Method for cultivating phototrophic microalgae

Abstract

The invention relates to the field of biotechnology, and more particularly to a method for cultivating marine microalgae in enclosed photobioreactors with optimized physical and chemical parameters, and to a device for carrying out said method. The proposed photobioreactor and cultivation method (in particular, the chemical composition of a medium, and the illumination and aeration) make it possible to provide optimal conditions for microalgae cultivation which allow good gains in both microalgal biomass and carotenoid mass.

Description

[0001] A METHOD FOR SUN-INDEPENDENT CULTIVATION OF MARINE PHOTOTROPHIC MICROALGAE AND MAXIMIZING THE QUANTITY OF CAROTENOIDS PRODUCED BY THEM

[0002] Field of technology

[0003] The invention relates to the field of biotechnology, namely to a method for growing marine microalgae in closed photobioreactors with optimized physical and chemical parameters and a device for implementing it, with the aim of increasing the efficiency of cultivation and obtaining the largest possible amount of carotenoid-enriched biomass.

[0004] State of the art

[0005] The cultivation of photosynthetic (phototrophic) microalgae that produce carotenoids is finding commercial application. Modern research demonstrates the crucial role of carotenoids in maintaining human and animal health throughout life. Therefore, carotenoids are widely used as dietary supplements for humans, as well as feed additives for animals and poultry, and in aquaculture.

[0006] Optimization of industrial carotenoid production through microbiological synthesis is aimed at selecting new carotenoid-producing strains. However, to fully realize the biosynthetic potential of microalgae, optimal cultivation conditions are necessary to ensure maximum yield of target substances. The productivity of photosynthetic microorganisms depends on light, dissolved CO2 in the water, aeration activity, nutrient salts present in the water, microelements such as iron, calcium, and magnesium, and other parameters. Photosynthetic microorganisms can be cultivated indoors using artificial lighting, but outdoor cultivation in sunlight is most common. Various photobioreactors designed for microalgae cultivation are available.

[0007] Photobioreactors from Phtoton Systems Instruments (Czech Republic) are among the simplest thin-walled photobioreactors (the width between the glass panels in the incubation chamber is only 6.5 cm). Aeration is primarily achieved using atmospheric air, supplied from the bottom of the photobioreactor. Active or natural lighting is used, so the photobioreactors can be used both outdoors and with artificial lighting.

[0008] Photobioreactors from Subitec, Germany (US6509188B1, published January 21, 2003) are also thin-walled photobioreactors, but have a curved geometry optimized for open-air cultivation under sunlight, although they can also be used with artificial lighting. The main advantage of these photobioreactors is that their geometry creates numerous small "vortices" within the photobioreactor, initiated by the passage of air bubbles through curved channels. The manufacturer cites experimental results that demonstrate the advantage of such "vortices" in terms of microalgae biomass growth compared to other designs.

[0009] The disadvantages of such thin-walled photobioreactor systems include their relatively small cultivation volume and high cost. They are designed primarily to utilize natural light and must be installed outdoors or at least in greenhouses. While this may be justified in some cases (for example, when electricity costs are very high), such cultivation also imposes significant limitations, primarily related to weather conditions. Therefore, active lighting using LED lamps is preferable in many cases.

[0010] Photobioreactors from Industrial Plankton Inc., Canada (US9688950B2, published 06 / 27 / 2017), like photobioreactors from Algotek, Russia (RU2540011C1, published 01 / 27 / 2015; RU2718515C1, published 04 / 08 / 2020) are not thin-walled, but rather large tanks (rectangular or star-shaped), in which lighting is installed inside in the form of central lanterns (Algotek) or lamps evenly spaced at the corners of the star (star-shaped tank). Such photobioreactors have exclusively active lighting, which can lead to the burnout of carotenoids and, as a result, prevents their accumulation in maximum quantities. Another feature of these photobioreactors is the presence of built-in CIP washers, which makes maintenance and cleaning of such photobioreactors extremely simple and is a significant advantage over thin-walled photobioreactors.In general, these photobioreactors are significantly cheaper than thin-walled designs and are much easier to maintain. However, the efficiency of aeration and mixing in these bioreactors is not always optimal.

