporting this conclusion is the absence of observed effects on phycocyanin. A total of 950 mL of 500 g / L solution was added. -1 Glycerin supplementation resulted in a final cell dry weight of 58 g / L. -1 The yield was 0.48, which is comparable to the figures reported in the literature.

[0385] like Figure 4 As shown in Table 3, using our airlift reactor system, phycocyanin remained at high intracellular levels (23–30 mg / g) throughout the 10-day extension period. -1 (cell dry weight), while even at 90-230 g / L -1 Even with high glycerol concentrations, the oxygen saturation in the reactor remains above 75%.

[0386] e) Continuous cultivation on other carbon sources

[0387] Figure 20 It showcases a series of studies using monosaccharides and disaccharides.

[0388] In a continuous airlift bioreactor, sulfur-loving primitive red algae were cultured sequentially with glucose, fructose, and sucrose. When oxygen was maintained at high saturation, a high phycocyanin production rate was achieved. Figure 20 (Table 4).

[0389] The culture was initiated in a semi-continuous mode and lasted for 6 days, during which the biomass reached 25.0 (±0.4) gL. -1 In this study, a computer control issue caused the pump rate to remain constant, so the feed pump rate was manually adjusted based on the current biomass each day. Therefore, a smooth growth curve was not observed. Figure 20 ). at 150 gL -1 D-glucose and 0.6 days -1 The dilution rate initiated continuous phased growth, resulting in rapid growth over two days until a stable concentration was reached (e.g., 64.7 (±1.8) g / L on days 7-10). -1Oxygen levels decreased and stabilized between 75-80% saturation, while glucose concentration increased to 200 g / L on day 11. -1 Intracellular phycocyanin levels remained at 44.2 (±2.3) mg / g between days 10 and 14. -1 The average phycocyanin production rate was 1796 (±151) mg·L·day. -1 The biomass yield relative to the substrate was 0.38 (±0.03) gg. -1 .

[0390] The growth substrate was converted into fructose (200 g / L) on day 14. -1 This caused a sharp drop in phycocyanin levels. This recovered after 24 hours and is likely the result of a brief lag during which the organism switched its metabolism to utilize the new substrate, leading to an increase in carbon in the reactor. Excess carbon is known to inhibit phycocyanin production (Sloth 2006). Some data were lost between days 16 and 19, but continuous culture continued running until day 19 when pump blockage caused a shutdown.

[0391] On day 21, the carbon substrate was changed back to sucrose (200 g / L). -1 Sucrose, as a disaccharide, is thought to require additional oxygen to overcome its higher energy density than glucose (i.e., sucrose is more reducing). Therefore, during the steady-state period of days 25–27, the oxygen saturation in the reactor stabilized at 65%, compared to 200 g / L. -1 The oxygen saturation at glucose levels is 75%+. This is because when oxygen levels are low (33.9 ± 2.04 mg / g), the oxygen saturation is lower. -1 The concentration of phycocyanin decreases, so testing is necessary to find the maximum growth rate for a given system. Higher biomass (81.2 ± 2.75 g / L) is produced from sucrose. -1 48.7 gL -1 .sky -1 This offset the reduced phycocyanin production, with a yield of 1540 ± 95 mg / L. -1 .sky -1 .

[0392] Table 4 – Continuous culture on monosaccharides and disaccharides

[0393]

[0394] Table 5A: Comparison of Phycocyanin Production Methods

[0395]

[0396] Table 5B: Further Comparison of Phycocyanin Production Methods

[0397]

[0398] As shown in Tables 5A and 5B, as well as the data currently provided, we are able to achieve very high productivity in heterotrophic mode by ensuring high oxygen saturation to produce phycocyanin from sulfur-loving primitive red algae (a strategy that is not feasible in stirred tank systems).

