Microbial culture method

Abstract

To provide a method for culturing microalgae that adjusts the nutritional form and culture conditions for microalgae, thereby dramatically improving the rate of their growth.SOLUTION: A method for culturing microalgae includes following steps 1 and 2. Step 1: Microalgae are cultured under darkness-dependent nutritional culture conditions to prepare multinucleated cells. Step 2: the resultant multinucleated cells are cultured under independent nutritional culture conditions or mixed nutritional culture conditions.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an improved method for culturing microorganisms, particularly microalgae, and more particularly to a culture method that dramatically improves the growth rate of microalgae. [Background technology]

[0002] Microalgae have higher productivity per unit area than terrestrial plants, and produce high-value-added substances that can be used as raw materials for pharmaceuticals, cosmetics, and highly functional feed, so they are expected to be used for industrial production [1, 2]. In particular, natural astaxanthin produced using the green alga Haematococcus lacustris has a strong antioxidant effect and is sold at a high price of tens of thousands to hundreds of thousands of yen per kilogram as a raw material for cosmetics, etc. [3, 4]. However, Haematococcus lacustris grows slowly, and high technology and expensive equipment are required for mass cultivation. Therefore, in recent years, the green alga Chromochloris zofingiensis, which is easier to mass-cultivate and has a high growth rate, has attracted attention.

[0003] There are three types of nutritional forms for microalgae cultivation: autotrophic culture, which uses light as the energy source and carbon dioxide as the carbon source; heterotrophic culture, which uses organic matter as the energy and carbon source; and mixotrophic culture, which uses both simultaneously. A limited number of species, including Chromochloris zofingiensis, can perform all three. In recent years, research has progressed on the physiological changes of Chromochloris zofingiensis in each nutritional form (Non-Patent Document 1). However, there have been very few studies that have examined the transition from one nutritional form to another, and knowledge is limited.

[0004] For example, Non-Patent Document 2 is an example of a study that tested the transition of trophic form in Chromochloris zofingiensis. In this study, a biomass production phase optimized for biomass production was carried out in heterotrophic culture, followed by an astaxanthin accumulation phase accompanied by light irradiation and nutrient deficiency stress in mixotrophic culture for astaxanthin accumulation. In this way, there are several studies that have changed the trophic form by dividing the objectives into biomass production and astaxanthin accumulation and combining them with stress conditions (e.g., Non-Patent Document 3). However, there is no study that has changed the trophic form without placing it under stress conditions for the purpose of biomass production. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Biotechnology for Biofuels,14(1),1-16 [Non-Patent Document 2] Journal of Agricultural and Food Chemistry,67(19),5579-5586 [Non-Patent Document 3] Bioresource Technology,224,515-522 [Non-Patent Document 4] Algal Research,39(October 2018),101455 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there has been a demand for a culture method that allows for easy mass cultivation of microalgae and that can be applied to the production of astaxanthin.

[0007] In the present invention, a dramatic improvement in the growth rate of microalgae, particularly microalgae of the genus Chromochloris, is achieved by changing the nutrient form in accordance with the culture stage.

[0008] That is, an object of the present invention is to provide a method for culturing microalgae, which dramatically improves the growth rate of microalgae by adjusting the nutritional form and culture conditions of the microalgae. [Means for solving the problem]

[0009] The inventors focused on developing promising nutritional conditions for the stock, pre-culture, and biomass production stages and compared cell growth of Chromochloris zofingiensis under various combinations of nutritional modes in the pre-culture and biomass production stages. They then performed a sequence of two pre-cultures under the same conditions to adapt the cells to each nutritional mode. The pre-cultured cells were then transferred to the biomass production stage under three nutritional modes, and the growth rates and chemical compositions of the various nutritional modes were compared in both the pre-culture and biomass production stages. Based on the findings from the results, the present invention was completed.

[0010] That is, the present invention provides the following: <1> ~ <14> to provide. <1> A method for culturing microalgae, comprising the following steps 1 and 2: Step 1: Cultivating microalgae under dark heterotrophic culture conditions to obtain multinucleated cells Step 2: Cultivating the resulting multinucleated cells under autotrophic or mixotrophic culture conditions.

