PROCESS FOR THE PRODUCTION OF BIOMASS AND PHYCOCYANIN IN A PHOTOBIOREACTOR FROM THE CULTIVATION OF SPIRULINA PLATENSIS

Description

1 / 25 PROCESS FOR THE PRODUCTION OF BIOMASS AND PHYCOCYANIN IN A PHOTOBIOREACTOR FROM THE CULTIVATION OF SPIRULINA PLATENSIS

[001] The present invention aims at the production of cyanobacteria biomass in a functional pilot-scale bioreactor, with low construction and operating costs, capable of cultivating various species, accelerating their growth, and thus obtaining biomass, supernatant, and phycocyanin pigment as products. To obtain the biomass, a culture medium with 0.35 g / L of NPK supplemented with 0.10 mL / L of micronutrient solution and water is used. The NPK consists of nitrogen: phosphorus: potassium in the following proportion 4:14:8. The cyanobacteria are optimized for the respective culture medium containing mineral salts and nutrients for the production of biomass and phycocyanin.

[002] Cyanobacteria are microorganisms with high photosynthetic efficiency, high biomass production, and rapid growth compared to other energy crops. Considering the immense biodiversity and consequent variability in the biochemical composition of the biomass obtained from microalgae cultures, coupled with the establishment of large-scale cultivation technology as well as the use of alternative cultivation sources, certain species have been allowed to be commercially exploited. Thus, this group of microorganisms represents one of the promising sources for new products, uses, and applications.

[003] The product portfolio extends from the simple production of biomass for human food and animal feed to value-added products obtained from biomass and cell-free culture medium (supernatant), including bioactive compounds that exhibit antimicrobial effects, Petition 870250079395, dated 04 / 09 / 2025, page 6 / 31 2 / 25 antioxidants as well as growth factors that can also be transformed and used for the production of biofertilizers. In addition to these, purified natural pigments can also be used in the cosmetic, food, medical and pharmaceutical fields. Fundamentals of the invention

[004] Cyanobacteria belong to the group of promising microorganisms that deserve attention within the vast range of new biotechnologies. An important characteristic of cyanobacterial systems is their versatility, making it possible to aggregate different applications within the same process, for example, in wastewater treatment, food production, animal feed, biochemical products with high commercial value, and recently in the area of ​​renewable energy. Another attractive characteristic of cyanobacteria, compared to other microorganisms, is their photosynthetic capacity to convert solar energy into biomass with a quite interesting biochemical composition. As such, cyanobacteria can play an important role in solar biotechnology (De la Noue and Pauw, 1988).

[005] Spirulina (Arthrospira) is a cyanobacterium that forms helical, multicellular filaments. These can be about 50 to 300 μm long and 10 μm in diameter (Costa et al., 2000; Vonshak & Maske, 1982). Species of this cyanobacterium have a cell wall composed of several layers of glucan and peptidoglycan polymers, covered by an outer sheath of acidic polysaccharides (Chen et al., 2020). These characteristics are beneficial for the preservation of intracellular components such as vitamins and fatty acids. Furthermore, they lack cellulose, which facilitates the digestion of this microalga by the human body (Tomaselli, 1997). This Petition 870250079395, dated 04 / 09 / 2025, page 7 / 31 3 / 25 Cyanobacteria is widely used as a food supplement and nutraceutical. Its taxonomic classification places it in the domain Bacteria, phylum Cyanobacteria, class Cyanophyceae, and family Oscillatoriaceae. (Alves de Oliveira et al., 2013; Pulz & Gross, 2004).

[006] Photosynthetic organisms possess organic pigments that absorb light energy. The main classes of pigments are chlorophylls (green), carotenoids (yellow or orange), and phycobilins (blue or pink), the first two being lipophilic pigments and the latter two hydrophilic (Sydney, 2009). Cyanobacteria of the genus Spirulina stand out as producers of phycobiliproteins, which are found in the thylakoid membranes of chloroplasts in the form of phycobilisomes. These are the main light-harvesting structures of photosystem II, transmitting light energy to the center of the photosynthesis reaction (Safaei et al., 2019; LA Silva et al., 2009).

[007] The phycobilisome is a protein complex composed of several bilin-type protein subunits, namely phycoerythrin (PE), which is pink or reddish in color and has a maximum absorption wavelength in the range of 565 to 575 nm, phycocyanin (PC), with an absorption spectrum in the wavelength range between 610 and 620 nm, and allophycocyanin (AC), which is blue-green in color and has an absorption spectrum between 650 and 665 nm (Figure 2). Phycocyanin is an oligomeric protein, composed of the same number of α and β subunits, both with the bilin chromophore. Due to its properties, phycocyanin is the most important pigment in this protein complex, with high added value and commercial interest. (Adjali et al., 2022; Safaei et al., 2019; Yan et al., 2014). Petition 870250079395, dated 04 / 09 / 2025, page 8 / 31 4 / 25

[008] There are different ways to produce microalgal biomass on a large scale, using formulated or diluted growth media, in open tanks or photobioreactors, with controlled lighting and temperatures or with ambient lighting and temperature (Zhou et al., 2022). The biomass composition and growth levels of Spirulina sp. can vary according to the nutritional conditions of the culture medium and the temperature, lighting, and aeration conditions to which they are subjected. Relevant concentrations of phosphorus and nitrogen are necessary in the culture media for biomass and bioproduct production. Nitrogen sources are important for the production of enzymes and proteins, while phosphorus is assimilated for ATP production, phospholipid synthesis, and nucleic acid synthesis (Muliterno et al., 2005; T. Zhou et al., 2022).

