UM MÉTODO PARA POTENCIALIZAR A CAROTENOGÊNESE EM MICROALGA USANDO SOLUÇÃO DE BICARBONATO E CARBONATO

Description

1 / 15 DESCRIPTIVE REPORT A METHOD TO ENHANCE CAROTENESIS IN MICROALGAE USING BICARBONATE AND CARBONATE SOLUTION Field of Invention

[001] . The present invention relates to a simple and high-performance process for inducing carotenogenesis in the microalga Haematococcus piuviaiis, belonging to the field of biotechnology, with application in the areas of processes for recovering biomolecules of pharmaceutical and food interest.

[002] . The proposed invention consists of a process to promote the accumulation of carotenoids in the microalga Haematococcus piuviaíis by means of the use of an alkaline solution of carbonate (CO32-) and bicarbonate (HCO3-) produced from the chemical fixation of gases rich in carbon dioxide (CO2).

[003] . According to the method proposed in this invention, the carotenoid content is notably increased, showing efficiency increases greater than 100% compared to a control group. Fundamentals of the Invention and State of the Art

[004] . Carotenoids are a group of phytochemicals important for being natural pigments responsible for colors ranging from yellow to orange and red. Carotenoids are widely distributed in nature, being biosynthesized by different organisms, including photosynthetic ones, such as cyanobacteria, microalgae, photosynthetic bacteria, and higher plants. Or even by Petition 870250006793, dated 01 / 28 / 2025, page 7 / 31 2 / 15 some non-photosynthetic organisms, such as fungi, yeasts and bacteria.

[005] . Interest in the carotenoid trade is growing, mainly in the food, beverage, pharmaceutical and cosmetic sectors. The increasing global demand is mainly associated with the antioxidant action of these compounds, which are used in daily food supplements or in the treatment of cataracts and reduction of the risks of diabetes, cancer and heart problems, in addition to being used as natural pigments for food and animal feed for crustaceans and fish.

[006] . The global carotenoid market is estimated at USD 1.84 billion (2023), projecting a CAGR of 4.64% between 2024 and 2029, highlighting types such as astaxanthin, beta-carotene, lutein, lycopene, and zeaxanthin. Table 1 lists carotenoids available on the market, indicating the sources from which they are extracted and the objectives of the products, which are mostly aimed at food supplementation. Microalgae are among the main organisms producing carotenoids on a large scale. These are microorganisms with high photosynthetic efficiency and ease of growth on different types of substrates, making it possible to produce biomass (rich in proteins, lipids and / or carbohydrates), biocompounds (e.g., carotenoids) and bioproducts (e.g., biodiesel, biomethane), which can be used in sectors such as food, pharmaceuticals, cosmetics and energy.

[007] . In microalgae metabolism, carotenoids have multifaceted functions, with groups that are fundamental to processes such as photosynthesis and others with specific functions, such as protection from oxidative damage and adaptation to environmental conditions. Petition 870250006793, dated 01 / 28 / 2025, page 8 / 31 3 / 15 In this concept, carotenoids can be divided into primary (e.g., beta-carotene, lycopene, lutein) and secondary (e.g., astaxanthin). Table 1 - Patents and scientific articles related to the subject of this patent application. Product Company Carotenoid Extraction Source Product Purpose Fuji Chemical Supplement AstaReal® Industries Astaxanthin Microalgae Food Supplement Co., Ltd. (Japan) Cosmetic AstaPure® Algatech (Israel) Astaxanthin Microalgae Food Supplement Xi'an Green BetaCarotene Spring Beta-Carotene Carrot Supplement Power Technology Food Supplement (China) BioAstin® Cyanotech® (Hawaii) Astaxanthin Microalgae Food Supplement Crystal Clear DOHLER Lutein Extract Food Supplement Colours 2.0 (Germany) Paprika Food Supplement Lycomate® 15% Lycored (Israel) Lycopene Tomato Food Supplement Xi'an Green Natural Power 5% Spring Technology Astaxanthin Microalgae Food Supplement (China) Hunan Natural Power Food Additive Huakang Biotech Inc. Astaxanthin Microalgae Aquaculture (China) Beta-Spirulina Pacifica™ Food Supplement Cyanotech (Hawaii) Carotene and Zeaxanthin Microalgae Food Supplement Xangold® BASF (Germany) Lutein Calendula Flowers Food Supplement Source: The Author (2024) Petition 870250006793, dated 01 / 28 / 2025, page 9 / 31 4 / 15

[008] . Among the secondary carotenoids, astaxanthin is the most widely produced, being mostly extracted from microalgae, and with a rapidly expanding economic market. On a large scale, this process is conducted in two reactors, one aimed at accumulating biomass with favorable conditions for cell development (green phase) and a second where environmental conditions are altered to induce stress and carotenoid production.