[0011] Disclosure of invention

[0012] The objective of the invention is to create a closed-type microalgae cultivation system consisting of photobioreactor chambers with active lighting and active aeration, as well as to develop a method for culturing in such a closed system for the purpose of optimal reproduction of microalgae and maximum production of carotenoids produced by these microalgae.

[0013] To solve the stated problem, a method for cultivating phototrophic microalgae producing carotenoids has been developed, including the preparation of a mineral medium, the cultivation of microalgae and the removal of the resulting suspension for flow centrifugation, followed by the discharge of the precipitated biomass as the target product, with the cultivation being carried out:

[0014] - in the photobioreactor chamber with constant (24-hour) illumination of 150 - 250 lumens per 1 liter of the culture mixture, with constant circulation of the culture mixture, preventing the settling of microalgae to the bottom, and its constant aeration, preventing the settling of microalgae to the bottom, and its constant aeration with atmospheric air at a rate of 0.4 - 0.6 liters of air per liter of culture mixture per minute using an aerator installed at the bottom of the photobioreactor chamber, providing vortex movements of the culture mixture by pumping atmospheric air with the formation of air bubbles,

[0015] - in a mineral medium having the following composition per liter of mineral medium:

[0016] NaNO3288.0 - 403.2 mg / l

[0017] MgSO4'7H2O 37.5 - 52.5 mg / l,

[0018] KH2PO4 8.5 - 11.9 mg / l

[0019] K2HPO4 18.75 - 26.25 mg / l

[0020] EDTA 63.7 mg / l,

[0021] H3BO3 11.42 mg / l,

[0022] FeSO4'7H2O 10.0 mg / l,

[0023] ZnSO4'7H2O 4.0 mg / l,

[0024] MnCl2'4H2O 4.57 mg / l,

[0025] CUSO4'5H2O 2.35 mg / l,

[0026] Na2Mo04'2H20 1,016 mg / l,

[0027] CO(N03)'6H20 I ,00 mg / l,

[0028] NaCI 0 - 45 g / l,

[0029] (NH4)2SO433.4 - 66.07 mg / l

[0030] succinic acid II.81 - 35.43 mg / l

[0031] water is the rest;

[0032] - in this case, the pH value of the culture mixture is maintained in the range of 6.0 - 7.0.

[0033] In some embodiments of the invention, illumination is provided with white light with a color temperature of 4000-4100 K or cold white light with a color temperature of 5000-6500 K. In particular embodiments, the size of the air bubbles for aeration is 0.8 - 2.1 mm.

[0034] In some embodiments of the invention, the circulation of the culture mixture is provided by at least two pumps, providing a flow of the culture mixture at a rate of 5000 - 7000 liters / hour and directing the flow of the culture mixture from the pumps along the bottom of the photobioreactor chamber.

[0035] In some embodiments of the invention, the mineral medium additionally contains thiamine - 1 mg / l and / or cyanocobalamin - 5 μg / l. In some embodiments of the invention, the pH level during the preparation of the mineral medium and throughout the cultivation is maintained by adding succinic acid and (N 4) 2804 to the mineral medium; more precise pH regulation during the cultivation process is achieved by adding 2M NaOH and / or 2M HCl.

[0036] In specific embodiments of the invention, cultivation is carried out for at least one cycle lasting 4-7 days. Cultivation cycles can be repeated an unlimited number of times, with the resulting suspension being partially removed for the next cultivation cycle, leaving 5-20% of the resulting suspension in the photobioreactor chamber.

[0037] The stated problem is also solved by developing a photobioreactor for cultivating phototrophic microalgae that produce carotenoids. The developed photobioreactor includes at least one photobioreactor chamber connected via a discharge line to a flow-through centrifuge, a mineral medium preparation unit connected at the outlet to the photobioreactor chamber, and an automatic cleaning system that ensures cleaning of the device modules without disassembling them.

[0038] - the photobioreactor chamber is equipped with lamps providing illumination of 150-250 lumens per 1 liter of culture mixture;

[0039] - the photobioreactor chamber is equipped with at least two pumps that ensure circulation of the culture mixture, preventing the microalgae from settling to the bottom, and an aerator installed at its bottom and providing vortex movements of the culture mixture by pumping atmospheric air with the formation of air bubbles with constant aeration of the culture mixture with atmospheric air at a rate of 0.4 - 0.6 liters of air per liter of culture mixture per minute.