[009] Companies that produce products from cyanobacteria have recently developed new technical systems for the production and recovery of biomass, thus obtaining differentiated products with high added value. Among these differentiated products with potential for commercial production are, in addition to the biomass itself, organic substances such as lipids, proteins, carbohydrates and natural pigments, for example, chlorophyll, carotenoids and phycocyanin. And such extracts obtained from microalgal biomass have diverse commercial applications.

[010] The “downstream” stage of Spirulina begins with the collection of biomass and its destination for the production procedures of a product from it, whether dry or lyophilized biomass, in powder, capsules, nibs and tablets or for the extraction of bioproducts from it, such as pigments (Costa et al., 2019; Trivedi et al., 2015). Petition 870250079395, dated 04 / 09 / 2025, page 9 / 31 5 / 25

[011] The search for bioactive compounds from cyanobacterial biomass is becoming increasingly promising, accompanied by the growing introduction of commercially cultivated species, facilitating and enabling the production of these molecules of interest. Controlled maintenance and production of cyanobacteria are essential factors in exploring them as an economically viable source of important products. Natural pigments of microbial origin are a class of pigments that offer advantages in terms of production compared to their counterparts extracted from plants or animals. The use of pigment production through bioprocesses involving microorganisms presents advantages such as: continuous cultivation and rapid multiplication of these microorganisms, which can guarantee such productivity for the process that it becomes advantageous (Rangel-Yagui et al., 2004).

[012] Natural pigments, among their various functions in the food, pharmaceutical, and biochemical fields, exhibit antioxidant activity. Microalgae are photoautotrophic organisms that are exposed to high oxygen levels and free radical stress and, consequently, have developed several efficient protection systems against reactive oxygen species and free radicals. The content and type of antioxidant compounds depend on the microalgae species and its growth conditions (Pulz and Gross, 2004).

[013] The commercial production of biomass and intracellular and extracellular metabolites from cyanobacteria requires several steps to be followed: (1) large-scale mono-aseptic production suitable for biomass production; (2) concentration of biomass from a culture medium, (3) extraction of the metabolite of interest from the biomass and / or culture supernatant and (4) purification of the metabolite. Petition 870250079395, dated 04 / 09 / 2025, page 10 / 31 6 / 25

[014] The concentration of biomass produced in cultures is achieved through tangential flow filtration. Tangential flow filtration, or cross-flow filtration, is a filtration system with the potential to recover large volumes of microalgae culture. In tangential flow filtration, the medium flows tangentially through a membrane. The retained biomass is recirculated through the membrane, keeping the cells in suspension and minimizing fouling (Uduman et al. 2010). Particles smaller than the membrane pores are able to pass through, while larger particles are retained. The type of membrane used in this process can be a microfiltration membrane or ultrafiltration membrane, both available in a wide range of pore sizes or molecular weights (Petrusevski et al., 1995). Grima et al. (2003) reported that microfiltration and ultrafiltration are alternatives that can be used to recover microalgae biomass.The advantage of tangential flow filtration over other recovery techniques such as conventional filtration, centrifugation, flocculation, and sedimentation is that better filtration rates can be achieved with the complete removal of debris and microalgae cells.

[015] After the biomass concentration stage, cell permeabilization can be performed. Cell permeabilization is an alternative technique aimed at accessing intracellular compounds. This method consists of using permeabilizing agents, which act by removing phospholipids from the layer and making the cell porous, allowing the passage of small molecules and solutes (Panesar et al., 2007). Membrane permeability can be altered without total destruction of its integrity, through a treatment known as cell membrane permeabilization. Solvents Petition 870250079395, dated 04 / 09 / 2025, page 11 / 31 7 / 25 Organic solvents and detergents have been studied in different concentrations and combinations as permeabilizing agents. However, organic solvents have proven to be more advantageous, as they present good results and have a lower cost than detergents (Panesar et al; 2007). The use of ethanol as a permeabilizing agent presents, among others, advantages such as its availability, its low price, the fact that it is a component of many fermented foods and beverages, and its use in cell permeabilization in the food industry is accepted (Siso and Doval, 1994).

[016] Obtaining phycocyanin is not a difficult procedure due to the pigment's high solubility in water. The most complicated issue is the stability of the protein in the medium (Adjali et al., 2022). The biomass used can be in its dry, frozen, or fresh form; however, for storage and preservation reasons, dry biomass is more commonly used for the extraction process (Devi et al., 2020). Extraction by the freeze-thaw method is based on freezing the intracellular fluid, creating ice crystals that can perforate the cell membrane, causing the intracellular contents to leak out. When thawing occurs, the cycles are repeated, increasing the chances of rupturing the cell due to the action of the crystals. Some authors assume a better yield of the pigment after 3 to 4 freeze-thaw cycles, reporting leakage of contaminating proteins after 5 cycles (Chentir et al., 2018).

