A species of dinoflagellate and a method for producing fucoxanthin from dinoflagellate.

Abstract

The application belongs to the field of microalgae biotechnology, and particularly relates to a brownish chrysochromulina and a method for producing fucoxanthin by using the brownish chrysochromulina. The brownish chrysochromulina is named PCSM-1, and was preserved in China Center for Type Culture Collection on January 11, 2024, with a preservation number of CCTCC NO: M 2024076. The content of fucoxanthin in the brownish chrysochromulina is as high as 1.58 wt%, and the brownish chrysochromulina can grow under photoautotrophic conditions and heterotrophic conditions. The brownish chrysochromulina is first subjected to heterotrophic fermentation to rapidly proliferate and ferment, and then is subjected to induction under photoautotrophic and heterotrophic conditions to rapidly accumulate fucoxanthin. The whole process has a short cycle, low energy consumption, and a far higher yield of fucoxanthin than existing microalgae, and can solve the problems of low production efficiency and high cost of fucoxanthin.

Description

Technical Field

[0001] This invention belongs to the field of microalgae biotechnology, specifically relating to a species of dinoflagellate algae and a method for producing fucoxanthin using dinoflagellate algae. Background Technology

[0002] Fucoxanthin is a bioactive pigment synthesized by algae, accounting for more than 10% of the total natural carotenoids. This compound has a polyene skeleton structure with a specific system of conjugated double bonds, interspersed with other single-bonded functional groups, such as epoxy, carbonyl, and hydroxyl groups. Fucoxanthin's unique molecular structure endows it with a variety of biological activities, including anti-inflammatory, anti-cancer, blood sugar regulating, antioxidant, and anti-obesity effects.

[0003] Currently, commercial fucoxanthin is mainly derived from macroalgae, such as wakame, kelp, and kelp japonica. However, there are several challenges to producing fucoxanthin from these macroalgae, including a long cultivation cycle (usually only 1-2 seasons per year), low fucoxanthin content (generally not exceeding 0.05% of dry weight), and the complexity of the extraction process. In contrast, single-celled microalgae, due to their high fucoxanthin content (between 0.2% and 2.2%) and ease of extraction, are gradually becoming an effective alternative source for commercial fucoxanthin production. However, the economic feasibility of using microalgae to produce fucoxanthin is not ideal at present. This is mainly because the microalgae currently used for fucoxanthin production primarily rely on photoautotrophic cultivation. Photoautotrophic cultivation has limitations such as large culture volume, insufficient light due to mutual shading between algal cells, energy deficiency, slow biomass accumulation, and low cell density, resulting in low yields and poor economic feasibility of fucoxanthin production from photoautotrophic microalgae.

[0004] Chinese patent document CN115820760A, published on March 21, 2023, discloses a method for efficiently producing fucoxanthin through fermentation-light coupling of golden algae. This method increases the biomass yield of golden algae by producing fucoxanthin through heterotrophic fermentation of golden algae. However, the fucoxanthin content disclosed in this method is extremely low (only 0.12%), resulting in a low final fucoxanthin yield and lacking economic feasibility. Therefore, it is necessary to explore more microalgal resources, especially those with high fucoxanthin productivity, and to optimize the fucoxanthin production model to improve the commercial viability of fucoxanthin production. Summary of the Invention

[0005] This invention discovers a new species of *Cyclocarya paliurus* (Cyclocarya paliurus). PoterioochromonasThis *Phaeocystis jirovecii* sp. contains up to 1.58 wt% fucoxanthin and can grow under both photoautotrophic and heterotrophic conditions. This invention first subjectes the *Phaeocystis jirovecii* sp. to heterotrophic fermentation to rapidly proliferate and ferment; then, it is induced under both photoautotrophic and heterotrophic conditions to rapidly accumulate fucoxanthin. The entire process is short in cycle and low in energy consumption, and the fucoxanthin yield is far higher than that of existing microalgae, solving the current problems of low fucoxanthin production efficiency and high cost.

[0006] The technical solution provided by this invention is as follows:

[0007] In a first aspect, the present invention provides a cup-shaped dinoflagellate algae named PCSM-1, which was deposited at the China Center for Type Culture Collection on January 11, 2024, with accession number CCTCC NO: M 2024076.

[0008] In conjunction with the first aspect of the present invention, the 18S rDNA gene sequence of the cuppyta is shown in SEQ ID NO:1.

[0009] Secondly, the present invention provides the application of the above-mentioned *Cyclocarya paliurus* in the preparation of fucoxanthin.