[0011] <2> Before step 1, the microalgae are mixed. nutrition Cultivating under culture conditions or autotrophic culture conditions, <1> The method according to

[0012] <3> In step 2, the obtained multinucleated cells are cultured under autotrophic culture conditions. <1> or <2> The method according to

[0013] <4> In step 2, the obtained multinucleated cells are cultured under mixotrophic culture conditions. <1> or <2> The method described above.

[0014] <5> Repeat steps 1 and 2. <1> ~ <4> 2. The method according to claim 1 ,

[0015] <6> The microalgae are algae belonging to the phylum Chlorophyta. <1> ~ <5> 2. The method according to claim 1 ,

[0016] <7> The algae belonging to the phylum Chlorophyta are algae belonging to the order Polytrichum. <6> The method described above.

[0017] <8> Chromochloris zofingiensis is an algae belonging to the order Polytrichum algae. <7> The method described above.

[0018] <9> A method for accelerating the growth rate of microalgae, comprising the steps of: Step 2: Cultivating the multinucleated cells under autotrophic or mixotrophic culture conditions

[0019] <10> The method further includes the following step 1 before step 2: <9> The method described above. Step 1: Cultivating microalgae under dark heterotrophic culture conditions to obtain multinucleated cells

[0020] <11> Before step 1, the microalgae are mixed. nutrition Cultivating under culture conditions or autotrophic culture conditions, <10> The method described above.

[0021] <12> The microalgae are algae belonging to the phylum Chlorophyta. <9> ~ <11> 2. The method according to claim 1 ,

[0022] <13> The algae belonging to the phylum Chlorophyta are algae belonging to the order Polytrichum. <12> The method described above.

[0023] <14> Chromochloris zofingiensis is an algae belonging to the order Polytrichum algae. <13> The method according to

[0024] Further, the present invention relates to Stock culture step: a step of culturing microalgae under mixotrophic or autotrophic culture conditions; Step 1: culturing microalgae under dark heterotrophic culture conditions to obtain multinucleated cells; and Step 2: Cultivating the resulting multinucleated cells under autotrophic or mixotrophic culture conditions. The present invention provides a method for culturing microalgae, comprising:

[0025] The present invention also provides Stock culture step: culturing microalgae under autotrophic culture conditions and then culturing them under heterotrophic culture conditions; Step 1: culturing microalgae under dark heterotrophic culture conditions to obtain multinucleated cells; and Step 2: Cultivating the resulting multinucleated cells under autotrophic or mixotrophic culture conditions. The present invention provides a method for culturing microalgae, comprising:

[0026] Furthermore, the present invention relates to Stock culture step: A step of culturing microalgae under repeated light irradiation and dark conditions to prepare an algae stock; Step 1: culturing microalgae under dark heterotrophic culture conditions to obtain multinucleated cells; and Step 2: Cultivating the resulting multinucleated cells under autotrophic or mixotrophic culture conditions. The present invention provides a method for culturing microalgae, comprising: Effect of the Invention

[0027] INDUSTRIAL APPLICABILITY The present invention is expected to achieve a dramatic improvement in the growth rate in the cultivation of microalgae such as Chromochloris zofingiensis, and thus improve the productivity of astaxanthin, which has antioxidant properties. [Brief description of the drawings]