[017] After the extraction processes, the biomass needs to be separated from the pigment. This step is usually carried out by filtration or centrifugation, and then the supernatant is removed and may undergo purification processes and Petition 870250079395, dated 04 / 09 / 2025, page 12 / 31 8 / 25 then phycocyanin can be dried or freeze-dried for commercialization (Chentir et al., 2018; Pez Jaeschke et al., 2021). During the cyanobacteria cultivation and phycocyanin extraction and purification processes, a circular bioeconomy can be promoted, for example, by reusing the medium after biomass recovery, utilizing solvents after pigment extraction and precipitation, and using residual biomass as animal feed, biofertilizers, biofuels, and bioplastics. In this way, all the waste generated can be reused (Thevarajah et al., 2022).

[018] In addition to known substances, the number of commercially interesting compounds that can be obtained from cyanobacteria seems unpredictable. Globally, the growing interest in clean, sustainable, and organic technologies for obtaining products for human consumption and other purposes demands a continuous search for species and / or varieties capable of synthesizing large quantities of specific compounds and how to enhance their biosynthesis (cultivation conditions, genetic improvement, etc.). Similarly, there is a need for research aimed at developing and, especially, improving production systems on a commercial scale, in order to make some of the known systems commercially viable.Finally, this research is also necessary to identify the products that can be extracted from microalgae, their potential biological activity (metabolic and toxicological studies), and the development of specific markets for them. STATE OF THE ART

[019] Cyanobacteria, known as blue-green algae, are the evolutionary link between bacteria and green plants and constitute a group Petition 870250079395, dated 04 / 09 / 2025, p. 13 / 31 9 / 25 considered as photosynthetic bacteria. These prokaryotic organisms have a morphological structure similar to that of bacteria, and a photosynthetic system similar to that of algae, thus comprising a great diversity of microorganisms with wide morphological, biochemical, and physiological characteristics. These photosynthetic microorganisms multiply rapidly, requiring only inorganic nutrients, water, and light for their growth. This relative ease of cultivation is what makes some species attractive sources for obtaining this biomass (Ashby and Houmard, 2006; Chronakis et al., 2000).

[020] For the cultivation of microalgae, even when exposed to light and nutrients for a period of time, different concepts have been developed. In this sense, the following prior art documents were found in the prior art search:

[021] Document WO2024108780 - METHOD FOR EXTRACTION AND PURIFICATION OF PHYCOCYANIN FROM Spirulina platensis discloses a process for the extraction and purification of phycocyanin from Spirulina platensis. Spirulina platensis cells dried with surface-enriched polysaccharide are obtained by spray drying with salt; using a salt solvent, the exudation of intracellular phycocyanin is promoted, improving the phycocyanin extraction rate; through homogenization of the extraction solution carrying Spirulina platensis cells, appropriate cell fragments are obtained, so that the Spirulina platensis cell debris exhibits the characteristics of a polysaccharide micropolymer, i.e., the Spirulina platensis cells migrate to the lower phase with dextran while promoting phase separation, through an aqueous biphasic system. Petition 870250079395, dated 04 / 09 / 2025, page 14 / 31 10 / 25 consisting of polyethylene glycol and solvent, enrichment and purification of the salt are carried out, so that finally, the extraction rate and purity of phycocyanin are greatly improved.

[022] Document CN117603867 - METHOD FOR EXTRACTING PHYCOCYANIN FROM Spirulina CULTIVATED BY MEANS OF FERMENTATION CULTURE discloses a process for extracting phycocyanin from Spirulina cultivated using a fermentation culture medium, belonging to the related technical fields of algal plant culture solutions and extraction of functional components from algal plants, and the pH value of the fermentation culture medium is 8.6-9.5; comprising 16 g / L to 20 g / L sodium bicarbonate, 2.3 g / L to 2.6 g / L sodium nitrate, 0.4 g / L to 0.5 g / L dipotassium phosphate, 0.05 g / L to 0. 15 g / L ethylenediamine tetraacetic acid, 0.9 g / L to 1.1 g / L sodium sulfate potassium, 0.9 g / L to 1.1 g / L sodium chloride, 0.1 g / L to 0.2 g / L magnesium sulfate heptahydrate, 0.04 g / L to 0.05 g / L crystallized calcium chloride and 0. 01 g / L ferrous sulfate heptahydrate.1 mL / L of microelement liquid A: 2.86 g / L peroxydiboronic acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, and 0.015 g / L molybdenum trioxide; microelement liquid B is prepared from 22.96 mg / L ammonium metavanadate, 96 mg / L potassium chromium sulfate, 47.85 mg / L nickel sulfate heptahydrate, 17.94 mg / L sodium tungstate dihydrate, 40 mg / L titanium sulfate, and 43.98 mg / L cobalt nitrate hexahydrate. The invention provides an optimal environment for the production of a high-quality culture medium for growing Spirulina, such that the amount of phycocyanin contained in Spirulina is... Petition 870250079395, dated 04 / 09 / 2025, p. 15 / 31 11 / 25 large, therefore, the amount of phycocyanin during extraction is also large.

