Aphanizomenon FLOS aquae biomass

Abstract

The present invention relates to Aphanizomenon flos aquae, or Afa, comprising compositions. Specifically, the present invention relates to Aphanizomenon flos aquae, or Afa, biomass. The present Aphanizomenon flos aquae, or Afa, biomass can be obtained by methods for producing an Aphanizomenon flos aquae biomass, the methods comprise the steps of: a) growing Aphanizomenon flos aquae in a closed reactor comprising a buffered growth medium until a dry biomass of between 1 to 10 grams / liter is obtained; b) harvesting the Aphanizomenon flos aquae biomass from the reactor thereby obtaining an Aphanizomenon flos aquae biomass; c) optionally, further processing of the Aphanizomenon flos aquae biomass. The present buffer of the growth medium can be comprised of NaHCO3, KHCO3 and CaCO3.

Description

[0001] APHANIZOMENON FLOS AQUAE BIOMASS

[0002] Description

[0003] The present invention relates to Aphanizomenon flos aquae, or Afa, compositions. Specifically, the present invention relates to Aphanizomenon flos aquae, or Afa, compositions comprised of lysed Aphanizomenon flos aquae, or Afa and to methods for providing the present compositions. The present compositions are only comprised of naturally occurring ingredients making them especially suitable for direct human consumption or as an additive for human food, beverages or feed supplements.

[0004] Aphanizomenon flos aquae, or Afa, belongs to the family of the cyanobacteria and is known for its beneficial properties especially in the field of human health because of the presence of, amongst others, proteins, pigments, magnesium, chlorophyl, omega-fatty acids, vitamins, essential fatty acids, essential amino acids and other useful cell constituents.

[0005] Non-toxic cyanobacteria such as Aphanizomenon flos aquae, Arthrospira platensis and Arthrospira maxima (the latter two are also designated spirulina) contain pigments such as chlorophyll, phycocyanin, porphyrin. Chlorophyll supports intestine peristalsis, normalizes secretion of digestive acids, decreases secretion of pepsin and soothes inflammation. Porphyrin forms a nucleus of hemoglobin, what makes it an important factor in the maintenance of healthy red blood cells. Phycocyanin is related to bilirubin, essential for liver functions and digestion of amino acids. Apart from standard functions carotenoids and phycocyanin are antioxidants, reduce free radicals and have potential anti-tumor properties.

[0006] Besides nutritional values, consumption of cyanobacteria as food supplements provide other advantages. Cyanobacteria biomass has a very low level of nucleic acids and is not easily contaminated by most human pathogens

[0007] Detailed research has been conducted to confirm the therapeutic effects of some compounds that are unique for cyanobacteria, including (I) calcium spirulan (II) immulina (III) C- phycocyanin and (IV) y-linolenic acid (GLA).

[0008] Calcium spirulan (Ca-SP) is a complex polysaccharide composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, galacturonic acid, sulfate, and calcium. It was shown to inhibit the replication of enveloped viruses from different genera. Immulina is also a high-molecular-weight polysaccharide and exhibits significant effects as an immunostimulator. C-Phycocyanin is an unusual nontoxic fluorescent protein having an antioxidative, anti-inflammatory and antitumor potential. Studies suggest it is more potent in colon cancer prevention than vitamin E and germanium- 132. The y-linolenic acid controls cholesterol levels and protects the cardiovascular system. Non-toxic cyanobacteria components have reported to be potent immunostimulating agents, they enhance the resistance to infections, have the capacity to influence hematopoiesis and stimulate the production of antibodies, especially IgA and cytokines. Compounds from cyanobacteria have been suggested to stimulate the immune system to inhibit carcinogenesis and assist natural healing mechanisms. Immulina is lOO-lOOOx more active as monocyte activation factor in vitro than polysaccharide preparations that were being used at the time in clinical settings for cancer immunotherapy. Immulina raises TNFa, IFNy, and IL-6 blood levels. Polysaccharide extracts from cyanobacteria significantly increased IL-1, IL-3, and TNFa levels.