[0040] In some embodiments of the invention, at least pumps are installed at the bottom of the photobioreactor chamber to ensure the flow of the culture mixture at a rate of 5000 - 7000 liters / hour, and the flow of the culture mixture is directed from the pumps along the bottom of the photobioreactor chamber.

[0041] In particular embodiments of the invention, the lamps provide illumination with white light with a color temperature of 4000 - 4100 K or cold white light with a color temperature of 5000 - 6500 K.

[0042] In some embodiments of the invention, the photobioreactor further has a system for adding water to the photobioreactor chamber during the cultivation process to maintain the volume of the culture mixture constant.

[0043] In some embodiments of the invention, the photobioreactor comprises two or more photobioreactor chambers. In particular embodiments of the invention, the photobioreactor is equipped with a programmable automatic monitoring and control system for at least specified parameters of temperature, pH, volume, dosage, loading of the mineral medium, removal of the resulting suspension into a flow centrifuge, and unloading of the precipitated biomass.

[0044] In some particular embodiments of the invention, the photobioreactor chamber is made in the shape of a parallelepiped, and the lamps are evenly distributed on panels installed along all side walls of the parallelepiped.

[0045] The developed photobioreactor and the cultivation method (especially the chemical composition of the medium, lighting and aeration) make it possible to achieve the technical result, which consists in creating optimal conditions for the cultivation of microalgae, ensuring a good increase in both biomass and carotenoid mass.

[0046] Brief description of the drawings

[0047] Fig. 1 A - B - Photobioreactor in the parallelepiped version: A - top view; B - front view; B - side view.

[0048] The numbers indicate: 1 - LED lamps, 2 - aerator tube, 3 - pump, 4 - direction of flow of air bubbles and liquid from the surface.

[0049] Fig. 2 - Photograph of a laboratory experimental sample, working volume 280 liters, without liquid, top view.

[0050] Terms and definitions

[0051] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement with equivalent techniques known to those skilled in the art.

[0052] In the documents of this invention, the terms "includes," "including," and the like, as well as "contains," "comprising," and the like, are interpreted to mean "includes, among other things" (or "contains, among other things"). These terms are not intended to be construed as meaning "consists solely of."

[0053] The term "and / or" means one, more than one, or all of the listed elements.

[0054] Numeric values ​​described in this document are intended to refer both to the stated value and to a functionally equivalent range around that value. Furthermore, enumerations of numerical ranges with endpoints described in this document include all numbers within that range.

[0055] The term "optional" or "optional" or "optionally" as used in this document means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0056] Within the context of the present invention, microalgae are understood to mean phototrophic microalgae that produce carotenoids. These include, but are not limited to, green microalgae (Chlorophyta) or ochrophyte microalgae (Ochrophyta). Examples of such microalgae include, but are not limited to, microalgae belonging to the genus Parachlorella or the genus Desmodesmus, or the class Xanthophyceae.

[0057] The term “water” (“purified water”) means water that has been maximally purified from impurities, such as distilled water, deionized water obtained by reverse osmosis, or water purified according to FS.2.2.0020.15.

[0058] By "vortex motion" we mean the movement of a liquid or gas in which its small elements (particles) move not only progressively, but also rotate around a certain instantaneous axis.

[0059] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

[0060] Detailed description of the invention

[0061] The design features, advantages, and disadvantages of existing photobioreactors were taken into account during the development of the photobioreactor. To ensure optimal conditions for culturing carotenoid-producing microalgae, significant modifications were made to the photobioreactor's design, primarily to lighting, aeration, and circulation of the culture mixture.

[0062] Thus, the photobioreactor chamber is equipped with lamps providing constant illumination of 150-250 lumens per 1 liter of culture mixture. Providing active illumination eliminates the need to use a translucent material for the photobioreactor chamber. It can be made of any material that ensures ease of use and the safety of the process and the resulting product, and there is no specific requirement for the volume or shape of the photobioreactor chamber. That is, the reactor can be, for example, parallelepiped, cube, or cylinder. In specific (but non-limiting) embodiments of the invention, the shape, dimensions, and arrangement of the photobioreactor chamber lamps are selected so as to ensure a light path length of 30-50 cm. In preferred embodiments, illumination is provided by LED lamps with white light with a color temperature of 4000-4100 K or cool white light with a color temperature of 5000-6500 K.In one particular, but non-limiting, embodiment of the invention, the photobioreactor chamber is shaped like a parallelepiped, with lamp panel systems arranged on all walls (see Example 1 and Figs. 1A-B). This arrangement of lamps along the entire perimeter of the walls forming the photobioreactor chamber ensures more uniform illumination of a larger volume of the culture mixture.