[0399] Under continuous conditions, we were able to produce more than 1.7 gL of glycerol. -1 .sky -1 ( Figure 4 or Figure 17 Using glucose to produce more than 1.75 gL -1 .sky -1 ( Figure 20 In contrast, the highest value found in the literature using glucose was 0.86 g / L. -1 .sky -1 (graver Holt and Eriksen, 2007). In fact, these data are at least comparable, if not superior, to processes requiring additional lighting for stirred tank reactors (WO 2017 / 050917 A1), to our 42 gL. -1 .sky -1 In comparison, it only achieved 24 gL -1 .sky -1 The biomass productivity was estimated, and its phycocyanin productivity was estimated to be between 0.7 and 2.4 g / L. -1 .sky -1 between.

[0400] These data show that phycocyanin production is 567 times higher than existing open pond technology based on industrial spirulina (Jimenez et al., 2003), and even at least 20 times higher than spirulina culture based on the largest-scale multi-trophic bioreactor (Chen and Zhang, 1997).

[0401] Ammonium and pH control in the production of phycocyanin from primitive red algae

[0402] e) pH changes with nitrogen absorption.

[0403] Figure 6 and Figure 7 The relationship between growth, low pH, and nitrogen uptake of the original red algae culture was shown. The flask cultures were heterotrophically grown in a dark water bath at 42°C with a pH of 20 g / L. -1 Glucose was used as a growth-limiting substrate. pH and medium nitrogen were measured throughout the experiment, and the cultures additionally contained glycyl-glycine as a pH buffer. Figure 6In this study, the concentration of protons added to the culture medium during cultivation was correlated with the intracellular phycocyanin concentration, indicating a relationship between protein production and culture medium acidification. Parallel studies were conducted with cultures grown at different initial pH values, while the control was at pH 2.0. Higher initial pH reduced the overall acidification of the culture medium, which was associated with a decrease in phycocyanin production, although... Figure 7 The study observed in A showed no significant effect on the growth rate.

[0404] It is unclear why increasing pH inhibits nitrogen uptake and subsequent proton release into the culture medium. It is possible that the acidity in the medium, along with uptake transport proteins, promotes nitrogen uptake, although the mechanism of this action has not yet been observed. Reduced phycocyanin expression implies that the culture process requires more stringent pH control.

[0405] f) The production of phycocyanin requires a large amount of nitrogen.

[0406] Studies optimizing the carbon (C:N ratio) of pristine red algae have identified a C:N ratio range of 10–15:1 that yields the highest phycocyanin productivity, with productivity declining at higher ratios (Sloth et al., 2006). Therefore, to support high-density cultivation, an extremely solute-rich feed solution—500 g / L—is required. -1 The glucose-based feed solution requires 110 g / L. -1 Ammonium sulfate (Graverholt and Eriksen, 2007) and other inorganic components. These amounts are very close to the solubility limit and usually require overnight heating to prepare for complete dissolution.

[0407] Switching to glycerol as the carbon source makes high-concentration solutions, as described above, impossible. Glycerol is hygroscopic, requiring the removal of uncomplexed water from a bulk volume necessary to solubilize large amounts of ammonium sulfate. Ammonium sulfate is insoluble in glycerol. Therefore, separation of the nitrogen source is necessary for glycerol, which presents a problem for the design of the culture system. In this invention, an automated control system is developed to address this problem, as described below.

[0408] g) Automatic control can maintain nitrogen levels

[0409] In addition to the aforementioned solubility issues in glycerol, the cultivation of *Pterocarya spp.* on ammonium led to a significant decrease in the pH of the culture medium. In fact, without control, typical high-density systems can easily drop below pH 1.0, thus affecting growth rates. There is evidence that *Pterocarya spp.* expresses the AMT ammonium uptake transporter system, which results in the protons being retained extracellularly due to the deprotonation of ammonium ions to ammonia, and ammonia being absorbed as a gas by facilitating transporters and then reprotonated intracellularly (Lamoureux et al., 2010).