[0009] C-phycocyanin also exhibits potent immunostimulatory effects by stimulating the generation of pro-inflammatory cytokines, which in turn boost the cytotoxic functions of CTL (cytolytic T lymphocytes) and NK (natural killer) cells activity. Moreover, pro-inflammatory cytokines activate macrophages, T, and B cells. Studies suggest that this multi-directional effect of cyanobacteria consumption is due to an involvement in signaling responses through toll-like receptors in blood cells.

[0010] Cyanobacteria extracts provide not only increased resistance to infectious diseases or natural healing mechanisms but also can modulate allergic responses. They sustain the functions of mucosal immunological mechanisms and reduce allergic inflammations by suppressing the antigen-specific IgE antibodies. Positive results were observed with “spirulina” dietary supplementation protecting against allergic rhinitis. Another anti-inflammatory effect of “spirulina” phycocyanin is the histamine release inhibition from mast cells and the reduction of functional neutrophils metabolic activity.

[0011] Although its exact mode of action is unknown, it is suspected that unique cyanobacteria nutrients play an important role as an antiviral. Calcium spirulan, isolated from hot water extracts, is reported to be very effective against a variety of viruses and it inhibits the replication of enveloped viruses such as Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), Human cytomegalovirus (HCMV), measles, mumps, influenza A, and HIV-1 in in vitro conditions Studies indicated that calcium spirulan selectively inhibits the penetration of the virus particle into the host cell and is more effective than dextran sulfate. The most probable mode of action is the conformational chelation of sulfate groups on the surface of the virus envelope.

[0012] Compounds isolated from cyanobacterium cells can also act as antimicrobial agents. Animal studies suggest that they enhance natural immunological clearance mechanisms after bacterial infections with Escherichia coli or Staphylococcus aureus. Studies were performed on the antimicrobial activity of various organic and aqueous extracts of cyanobacteria. In agarsolid diffusion tests, a water extract of Arthrospira (WE A) showed a maximal antimicrobial activity (18.00 mm inhibition zone) in case of Klebsiella pneumoniae and a minimum activity against Proteus vulgaris (10.0 mm). A propanol extract (PEA) exhibited at least a 7.0 mm inhibition zone for Pseudomonas aeruginosa and 8.0 mm for E. coli. The acetone extract (AEA) displayed the highest biological activity with 17 mm inhibition zone against K. pneumonia, moderate activity in case of Salmonella typhi and a 10.0 mm inhibition zone with P. aeruginosa, E. coli, and S. aureus.

[0013] Microalgal cultures of cyanobacteria displayed significant antimicrobial activity against six Vibrio strains. Phycobiliproteins isolated from A. fusiformis exhibited antibacterial activity against Streptococcus pyogenes. The C-phycocyanin purified from A. platensis was able to inhibit the bacterial growth in many drug-resistant strains such as E. coli, K. pneumoniae, P. aeruginosa, and S. aureus. Besides antimicrobial and antiviral properties, non-toxic cyanobacteria formulations show antifungal characteristics. Butanol extract from “spirulina” powder was tested as a potential antifungal agent against Candida glabrata and caused a 13 mm growth inhibition zone in test plates.

[0014] Non-toxic cyanobacteria extracts and compounds may play an important role in the prevention of cardiovascular diseases. They lower the blood pressure, plasma lipid concentration, especially triacylglycerols. They indirectly modify total cholesterol and HDL / LDL (high- to low- density lipoprotein rates). Animal studies indicate that cyanobacteria could be a novel preventive tonic for the heart and whole cardiovascular system. “Spirulina” may be a prominent dietary supplement for patients with ischemic heart disease as it improves patients' lipid profiles and helps to lower blood pressure.

[0015] Phycocyanin has a high bile binding capacity, lowers cholesterol solubility and intake by Caco-2 intestinal cells (in comparison to casein). In mice, C-phycocyanin can promote the CD59 gene expression. This activity prevents smooth muscle cell proliferation and the apoptosis of endothelial cells, reducing blood fat levels. In consequence, C-phycocyanin inhibits the development of atherosclerosis. The GLA reduces cholesterol accumulation in the hypolipidemic nephrotic syndrome.