[0063] Experiments have shown that continuous (rather than intermittent) illumination of 200-250 lumens per second per liter of culture medium creates optimal conditions for microalgae to accumulate carotenoids. Using brighter lamps can cause carotenoids to burn out, thus preventing their accumulation in maximum quantities. Cool white light is somewhat preferable, as it contains more blue light, which activates photosynthesis and promotes carotenoid production. When using cool white light, the lumens per liter can be set at the lower end of the recommended range or even 10-20% lower.

[0064] Furthermore, unlike existing designs, the developed photobioreactor features an exceptionally effective aeration system, capable of meeting the microalgae's carbon dioxide and oxygen needs without the need for additional gas. This aeration is achieved by passing air bubbles through the water using an aerator powered by an air pump. Calculations and experiments have shown that the optimal aeration rate is approximately 0.4-0.6 liters of air per liter of culture medium per minute. It is also crucial to use an aerator that creates a vortex effect throughout the photobioreactor chamber—the formation of vortex motions in the culture mixture by pumping in atmospheric air, creating air bubbles. The preferred air bubble size is 0.8-2.1 mm. Oxygen transfer efficiency is 0.25-0.45 kg O2 / h.Since such an air flow leads to strong bubbling and evaporation in photobioreactor chambers with a volume of 250 liters or more, the design of a large-volume photobioreactor chamber additionally includes a system for adding water to the culture medium to maintain a constant volume. To achieve optimal aeration parameters throughout the entire photobioreactor chamber, an aerator is installed at the bottom of the chamber. In one specific, but non-limiting, embodiment of the invention, the aerator is a tube with microscopic holes that is attached to the bottom of the parallelepiped-shaped photobioreactor chamber at opposite corners, thereby generating a strong air flow diagonally across the parallelepiped (see Example 1 and Fig. 1A).

[0065] The design of the photobioreactor according to the invention provides for constant circulation of the culture mixture, preventing the microalgae from settling to the bottom. Such circulation can be ensured, for example, by pumps that ensure the movement (flow) of the culture mixture from the pumps along the bottom of the photobioreactor chamber. In particular embodiments of the invention, two or more pumps can be installed to ensure circulation, providing a flow rate of 5000-7000 liters / hour, directing the flow of liquid from the pumps along the bottom of the container. In particular, for example, in a parallelepiped-shaped photobioreactor chamber, two pumps can be installed at corners different from the corners along which the aerator is oriented (see Example 1 and Figs. 1A-B).

[0066] The developed photobioreactor includes at least one chamber. Each chamber is connected via a drain line to a continuous-flow centrifuge, with the drain valve located near the bottom of the chamber. The photobioreactor also includes an automatic cleaning system (CIP) that allows for cleaning of the device modules without disassembling them. The size of the photobioreactor chamber can vary widely, depending on production requirements. For example, the working volume of a photobioreactor chamber can be 250 liters, 500 liters, or 1000 liters, but is not limited to these. A photobioreactor can have a single chamber or more than one, such as two, three, or more chambers.

[0067] Thus, the developed photobioreactor provides cultivation parameters such as optimal (not maximum) illumination and exceptionally good aeration and circulation of the culture mixture, which ensure both good biomass and carotenoid growth, significantly increasing the efficiency of biotechnological carotenoid production. The design features of the photobioreactor are complemented by chemical properties implemented in the culture medium used. These are presented below.

[0068] The culture medium was developed using Bold's Basal Medium (BBM) as the basis, with modifications to provide optimal conditions for carotenoid-producing microalgae. The medium consists of four components: (1) Basic Salinity, (2) Macronutrients, (3) Micronutrients, and (4) pH Regulators.