[023] Document RU2799540 - METHOD FOR OBTAINING A PRODUCT WITH S-PHYCOCYANIN FROM Spirulina BIOMASS refers to the field of biotechnology and the food industry and, in particular, to methods for obtaining a powdered product that can subsequently be used as a biologically active food additive, in medicine, cosmetics, etc. The extraction is carried out with distilled water, 3 to 4 times, in a 1:6 ratio of dry biomass: water should be heated to a temperature of 39±1 °C for 40 minutes, then the resulting extracts are combined and precipitated by centrifugation at 8,000 rpm for 15 minutes, the particles of the destroyed biomass are dried by lyophilization to obtain a product with a high content of phycocyanin.

[024] Document CN116199770 - PROCESS FOR EXTRACTION AND PURIFICATION OF PHYCOCYANIN FROM Spirulina platensis discloses a process for the extraction and purification of phycocyanin from Spirulina platensis, comprising the steps of: adding salt and spray drying to obtain dry cells, the surfaces of which are enriched in polysaccharide, promoting the exudation of intracellular phycocyanin through a salt solvent to improve the phycocyanin extraction rate, and homogenizing an extraction solution containing the cells to obtain purified phycocyanin. The process comprises the steps of: taking glucan as raw material, performing ultrasonic extraction on Spirulina to obtain suitable cell fragments, so that the cell fragments exhibit the characteristics of polysaccharide micropolymers, and Petition 870250079395, dated 04 / 09 / 2025, page 16 / 31 12 / 25 while promoting phase separation, the cells migrate downwards along with the glucan, and so that the separation and purification of phycocyanin are carried out, and finally, forming two aqueous phases through polyethylene glycol and a salt solvent, and carrying out the enrichment and purification. Finally, the extraction rate and purity of phycocyanin are greatly improved.

[025] Document CN115894669 - METHOD FOR EXTRACTION AND PURIFICATION OF PHYCOCYANIN FROM Spirulina discloses a process for the extraction and purification of phycocyanin from Spirulina. The extraction and purification process consists of three steps: 1) wall breaking by a swelling method; 2) extraction by an activated carbon adsorption method; and 3) purification by hydrophobic chromatography. Food-grade phycocyanin (P ≥ 0.7) and pharmaceutical-grade phycocyanin (P ≥ 3.0) are obtained by purification using a hydrophobic chromatography method, and the total recovery rate is 61.15%. The operation to break down the cell wall, using the swelling method, is carried out as follows: the dry spirulina powder is precisely weighed, pure water is added according to the proportion of material to liquid to swell the wall, and the swelling is repeated several times.Separate the algae liquid obtained from each swelling and then subject it to ultrasonic static centrifugation to obtain the supernatant. Extraction by the activated carbon adsorption method is performed as follows: 1) the supernatant obtained is removed by breaking the wall; 2) activated carbon is added and allowed to stand for adsorption; and 3) the supernatant is obtained by centrifugation. Purification by hydrophobic chromatography is performed as follows: the supernatant obtained after extraction. Petition 870250079395, dated 04 / 09 / 2025, page 17 / 31 13 / 25 by the activated carbon adsorption method is concentrated by ultrafiltration, and hydrophobic chromatography is pre-equilibrated with 0.05 mol / L PBS phosphate buffer containing 0.6 mol / L ammonium sulfate. The packing was subjected to gradient elution, the flow rate was controlled at 2 mL / min and 3 min / tube, and protein liquids with purities of 1-2, 2-3 and greater than 3 were combined respectively.

[026] Document WO2023015792 - RAPID EXTRACTION METHOD OF PHYCOCYANIN discloses the steps of: mixing aquaculture water containing microalgae with a protein protectant and performing a first centrifugation to obtain concentrated algae liquid; mixing the concentrated algae liquid with a cell cryoprotectant, and performing repeated freezing and thawing to obtain thawed concentrated algae liquid; mixing the thawed concentrated algae liquid with the protein protectant and a surfactant, and crushing cells to obtain crushed algae liquid; subjecting the crushed algae liquid to a second centrifugation to obtain the supernatant; mixing the supernatant with ammonium sulfate and polypropylene glycol, and performing agitation; subjecting the agitated solution to a third centrifugation to obtain a precipitate; and washing the precipitate with aqueous ethanol solution, performing a fourth centrifugation, and freeze-drying the precipitate.

[27] Studies show that as salinity increases, carbohydrate content increases, while protein, phycocyanin, carotenoids, and total phenolics decrease. Thus, the present invention proposes increasing the production of cyanobacteria biomass in a functional bioreactor capable of carrying out cultures of various species, making the growth of biomass accelerated, by means of Petition 870250079395, dated 04 / 09 / 2025, page 18 / 31 14 / 25 cultivation with 0.35 g / L of NPK supplemented with 0.10 mL / L of micronutrient solution and water, consequently with an increase in supernatant and phycocyanin pigment as a product. DESCRIPTION OF THE FIGURES

[028] Figure 1 - Bioreactor flowchart;

[029] Figure 2 - Flowchart of microalgae fermentation for biomass production.

[030] Figure 3 - Flowchart of microalgae biomass separation and concentration.