[0010] Thirdly, the present invention provides a method for producing fucoxanthin using *Cyclocarya paliurus*, comprising:

[0011] The above-mentioned *Cyclocarya paliurus* was proliferated and fermented under light or no light conditions;

[0012] Under light conditions, the fermented and proliferated *Cyclocarya paliurus* was placed in a heterotrophic culture medium for induction culture;

[0013] Fucoxanthin was extracted from *Cyclocarya paliurus* after induced culture.

[0014] In conjunction with the third aspect of the present invention, in some embodiments, the proliferation and fermentation medium used for the proliferation and fermentation of *Cyclocarya paliurus* contains a nitrogen source, a carbon source and auxiliary nutrients, a dissolved oxygen level of more than 20%, a temperature of 24-28°C, and a pH value of 5.0-7.0.

[0015] In conjunction with the third aspect of the present invention, in some embodiments, the proliferation fermentation medium contains: a nitrogen source concentration of 0.0158~0.021M based on molar amounts of nitrogen; and / or a carbon source concentration of 0.3~0.4M based on molar amounts of carbon; and / or auxiliary nutrients including KH2PO4, MgSO4, EDTA-2Na, CaCl2, vitamin B12, and vitamin B1, wherein the concentration of KH2PO4 is 0.2~0.4 g / L, the concentration of MgSO4 is 0.2~0.3 g / L, the concentration of EDTA-2Na is 4.0~5.0 g / L, the concentration of CaCl2 is 0.055~0.065 g / L, the concentration of vitamin B12 is 0.12~0.13 g / L, and the concentration of vitamin B1 is 0.37~0.38 g / L.

[0016] In conjunction with the third aspect of the present invention, in some embodiments, the fermentation time of *Cyclocarya paliurus* is 3 to 5 days.

[0017] In conjunction with a third aspect of the invention, in some embodiments, the light conditions for induced culture are 50-70 µmol photons·m⁻¹ of photosynthetically active radiation. -2 ·s -1 Continuous light exposure; temperature of 24±2°C; CO2 gas was introduced into the heterotrophic medium during induction culture.

[0018] In conjunction with the third aspect of the present invention, in some embodiments, the induction culture time is 3 to 4 days.

[0019] In conjunction with the third aspect of the present invention, in some embodiments, the basal medium of the heterotrophic medium is AF-6 medium, and the heterotrophic medium further comprises yeast extract at a concentration of 2-4 g / L, beef extract at a concentration of 0.5-1.5 g / L, glutamic acid at a concentration of 0.5-1.5 g / L, and glycine at a concentration of 0.5-1.5 g / L.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] 1. The fucoxanthin content of the novel *Cyclocarya calicifolia* species provided by this invention is higher than that of known microalgae. 2. The method for producing fucoxanthin using *Cyclocarya calicifolia* provided by this invention is low-cost and highly efficient. Attached Figure Description

[0022] Figure 1 The alignment results of the 18S rDNA gene sequence of *Cyclocarya caliciformis* PCSM-1 in GenBank.

[0023] Figure 2The image shows a comparison of cell morphology of *Cyclocarya paliurus* under different induction conditions. From left to right, the images show cell morphology after 1 day, 2 days, 3 days, and 4 days of culture.

[0024] Figure 3 Cell concentrations of *Cyclocarya paliurus* under different induction conditions ( Figure 3 A) Dry weight ( Figure 3 (B) Chlorophyll a concentration ( Figure 3 C), Fv / Fm ( Figure 3 The changes in the value of D in the data. Figure 4 The content of fucoxanthin in *Cyclocarya paliurus* under different induction conditions ( Figure 4 A) Production ( Figure 4 B) Yield ( Figure 4 The changes in C). Detailed Implementation

[0025] The present invention will be further described below through embodiments.

[0026] The inventors discovered a new species of *Cyclocarya caliciformis* from laboratory-cultured *Chlorella vulgaris*. Gene sequencing revealed that the 18S rDNA sequence of this *Cyclocarya caliciformis* is shown in SEQ ID NO:1, and its evolutionary distance is... Poterioochromonas The closest sp. is named PCSM-1 by the inventors, and it was deposited at the China Center for Type Culture Collection on January 11, 2024, with accession number CCTCC M 2024076.

[0027] This algal dinoflagellate can undergo both heterotrophic and photoautotrophic cultivation. In heterotrophic cultivation, each algal cell can fully contact and utilize organic matter, avoiding the problems of insufficient light and energy caused by mutual shading between algal cells in photoautotrophic cultivation. The algal cells can grow rapidly and accumulate a large amount of biomass in a short period of time, providing a large number of cells for the subsequent accumulation of fucoxanthin.

[0028] The proliferation and fermentation experiments of this invention show that after 3-5 days of proliferation and fermentation, the algal cell density can reach 30 g / L from the initial 2 g / L, demonstrating an astonishing proliferation and fermentation rate. In the experiments, this invention observed that under mixed-culture induction conditions, the maximum fucoxanthin content and production efficiency of this *Cyclocarya paliurus* were 1.58% and 12.4 mg / L / d, respectively, far exceeding the production potential of existing *Cyclocarya paliurus* or *Phaeodactylum tricornutum* species (see Table 1).