[0069] (1) The base salinity is determined by the final NaCl concentration, which can vary between 0 and 4.5 wt% for different microalgae (i.e., no more than 45 g / L). Salinity is selected based on the NaCl concentration requirements of a specific microalgae species.

[0070] (2) Macronutrients

[0071] In the context of the invention, macronutrients include NaNO3, MgBO2H2O, KH2PO4, and K2HPO4. The final concentration of macronutrients in the medium may vary within the following limits:

[0072] NaNO3288.0 - 403.2 mg / l,

[0073] MgSO4'7H2O 37.5 - 52.5 mg / l,

[0074] KH2PO4 8.5 - 11.9 mg / l K2HPO418.75 - 26.25 mg / l.

[0075] (3) The content of microelements in the medium is controlled at a precisely defined level (final concentration):

[0076] EDTA 63.7 mg / l,

[0077] H3VO3 11.42 mg / l,

[0078] FeSO4-7H2O 10.0 mg / l (prepared together with EDTA),

[0079] ZnSO4-7H2O 4.0 mg / l,

[0080] MnCI2-4H2O 4.57 mg / l,

[0081] CUSO4-5H2O 2.35 mg / l,

[0082] Na2Mo042H20 1 .016 mg / l,

[0083] CO(N03)'6H20 1.0 mg / l.

[0084] (4) pH regulator

[0085] During cultivation, the pH must be maintained within the range of 6.0 to 7.0. The required pH level can be achieved by various means known in the art and suitable for microalgae cultivation. In some non-limiting embodiments, the pH level during the preparation of the mineral medium and throughout cultivation is maintained by adding succinic acid and ammonium sulfate to the mineral medium, while more precise pH regulation during cultivation is achieved by adding 2 M NaOH and / or 2 M HCl. Since succinic acid is a weak acid, it increases its dissociation as sodium ions accumulate, which occurs during the consumption of nitrates by the microalgae. Ammonium sulfate ((NH4)2SO4) releases ammonium ions to the microalgae as a source of nitrogen.During cultivation, sulfate ions accumulate, which also compensate for the accumulation of sodium ions resulting from the microalgae's consumption of nitrates. Therefore, to maintain the pH at the desired level, succinic acid and ammonium sulfate are added gradually during cultivation. Succinic acid concentrations in the culture mixture range from 11.81 to 35.43 mg / L, and ammonium sulfate concentrations range from 33.4 to 66.07 mg / L.

[0086] In some embodiments of the invention, in order to accelerate the specific growth of the culture and / or the release of carotenoids, thiamine (in an amount of 1 mg / l) and / or cyanocobalamin (5 μg / l) can be added to the culture medium, which have a stimulating effect on the growth of microalgae.

[0087] Cultivation in the photobioreactor according to the invention is carried out until a cell density of approximately 50-70 million / L is reached, depending on the strain characteristics. At the end of the cultivation cycle, the majority of the culture medium (80-95%) containing the microalgae is collected to obtain the final biomass. The stock culture (5-20%) is retained, and new medium is added to resume cultivation. For various microalgae, cultivation cycles last 2-8 days, most commonly 4 to 7 days, and can be repeated virtually indefinitely.

[0088] After harvesting, the biomass is dried to obtain the final product, sterilized and freeze-dried if necessary, and its qualitative and quantitative composition is analyzed. The resulting product can be used to produce dietary supplements containing carotenoids, enrich foods with carotenoids, serve as a feed additive for animals and poultry, be used in aquaculture, serve as a food coloring and pigment, and more.

[0089] The possibility of objectively demonstrating the technical result when using the invention is confirmed by reliable data given in examples containing experimental information obtained in the process of conducting research using methods accepted in this field.

[0090] It should be understood that the examples given in the application materials are not limiting and are given only to illustrate the present invention.