[031] Figure 4 - Phycocyanin purification flowchart. DESCRIPTION OF THE INVENTION

[032] Studies on the application of cyanobacteria in different biotechnological processes have been spreading due to the growing need for renewal of energy, inputs and alternative foods. Furthermore, numerous scientific data already demonstrate the potential of cyanobacteria for bioremediation, biofuel production, pharmaceuticals using their bioactive compounds, food supplementation with their biomass, among many other applications, especially the obtaining of natural pigments. Thus, the focus is on standardizing the production of biomass and phycocyanin (blue pigment) from the cultivation of Spirulina platensis. Through the extracts obtained, it is possible to develop several other studies to demonstrate the potential of microalgae in the most diverse biotechnological applications.

[033] The fermentation of Spirulina (Arthrospira) platensis is carried out in a culture medium containing micro and macronutrients in a pilot-scale bioreactor (4) with a working volume of 400 Liters, containing 10% (v / v) of initial inoculum. Petition 870250079395, dated 04 / 09 / 2025, page 19 / 31 15 / 25 of the cyanobacteria, in Paoletti medium (TABLE 01) or adapted culture medium. The preparation of the culture medium for the microalga Spirulina (Arthrospira) platensis is obtained in a culture medium containing 0.35 g / L of macronutrients such as nitrogen:phosphorus:potassium (NPK) in the proportion of (4:14:8) supplemented with 0.10 mL / L of micronutrient solution such as Na2MoO4.2H2O (0.39 g / L), ZnSO4.7H2O (0.22 g / L), CuSO4.5H2O (0.079 g / L), MnSO4.4H2O (1.54 g / L), H3BO3 (2.86 g / L), CoCl2.6H2O (0.038 g / L). In this case, nitrogen (N) will act as a structural component of macromolecules. Phosphorus (P) will carry out the energy exchange process (ATP) and potassium (K) osmotic regulation. The cultivation temperature is 28°C ± 2°C with a photoperiod of 12 h in the light and 12 h in the dark. The temperature of the medium is controlled depending on the cyanobacterium in order to obtain appropriate conditions for the growth of the cyanobacteria. The temperature is controlled by an electric immersion heating system (1) of the “serpentine” type.The cultivation condition is autotrophic, i.e., with luminosity (5) of 4000 lux for the production of biomass for the cell permeabilization process in order to obtain permeabilized cells and a greater quantity of phycocyanin in the extraction process. The permeabilized cyanobacteria cells and phycocyanin have potential for application as a protein and antioxidant source, respectively. Microalgae and preparation of the culture medium

[034] From the non-axene stock culture tubes (test tubes), cultures are developed in 100 mL Erlenmeyer flasks, in which chemical and physical acclimatizations to the new cultivation conditions are carried out. The temperature is controlled at 28 ± 2 °C, under constant aeration and continuous illumination of 4,000 lux from 15W LED daylight lamps. Subcultures Petition 870250079395, dated 04 / 09 / 2025, page 20 / 31 16 / 25 are always performed in the exponential growth phase or log phase, and are subsequently transferred to 1000 mL Erlenmeyer flasks (experimental units).

[035] For the test, microalgae with potential for biomass production were used, such as the cyanobacterium Spirulina (Arthrospira) platensis, a strain obtained from the microalgae collection of the Rural Development Institute of Paraná (IDR), Londrina, Paraná, and maintained in the Biotechnology Laboratory of the Anhanguera Pitágoras Unopar University, Londrina, Paraná. These are maintained in Paoletti medium (Table 01) and kept in an incubator under controlled conditions with a photoperiod of 12h in the light and 12h in the dark.

[036] Cyanobacterial culture maintenance is carried out in 5-liter (L) transparent plastic containers with a working volume of 4 liters (L), containing 10% (v / v) initial inoculum of Spirulina platensis at 1 x 10⁵ cells / mL, in NPK medium supplemented with micronutrient solution. The containers are kept at 28 ± 2 °C with a 12h light / 12h dark photoperiod in the laboratory. After 7-10 days of cultivation, culture samples (10 mL) are collected and centrifuged at 3,500 rpm for 20 min, and two fractions are formed: the sediment or pellet (biomass) and the supernatant. Both fractions are collected separately and stored at 4°C for later analysis, as per

[000] . Fermentation of the microalgae strain

[037] The cyanobacterium Spirulina platensis is used for the study. The cyanobacterium is stored and maintained in the laboratory. As a culture control, the cyanobacterium is maintained in Paoletti mineral salt agar medium. The initial inoculum is 10% (v / v) in relation to the total volume of the culture medium. At the end of the Petition 870250079395, dated 04 / 09 / 2025, page 21 / 31 17 / 25 After fermentation, the culture is microfiltered to obtain biomass. The cyanobacteria are cultivated in a medium with 0.35 g / L of NPK supplemented with 0.10 mL / L of micronutrient solution for biomass and phycocyanin production. The cyanobacteria are cultivated under autotrophic conditions in 2000 mL Erlenmeyer flasks, with a working volume of 1000 mL, under constant aeration. The experiments are performed with continuous illumination provided by an LED lamp with a luminous intensity of 4000 lux, with a 12 / 12 h photoperiod (light / dark) and a temperature of 28 ± 2°C. Biomass growth is monitored daily by the optical density of the cultures at 670 nm in a UV / VIS spectrophotometer using a previously constructed standard curve relating dry weight and optical density; the initial biomass concentration is also determined by this procedure. The pH of the crops is determined every 3 days using a digital pH meter. The maximum biomass concentration (Xmax, g) is evaluated.L-1), the maximum productivity (Pmax, gL-1.day-1) obtained according to the equation P = (Xt - Xc) / (t - tc), where Xt is the biomass concentration (gL-1) at time t (day), and X0 is the biomass concentration (gL-1) at time t0 (day)); and the maximum specific growth rate (pmax, day-1) by exponential regression applied to the logarithmic growth phase.