[0029] Table 1 Comparison of fucoxanthin yield and productivity among different microalgae

[0030]

[0031] Note: The references listed in the table above are from the following sources:

[0032] [1]Lu,

[0033] [2]Yang, R., Wei, D., Pohnert, G. 2022. Nitrogen utilization analysis reveals the synergetic effect of arginine and urea in promoting fucoxanthinbiosynthesis in the mixotrophic marine diatom Phaeodactylum tricornutum.Frontiers in Marine Science, 9, 947726

[0034] [3]Guo, B., Liu, B., Yang, B., Sun, P., Lu, X., Liu, J., Chen, F.2016. Screening of diatom strains and characterization of Cyclotella crypticaas a potential fucoxanthin producer. Marine Drugs, 14(7), 125.

[0035] The above findings indicate that this *Cercospora chinensis* can replace existing microalgae for the preparation of fucoxanthin, thus solving the problems of low fucoxanthin content and low production efficiency in existing microalgae.

[0036] Based on this, the present invention provides a method for producing fucoxanthin using *Cyclocarya paliurus*. The method is divided into two stages: a proliferation and fermentation stage and an induction stage. The purpose of the proliferation and fermentation stage is to obtain a sufficient number of *Cyclocarya paliurus* cells, and a culture medium containing organic nitrogen sources and / or inorganic nitrogen sources can be selected for fermentation of *Cyclocarya paliurus*. The purpose of the induction stage is to obtain a sufficient number of fucoxanthin, and a heterotrophic culture medium containing a large amount of organic nitrogen sources is used for nutrient supply.

[0037] Specifically, the method for producing fucoxanthin using *Cyclocarya paliurus* provided by this invention includes:

[0038] The above-mentioned *Cyclocarya paliurus* was proliferated and fermented under light or no light conditions;

[0039] Under light conditions, the fermented and proliferated *Cyclocarya paliurus* was placed in a heterotrophic culture medium for induction culture;

[0040] Fucoxanthin was extracted from *Cyclocarya paliurus* after induced culture.

[0041] This method first accumulates biomass through proliferation and fermentation, providing a large number of cells for the accumulation of fucoxanthin. Then, it induces the accumulation of fucoxanthin in algal cells through polyculture, which can produce fucoxanthin rapidly, in large quantities, and economically. It is expected to solve the current problems of low fucoxanthin yield and high cost, and promote the industrialization of microalgal fucoxanthin.

[0042] In some implementations, the culture medium used for the proliferation and fermentation of *Cyclocarya paliurus* contains nitrogen source, carbon source and auxiliary nutrients, with a dissolved oxygen level of over 20%, a temperature of 24-28°C, and a pH of 5.0-7.0.

[0043] In some embodiments, the proliferation fermentation medium contains: a nitrogen source concentration of 0.0158–0.021 M (based on molar amounts of nitrogen); and / or a carbon source concentration of 0.3–0.4 M (based on molar amounts of carbon); and / or auxiliary nutrients including KH₂PO₄, MgSO₄, EDTA-2Na, CaCl₂, vitamin B12, and vitamin B1, wherein the concentration of KH₂PO₄ is 0.2–0.4 g / L, the concentration of MgSO₄ is 0.2–0.3 g / L, the concentration of EDTA-2Na is 4.0–5.0 g / L, the concentration of CaCl₂ is 0.055–0.065 g / L, the concentration of vitamin B12 is 0.12–0.13 g / L, and the concentration of vitamin B1 is 0.37–0.38 g / L.

[0044] In some implementation methods, the fermentation time for *Cyclocarya paliurus* is 3-5 days. Fermentation experiments show that after 3-5 days of fermentation, the algal cell density can reach 30 g / L from the initial 2 g / L.

[0045] In some implementations, the light conditions for induction culture are 50–70 µmol photons·m⁻¹ of photosynthetically active radiation. -2 ·s -1 Under continuous light and at a temperature of 24±2°C, CO2 gas is bubbled into the heterotrophic medium during the induction culture period. Preferably, the CO2 gas is a mixture of air and CO2, with a CO2 volume ratio of 1% to 2%.

[0046] In some implementation methods, the induction culture time is 3 to 4 days. Experiments have shown that the fucoxanthin content and production efficiency of *Cyclocarya paliurus* reach their maximum after 3 to 4 days of induction culture.