[0016] Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polymers for tissue or organ scaffold construction in regenerative medicine. They can be extracted from different microorganisms including cyanobacteria. Replacement of commercially available PHA biopolymers with equivalents obtained from cyanobacteria or / and containing its biomass significantly increases the eukaryotic cell proliferation and decreases the risk of transplant rejection The development of nanofibers produced from PL A (polylactic acid), polyethylene oxide and PHB (polyhydroxybutyrate) extracted from Arthrospira LEB 18 strain and incorporation of its compounds in artificial extracellular matrices has been studied. Observations reveal that addition of LEB 18 biomass increases nanofibers conductivity. PHB nanofibers from LEB 18 have higher mechanical durability with enhanced elasticity, tensile strength, and breaking elongation. Those characteristics support nutrient, growth factors, and metabolism byproducts.

[0017] Some observed beneficial effects of cyanobacteria supplementation cannot be classified, but their existence underlines the multiple therapeutical possibilities and how to take advantage of those unusual bacteria.

[0018] Studies worth mentioning show that cyanobacteria can promote growth of probiotic microbiotica boosting the production of B6 vitamin that aids in energy release. Also the high content of rhamnose, glycogen, and GLA aid in energy release.

[0019] The abundance of natural bioactive compounds makes non-toxic cyanobacterium extracts perfect for use in commercial as well homemade cosmetics. Formulas containing “spirulina” are mostly sold as anti-aging products that combat the action of free radicals, provide hydration, and protection to the skin. Due to its antimicrobial activity, cosmetics against acne, and other bacterial skin infections are enriched with “spirulina” extracts.

[0020] Previously, animals were fed with cyanobacteria biomass to examine the effect on a living organisms, which was a preliminary model for human nutrition. Nowadays, “spirulina” is treated as a valuable supplement that supports animal well-being and their products quality.

[0021] Dietary “spirulina” supplementation in poultry influences both the yellowness and redness of broiler flesh, increases several immunological functions, rising resistance to infections and could be as effective as the diet with synthetic pigment in producing an agreeable egg yolk color.

[0022] Because of the presence of health beneficial nutrients, Aphanizomenon biomass has become very popular as a food supplement for, particularly, the human diet. Beneficial effects are reported with regard to inflammation, cancer, skin harm, oxidative harm, eye illnesses; stem cell trafficking, collagen formation; hair loss; neurological disorder; skin wrinkles; suppression of harmful bacteria; water purification; inflammation control; mobilizing hematopoietic stem; dietary weight loss formulation; painful joints and bad temper; prevention of color changes of skin after inflammation; and skin aging.

[0023] Presently, the majority of the Aphanizomenon flos aquae biomass is obtained from the lake Klamath, Oregon, USA, where the biomass is harvested, or is obtained by cultivation in open ponds. Biomass harvesting takes only place two times per year, is not efficient and dependent on the weather conditions. Furthermore, Lake Klamath is eu-trophied from the surrounding agricultural regions and agricultural pesticides are expected and may accumulate in the biomass. From algae it is also known that they may accumulate ions of heavy metals as lead, mercury, cadmium, barium and thallium. Besides, the harvest is too low to meet the yearly world demand. Moreover, Aphanizomenon biomass is nearly always, if not, always polluted with specimens of other (harmful) algae, bacteria, yeasts or fungi. Furthermore, it is known that Aphanizomenon flos aquae is capable of synthesizing phytotoxins under natural (uncontrolled) conditions.

[0024] Considering the beneficial properties of cyanobacterium preparations, and especially Aphanizomenon flos aquae, it is an object of the present invention, amongst other objects, to provide Aphanizomenon flos aquae compositions or preparations.

[0025] The above object, amongst other objects, is met according to the present invention as outlined in the appended claims.

[0026] Specifically, the above object, amongst other objects, is met by a method for producing an Aphanizomenon flos aquae biomass, the method comprises the steps of: a) growing Aphanizomenon flos aquae in a closed reactor comprising a buffered growth medium until a dry biomass of between 1 to 10 grams / liter is obtained, preferably at an average day temperature below 23 °C; b) harvesting the Aphanizomenon flos aquae biomass from the reactor thereby obtaining an Aphanizomenon flos aquae biomass; c) optionally, further processing of the Aphanizomenon flos aquae biomass, preferably selected from one or more of the group consisting of induction of Aphanizomenon flos aquae biomass to form compounds, drying, and concentrating.