[0091] Example 1. Photobioreactor with a working volume of 280 liters

[0092] As a test sample, a laboratory experimental photobioreactor was designed, a detailed diagram of which is shown in Fig. 1, and a photograph is presented in Fig. 2, having a chamber with the following parameters:

[0093] • The basis was a plastic container with dimensions of 1017 x 636 x 793 mm (with wheels attached). The actual internal dimensions of the container are approximately 1000 x 600 x 500 mm, the total volume (excluding stiffeners, etc.) is approximately 280 liters;

[0094] • LED lamps (position 1 in Fig. 1) on the genuine side, approximately 1400 lumens (10 pieces each), combined into a panel;

[0095] • LED lamps (position 1 in Fig. 1) on the short side, approximately 1200 lumens (10 pieces each), combined into a panel;

[0096] • An aerator tube (aerator-spray) 75 cm long (position 2 in Fig. 1), installed at the bottom of the photobioreactor chamber and providing constant aeration with atmospheric air at a rate of 0.4 - 0.6 liters of air per l / min, and also providing vortex movements of the culture mixture by pumping atmospheric air with the formation of air bubbles of 0.8-2.1 mm in size, with an oxygen transfer efficiency of 0.25-0.45 kg Og / h;

[0097] • Air pump with a capacity of 120 liters / minute;

[0098] • Two pumps (position 3 in Fig. 1), providing a flow of 5000 - 7000 liters / hour (the direction of flow is shown by arrows (marked with number 4) in Fig. 1);

[0099] • SIP-washing. Example 2. Carrying out cultivation in a photobioreactor

[0100] The cultivation was carried out in a photobioreactor chamber constructed according to Example 1. The following microalgae were used as test phototrophic microalgae producing carotenoids: Desmodesmus subspicartus (Dia 5), ​​Parachlorella sp. (Nanno 1), Tetraselmis suecica, Tisochrysis lutea, Chaetoseros muellery, Chaetoceros gracilis and Pavlova lutheri, with each of them undergoing a series of cultivation experiments under different conditions, both in terms of the composition of the mineral medium and in terms of physical parameters such as temperature, illumination, circulation and aeration of the culture mixture.

[0101] The operating temperature in the chamber was selected depending on the microalgae species, ranging from 25 to 29°C; for some microalgae, it ranged from 8 to 35°C. As experiments showed, although ambient temperature influences the productivity of carotenoid-synthesizing microalgae, once other cultivation conditions are established as optimal, the operating temperature in the chamber for many microalgae can vary widely.

[0102] Lighting was provided 24 hours a day, ranging from 150 to 250 lm / L, depending on the needs of the individual species. Cultivation was carried out with constant circulation of the culture mixture containing the microalgae, preventing them from settling to the bottom, and with constant aeration of atmospheric air at a rate of 0.4 to 0.6 liters of air per L / min.

[0103] The cultivation was carried out in a modified mineral medium VVM of the following composition (per liter of water): NaNOs (288.0 - 403.2 mg / l), MgSO4 7H2O (37.5 -52.5 mg / l), KH2PO4 (8.5 - 11.9 mg / l), K2HPO4 (18.75 - 26.25 mg / l), EDTA (63.7 mg / l), H3BO3 (11.42 mg / l), FeSO4 7H2O (10.0 mg / l), ZnSO4 7H2O (4.0 mg / l), MnCl2 4H2O (4.57 mg / l), CUSO4 5H2O (2.35 mg / l), Na2MoO4 2H20 (1.016 mg / l), Co (NO3) 6H20 (1.0 mg / l). In this case, the concentration of macroelements (NaNOs, MgSO4'7H2O, KH2PC>4 and K2HPC>4) was selected in different experiments from the minimum to achieving the optimal value for a specific microalgae, determined by the rate of biomass growth.

[0104] The salinity of the medium was selected for each microalgae species from 0 to 4.5%, depending on the salinity requirements of specific microalgae. Thus, for Desmodesmus subspicartus (Dia 5), ​​the salinity was 1.5%, for Parachlorella sp. (Nanno 1), the salinity was 2%, for Tetraselmis suecica, the salinity was 2%, for Tisochrysis lutea, the salinity was -2%, for Chaetoseros muellery, the salinity was 2%, for Chaetoceros gracilis, the salinity was 2%, and for Pavlova lutheri, the salinity was 2%.

[0105] The pH of the culture mixture was maintained in the range of 6.0–7.0 throughout cultivation (the limits of succinic acid content in the culture mixture varied within 11.81–35.43 mg / l, ammonium sulfate within 33.4–66.07 mg / l; more precise pH regulation during cultivation was achieved by adding 2M NaOH and / or 2M HCl).