[0047] In some embodiments, the basal medium of the heterotrophic medium is AF-6 medium, which further contains yeast extract at a concentration of 2-4 g / L, beef extract at a concentration of 0.5-1.5 g / L, glutamic acid at a concentration of 0.5-1.5 g / L, and glycine at a concentration of 0.5-1.5 g / L. The AF-6 medium is purchased from a third-party company or prepared in-house. Preferably, the composition of the AF-6 medium is: 0.14 g / L NaNO3, 0.022 g / L NH4NO3, 0.03 g / L MgSO4, 0.01 g / L KH2PO4, 0.005 g / L K2HPO4, 0.01 g / L CaCl2·2H2O, 0.01 g / L CaCO3, 0.002 g / L citric acid, 0.002 g / L Fe-citric acid complex, 2×10 -2 µg / L biotin, 5×10 -3 µg / L thiamine hydrochloride, 1 µg / L vitamin B6, 1 µg / L vitamin B12, 3.75 mg / L EDTA-2Na·2H2O, 0.485 mg / L FeCl3·6H2O, 0.205 mg / L LmnCl2, 0.025 mg / L ZnCl2·7H2O, 0.01 mg / L CoCl2·6H2O, and 0.02 mg / L Na2MoO4·2H2O.

[0048] Specifically, the present invention provides a method for producing fucoxanthin using *Cyclocarya paliurus*, comprising the following steps:

[0049] S1. Inoculate *Cyclocarya paliurus* into a fermenter for batch fermentation culture. The fermentation time for a single batch is 3-5 days, and the temperature is maintained at 0-200 µmol photons·m⁻¹ during fermentation. -2 ·s -1The light intensity (including both light and no light conditions), dissolved oxygen level maintained above 20%, temperature 24-28℃, and pH value stable at 5.0-7.0; the substrate culture medium for fermentation contains nitrogen source, carbon source, and auxiliary nutrients; the nitrogen source concentration is 0.0158-0.021M (calculated as molar amount of nitrogen), and the carbon source concentration is 0.3-0.4M (calculated as molar amount of carbon); the auxiliary nutrients include 0.2-0.4 g / L KH₂PO₄, 0.2-0.3 g / L MgSO₄, 4.0-5.0 g / L EDTA-2Na, 0.055-0.065 g / L CaCl₂, 0.12-0.13 g / L vitamin B12, and 0.37-0.38 g / L vitamin B1;

[0050] S2. After fermentation, *Cyclocarya paliurus* was transferred to a photobioreactor for fucoxanthin accumulation induction culture. The induction culture time was 3-4 days, at a temperature of 24±2°C and photosynthetically active radiation of 50-70 µmol photons·m⁻¹. -2 ·s -1 The induction culture was carried out under continuous light. During the induction culture, a mixture of air and CO2 was continuously introduced at a rate of 2-10 L / min per 5 L of culture medium, with a CO2 volume fraction of 1%-2%. The basal medium for induction culture was AF-6 medium supplemented with 2-4 g / L yeast extract, 0.5-1.5 g / L beef extract, 0.5-1.5 g / L glutamic acid, and 0.5-1.5 g / L glycine.

[0051] Preferably, in step S1, the inoculum concentration for fermentation culture is 0.5~2.0 g / L.

[0052] Preferably, in step S1, the carbon source is one or a combination of two or more of glucose, glycerol, sucrose and molasses; the nitrogen source is one or a combination of two or more of NH4Cl, bovine liver extract, yeast extract and ammonium nitrate.

[0053] Preferably, in step S1, the substrate culture medium for fermentation contains: 1.0 g / L NH4Cl, 10.0 g / L glucose, 0.3 g / L KH2PO4, 0.244 g / L MgSO4, 4.40 g / L EDTA-2Na, 0.0559 g / L CaCl2, 0.125 g / L vitamin B12 and 0.375 g / L vitamin B1.

[0054] Preferably, in step S1, the substrate culture medium for fermentation further includes: 0.96~0.99 g / L H3BO3, 0.114~0.115 g / L MnCl2, 0.011~0.012 g / L ZnSO4, 1.8~2.0 g / L FeCl3, 0.005~0.006 g / L Na2MoO4, and 0.007~0.008 g / L Co(NO3)2; more preferably, the substrate culture medium for fermentation includes: 0.97 g / L H3BO3, 0.18 g / L MnCl2·4H2O, 0.02 g / L ZnSO4·7H2O, 3.15 g / L FeCl3·6H2O, 0.006 g / L Na2MoO4·2H2O, and 0.012 g / L Co(NO3)2·6H2O.

[0055] Preferably, in step S2, the initial cell concentration of *Callicarpa chinensis* in the photobioreactor is (2~6) × 10⁻⁶. 6 per mL.

[0056] In step S2, before transferring the dinoflagellates to the photobioreactor, the dinoflagellates cells are washed 1-3 times with the culture medium used for induction culture.