[0027] According to a preferred embodiment the buffer of the growth medium is comprised of NaHCCh. KHCO3 and CaC'Ch. optionally further supplemented with NaiCCh. preferably wherein the buffer of the growth medium is comprised of 0.01 to 0.04 grams / liter NaHCCh, 0.2 to 0.4 grams / liter KHCO3 and 0.01 to 0.1 grams / liter CaC'Ch. optionally further supplemented with 0.002 to 0.025 Na^C'Ch grams / liter, wherein the total amounts NaHCCh and NaiCC are in the range of 0.01 to 0.75 grams / liter.

[0028] According to the present invention, preferably, in step (a) the pH is between 4 to 9, preferably 6 and 8, most preferably 7 and / or the growth medium further comprises 0.5 to 5 mmol / liter Na+.

[0029] According to another preferred embodiment, the present 0.5 to 5 mmol / liter Na+is provided by adding 0.03 to 0.2 grams / liter dried sea salt, or equivalent, to the growth medium and / or the pH is maintained between 6.5 and 8 by using a CO2 flow.

[0030] Preferably, in the present method, the growing Aphanizomenon flos aquae is aerated in step (a) and / or step (a) is performed in a continue reactor.

[0031] According to the present invention, the biomass or processed biomass preferably is further supplemented with nutrients, colorings, flavorings, edible microorganisms, plant parts, plant extracts, seeds, nuts, buffers, dairy products, vitamins, minerals, foods or processed foods and / or wherein the biomass or processed biomass is further supplemented with Haematococcus pluvialis and / or the biomass or processed biomass is processed into beverages, human and animal food supplements, food additives, powders, suspensions, pharmaceuticals, cosmetics or tablets.

[0032] According to an especially preferred embodiment step (a) comprises: al) growing Aphanizomenon flos aquae in a first reactor comprising a growth medium and a light source until a dry biomass of between 0.1 to 10 grams / liter is obtained; a2) optionally, transferring the Aphanizomenon flos aquae obtained in step (al) to a second reactor; a3) exposing the Aphanizomenon flos aquae of step (al) or step (a2) to UV radiation with a wavelength between 50 to 400 nm; wherein preferably, the light source in step (al) exposes the growing Aphanizomenon flos aquae to red light with a wavelength of circa 640-660, optionally supplemented with white light of 2300 to 6000K and blue light with a wavelength of circa 440-460 nm, more preferably, wherein the light source in step (al) exposes the growing Aphanizomenon flos aquae to 50 to 100% red light with a wavelength of circa 640-660 nm.

[0033] The present invention relates o Aphanizomenon flos aquae biomass obtainable by the present methods.

[0034] According to the present invention, the above object, amongst other objects, is also met by a method for producing an Aphanizomenon flos aquae derived composition, the method comprises the steps of: a) growing Aphanizomenon flos aquae in a closed reactor comprising a growth medium at a light intensity of less than 100 pm / m2 / s until a dry biomass of between 1 to 10 grams / liter is obtained; b) increasing the light intensity to more than 100 pm / m2 / s and / or increasing the growth temperature to induce lysis of Aphanizomenon flos aquae biomass in the reactor; c) harvesting the lysed Aphanizomenon flos aquae biomass from the reactor thereby obtaining an Aphanizomenon flos aquae derived composition.

[0035] The present inventors have surprisingly discovered that exposing an Aphanizomenon flos aquae culture to increased light intensity, generally 2 to 10 times increased light intensity, and / or increasing the temperature in the closed reactor induced spontaneous lysis of Aphanizomenon flos aquae without further process steps or the addition of lysis inducing agents such as high salt or detergents. This yields a composition, or lysate, only comprising naturally in Aphanizomenon flos aquae present proteins, lipids, sugars and other molecules. Further, the absence of any further lysis inducing process steps provides these naturally in Aphanizomenon flos aquae present proteins, lipids, sugars and other molecules without little and no degradation or modification caused by, for example, mechanical lysis methods such as sonification or grinding. The presently obtained Aphanizomenon flos aquae derived composition is a clear lysate, optionally after removal of residual insoluble components by, for example, centrifugation or sedimentation.

[0036] According to a preferred embodiment of the present invention the light intensity in step (b) is between 100 to 1000 pm / m2 / s under the condition that the light intensity is increased, preferably 2 to 10 times, as compared to the light intensity used in step (a).