[0106] Microalgae were cultured under the selected conditions for at least two 7-day cycles. After each cycle, the resulting suspension was removed for continuous-flow centrifugation, followed by the removal of 50-90% of the precipitated biomass (preferably 80-95%). The remaining biomass was used to start the next cultivation cycle.

[0107] Productivity was assessed based on the results of each cultivation cycle. Table 1 presents the results of the evaluation of the cultivation conditions used and their impact on the productivity of the tested microalgae (productivity is presented as an average value for several cultivation cycles).

[0108] Table 1. Cultivation of phototrophic microalgae of different species producing carotenoids according to the developed method

[0109]

[0110] As shown by the measurement results presented in Table 1, cultivation of all tested species of carotenoid-producing phototrophic marine microalgae using the proposed method resulted in biomass gains ranging from 0.4 g / L dry biomass for Chaetoseros muelleri to 0.9 g / L dry biomass for Chaetoceros gracilis. According to literature data, such biomass gains are significant, confirming the effectiveness of the proposed method and the optimality of the developed cultivation modes achieved using the developed photobioreactor.

[0111] Example 3. Collection and preparation of biomass for final formulation, assessment of the amount of carotenoids

[0112] The assessment of the amount of produced carotenoids is shown using the example of the microalga Parachlorella sp. strain Nannol (IPPAS C-2073).

[0113] The readiness of microalgae biomass for harvesting was assessed using the following methods:

[0114] 1. Visually, based on the degree of darkening of the culture fluid. Used only for a quick preliminary assessment.

[0115] 2. Using a microscope, collecting 20 µl of sample and counting the number of cells per ml using a Goryaev chamber. For the microalgae strain Parachlorella sp. Nannol, the cell density was approximately 50-10 6 / ml.

[0116] A flow-through centrifuge was used to collect the biomass. The resulting microalgae concentrate was sterilized to remove foreign microorganisms. This is especially important before preparing the microalgae for the creation of biologically active additives (BAA) or food supplements for humans. For this purpose, a thick paste (containing 25% dry microalgae) is diluted with water or saline. This is followed by sterilization by heating, followed by freezing and sublimation. The treatment is performed at a rate of 2 W / ml. The microalgae are heated until they begin to boil (usually 3-4 minutes). The treatment is then immediately stopped, and the microalgae concentrate is frozen before sublimation. After sublimation, the resulting product can be packaged for dietary supplement formulations or used as food.

[0117] The collected Parachlorella sp. Nannol microalgae biomass was analyzed qualitatively and quantitatively for carotenoid and chlorophyll content after harvesting, sterilization, and freeze-drying. The results are presented in Table 2.

[0118] Table 2. Content of photosynthetic pigments

[0119]

[0120] As can be seen from Table 2, the yield of the useful product, carotenoids, was over 13 mg per g of dry biomass per cultivation cycle. However, culturing this strain under different conditions (more intense but not constant illumination, lack of continuous circulation of the culture medium with vortex motion, and a different mineral composition) does not allow for such a carotenoid yield.

[0121] Table 3 shows the results of the analysis of carotenoids contained in the obtained sublimate.

[0122]

[0123] Thus, the developed photobioreactor and the cultivation method (especially the chemical composition of the medium, lighting and aeration) make it possible to provide optimal conditions for the cultivation of microalgae, ensuring a good increase in both the biomass of microalgae and the mass of carotenoids.

[0124] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific, detailed examples are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

Claims

Invention formula 1. A method for cultivating phototrophic microalgae producing carotenoids, comprising preparing a mineral medium, cultivating the microalgae and removing the resulting suspension for flow centrifugation followed by unloading the precipitated biomass as the target product, wherein the cultivation is carried out: - in the photobioreactor chamber with constant illumination of 150 - 250 lumens per 1 liter of the culture mixture, with constant circulation of the culture mixture, preventing the microalgae from settling on the bottom, and its constant aeration with atmospheric air at a rate of 0.4 - 0.6 liters of air per liter of culture mixture per minute using an aerator installed at the bottom of the photobioreactor chamber, providing vortex movements of the culture mixture by pumping atmospheric air with the formation of air bubbles, - in a mineral medium having the following composition per liter of mineral medium: NaNO3288.0 - 403.2 mg / l MgSO4'7H2O 37.5 - 52.5 mg / l, KH2PO4 8.5 - 11.9 mg / l K2HPO4 18.75 - 26.25 mg / l EDTA 63.7 mg / l, H3BO3 11.42 mg / l, FeSO4'7H2O 10.0 mg / l, ZnSO4'7H2O 4.0 mg / l, MnCl2'4H2O 4.57 mg / l, CUSO4'5H2O 2.35 mg / l, Ma2Mo04-2HgO 1.016 mg / l, CO(N03)'6H20 I, 0 mg / l, NaCI 0 - 45 g / l, (NH4)2SO433.4 - 66.07 mg / l succinic acid II.81 - 35.43 mg / l water is the rest; - in this case, the pH value of the culture mixture is maintained in the range of 6.0 - 7.