[009] . Commercially, astaxanthin is produced by the microalga Haematococcus piuviaiis, which induces accumulation through stress conditions. This process is generally conducted in two stages: (i) growth phase, in which there is an increase in the number of cells and accumulation of green biomass through conditions favorable to support growth; (ii) accumulation phase or encystment phase, in which there are limited nutrient conditions (e.g., removal of nitrogen, phosphorus) or environmental conditions (e.g., light, pH, and temperature) and the cells lose their reproductive capacity and form cysts (Debnath et al., Astaxanthin from microalgae: A review on structure, biosynthesis, production strategies and application. Food Research International 176, 113841, 2024).

[010] . In terms of technologies used to obtain carotenoids from Haematococcus pluvials, saline stress using NaCl is widely applied, in addition to the use of abiotic conditions such as light and temperature, chemical reagents and / or the combination of methods with the aim of reducing production time, improving process performance and reducing costs in obtaining the bioproduct.

[011] . For example, patent CN115627237 details the use of proline as a stress agent in Haematococcus cells. Petition 870250006793, dated 01 / 28 / 2025, page 10 / 31 5 / 15 pluvialis, exploring concentrations from 50 to 150 pmol.L⁻¹ and showing increases greater than 60% compared to the control group. Similarly, a study used GABA, melatonin, proline, and arginine as stress agents for Haematococcus pluvialis aiming at astaxanthin production. In patent CN107058441, the application of alcoholic solutions (methanol, butanol, and ethanol) is observed (Acheampong et al., Exogenous arginine promotes the coproduction of biomass and astaxanthin under high-light conditions in Haematococcus pluvialis. Bioresource Technology 393, 130001, 2024). The company FUJIAN KANG SHI MEI BIOTECHNOLOGY CO LTDA. The innovation CN109609385 describes processes for controlling biological contaminants by adding sodium bicarbonate (0.01 ~ 0.02 gL-1) and ampicillin, however, it is still limited to the growth phase of the microalga Haematococcus pluvialis. The patent in question (CN109609385) describes the addition of NaAc and gibberellin in the astaxanthin induction phase.

[012] . Additionally, some processes using carbonate solutions are described in patents CN106399108 and WO2018182100, in which these solutions are used as buffers to improve performance in the growth phase of the alga Haematococcus pluvialis, exhibiting smaller ranges than those proposed in this invention. The technologies can also combine the use of combined techniques, as demonstrated in patent KR101841917B1, which uses a combination of strong bases and spraying of CO2-rich gases for conversion into bicarbonate, which is used as a substrate for microalgae growth.

[013] . Nevertheless, the methodology reported in this innovation stands out from the others by using an inexpensive culture medium, with a low amount of reagent, efficient and with a short stress time. Petition 870250006793, dated 01 / 28 / 2025, page 11 / 31 6 / 15

[014] . It should be noted that culture media containing carbonate and bicarbonate can come from chemical fixation processes of CO2 and combustion gases which, from the reaction with strong bases, such as sodium hydroxide, generate solutions rich in CO32- and HCO3- that can be directed to the carotenogenesis induction processes described in this innovation.

[015] . At the time of writing this patent application, only one published document (CN103881921) was found concerning the use of bicarbonate solution for astaxanthin induction by Haematococcus pluviais. Patent CN103881921 describes the process of applying NaHCO3 combined with vitamins and iron for astaxanthin induction in Haematococcus pluviais in BBM medium.