[0037] According to a more preferred embodiment the light intensity in step (b) is between 200 to 700 pm / m2 / s, preferably between 400 to 600 pm / m2 / s.

[0038] According to the present invention, the pH is maintained between 4 to 9, preferably 6 and 8, in step (a) using generally known pH adjusting agents and buffers, preferably CO2. More preferably, the pH is maintained between 7 and 8, such as 7.5 using a CO2 flow in step

[0039] (a).

[0040] According to another preferred embodiment of the present invention, the growing Aphanizomenon flos aquae is aerated in step (a). The aeration can be suitably performed using ambient air both providing CO2 and O2 however specially composed gas mixtures comprising one or both gasses, and optionally other gasses, are contemplated within the context of the present invention.

[0041] According to an especially preferred embodiment of the present invention, in step

[0042] (b) the growing Aphanizomenon flos aquae is not aerated and / or the pH is not adjusted. Not adjusting the pH generally results in an increased pH increasing lysis of the Aphanizomenon flos aquae culture. According to the present invention, increasing the average temperature in step (b), as compared to the average temperature in step (a) further aids the lysis of Aphanizomenon flos aquae culture. For example, in step (a), the average day temperature can be below 23 °C such as , 22 °C, 21°C, 20°C, 19°C, 18°C, 17°C or 16°C and, in step (b), the average day temperature can be above 22 °C such as 23°C, 24°C, 25°C, 26°C, 27°C or 28°C.

[0043] The Aphanizomenon flos aquae derived composition, in the form of a lysate preferably a clear lysate, conveniently provides valuable Aphanizomenon flos aquae compounds. According, the present invention relates o Aphanizomenon flos aquae compositions obtainable by the present methods.

[0044] The present Aphanizomenon flos aquae derived composition is preferably dried or concentrated and optionally further supplemented with nutrients, colorings, flavorings, edible microorganisms, such as Haematococcus pluvialis, plant parts, plant extracts, seeds, nuts, buffers, dairy products, vitamins, minerals, foods or processed foods.

[0045] Using generally known process techniques such as drying, diluting, concentrating or purifying of the present Aphanizomenon flos aquae derived composition, the present Aphanizomenon flos aquae derived composition, either directly or indirectly, can be used for the preparation of beverages, human and animal food supplements, food additives, powders, suspensions, pharmaceuticals, cosmetics or tablets.

[0046] A preferred embodiment of the method according to the present invention comprises in step (a) the steps of: al) growing Aphanizomenon flos aquae in a closed reactor comprising a growth medium at a light intensity of less than 100 pm / m2 / s until a dry biomass of between 1 to 10 grams / liter is obtained; a2) increasing the light intensity to more than 100 pm / m2 / s and / or increasing the growth temperature to induce lysis of Aphanizomenon flos aquae biomass in the reactor;

[0047] The present inventors have surprisingly discovered that exposing an Aphanizomenon flos aquae culture to increased light intensity, generally 2 to 10 times increased light intensity, and / or increasing the temperature in the closed reactor induced spontaneous lysis of Aphanizomenon flos aquae without further process steps or the addition of lysis inducing agents such as high salt or detergents. This yields a composition, or lysate, only comprising naturally in Aphanizomenon flos aquae present proteins, lipids, sugars and other molecules. Further, the absence of any further lysis inducing process steps provides these naturally in Aphanizomenon flos aquae present proteins, lipids, sugars and other molecules without little and no degradation or modification caused by, for example, mechanical lysis methods such as sonification or grinding.

[0048] The presently obtained Aphanizomenon flos aquae derived composition is a clear lysate, optionally after removal of residual insoluble components by, for example, centrifugation or sedimentation.

[0049] According to a preferred embodiment of the present invention the light intensity in step (a2) is between 100 to 1000 pm / m2 / s under the condition that the light intensity is increased, preferably 2 to 10 times, as compared to the light intensity used in step (al).

[0050] According to a more preferred embodiment the light intensity in step (b) is between 200 to 700 pm / m2 / s, preferably between 400 to 600 ,u m / nr / s.