[0057] Preferably, in step S2, the culture medium for induction culture is AF-6 medium supplemented with 3 g / L yeast extract, 1 g / L beef extract, 1 g / L glutamic acid and 1 g / L glycine.

[0058] The technical solution of the present invention will be described in detail below through specific embodiments.

[0059] Example 1

[0060] The inventors used a Pasteur pipette to select a single clone of *Chlorella vulgaris* cell under a microscope from a *Chlorella* culture in the laboratory. The single-cell clone was then transferred to a 24-well plate containing 1 mL of AF-6 medium for expansion culture. The bacteria in the algal culture were then treated with a mixed antibiotic consisting of streptomycin, chloramphenicol, gentamicin, and ciprofloxacin, with each antibiotic at a concentration of 20 mg / L. After 3-5 generations of treatment, a strain-free line was obtained.

[0061] Genomic DNA was extracted from the strain-free line, and its 18S rDNA was amplified by PCR. The forward primer used for PCR amplification was: F: 5'-AACCTGGTTGATCCTGCCAGT-3' (as shown in SEQ ID NO:2); the reverse primer was: R: 5'-TGATCCTTCTGCAGGTTCACCTAC-3' (as shown in SEQ ID NO:3). The amplification products were subjected to 1% agarose gel electrophoresis. The PCR products were recovered using a kit (OMEGA, USA), and the gel-recovered products were directly sent to a gene company for sequencing (Shanghai Sangon Biotech Co., Ltd.). The 18S rDNA gene sequence of the strain-free line was obtained, as shown in SEQ ID NO:1. Figure 1 As shown, the 18S rDNA gene sequence of the strain-free line was compared with that in GenBank, revealing that the evolutionary distance of the strain-free line was related to... Poterioochromonas The closest sp. was identified based on molecular sequence information as *Calibrachoidea*. Poterioochromonas The specimen, named PCSM-1, was deposited on January 11, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2024076.

[0062] Finally, the purified algal strain PCSM-1 was stored in a specific culture medium at 25°C and with a photosynthetically active radiation of 60 µmol photons·m -2 ·s -1 The specific culture medium consisted of 10 g / L glucose, 3 g / L yeast extract (OXOID, LP0021B), 1 g / L beef extract (Beijing Aoboxing Biotechnology Co., Ltd., 01-085B), 0.5 g / L KH2PO4 and 0.5 g / L MgSO4, with the pH maintained at around 6.

[0063] Example 2: Experiment on fucoxanthin production by *Calibrachoa var. cristatum* PCSM-1

[0064] 1 Experimental Design

[0065] The dinoflagellate dinoflagellate PCSM-1 was cultured in batches using a 5 L fermenter, with each batch fermentation lasting 4 days.

[0066] The specific formulation of the substrate culture medium in the fermenter is as follows: 1.0 g / L NH4Cl, 10.0 g / L glucose, 0.3 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 4.40 g / L EDTA-2Na, 0.074 g / L CaCl2·2H2O, 0.97 g / L H3BO3, 0.18 g / L MnCl2·4H2O, 0.02 g / L ZnSO4·7H2O, 3.15 g / L FeCl3·6H2O, 0.006 g / L Na2MoO4·2H2O, 0.012 g / L Co(NO3)2·6H2O, 0.125 g / L vitamin B12, and 0.375 g / L vitamin B1. In the above substrate culture medium, NH4Cl is the main nitrogen source, and glucose is the main carbon source.

[0067] The fermentation seed was obtained through shake-flask culture. The propagation fermentation medium used in the shake-flask culture contained: 10 g / L glucose, 3 g / L yeast extract, 1 g / L beef extract, 0.5 g / L KH₂PO₄, and 0.5 g / L MgSO₄, with a pH of 6.0. The Erlenmeyer flasks were calibrated to 1 L, with a liquid volume of 400 mL. The shaker speed was 180 rpm, and the culture temperature was 26℃. The initial inoculum concentration in the fermenter was 0.5 g / L. An automatic control system continuously monitored the fermentation process, ensuring that the pH remained stable between 5.0 and 7.0, the dissolved oxygen (DO) level was maintained above 20%, and the temperature between 24 and 28℃.

[0068] During the fermentation process, six LED lights were evenly arranged around each fermenter to provide an average photosynthetically active radiation of 60 µmol photons·m⁻¹. -2 ·s -1 The lighting provides continuous illumination for the fermentation tank.