[016] . However, the novelty presented in this innovation is the use of solutions containing combinations of carbonate and bicarbonate derived from the chemical fixation of CO2 and / or combustion gases to induce stress in the microalga, with a high content of carotenoids accumulated in the cells. Description of the approach to the technical problem

[017] In recent years, climate change has been a widely discussed topic, mainly due to the increasing concentration of greenhouse gases (GHGs) from the burning of fossil fuels that are emitted into the atmosphere. According to recent data (NOAA. Broken record: Atmospheric carbon dioxide levels jump again. National Oceanic and Atmospheric Administration, 2023), CO2 levels in the atmosphere reached 424 ppm in May 2023, marking a staggering 50% increase compared to levels documented at the beginning of the industrial era. Industrial activities and burning Petition 870250006793, dated 01 / 28 / 2025, p. 12 / 31 7 / 15 of fossil fuels account for 76% of GHG emissions, of which 72% are used for energy production, such as burning coal, diesel or natural gas, emitting 36.3 Gt of CO2 in combustion gases per year (Center for Climate and Energy Solutions. Global Emissions, 2021).

[018] . The capture and use of CO2 in industrial processes can effectively reduce carbon emissions in industry, also integrating bioproduct recovery processes. This process can be carried out through various pathways, including chemical absorption using strong bases such as sodium, potassium, and calcium hydroxide. Briefly, the catalytic chemical conversion of CO2 in contact with strong bases occurs through chemical reactions in distinct phases, the first being conversion to carbonate, followed by the conversion of CO32- to HCO3-. This reaction directly influences the pH of the solution, which starts above 12 and in the last reaction (HCO3-) reaches a pH close to 8.

[019] . Considering energy consumption and process integrity, an alternative may be the coupling of chemical absorption and biotransformation. For example, carbonate and bicarbonate are intermediate products of the conventional chemical absorption process with hydroxides, and can be used as a carbon source by microalgae, or as stress-inducing compounds for conversion into bioproducts.

[020] . Systems with bicarbonate and carbonate in microalgal cultures can reduce energy consumption and reagent costs, proving to be an economical and environmentally sound carbon capture technology, enhancing the absorption of combustion gases and converting carbon into biomass or bioproduct. Petition 870250006793, dated 01 / 28 / 2025, page 13 / 31 8 / 15

[021] . Hybrid systems (coupling gas fixation with chemical and biological absorption) are commonly used for the production of carbon sources for microalgae, such as cyanobacteria (Rosa et al., Chemical absorption and CO2 biofixation via the cultivation of Spirulina in semicontinuous mode with nutrient recycle. Bioresource Technology 192, 321-327, 2015; Zhang, et al., Effects of different bicarbonate on Spirulina in CO2 absorption and microalgae conversion hybrid system. Frontiers in Bioengineering and Biotechnology, 10, 2023). However, there are no reports of their use as stress inducers for the production of secondary carotenoids. Although well-established large-scale processes for carotenoid production already exist, varying environmental conditions and osmotic stress, the development of processes with lower economic cost and greater efficiency is of interest to the market, such as the one presented in this study.

[022] . In summary, the developed process presents a series of innovative advantages, in addition to those mentioned, such as: a. The stress medium is a stable byproduct, produced in large quantities in chemical absorption systems. Furthermore, carotenoids can be one of the products of this chain, and bicarbonate precipitation may also occur in the chemical fixation system; b. It is a simplified process, with few unit operations, environmentally safe and with lower reagent consumption; c. The process is designed to emit fewer GHGs and generate high-value-added bioproducts from chemical fixation; d. The process reduces the amount of reagents and gaseous effluents generated, contributing to a circular economy; Petition 870250006793, dated 01 / 28 / 2025, page 14 / 31 9 / 15 e. The process developed is innovative, not described in the literature, and has a high yield.

[023] . Based on this information, the present invention offers the advantage of using chemical fixation waters of gases rich in CO2 or industrial effluents rich in bicarbonate and carbonate in the culture medium of microalgae that produce secondary carotenoids, unlike the works cited in the prior art, in which these effluents were used for the growth of microorganisms. List of figures

[024] . Figure 1: Flowchart of a method to increase carotenoid production from Haematococcus pluvialis using a carbonate and bicarbonate solution.

[025] . Figure 2: Samples relating to example 1, showing encysted cells (a), control sample (b), comparing both treatments (c).

[026] . Figure 3: Flowchart of pilot proposal according to the process of the present invention. Detailed description of the invention.