[0410] By stoichiometrically balancing the proton content in continuous culture media, or by ensuring equal pH levels in both batch and fed-batch systems, we can maintain stable nitrogen concentrations in the reactor without intervention from an automated control system that automatically responds to pH decreases. Figure 8 ).

[0411] 1. Semi-continuous

[0412] a) NH4OH

[0413] Figure 9 Successful glycerol-based culture was demonstrated, with ammonium hydroxide used to maintain nitrogen levels in the reactor during feeding. Balancing the pH of the feed and the reactor was important, as evidenced by the downward trend in medium nitrogen throughout the study. The pH difference between the feed (2.0) and the bioreactor (1.9) is believed to be the primary cause of this decrease, as the feed produces a dilution effect under semi-continuous conditions. However, despite rapid growth to 75 g / L within 6 days... -1 High cell density was maintained, and 150 mL of 10 M NH4OH was added during feeding, while nitrogen in the reactor was maintained at 1.4–0.6 g / L. -1 This is much faster than the growth reported in the literature, where the highest reported growth was 109 g / L after 16 days. -1 Cell dry weight. These data also support the requirement for high oxygen levels to promote phycocyanin production—oxygen reached a low saturation of 28% on day 5.5, compared to a decrease in intracellular phycocyanin to 23 mg / g near the end of culture. -1 This is consistent with the reported highest productivity of 26 mg / g. -1 Similar. The reduction in oxygen in this MK 3 airlift bioreactor revealed problems with oxygen transport, prompting the construction of the MK 4 airlift reactor with an improved gas distributor. Figure 12 This experiment uses NH3 gas, but importantly, the oxygen saturation does not drop below 80%, and 45 mg / g is produced in the final sample. -1 Intracellular phycocyanin.

[0414] b) NH3

[0415] We have found that using ammonia is a more reliable technique for adding basic nitrogen to the system without creating localized high pH areas due to insufficient mixing, which is especially important for scalability. Figure 10 The system setup for this type of control is outlined. Instead of a peristaltic pump, a solenoid valve controlled by a pH-sensing computer controls the flow of ammonia into the bioreactor. Figure 11The safety features of the automated system are outlined. By introducing a lockout for abnormally high or low pH measurements, the addition of ammonia to the system under fault conditions is prevented: assuming a reactor malfunction and culture leakage, the system is locked at the point when the pH probe is no longer in contact with the culture medium and the pH measurement rises.

[0416] Two process benefits were observed by using ammonia gas instead of ammonium hydroxide. First, the addition of gas does not readily alter the internal volume of the reactor compared to a hydroxide solution, simplifying process kinetics. Second, insufficient mixing is overcome due to the significantly larger contact area of ​​the bubbles compared to liquid hydroxide tubes. Third, the high-speed gas exchange inherent in the airlift design can be utilized because the airlift reactor of this invention has a slower mixing time than a stirred tank, preventing excessive shearing of the cells.

[0417] In fact, despite the addition of a total of 43 g of ammonia over 7 days, the system operated as expected, maintaining a stable pH and a concentration of 0.5–1.5 g / L. -1 medium nitrogen concentration ( Figure 12 The pH was observed to decrease at the start of the study until day 3, when the ammonia control system was activated and the pH stabilized.

[0418] 2. Continuous

[0419] a) NH4OH

[0420] In continuous systems, the relationship between pH and the addition of alkaline nitrogen differs from that in the semi-continuous systems described above because an acidic cell suspension is leaving the system and must be taken into account. In this case, additional acid is needed in flowing continuous feeds to replenish the acid lost from the system. Acid is more soluble in glycerol solution than ammonium sulfate and has the added benefits of inhibiting glycerol contamination, simplifying capital equipment, and ensuring sterility. In the experiments described here, ammonium sulfate was used to provide the initial nitrogen source at inoculation. A better strategy to further minimize the risk of contamination (which is highest at the start of culture) is to prepare a nitrogen-free starting batch medium with a very low pH and raise the pH by adding alkaline nitrogen during the first few days after inoculation.