[038] Pilot-scale cultivation of the cyanobacterium strain Spirulina platensis for biomass production is carried out in a 500 Liter (4) photobioreactor with a working volume of 400 Liter. For inoculum preparation, the cyanobacterium is cultivated in a medium containing 0.35 g / L of NPK supplemented with 0.10 mL / L of micronutrient solution for biomass production and phycocyanin. The cyanobacterium is cultivated under autotrophic conditions in 5 liter (L) transparent plastic containers with a working volume of 4 liters (L), with aeration. Petition 870250079395, dated 04 / 09 / 2025, page 22 / 31 18 / 25 constant. The experiments are carried out with 10% (v / v) inoculum, with continuous illumination (5) provided by an LED lamp with a luminous intensity of 4,000 lux, with a photoperiod of 12 / 12 h (light / dark) and a temperature of 28 ± 2°C. Biomass growth is monitored daily by the optical density of the cultures at 670 nm in a UV / VIS spectrophotometer using a previously constructed standard curve relating dry weight and optical density; the initial biomass concentration is also determined by this procedure.

[039] The pH of the cultures is determined every 3 days using a digital pH meter. The maximum biomass concentration (Xmax, gL-1) and the maximum productivity (Pmax, gL-1.day-1) are evaluated, obtained according to the equation P = (Xt Xü) / (t - tc), where Xt is the biomass concentration (gL-1) at time t (day), and X0 is the biomass concentration (g.L-1) at time t0 (day)); and the maximum specific growth rate (pmax, day-1) by exponential regression applied to the logarithmic growth phase. Determination of biomass and analysis of microalgae growth.

[040] Biomass determination is performed by taking 10 mL aliquots of the culture medium. The samples are centrifuged at 3500 rpm for 20 minutes. The precipitate is resuspended in saline solution (0.85%) and centrifuged again.

[041] This operation is repeated twice for complete washing of the cells.

[042] Biomass analysis is quantified gravimetrically using the dry weight of the cells. The centrifuged material is transferred to pre-weighed porcelain flasks and placed in an oven at 105°C for 4-5 hours until a constant weight is reached. The value (g / L) is given by the difference in initial weight of the pre-weighed porcelain flasks. Petition 870250079395, dated 04 / 09 / 2025, pages 23 / 31 19 / 25 Determination of absorbance

[043] 5.0 mL of sample are collected to determine the optical density (OD) in absorbance at a wavelength of 670 nm (Costa et al., 2004; Andrade and Costa, 2007; Silveira et al., 2007) using a UV / VIS spectrophotometer. Calculation of cell concentration

[044] Cell concentration is obtained through a mathematical formula using the standard microalgae growth curve, correlating the dry mass of biomass with the optical density (OD) in absorbance at 670 nm. Productivity

[045] The productivity of cyanobacteria is calculated using the equation: P = (Xi - Xo) / ti where: P = productivity (mg.L-1 day-1); Xo = initial cell concentration (mg.L-1); Xi = cellular concentration at time i (mg.L-1) and ti = time interval (day) between Xo and Xi. Biomass concentration

[046] Biomass concentration is achieved by tangential flow microfiltration (6). After sedimentation, the collected biomass is concentrated by tangential flow filtration in a pilot microfiltration unit (6) coupled to a membrane with a porosity of 0.22 μm (TIA - Tecnologia Industrial Aplicada Ltda, Brazil) until the desired concentrate is reached. The liquid biomass passes through the microfiltration unit (6). The cells pass through the membrane surface and return to the unit's reservoir. Petition 870250079395, dated 04 / 09 / 2025, pages 24 / 31 20 / 25 pilot. Water, mineral salts and other smaller particles are filtered through the membrane and will constitute the permeate, also called supernatant, which is stored in another container. The microfiltration unit (6) continues processing the biomass until the desired concentration is reached. The concentrate obtained can be dehydrated in two ways: either by freeze-drying or by drying in a circulating air oven at 45°C for 2 days. Cell permeation of cyanobacteria

[047] In order to obtain the intracellular pigment phycocyanin, Spirulina platensis cells are permeabilized. The permeabilization of Spirulina platensis cells is carried out using the organic solvent ethanol, as described by Morioka et al. (2016), with modifications. 40 mL of the culture are collected after fermentation. The cells are centrifuged and resuspended in a final volume of 10 mL at a concentration of 1% ethanol in 0.1M potassium phosphate buffer (pH 6.8). The suspension is incubated at room temperature for 12 minutes. After the incubation time, centrifugation is performed, obtaining the supernatant and the concentrate consisting of the permeabilized cells, which are washed (2x) with distilled water. The permeabilized cells are resuspended in 10 mL of 0.1M potassium phosphate buffer (pH 6.8) for the extraction step. Phycocyanin extraction