[027] . The process for producing carotenoids from microalgae, according to the present invention, consists of the process steps described below (Figure 03): a. Chemical fixation of CO2-rich gases using strong bases, such as sodium and potassium hydroxide, with concentrations of 0.1 to 2 mol.L-1, in 800 mL vertical bubble column reactors, with injection of CO2-rich gases at concentrations of 1 to 15% (v.v1) and flow rates of 1.5 to 6 vvm; Petition 870250006793, dated 01 / 28 / 2025, page 15 / 31 10 / 15 b. Photobioreactors for biomass production for vegetative cell culture: 20 to 30°C, illuminated at 50 to 85 μmol.m-2.s-1 in a 2,000 mL conical flask using ES culture medium (0.2 gL-1 KNO3, 0.2 gL-1 K2HPO4, 0.2 gL-1 MgSO4.7H2O, 30 mL.L-1 of soil extract, 5 mL.L-1 micronutrient solution, 1 mL vitamins), with CO2 injection at concentrations of 0.04% to 4% (vv-1), with Haematococcus pluvialis SAG culture cells; c. Preparation of the carotenoid induction medium composed of nitrogen-free ES culture medium (KNO3) and medium resulting from chemical fixation obtained in step (a) with a dilution factor suitable to achieve HCO3- concentrations of 15 to 50 mM while carbonate (CO32-) is in the range of 2 to 10 mM, and sodium chloride (NaCl) at a concentration of up to 1 gL-1; d. Carotenogenesis induction photobioreactor with 50 mL of induction medium (step c), static or stirred up to 50 rpm, inoculum of 0.3 to 0.6 gL-1 obtained in step (b), temperature of 28 to 36°C and light intensity of 100 to 250 pmol.m-2.s-1; e. Extraction of carotenoids from encysted cells in the solution described in step (d) by centrifuging the final culture at 2700 rpm for 10 min, successive washing with distilled water and treatment with the organic solvent dimethyl sulfoxide (DMSO) at 50 to 70°C for 10 to 30 min in an ultrasonic system with a power of 160 W, centrifuged to remove cellular debris. The extraction process was repeated twice. The carotenoid content was measured by absorbance at 480 nm, with a production of 110.97 mg.L-1 and a yield of 22.2 mg.L-1.d1, showing a significant increase compared to the control group. Petition 870250006793, dated 01 / 28 / 2025, page 16 / 31 11 / 15 The present invention consists of a simple method for promoting the induction of secondary carotenoid accumulation in the microalga Haematococcus piuviaíis, encompassing evidence of positive effects in the research and development process, and has significant advantages over already established techniques. Examples Example 1

[028] . The method provided by the invention embodiment is used to induce and extract carotenoids by providing a composition containing 0.5 gL-1 of NaCl, 50 mM of HCO3·, 10 mM of CO32-, comprising the following specific steps:

[029] . CO2 was sprayed onto 1.35 mol.L-1 sodium hydroxide at a flow rate of 5.45 vvm. The process was carried out at room temperature. The medium was diluted 25 times in sterile ES medium, without KNO3, supplemented with 0.5 gL-1 of NaCl, to achieve concentrations of 50 mM and 10 mM of HCO3· and CO32-, respectively.

[030] . Liquid cultures of Haematococcus piuviaiis SAG cells were grown in the vegetative phase in ES medium with 0.04% CO2 spray, temperature of 20 to 25°C and illumination of 50-70 pmol.m-2.s-1, photoperiod 12:12 for 14 days.

[031] . Insertion of vegetative state cells at 0.50 gL-1 with a volume of 50 mL of medium formulated in the previous step (sterile ES medium, without KNO3, 0.5 gL-1 of NaCl, 50 mM HCO3- and 10 mM CO32-), at a light intensity of 250 pmol.m-2.s-1, a temperature of 30-34°C and agitation at 50 rpm for 120 hours. Petition 870250006793, dated 01 / 28 / 2025, page 17 / 31 12 / 15

[032] . Extraction of carotenoids from algal cells from culture obtained in the previous description where the culture is centrifuged at 2700 rpm for 10 min and successively washed with distilled water. The cells are treated with DMSO for 15 min at 60°C in an ultrasound system at 160 W, centrifuged at 2700 rpm for 10 min. The extraction process is repeated twice. The carotenoid content was determined by absorbance measurement (480 nm), with a production of 73.15 ± 1.49 mg.L-1 and a productivity of 14.64 ± 0.30 mg.L-1.d-1, with an increase of more than 120% compared to the control group (see Figure 2). Example 2

[033] . The method provided by the invention embodiment is used to induce and extract carotenoids by providing a composition containing 1.0 gL-1 of NaCl, 35 mM of HCO3-, 5 mM of CO32-, comprising the following specific steps:

[034] . CO2 was sprayed onto 2 mol.L-1 sodium hydroxide at a flow rate of 2.4 vvm. The process was carried out at room temperature. The medium was diluted 35 times in sterile ES medium, without KNO3, supplemented with 1.0 gL-1 of NaCl, to achieve concentrations of 35 mM and 5 mM of HCO3- and CO32-, respectively.