[0069] After 4 days of fermentation culture, the *Callicarpa chinensis* cells were transferred to a 700 mL glass column photobioreactor. φ Fucoxanthin accumulation was induced in a medium (5 cm²) for culture. During the induction culture phase, the effects of three different culture media on fucoxanthin content, yield, and productivity were tested. These three culture media were:

[0070] 1) Autotrophic induction medium: Simple AF-6 medium; AF-6 medium formula: 0.14 g / L NaNO3, 0.022 g / L NH4NO3, 0.03 g / L MgSO4, 0.01 g / L KH2PO4, 0.005 g / L K2HPO4, 0.01 g / L CaCl2·2H2O, 0.01 g / L CaCO3, 0.002 g / L citric acid, 0.002 g / L Fe-citric acid complex, 2 × 10 -2 µg / L biotin, 5×10 -3 µg / L thiamine hydrochloride, 1µg / L vitamin B6, 1µg / L vitamin B12, 3.75mg / L EDTA-2Na·2H2O, 0.485mg / L FeCl3·6H2O, 0.205mg / L MnCl2, 0.025mg / L ZnCl2·7H2O, 0.01mg / L CoCl2·6H2O and 0.02mg / L Na2MoO4·2H2O;

[0071] 2) Phagocytosis induction medium: AF-6 medium + heat-inactivated Chlorella cells, wherein the concentration of heat-inactivated Chlorella cells is 1×10⁻⁶. 7 cells / mL;

[0072] 3) Mixed culture induction medium: AF-6 medium + 3g / L yeast extract + 1g / L beef extract + 1g / L glutamic acid + 1g / L glycine.

[0073] Before being added to the glass column photobioreactor, *Cyclocarya calicifolia* was washed three times with the appropriate culture medium. The initial cell concentration of *Cyclocarya calicifolia* in the glass column photobioreactor was 6 × 10⁻⁶. 6 Cells / mL. Induction culture time was 4 days, temperature was 24±1°C, and photosynthetically active radiation was 60 µmol photons·m⁻¹. -2 ·s -1 The culture was continuously illuminated and a mixed gas (CO2 volume fraction of 1%~2%, with the mixed gas introduced at a rate of 0.6 L / min per 500 mL of culture medium) was continuously introduced. Each treatment was set up in 3 replicates, and samples were collected and analyzed daily.

[0074] 2. Analytical Methods

[0075] 2.1 Cell growth and cell morphology

[0076] The growth of *Cyclocarya caliciformis* was quantified by measuring dry weight and cell concentration. Dry weight (g / L) was measured according to the method described by Jin et al. (2022). Simply put, 1 mL of culture was centrifuged at 1500 × g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 200 µL of deionized water. The resuspended solution was transferred to a pre-weighed and dried GF / C membrane (47 mm, Whatman, UK), dried at 105 °C for 12 h, cooled to room temperature, and weighed again. Cell concentration of *Cyclocarya caliciformis* was pre-fixed with 1% Lugol's iodine solution and counted using a hemocytometer (Improved Neubauer, USA) under an optical microscope (BX53, Olympus, Japan). Cell morphology of *Cyclocarya caliciformis* in different culture media was observed and recorded using a BX53 microscope. To record the color changes of the algal solution, 3 mL of *Cyclocarya paliurus* culture medium was filtered onto a GF / C membrane and photographed using a 6D Mark II camera (Canon, Japan).

[0077] 2.2 Chlorophyll fluorescence determination of chlorophyll a and PSII activities

[0078] Algal cells were placed in pre-cooled methanol at 4°C for low-temperature pigment extraction for 12 hours. The extract was then centrifuged at 10,000 g for 1 minute. The absorbance of the supernatant at wavelengths of 653 nm (A653) and 666 nm (A666) was measured using a fluorescence spectrophotometer (DR6000, HACH, USA).

[0079] Chlorophyll a (Chl-a) concentration is calculated using the following formula: Chl-a (mg / L) = (16.72 × A666 – 9.72 × A653) × V MeOH ÷ V algae V MeOH V is the volume of methanol. algae This is the volume of the microalgal suspension used for pigment extraction. The potential quantum efficiency of Photosystem II, expressed as Fv / Fm, was measured using an AquaPen AP110-C (Czech PS I). Fresh algal samples were placed in complete darkness for 20 min, and then 3 mL of culture medium was transferred to a pre-cleaned cuvette. The spectra were analyzed and data were acquired using FluorPen software.

[0080] 2.3 Determination of fucoxanthin content

[0081] The algal cells collected by centrifugation were stored in the dark at -80℃ and then freeze-dried using a vacuum freeze dryer. The freeze-dried microalgae powder was then used for pigment extraction using a methanol / dichloromethane mixture (3:1, v / v). The fucoxanthin content in the extracted samples was analyzed by high performance liquid chromatography.