[0051] The present invention will be further detailed in the following example. In the examples, reference is made to figures wherein: Figure 1: shows growth of the algae in the PBR as measured in OD700, OD750 or dry weight in grams / liter;

[0052] Figure 2: shows growth of the algae AFA 762 after restart;

[0053] Figure 3: shows a flow diagram for growing AFA and subsequent processing steps.

[0054] Example 1: Culturing Aphanizomenonflos aquae (AFA 762) in a photobioreactor

[0055] Method

[0056] Parameters of the photobioreactor (PBR) pH 7.5, this is maintained by the addition of CO2 via the software of the PBR.

[0057] Temperature: 22°C, a heater and cooler are installed to maintain this average temperature.

[0058] Volume: total is 20.5 liters, when a sample is taken the volume is restored by adding fresh medium or concentrated nutrient solution.

[0059] Airflow: 2 liter s / minute

[0060] CO2 flow: steered by pH (0.05 1 / min)

[0061] Duration: Minimum of 4 weeks, maximum of 8 weeks, depending on the experiment.

[0062] Nutrient additions: When NO3 concentration reaches zero nutrients are supplied.

[0063] Light: The experiment is initiated with a light intensity of 10%. This is gradually increased during the experiment

[0064] Measurements

[0065] During the cultivation NO3, NH4 and other element levels were monitored, after the cultivation the following analysis was conducted.

[0066] Vitamin B 12 concentration

[0067] Protein concentration

[0068] Nutritional values

[0069] Amino acid composition

[0070] Lysis

[0071] After initial cultivation of the algae a lysis experiment was performed. To induce lysis of the algae the following conditions were tested: Normal conditions (light and salt as in during initial cultivation)

[0072] Normal light, increased salt concentration

[0073] Normal light, increased salt concentration, high day temperature (26°C) low night temperature (16°C) Maximum light

[0074] Maximum light, high day temperature (26°C) low night temperature (16°C)

[0075] Maximum light, high salt, high day temperature (26°C) low night temperature (16°C)

[0076] Lysis was determined by microscopic analysis of the cultures. After lysis, the resulting lysate was analyzed for protein contents, vitamin bl2, amino acid composition and nutritional values.

[0077] Analysis with water fleas

[0078] To determine toxicity of the algae an assay was performed in which water fleas (Daphnia) were exposed to the algae. In this assay the water fleas were exposed to a live or lysed culture of the algae diluted 5 or 50 times with water. After dilution 10 adult water fleas were added to for analysis of toxicity.

[0079] Results

[0080] The PBR was inoculated with 3 liters of mature culture. With these 3 liters, 17.5 liters of fresh medium was inoculated obtaining a total volume of 20.5 liters in the PBR. The intensity of the lights on the PBR was set at a low setting. At the start of the cultivation a plastic sheet was hung over the PBR to minimize environmental light, since this had a greater impact at this stage of cultivation. At higher light intensities the incoming environmental light was neglectable. Temperature during cultivation was set at 22°C. When needed cooling was used during the day and heating during the night.

[0081] The final biomass produced was + 7grams / liter, with a maximum production of 0.7 grams / day, with a few days with a steady growth of 0.5 gr / day. The weekend before the termination of the experiment the growth declined due to a hold in nutrient supply. Nitrate levels went to 0 on day 4 and from this day onward extra nutrients were supplied, with an average of 100 ml / day. Due to weekends and holidays there was a higher supply on certain days to reach the average of 100 ml / day. Because the nutrients supply contained only extra high amounts of nitrogen and phosphate there was no build up of salts. The growth curve observed during the cultivation period is shown in Figure 1. After harvesting 80% of the algae for further analysis a restart was performed. The PBR was supplied with fresh medium to reach a total volume of 20 liters. Due to the high light intensity and low concentration of algae the culture initially turned yellow. After a couple of days the culture recovered and turned green again. In the first few days there was also a slight decrease in biomass indicating lysis. During this growth period (see Figure 2) the algae didn’t grow as fast as it did in the initial experiment, reasons for this could be the high initial light intensity, and a few very warm days. The total biomass concentration obtained during this experiment was 6.5 grams / liter.