0.

2. The method according to claim 1, in which illumination is provided with white light with a color temperature of 4000 - 4100 K or cold white light with a color temperature of 5000 - 6500 K.

3. The method according to any one of paragraphs 1-2, in which the size of the air bubbles formed by the aerator during aeration is 0.8 - 2.1 mm.

4. The method according to any one of paragraphs 1-3, in which the circulation of the culture mixture is provided by at least two pumps that ensure the flow of the culture mixture at a rate of 5000 - 7000 liters / hour and direct the flow of the culture mixture from the pumps along the bottom of the photobioreactor chamber.

5. The method according to any one of paragraphs 1-4, in which the mineral medium additionally contains thiamine - 1 mg / l and / or cyanocobalamin - 5 μg / l.

6. The method according to any of paragraphs 1-5, in which the pH level during the preparation of the mineral medium and throughout the cultivation is ensured by adding succinic acid and (N 4)2804 to the mineral medium, more precise regulation of the pH during the cultivation process is carried out by adding 2M NaOH and / or 2M HCl.

7. The method according to any one of paragraphs 1-6, characterized in that the cultivation is carried out for at least one cycle of 4-7 days.

8. The method according to item 7, characterized in that the cultivation cycles are repeated an unlimited number of times, while, for the next cultivation cycle, the resulting suspension is not completely removed, leaving 5 - 20% in the photobioreactor chamber.

9. A photobioreactor for cultivating phototrophic microalgae producing carotenoids using the method according to any one of paragraphs 1-8, comprising at least one chamber of the photobioreactor connected by a discharge line to a flow centrifuge, a mineral medium preparation unit connected at the outlet to the chamber of the photobioreactor, and an automatic washing system that ensures washing of the modules of the device without disassembling them, characterized in that - the photobioreactor chamber is equipped with lamps providing illumination of 150 - 250 lumens per 1 liter of culture mixture; - the photobioreactor chamber is equipped with at least two pumps that ensure circulation of the culture mixture, preventing the microalgae from settling to the bottom, and an aerator installed at its bottom and providing vortex movements of the culture mixture by pumping atmospheric air with the formation of air bubbles with constant aeration of the culture mixture with atmospheric air at a rate of 0.4 - 0.6 liters of air per liter of culture mixture per minute.

10. The photobioreactor according to paragraph 9, characterized in that the pumps provide a flow of the culture mixture at a rate of 5000 - 7000 liters / hour, and direct the flow of the culture mixture from the pumps along the bottom of the chamber of the photobioreactor.

11. The photobioreactor according to any of paragraphs 9-10, characterized in that the lamps provide illumination with white light with a light temperature of 4000 - 4100 K or cold white light with a light temperature of 5000 - 6500 K.

12. A photobioreactor according to any of paragraphs 9-11, characterized in that it additionally has a system for adding water to the photobioreactor chamber during the cultivation process to maintain the volume of the culture mixture constant.

13. A photobioreactor according to any of paragraphs 9-12, characterized in that it contains two or more photobioreactor chambers.

14. A photobioreactor according to any of paragraphs 9-13, characterized in that it is equipped with a programmable automatic control and management system for at least specified parameters of temperature, pH, volume, dosage, loading of mineral medium, removal of the resulting suspension into a flow centrifuge, and unloading of sedimented biomass.

15. A photobioreactor according to any of paragraphs 9-14, characterized in that the photobioreactor chamber is made in the shape of a parallelepiped, and the lamps are evenly distributed on panels installed along all side walls of the parallelepiped.

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