[048] The cyanobacterial culture is collected after 7-10 days of cultivation, under controlled conditions (temperature, light, and agitation and / or aeration), by filtration through a nylon membrane. The collected biomass is washed with distilled water. After the cell permeabilization process, 100 mL of distilled water is added to the biomass and it is subjected to freezing at -20°C. Petition 870250079395, dated 04 / 09 / 2025, pages 25 / 31 21 / 25 overnight and thawing. The freeze / thaw process can be repeated twice. The cell suspension is thawed and centrifuged at 4000 rpm for 20 minutes, thus obtaining the crude extract containing phycocyanin in the supernatant. Phycocyanin quantification

[049] The absorbance of phycocyanin present in the crude extract will be measured using a Spectrum UV / VIS spectrophotometer at wavelengths of 620 and 652 nm to calculate the phycocyanin (FC) concentration, according to the equation: A620 - 0.474 x A652 Phycocyanin (mg / mL) =-------—-------5.34 Where: A620 = absorbance at 620nm A652 = absorbance at 652nm

[050] The phycocyanin yield in the sample is given by the equation: FC x V Yield (mg / g~) = ——— BS Where: FC = phycocyanin concentration (mg / mL) V = volume of the extract (mL) BS = dry biomass of Spirulina (g) Purification of phycocyanin

[051] Phycocyanin is purified in two stages by tangential flow filtration. In the first stage, microfiltration will be carried out in a Minitan™ Microfiltration / Ultrafiltration System (MILLIPORE) unit to remove cells and debris. The unit is equipped with plastic plates where each Petition 870250079395, dated 04 / 09 / 2025, pages 26 / 31 On the 22 / 25 side, membranes with an average pore diameter of 0.2 μm are fixed. Each plate has a total filter surface area of ​​60 cm². Up to ten plates can be used simultaneously, making a total filter surface area of ​​600 cm². The filtration capacity varies between 500 mL and 2000 mL. A peristaltic pump (3) creates a flow in which the sample is pumped from a non-pressurized reservoir to the filtration unit. The sample passes through the Minitan filtration unit. Large particles pass through the surface of the membranes and return to the sample contained in the reservoir. Water, mineral salts, and other smaller particles are filtered through the membrane and constitute the permeate, also called extract, which is stored in another container. The Minitan continues processing the sample until the desired concentration is reached.

[052] The second stage is performed by ultrafiltration in a Vivaflow 200 Ultrafiltration unit (Sartorius), for the purification of phycocyanin. The unit is equipped with a polyethersulfone membrane with a pore diameter of 10000 MWCO (10 kDa). The phycocyanin is purified and concentrated. A peristaltic pump (3) creates a flow in which the sample is pumped from an unpressurized reservoir to the filtration unit. The sample passes through the Vivaflow 200 ultrafiltration unit. Large particles pass through the surface of the membranes and return to the sample contained in the reservoir. Water, mineral salts and other smaller particles are filtered through the membrane and will constitute the permeate, also called extract, which is stored in another container. The ultrafiltration unit continues processing the sample until a desired concentration is reached. Petition 870250079395, dated 04 / 09 / 2025, pages 27 / 31 23 / 25

[053] The purification of phycocyanin on a pilot scale is carried out in a pilot-scale tangential flow filtration unit which operates independently of the bioreactor. In this pilot tangential flow filtration unit (6), a microfiltration membrane with an average pore diameter of 1.2 μm can be used to remove cells and debris. The membrane is then changed to an ultrafiltration membrane with an average pore diameter of 10000 MWCO (10 kDa). The filtered pigment is passed through the ultrafiltration membrane to obtain the concentrated and purified pigment.

[054] TABLE 01 - Liquid Paoletti Half NaCl 0.92 g Na2SO4 1.88 g K2HPO4 0.50 g Na2CO3 8.89 g NaHCO3 15.15 g KNO3 2.57 g MgSO4.7H2O 0.25 g Distilled water 1 L Fe-EDTA solution - Table 02 1 mL Micronutrient solution - Table 03 1 mL CaCl2 solution 0.5 g / 10 mL - Table 04 1 mL pH « 9.5 Weigh the first reagent, dissolve it in a little distilled water, stirring with a stirring rod in a beaker; 1. Repeat step 1 for all reagents; 2. Top up the rest with distilled water; Petition 870250079395, dated 04 / 09 / 2025, pp. 28 / 31 24 / 25 3. Transfer to a glass jar with a lid and autoclave; 4. After sterilization, add the Fe-EDTA solution, the micronutrient solution, and the CaCl2 solution in the aseptic room.

[055] TABLE 02 - Fe-EDTA Solution EDTA-Na2 2.98 g FeSO4.7H2O 2.49 g Citric acid 2.49 g Distilled water 100 mL pH « 4.5 1. Weigh the first reagent, dissolve it in a little distilled water using a magnetic stirrer, and set it aside in a beaker; 2. Repeat step 1 for all reagents; 3. Top up the rest with distilled water; 4. Transfer to a light-protected, lidded glass container and autoclave.