[035] . Liquid cultures of Haematococcus pluvialis SAG cells were grown in the vegetative phase in ES medium with 2% CO2 spray, a temperature of 25 to 30°C and illumination of 6085 pmol.m-2.s-1, a photoperiod of 12 hours for 12 days.

[036] . Insertion of vegetative state cells at 0.30 gL-1 with a volume of 50 mL of medium formulated in the previous step (sterile ES medium, without KNO3, 1.0 gL-1 of NaCl, 35 mM HCO3- and 5 mM CO32-), a Petition 870250006793, dated 01 / 28 / 2025, page 18 / 31 13 / 15 light intensity of 200 pmol.m-2.s-1, temperature of 28-34°C and agitation of 50 rpm for 120 hours.

[037] . Extraction of carotenoids from algal cells from culture obtained as described above, where the culture is centrifuged at 2700 rpm for 10 min and successively washed with distilled water. The cells are treated with DMSO for 15 min at 60°C in an ultrasound system at 160 W, centrifuged at 2700 rpm for 10 min. The extraction process is repeated twice. The carotenoid content was determined by absorbance measurement (480 nm), with a production of 110.97 ± 0.70 mg.L-1 and a productivity of 22.19 ± 0.15 mg.L-1.d-1, with an increase of more than 140% compared to the control group.

[038] . Figure 3 shows a pilot plant based on the data of the present invention, specifically estimates of results from Example 2. Example 3

[039] . The method provided by the invention embodiment is used to induce and extract carotenoids, providing a composition containing 35 mM HCO3-, 10 mM CO32-, comprising the following specific steps:

[040] . CO2 was sprayed onto 1.35 mol.L-1 sodium hydroxide at a flow rate of 5 vvm. The process was carried out at room temperature. The medium was diluted 20 times in sterile ES medium, without KNO3, to achieve concentrations of 35 mM and 10 mM of HCO3- and CO32-, respectively.

[041] . Liquid cultures of Haematococcus pluvialis SAG cells were grown in the vegetative phase in ES medium with Petition 870250006793, dated 01 / 28 / 2025, page 19 / 31 14 / 15 spraying with 1.5% CO2, temperature of 23 to 28°C and illumination of 7085 pmol.m-2.s-1, photoperiod of 12 hours for 10 days.

[042] . Insertion of vegetative state cells at 0.6 gL-1 with a volume of 50 mL of medium formulated in the previous step (sterile ES medium, without KNO3, 35 mM HCO3- and 10 mM CO32-), at a light intensity of 100 pmol.m-2.s-1, photoperiod 16:0, temperature of 32-36°C for 120 hours.

[043] . Extraction of carotenoids from algal cells from culture obtained as described above, where the culture is centrifuged at 2700 rpm for 10 min and successively washed with distilled water. The cells are treated with DMSO for 15 min at 60°C in an ultrasound system at 160 W, centrifuged at 2700 rpm for 10 min. The extraction process is repeated twice. The carotenoid content was determined by absorbance measurement (480 nm), with a production of 70.79 ± 2.37 mg.L-1 and a productivity of 14.16 ± 0.15 mg.L-1.d-1, with an increase greater than 21% compared to the control group. Example 4

[044] . The method provided by the invention embodiment is used to induce and extract carotenoids, providing a composition containing 20 mM HCO3-, 5 mM CO32-, comprising the following specific steps:

[045] . CO2 was sprayed onto 0.5 mol.L- sodium hydroxide. 1, flow rate of 5.45 vvm. The process was carried out at room temperature. The medium was diluted 10 times in sterile ES medium, without KNO3, to achieve concentrations of 20 mM and 5 mM of HCO3- and CO32-, respectively.

[046] . Liquid cultures of Haematococcus pluvialis SAG cells were grown in the vegetative phase in ES medium with Petition 870250006793, dated 01 / 28 / 2025, page 20 / 31 15 / 15 spraying with 4% CO2 for 4 hours followed by spraying with sterile air (0.04% CO2), temperature of 23 to 28°C and illumination of 70-85 pmol.m-2.s-1, with a photoperiod of 12 hours, for 12 days.