[0421] Use an acid concentration proportional to the carbon concentration in the continuous feed, as done for nitrogen, to ensure proper stoichiometric addition of balanced nitrogen to support the growth of primary red algae and phycocyanin production (Table 1).

[0422] Figure 13 and Figure 14 Two examples are provided in which acidic, nitrogen-free continuous culture media can maintain stable nitrogen concentrations for the cultivation of pristine red algae in bioreactors. Figure 13 In the reactor, nitrogen and pH were maintained at 5.0 g / L. -1Around day 7, except for the MK 2 airlift reactor, where a problem caused some culture loss. Figure 14 The results showed a more stable nitrogen concentration of approximately 1.0 g / L when using the MK 4 bioreactor. -1 In this experiment, further evidence supporting the high oxygen requirement of phycocyanin was observed; at 50% O2, the intracellular concentration of phycocyanin began to decrease from days 9–11. Mixing was also affected because the airflow rate had to be reduced to lower the oxygen saturation in the airlift bioreactor, leading to a positive feedback effect of excess glycerol inhibiting cell growth near the end of the experiment. Approximately 10 g / L of glycerol was used throughout the experiment. -1 The baseline glycerol concentration also resulted in 25-30 mg / g. -1 The overall phycocyanin concentration in cell dry weight decreased.

[0423] Comparison of pigments produced by primitive red algae heterotrophically grown on glycerol and by spirulina autotrophically cultured.

[0424] Figure 15 The absorbance measurements of pigments extracted in the same manner from primitive red algae grown heterotrophically on glycerol (B) compared to Spirulina grown in a conventional autotrophic manner (A). C-phycocyanin (CPC) showed the strongest absorption at approximately 620 nm, while the associated protein, allophycocyanin (APC), showed the strongest absorption at 652 nm. Characteristic shoulders of the absorbance curves were observed when both proteins were in a mixture, as shown in (A), confirming the obtained absorbance spectra. As shown in (B), highly purified CPC can be extracted from the heterotrophic culture of the primitive red algae; the absence of such a shoulder at 652 nm in Figure (B) indicates the lack of the allophycocyanin pigment impurity. The Spirulina sample contained 2.06 mg / ml. -1 ±0.024 mg / mL of phycocyanin and 0.037 mg / mL -1 ±0.001 mg / mL of heterotrophic phycocyanin, while the heterotrophic primitive red algae sample contained 1.72 mg / mL. -1 Phycocyanin and undetectable levels of isophycocyanin both underwent the same purification process.

[0425] CARBONE, D. A., OLIVIERI, G.g., POLLIO, A. & MELKONIAN, M. 2020.Biomass and phycobiliprotein production of Galdieria sulphuraria, immobilizedon a twin-layer porous substrate photobioreactor. Applied Microbiology andBiotechnology, 104, 3109-3119.

[0426] GRAVERHOLT, O. S. & ERIKSEN, N. T. 2007. Heterotrophic high-cell-density fed-batch and continuous-flow cultures of Galdieria sulphuraria andproduction of phycocyanin. Applied Microbiology and Biotechnology, 77, 69-75.

[0427] GRAZIANI, G.g., SCHIAVO, S., NICOLAI, M. A., BUONO, S., FOGLIANO, V.,PINTO, G. & POLLIO, A. 2013. Microalgae as human food: chemical andnutritional characteristics of the thermo-acidophilic microalga Galdieriasulphuraria. Food Funct, 4, 144-52.

[0428] GROSS, W., KÜVER, J., TISCHENDORF, G., BOUCHAALA, N. & BÜSCH, W.1998. Cryptoendolithic growth of the red alga Galdieria sulphuraria involcanic areas. European Journal of Phycology, 33, 25-31.