[0082] At the end of the cultivation period, an attempt to force lysis was done. At that moment there was a warm period with high light intensity and high day temperatures (±25 °C) and low night temperatures (±17 °C). After a few days the CO2 ran out it was noted that this caused the pH to quickly rise to ±9.5. It was then decided to not reconnect the CO2 to determine if this rise in pH could lead to lysis, since there wasn’t a clear sign of lysis yet. After a few days under these conditions (high pH, high light intensity, high day temperature, low night temperature) microscopic analysis of the culture showed only akinites left in the medium and no vegetative cells.

[0083] In all set-ups of the lysis experiments, after time lysis was observed. The set-ups with high light intensity and day temperature of 26°C and night temperature of 16°C showed the fastest sign of lysis. After 5 days no more intact cells were visible under the microscope. There was also a clear formation of foam on the surface of the culture indicating lysis. The lysis caused an unpleasant smell, this smell was readily removed through aeration of the culture.

[0084] Analysis with the water fleas showed that the lysed culture is more toxic to water fleas than the live culture although the observed toxicity is believed to be an indirect effect of additional bacterial growth in the lysate due to high concentrations of nutrients available. At 5x dilution all water fleas treated with the lysed culture die, this dilution of live algae doesn’t have this effect. At a 50x dilution, there is survival of water fleas in all treatments. When small water fleas are observed it means that the water fleas are capable of reproduction in the presence of the algae. These small water fleas are difficult to count due to their small size.

[0085] The following nutrients produced were measured:

[0086] Vitamin bl2: The concentration of vitamin bl2 was approximately 0.59pg / 100g. The amount of vitamin bl2 in the medium was 0.05pg / 100g. The concentration of vitamin bl2 in the lysed cultures was comparable to the freshly harvested culture, 0.50 and 0.59 pg / lOOg. These measurements were performed on fresh cultures, not on dry matter. The biomass at the moment of harvesting was 6.91 grams / liter. On the basis of dry weight it can be concluded that 0.85pg vitamin bl2 is produced per gram biomass. These analyses have been performed according to the AOAC 952.20 method.

[0087] Protein contents: Directly after harvesting the protein concentration in the algae was 30.8%.

[0088] After lysis without salt a protein concentration of 32.1% was measured, and after lysis with salt 14.8 was measured. The reason why the protein concentration is higher after lysis without salt might be evaporation, but this is speculation. The lower protein concentration in the lysis with salt is due to the fact that there was almost as much salt added as there was biomass (6 grams / liter) and the protein measurement was done on dry matter which would have contained the salt. The protein measurement was done according to Dumas method, dry weight was determined via the ISO- 1442 method.

[0089] Amino acids: The determination of amino acid composition was only partially successful when a lOx concentrated culture was used. However the concentration of some amino acids was still to low to be accurately quantified. Due to these low concentrations it was not possible to get a complete picture of amino acid make up of the samples. The method used to determine the amino acid ratio was 13903:2005 and EU 152 / 2009.

[0090] Conclusion

[0091] This example shows that with the used cultivation method a minimal biomass of 6.91 grams / liter is obtainable, in which the biomass has a protein contents of at least 30.8%. The used algae produces vitamin bl2 with a maximum production of 5.9 pg / liter. At this moment the biomass was 6.91 grams / liter meaning that 1 gram of biomass can produce 0.85pg of vitamin bl2.

[0092] Lysis of the algae can be induced with high light intensity and / or by increased temperatures. Addition of salt speeds up the lysis process minimally and is not necessary. Lysis does not have negative effects on the concentration of vitamin bl 2. Unpleasant smells formed during lysis are easily removed with aeration of the lysed culture. The algae are not lethal to water fleas at 5x dilution, when the algae are lysed it is lethal, possibly as an indirect effect, to water fleas at a 5x dilution. Example 2: Culturing Aphanizomenonflos aquae (AFA 762 ) in a photobioreactor using a buffered medium

[0093] The above example was repeated using a buffered growth medium wherein the buffer allows for increased acidic CO2 suppletion while maintaining the pH within a desired range of above 6.5, preferably between 7 and 8 to allow optimal growth of the algae.

[0094] The buffer consisted of (per 1000 liter growth medium) 200 to 400 grams KHCO3 and

[0095] 10 to 100 grams CaCO3

[0096] NaHCO3and / or Na2CO3*

[0097] The total amounts of NaHCO3and Na2CO3are in the range of 1 to 75 grams Despite the continuous supplementation of CO2during growth of 27 days, the desired pH range of above 6.5 was maintained.