[0082] 3 Results

[0083] 3.1 Cell growth and cell morphology

[0084] Observe the cells of *Callicarpa chinensis* under a microscope, such as... Figure 2 As shown, *Cyclocarya caliciformis* cells cultured in autotrophic and phagocytic induction media had very little pigment, and chloroplasts remained incomplete throughout the four-day culture period. In contrast, *Cyclocarya caliciformis* cells grown in the mixed-culture induction medium accumulated more pigment, and their chloroplast morphology was extremely complete. Especially on the third and fourth days, chloroplasts accounted for more than half of the total cell size in the mixed-culture induction medium.

[0085] 3.2 Cell concentration, dry weight, chlorophyll a concentration, Fv / Fm value

[0086] Figure 3 The cell concentrations of *Cyclocarya paliurus* under different induction conditions were shown. Figure 3 A) Dry weight ( Figure 3 (B) Chlorophyll a concentration ( Figure 3 C), Fv / Fm ( Figure 3 The change in the D value in the figure, Fv / Fm is the core indicator of the light-harvesting efficiency of the photosystem II, reflecting the performance of photosynthesis.

[0087] It can be seen that under autotrophic induction conditions, the cell concentration of *Cyclocarya paliurus* is relatively stable, close to the initial inoculum concentration of 6.0 × 10⁻⁶. 6 Cells / mL; under phagocytosis-induced conditions, the cell concentration of *Cyclocarya paliurus* increased slightly, reaching a maximum of 7.2 × 10⁻⁶ cells / mL after three days of culture. 6 Cells / mL; under polyculture induction conditions, the cell concentration of *Cyclocarya paliurus* increased significantly, reaching a maximum of 12.2 × 10⁻⁶ cells / mL. 6 The biomass per mL was similarly high, with the maximum biomass under polytrophic induction conditions being 2.7 g / L, the maximum biomass under phagocytic induction conditions being 1.6 g / L, and the maximum biomass under autotrophic induction conditions being 1.9 g / L.

[0088] Chlorophyll a concentration showed a similar trend to cell concentration and dry weight, with the highest chlorophyll a concentration (18.6 mg / L) occurring under polytrophic induction conditions, which was more than 5 times higher than that under autotrophic induction conditions (3.4 mg / L). Furthermore, the Fv / Fm results also indicated that the photosynthetic capacity of *Cyclocarya paliurus* was significantly enhanced under polytrophic induction conditions.

[0089] 3.3 Fucoxanthin content, yield and productivity

[0090] Figure 4The content of fucoxanthin in *Cyclocarya paliurus* under different induction conditions was shown. Figure 4 A) Production ( Figure 4 B) Yield ( Figure 4 The changes in C).

[0091] Under both autotrophic and phagocytic induction conditions, the fucoxanthin content of *Cyclocarya paliurus* increased slowly throughout the induction phase, from 0.1 wt% to 0.2 wt%. Under polytrophic induction conditions, the fucoxanthin content of *Cyclocarya paliurus* increased significantly, from an initial 0.06 wt% to a maximum of 1.58 wt% after four days. Under polytrophic induction conditions, the maximum fucoxanthin yield and productivity were observed to be 37.8 mg / L and 12.4 mg / L / d, respectively. The fucoxanthin productivity under polytrophic induction conditions exceeded the production potential of other known fucoxanthin-producing microalgae.

[0092] 4. Economic Feasibility Analysis

[0093] The market price of high-quality fucoxanthin has risen to US$40 million per ton, attracting widespread attention in commercial production. Comparing the production cost of the method for producing fucoxanthin using *Cyclocarya paliurus* provided in this invention with the production cost of producing fucoxanthin using *Phaeodactylum tricornutum* (currently the most promising microalgae for fucoxanthin production), the results show that the production cost of the method using *Cyclocarya paliurus* provided in this invention is only US$2.39 million per ton, far lower than the cost of producing fucoxanthin using *Phaeodactylum tricornutum* (nearly US$10 million per ton). Analysis reveals that the key factor for the significant cost reduction is the reduction in electricity demand. The electricity cost required to produce one ton of algal powder in this invention is US$290,000, while the electricity cost required to produce one ton of *Phaeodactylum tricornutum* is US$5.7 million. Compared to the photoautotrophic cultivation mode of *Phaeodactylum tricornutum*, the fermentation mode of *Cyclocarya paliurus* has a higher biomass yield and lower land requirements. Therefore, the land and construction costs required for the method for producing fucoxanthin using *Cyclocarya paliurus* provided in this invention are more than ten times lower than the autotrophic production mode of *Phaeodactylum tricornutum*.