[0429] KURSAR, T. A. & ALBERTE, R. S. 1983. Photosynthetic Unit Organizationin a Red Alga : Relationships between Light-Harvesting Pigments and ReactionCenters. Plant Physiol, 72, 409-14.

[0430] LAMOUREUX, G., JAVELLE, A., BADAY, S., WANG, S. & BERNECHE, S. 2010.Transport mechanisms in the ammonium transporter family. Transfus Clin Biol,17, 168-75.

[0431] MINODA, A., SAKAGAMI, R., YAGISAWA, F., KUROIWA, T. & TANAKA, K.2004. Improvement of culture conditions and evidence for nucleartransformation by homologous recombination in a red alga, Cyanidioschyzonmerolae 10D. Plant Cell Physiol, 45, 667-71.

[0432] RHINE, E. D., MULVANEY, R. L., PRATT, E. J. & SIMS, G. K. 1998.Improving the Berthelot Reaction for Determining Ammonium in Soil Extractsand Water. Soil Science Society of America Journal, 62, 473-480.

[0433] SLOTH, J. K., WIEBE, M. G. & ERIKSEN, N. T. 2006. Accumulation ofphycocyanin in heterotrophic and mixotrophic cultures of the acidophilic redalga Galdieria sulphuraria. Enzyme and Microbial Technology, 38, 168-175.

[0434] LEMASSON, C., TANDEAUD.N & COHENBAZ.G 1973. ROLE OF ALLOPHYCOCYANINAS A LIGHT-HARVESTING PIGMENT IN CYANOBACTERIA. Proceedings of the NationalAcademy of Sciences of the United States of America, 70, 3130-3133.

Claims

1. A heterotrophic method for culturing algae, comprising: (a) Culture algae in a medium containing a carbon source and a nitrogen source, wherein the oxygen saturation of the medium is greater than 75%.

2. The method according to claim 1, wherein the carbon source comprises sugar, carbohydrate, or polyol, or any combination thereof.

3. The method according to claim 2, wherein the sugar is selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides or any combination thereof; preferably, the sugar is selected from glucose, mannose, ribose, xylose, fructose, galactose, sucrose, lactose, isomaltose, trehalose, maltose, maltotriose, raffinose, stachyose, verbascose, maltodextrose, cellulose glucose or any combination thereof.

4. The method according to claim 2, wherein the polyol is selected from glycerol, erythritol, inositol, lactitol, mannitol, sorbitol, xylitol or any combination thereof, and wherein the carbohydrate is selected from isomaltitol, cellulose, hemicellulose, pectin, starch, glycogen, chitin, chitosan, guar gum, β-glucan, alginate, gum arabic, β-mannan, inulin, tara gum, xanthan gum, carrageenan, polydextrose, glucomannan or any combination thereof.

5. The method according to claim 1, wherein the carbon source comprises glucose, fructose, sucrose, or glycerol, or any combination thereof.

6. A heterotrophic method for culturing algae, comprising: (b) Culture algae in a medium containing a carbon source and a nitrogen source, wherein the majority of the carbon source is glycerol, and wherein the oxygen saturation of the medium is above 75%.

7. The method according to any one of claims 1-6, wherein the culture medium is acidic.

8. The method according to any one of the preceding claims, wherein the nitrogen source is the only nitrogen source, preferably wherein the only nitrogen source is a base.

9. A heterotrophic method for culturing algae, comprising: (a) Algae are cultured in an acidic medium containing a carbon source and a single nitrogen source, wherein the single nitrogen source is a base, which is added to the acidic medium during the culture process and alters the pH of the acidic medium.

10. The method of claim 9, wherein the carbon source is defined as any one of claims 2-5.

11. A heterotrophic method for culturing algae, comprising: (a) Algae are cultured in an acidic medium containing a carbon source and a single nitrogen source, wherein the majority of the carbon source is glycerol; and wherein the single nitrogen source is a base, which is added to the acidic medium during the culture process and alters the pH of the acidic medium.