[056] TABLE 03 - Micronutrient solution Na2MoO4.2H2O 0.390 g ZnSO4.7H2O 0.220 g CuSO4.5H2O 0.079 g MnSO4.4H2O 1.544 g H3BO3 2.860 g COCl2.6H2O 0.038 g Distilled water 1 L Petition 870250079395, dated 04 / 09 / 2025, pages 29 / 31 25 / 25 1. Weigh the first reagent, dissolve it in a little distilled water, stirring with the stirring rod, and set aside in a beaker; 2. Repeat step 1 for all reagents; 3. Top up the rest with distilled water; 4. Transfer to a glass jar with a lid and autoclave.

[057] TABLE 04 - CaCl2 solution CaCl2.2H2O 0.5 g Distilled water 10 mL 1. Weigh the reagent, dissolve it in a little distilled water, stirring with the stirring rod, and set aside in a beaker; 2. Top up the rest with distilled water; 3. Transfer to a glass jar with a lid and autoclave. Petition 870250079395, dated 04 / 09 / 2025, pages 30 / 31

Claims

1 / 3 CLAIMS 1) PROCESS FOR PRODUCING BIOMASS AND PHYCOCYANIN IN A PHOTOBIOREACTOR FROM THE CULTIVATION OF SPIRULINA PLATENSIS, characterized by comprising the steps of: a) preparing the culture medium of the microalga Spirulina (Arthrospira) platensis containing 0.35 g / L of the macronutrients nitrogen, phosphorus and potassium (NPK) in the proportion of (4:14:8) supplemented with 0.10 mL / L of solution of the micronutrients Na2MoO4.2H2O (0.39 g / L), ZnSO4.7H2O (0.22 g / L), CuSO4.5H2O (0.079 g / L), MnSO4.4H2O (1.54 g / L), H3BO3 (2.86 g / L), CoCl2.6H2O (0.038 g / L); b) fermentation of Spirulina (Arthrospira) platensis in a culture medium containing micro and macronutrients containing 10% (v / v) initial inoculum of the cyanobacteria, in Paoletti medium to obtain biomass; c) concentration of biomass by means of tangential flow microfiltration in a pilot microfiltration unit coupled to a membrane with a porosity of 0.22 μm until the desired concentrate is reached, and the concentrate obtained is dehydrated by lyophilization or by drying in an oven; d) permeabilization of Spirulina platensis cells using the organic solvent ethanol; e) extraction of phycocyanin; f) purification of phycocyanin. Petition 870250052367, dated 06 / 23 / 2025, pp. 30 / 39 2 / 3. 2) PROCESS FOR PRODUCING BIOMASS AND PHYCOCYANIN IN A PHOTOBIOREACTOR FROM SPIRULINA PLATENSIS CULTIVATION, as claimed in claim 1, characterized by the preparation of the culture medium of the microalga Spirulina (Arthrospira) platensis under autotrophic conditions in 5-liter (L) transparent plastic containers with a working volume of 4 liters (L), with constant aeration, carried out with 10% (v / v) inoculum, with continuous illumination provided by an LED lamp with a luminous intensity of 4,000 lux, with a photoperiod of 12 / 12 h (light / dark) and a temperature of 28 ± 2°C. 3) PROCESS FOR PRODUCING BIOMASS AND PHYCOCYANIN IN A PHOTOBIOREACTOR FROM SPIRULINA PLATENSIS CULTIVATION, as per claim 1, characterized by the permeabilization being carried out using an organic solvent with 1% ethanol, the suspension being incubated at room temperature for 12 minutes, the cells being centrifuged and resuspended in a final volume of 10 mL in 0.1M potassium phosphate buffer (pH 6.8), obtaining the supernatant and the concentrate consisting of the permeabilized cells which are washed in distilled water twice and resuspended in 10 mL of 0.1M potassium phosphate buffer (pH 6.8). 4) PROCESS FOR PRODUCING BIOMASS AND PHYCOCYANIN IN A PHOTOBIOREACTOR FROM SPIRULINA PLATENSIS CULTIVATION, as per claim 1, characterized by the extraction being carried out by means of the permeabilized biomass being washed with distilled water, adding 100 mL of distilled water and being subjected to freezing at -20°C overnight and thawing, which may be repeated 2 times, and the cell suspension being thawed and centrifuged at 4000 rpm for 20 minutes, thus obtaining in the supernatant the crude extract containing phycocyanin. 5) PROCESS FOR PRODUCING BIOMASS AND PHYCOCYANIN IN A PHOTOBIOREACTOR FROM SPIRULINA PLATENSIS CULTIVATION, as claimed in claim 1, characterized by the purification being in two stages: a) Tangential flow filtration for the removal of cells and residues, equipped with plastic plates where membranes with an average pore diameter of 0.2 μm are fixed on each side, each plate having a total filter surface area of ​​60 cm2, with up to ten plates that can be used simultaneously, making a total filter surface area of ​​600 cm2, with a filtration capacity between 500 mL and 2000 mL, until the desired concentration is reached; b) Ultrafiltration, for the purification of phycocyanin, equipped with a polyethersulfone membrane with a pore diameter of 10000 MWCO (10 KDa), until a desired concentration is reached. Petition 870250052367, dated 06 / 23 / 2025, pages 32 / 39

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