[0094] Example 3

[0095] This embodiment compares the effects of fermentation broths with different carbon and nitrogen sources on the growth rate of *Cyclocarya paliurus* PCSM-1. The experiment was conducted as follows: Heterotrophic fermentation batch culture of *Cyclocarya calicifolia* PCSM-1 was carried out. The culture medium formula was as follows: nitrogen source (0.02 M molar nitrogen concentration), carbon source (0.3 M molar carbon concentration), 0.3 g / L KH₂PO₄, 0.5 g / L MgSO₄·7H₂O, 4.40 g / L EDTA-2Na, 0.074 g / L CaCl₂·2H₂O, 0.97 g / L H₃BO₃, 0.18 g / L MnCl₂·4H₂O, 0.02 g / L ZnSO₄·7H₂O, 3.15 g / L FeCl₃·6H₂O, 0.006 g / L Na₂MoO₄·2H₂O, 0.012 g / L Co(NO₃)₂·6H₂O, 0.125 g / L vitamin B12 and 0.375 g / L vitamin B1.

[0096] In this embodiment, when comparing the effects of fermentation broths with different carbon sources on the growth of *Phaeodactylum calicifolium* PCSM-1, the nitrogen sources used were fixed as yeast extract and liver extract powder, and the carbon sources were selected in sequence as a mixture of glycerol, sucrose, molasses, glucose and glycerol.

[0097] In this embodiment, when comparing the effects of fermentation broths with different nitrogen sources on the growth of *Phaeocystis chinensis* PCSM-1, the carbon source used was fixed as glucose, and the nitrogen sources were selected sequentially as bovine liver extract powder, yeast extract, ammonium nitrate, and a mixture of bovine liver extract powder and yeast extract.

[0098] Following the conditions in Example 2, with an average photosynthetically active radiation of 60 µmol photons·m -2 ·s -1 The cells were cultured under fermentation conditions for 4 days. Experimental results showed that the *Phaeodinium calicifolium* PCSM-1 provided by this invention was not sensitive to changes in carbon and nitrogen sources, and exhibited strong adaptability and utilization capabilities to different nitrogen and carbon sources. After 4 days of culture, the cell density of *Phaeodinium calicifolium* increased from the initial 0.5 g / L to 11.6–14.2 g / L. Furthermore, *Phaeodinium calicifolium* cultured with different nitrogen and carbon sources showed similar results in subsequent fucoxanthin accumulation.

[0099] In summary, this invention provides a novel species of *Cyclocarya calicifolia* capable of heterotrophic and photoautotrophic cultivation. This *Cyclocarya calicifolia* exhibits higher fucoxanthin content and maximum fucoxanthin yield than known microalgae, and demonstrates strong adaptability and utilization to different nitrogen and carbon sources. It grows rapidly and accumulates large amounts of biomass in a short time, providing a large cell base for subsequent fucoxanthin accumulation. Utilizing this *Cyclocarya calicifolia* for fucoxanthin production holds promise for solving the current problems of low fucoxanthin yield and high cost. The above descriptions are merely preferred embodiments of this invention and are not intended to limit the invention. Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for producing fucoxanthin using botryococcus braunii, characterized by, The cupuladriatis porphyridium is named PCSM-1, which is preserved in China Center for Type Culture Collection on January 11, 2024, and the preservation number is CCTCC NO: M 2024076; the method comprises the following steps: S1. The cupraea is inoculated into a fermenter for batch fermentation culture, the single batch fermentation culture time is 3-5 days, the illumination intensity is 2000-4000 lux, the dissolved oxygen level is maintained above 20%, the temperature is 24-28℃, the pH value is stabilized at 5.0-7.0; the substrate culture medium of the fermentation culture contains a nitrogen source, a carbon source and auxiliary nutritional ingredients; the concentration of the nitrogen source is 0.0158-0.021M in terms of the molar amount of nitrogen element, the concentration of the carbon source is 0.3-0.4M in terms of the molar amount of carbon element; the auxiliary nutritional ingredients include 0.2-0.4g / L of KH2PO4, 0.2-0.3g / L of MgSO4, 4.0-5.0g / L of EDTA-2Na, 0.055-0.065g / L of CaCl2, 0.12-0.13g / L of vitamin B12 and 0.37-0.38g / L of vitamin B1. -2 · -1 ​ S2. After the end of the fermentation culture, the b. diatomea is transferred to a photobioreactor for fucoxanthin accumulation induction culture. The induction culture time is 3-4 days, and the temperature is 24±2°C, the photosynthetic active radiation is 50-70 µmol photons·m -2 ·s -1 under continuous light; during the induction culture, a mixture of air and CO2 is continuously introduced at 2-10 L / min per 5 L of culture volume, and the volume fraction of CO2 in the mixed gas is 1%-2%; the base medium for the induction culture is AF-6 medium, and 2-4 g / L of yeast extract, 0.5-1.5 g / L of beef extract, 0.5-1.5 g / L of glutamic acid, and 0.5-1.5 g / L of glycine are added.

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