[047] . Insertion of vegetative state cells at 0.30 gL-1 with a volume of 50 mL of medium formulated in the previous step (sterile ES medium, without KNO3, 20 mM HCO3- and 5 mM CO32-), at a light intensity of 100 pmol.m-2.s-1, photoperiod 16:0, temperature of 30-35°C for 120 hours.

[048] . Extraction of carotenoids from algal cells from culture obtained as described above, where the culture is centrifuged at 2700 rpm for 10 min and successively washed with distilled water. The cells are treated with DMSO for 15 min at 60°C in an ultrasound system at 160 W, centrifuged at 2700 rpm for 10 min. The extraction process is repeated twice. The carotenoid content was determined by absorbance measurement (480 nm), with a production of 78.04 ± 1.89 mg.L-1 and a productivity of 15.61 ± 0.38 mg.L-1.d-1, with an increase greater than 29% compared to the control group. Petition 870250006793, dated 01 / 28 / 2025, page 21 / 31

Claims

1 / 3 CLAIMS 1. PROCESS FOR INDUCING CAROTENOGENESIS IN MICROALGAE characterized by using a bicarbonate and carbonate solution derived from the fixation of carbon dioxide-rich gases and comprising the steps of: a. Chemical fixation of CO2-rich gases using a strong base; b. Cultivation in photobioreactors for the production of microalgae biomass; c. Preparation of the carotenoid induction medium with a bicarbonate (HCO3·) and carbonate (CO32) solution; d. Cultivation in a carotenogenesis induction photobioreactor, with an inoculum of cells obtained in step (b), with controlled temperature and light intensity; e. Extraction of carotenoids from the encysted cells in the solution obtained in step (d); f. Harvesting of the biomass produced; g. Drying of the biomass produced.

2. PROCESS, according to claim 1, characterized by carrying out step (a), using more specifically sodium hydroxide or potassium hydroxide solutions, with concentrations of 0.1 to 2 mol.L-1, in a bubble column type reactor, with an injection flow rate of 1.5 to 6 vvm and a CO2 concentration of 1 to 15% (vv '), with an attached pH reader and a gas outlet system coupled to the photobioreactor inlet of step (b).

3. PROCESS, according to claim 1, characterized by carrying out step (b), using a photobioreactor for cultivation Petition 870250066267, dated 07 / 30 / 2025, page. 6 / 9 2 / 3 of vegetative cells with piping coupled to the reactor outlet described in claim 2 for CO2 injection at concentrations of 0.04 to 4.5% (vv-1), with a temperature of 20 to 30°C, illumination of 50 to 85 pmol.m-2.s-1 using ES culture medium (0.2 gL-1 KNO3, 0.2 gL-1 K2HPO4, 0.2 gL-1 MgSO4.7H2O, 30 mL.L-1 of soil extract, 5 mL.L-1 micronutrient solution, 1 mL vitamins), with Haematococcus pluvialis SAG culture cells.

4. PROCESS, according to claim 1, characterized by carrying out step (c), by preparing the carotenoid induction medium composed of nitrogen-free ES culture medium (KNO3) and medium resulting from chemical fixation obtained in step (a) with a dilution factor of 10 to 100 times to achieve HCO3- concentrations of 15 to 50 mM while carbonate (CO32-) is in the range of 2 to 10 mM, with the addition of sodium chloride (NaCl) at a concentration of up to 0.1 to 1 gL-1.

5. PROCESS, according to claim 1, characterized by step (d), using a carotenogenesis induction photobioreactor with induction medium from step (c), static or stirred from 1 to 50 rpm, inoculum of 0.25 to 1 gL-1 obtained in step (b), temperature of 28 to 36°C and light intensity of 100 to 250 pmol.m-2.s-1.

6. PROCESS, according to claim 1, characterized by carrying out step (e), conducting the extraction of carotenoids from encysted cells in the solution obtained in step (d) from centrifugation of the final culture, successive washing with distilled water and treatment with the organic solvent dimethyl sulfoxide (DMSO) at 50 to 70°C for 10 to 30 min in an ultrasonic system with a power of 160 W, repeating the extraction process twice and measuring the carotenoid content by absorbance measurement at 480 nm.

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