[0098] Example 3

[0099] Example 2 was repeated under the following light conditions and the exposure of the algae after the log phase to UV-C light.

[0100] 1) LOG phase, exponential growth between LAG phase and steady state

[0101] 2) LOG phase in steady state in continuous culture

[0102] 3) LOG phase in steady state in continuous culture, biomass with increased compound production, optimally when 75% to 90% is refreshed very couple of days.

Claims

CLAIMS1. Method for producing an Aphanizomenon flos aquae biomass, the method comprises the steps of: a) growing Aphanizomenon flos aquae in a closed reactor comprising a buffered growth medium until a dry biomass of between 1 to 10 grams / liter is obtained, preferably at an average day temperature below 23 °C; b) harvesting the Aphanizomenon flos aquae biomass from the reactor thereby obtaining an Aphanizomenon flos aquae biomass; c) optionally, further processing of the Aphanizomenon flos aquae biomass, preferably selected from one or more of the group consisting of induction of Aphanizomenon flos aquae biomass to form compounds, drying, and concentrating.

2. Method according to claim 1 , wherein the buffer of the growth medium is comprised of NaHCCh. KHCO3 and CaC'Ch. optionally further supplemented with NaiCCh.

3. Method according to claim 2, wherein the wherein the buffer of the growth medium is comprised of 0.01 to 0.04 grams / liter NaHCCh. 0.2 to 0.4 grams / liter KHCO3 and 0.01 to 0.1 grams / liter CaCCh, optionally further supplemented with 0.002 to 0.025 NaHCCh grams / liter, wherein the total amounts NaHCCh and NaHCCh are in the range of 0.01 to 0.75 grams / liter.

4. Method according to any one of the claims 1 to 3, wherein in step (a) the pH is between 4 to 9, preferably 6 and 8, most preferably 7.

5. Method according to any one of the claims 1 to 4, wherein the growth medium further comprises 0.5 to 5 mmol / liter Na+.

6. Method according to any one of the claims 5, wherein 0.5 to 5 mmol / liter Na+is provided by adding 0.03 to 0.2 grams / liter dried sea salt, or equivalent, to the growth medium.

7. Method according to claim 4, wherein the pH is maintained between 6.5 and 8 by using a CO2 flow.

8. Method according to any one of the claims 1 to 7, wherein, in step (a) the growing Aphanizomenon flos aquae is aerated.

9. Method according to any one of the claims 1 to 8, wherein step (a) is performed in a continue reactor.

10. Method according to any one of the claims 1 to 9, wherein the biomass or processed biomass is further supplemented with nutrients, colorings, flavorings, edible microorganisms, plant parts, plant extracts, seeds, nuts, buffers, dairy products, vitamins, minerals, foods or processed foods and / or wherein the biomass or processed biomass is further supplemented with Haematococcus pluvialis.

11. Method according to any one of the claims 1 to 10, wherein the biomass or processed biomass is processed into beverages, human and animal food supplements, food additives, powders, suspensions, pharmaceuticals, cosmetics or tablets.

12. Method according to any one of the claims 1 to 11, wherein step (a) comprises: al) growing Aphanizomenon flos aquae in a first reactor comprising a growth medium and a light source until a dry biomass of between 0.1 to 10 grams / liter is obtained; a2) optionally, transferring the Aphanizomenon flos aquae obtained in step (al) to a second reactor; a3) exposing the Aphanizomenon flos aquae of step (al) or step (a2) to UV radiation with a wavelength between 50 to 400 nm.

13. Method according to claim 12, wherein the light source in step (al) exposes the growing Aphanizomenon flos aquae to red light with a wavelength of circa 640-660, optionally supplemented with white light of 2300 to 6000K and blue light with a wavelength of circa 440-460 nm.

14. Method according to claim 13, wherein the light source in step (al) exposes the growing Aphanizomenon flos aquae to 50 to 100% red light with a wavelength of circa 640-660 nm.

15. Aphanizomenon flos aquae biomass obtainable by a method according to any one of the claims 1 to 14.

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