12. The method according to any one of claims 9-11, wherein the oxygen saturation of the culture medium is higher than 75%.

13. The method according to any one of claims 1-12, wherein the oxygen saturation of the culture medium is higher than 80%, higher than 85%, higher than 90%, and higher than 95%.

14. The method according to any one of claims 1-13, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% of the carbon source is glycerol, preferably the carbon source is composed of glycerol.

15. The method according to any one of claims 6-8 or 11-14, wherein the purity of the glycerol is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

16. The method according to any one of claims 8-15, wherein the nitrogen source comprises an ammonia source, preferably wherein the sole nitrogen source is selected from ammonia, ammonium hydroxide, ammonium sulfate and ammonium phosphate.

17. The method according to any one of claims 8-16, wherein the nitrogen source is ammonia.

18. The method according to any one of claims 1-17, wherein the addition of the nitrogen source to the acidic culture medium is automated, preferably by a pH control system.

19. The method according to any one of claims 7-18, wherein the pH of the acidic culture medium is about 2.

20. The method according to any one of the preceding claims, wherein the algae is a red algae of the class Rhodophyta, preferably a red algae of the order Rhodophyta, preferably a red algae of the family Cyanidiaceae or Galdieriaceae, preferably a red algae of the genera Cyanidioschyzon, Cyanidium, or Primitive Rhodophyta, preferably a species of Cyanidioschyzonmerolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, or a sulfur-loving Primitive Rhodophyta species.

21. The method according to claim 20, wherein the algae is a sulfur-loving primitive red algae, preferably selected from the following strains: ACUF141, SAG 108.79, 074G, 074G-G1, 074G-G2, CCMEE 5587.1, SAG 108.71 and UTEX#2919, and preferably the algae is the sulfur-loving primitive red algae strain ACUF141.

22. The method according to any one of the preceding claims, wherein the method is continuous.

23. The method according to any one of the preceding claims, wherein the temperature of the culture medium is between 35 and 55°C, preferably the temperature of the culture medium is about 42°C.

24. The method according to any one of the preceding claims, wherein the method further comprises the step of collecting algal biomass from the culture medium.

25. The method according to any one of the preceding claims, wherein the method produces 35 to 45 g / L. -1 .sky -1 Algal biomass.

26. A method for producing phycocyanin, comprising culturing algae according to any one of claims 1-25.

27. The method according to any one of the preceding claims, wherein the algal biomass has a content of 25-50 mg / g. -1 The average intracellular concentration of phycocyanin per cell dry weight.

28. The method according to claim 26 or 27, wherein the method produces 1-2.5 gL -1 .sky -1 Phycocyanin.

29. Algal biomass produced by the method of any one of claims 1-28.

30. An algal biomass having a content of at least 25 mg / g -1 The average intracellular concentration of phycocyanin per cell dry weight is less than 1.0 mg.g -1 The average intracellular concentration of phycocyanin by cell dry weight.

31. A method for preparing valuable chemicals from algal biomass according to claim 29 or 30.

32. The method according to claim 31, comprising the following steps: (a) Lysing algal cells and (ii) Purifying valuable chemicals from the lysate.

33. The method according to claim 31 or 32, wherein the valuable chemical comprises phycocyanin.

34. A reactor for culturing algae, comprising a container for containing a culture medium, wherein the container has a total volume and a working volume, wherein the working volume is a portion of the total volume operable for containing the culture medium, and wherein the ratio of the total volume to the working volume of the container is at least 2:

1.

35. The reactor of claim 34, further comprising an automated pH control system.

36. The reactor according to claim 34 or 35, wherein the reactor further comprises a pair of baffles located within the container.

37. The reactor of claim 36, wherein the baffle comprises a triangular profile.

38. The reactor according to any one of claims 34-37, wherein the ratio of the total volume of the container to the working volume is at least 3:1, preferably at least 4:1.

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