[0028] [Figure 1] Growth curves of Chromochloris zofingiensis in three nutritional modes: first preculture followed by a second preculture, (a) autotrophic, (b) mixotrophic, and (c) heterotrophic culture. Data are expressed as mean ± standard deviation, n = 3. [Diagram 2] Figure 1 shows the biomass production stages of Chromochloris zofingiensis under three pre-culture conditions, autotrophic, mixotrophic, and heterotrophic. (a) Autotrophic, (b) Mixotrophic, and (c) Heterotrophic conditions. Data are expressed as mean ± standard deviation, n = 3. [Diagram 3] Figure 1 shows the cell size of Chromochloris zofingiensis under three pre-culture conditions, autotrophic, mixotrophic, and heterotrophic, (a) autotrophic (b) mixotrophic, and (c) heterotrophic conditions. Data are expressed as mean ± standard deviation, n = 3. [Figure 4] Figure 1 shows the carotenoid content of Chromochloris zofingiensis under three pre-culture conditions, autotrophic, mixotrophic, and heterotrophic. (a) Autotrophic, (b) Mixotrophic, and (c) Heterotrophic conditions. Astaxanthin content shown is the sum of free, mono-, and diesters. Data are expressed as mean ± standard deviation, n=3. [Diagram 5] The generation of multinucleated cells by heterotrophic pre-cultivation and their transformation to mononuclear cells through rapid cell division upon transition to autotrophic culture are shown in nuclear stained micrographs. [Figure 6] This is a nuclear stained micrograph showing that mononuclear cells precultured autotrophically rapidly become multinucleated after the transition to heterotrophic culture. [Figure 7]Average number of nuclei per cell upon transfer to autotrophy from two pre-cultivation conditions (autotrophic and heterotrophic), respectively. Data are expressed as mean ± standard deviation, n = 3, except for the pre-cultivation heterotrophic condition. [Figure 8] Average number of nuclei per cell upon shifting from two pre-cultivation conditions (autotrophic and heterotrophic) to heterotrophic mode, respectively. Data are expressed as mean ± standard deviation, n = 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention will be described in detail below.

[0030] Definition of Terms In the present invention, autotrophy means that an organism uses carbon dioxide (CO2) in the atmosphere as the only carbon source and synthesizes the carbon skeletons of all organic compounds in the organism from carbon dioxide, and culturing microalgae under such conditions is called autotrophic culture. Since carbon dioxide is used as the carbon source, photosynthesis is utilized and light is essential.

[0031] In the present invention, heterotrophy refers to an organism utilizing organic matter produced by other plants and animals to obtain the carbon compounds it needs, and deriving the carbon skeletons of all organic compounds in the organism from organic matter present in the environment. Heterotrophic culture refers to culturing microalgae in the dark after adding organic matter, such as glucose.

[0032] In the present invention, mixotrophy refers to a combination of autotrophy and heterotrophy. ,light It uses organic matter as a source of synthesis and other nutrients.

[0033] In the present invention, microalgae refers to algae that exist in water such as freshwater and seawater, and the size of each individual is mainly several μm to several tens of μm, and includes cyanobacteria and chlorophytes, with chlorophytes being particularly preferred.

[0034] In the present invention, examples of green algae include the Mammielopoeceae (e.g., Mammietales, Monomastyales, Dolichomasticles), Nephroselmisphyceae (e.g., Nephroselmisdales), Pedinophyceae (e.g., Pedinomonadales), Chlorodendronyceae (e.g., Chlorodendronyceae), Trebouxiophyceae (e.g., Chlorellales, Microtamnionales, Trebouxiales, Enzophyticles), Ulva algae, and the like. rope Examples of phyla include the order Cladophorales, Order Dermestales, Order Creepingales, Order Violales, Order Hibiscales, Order Ulvales, Order Creepingales, and Order Scotinosphaeriales, the order Chlorophyceae (e.g., the order Vulgariales, the order Amplexicales (particularly the families Chromochloridaceae, Hydrodictyaceae, Scenedesmaceae, and Neochloridaceae), the order Heterogeneales, the order Chaetophorales, and the order Chaetopeltis, as well as the order Pyramimonadales, the order Scoulfieldiales, and the order Pseudoscoulfieldiales. Among these, the families Hydrodictyaceae, Scenedesmaceae, and Neochloridaceae of the order Amplexicales, and in particular the genus Chromochloris, such as Chromochloris zofingiensis, are exemplified.

[0035] Alternatively, the microalgae of the present invention are preferably those belonging to the phylum Chlorophyta, more preferably those belonging to the class Chlorophyceae, order Polytrichumales, etc. Alternatively, the microalgae of the present invention are preferably those forming multinucleate cells, specifically, Chromochloris zofingiensis, Scenedesmus quadricauda, ​​Chlorella ellipsoidea, Chlamydomonas reinhardtii, Parachlorella kessleri, etc.

[0036] In the present invention, a particularly preferred microphytophyte is Chromochloris zofingiensis.

[0037] The present invention provides a method for culturing microalgae, comprising the following steps 1 and 2: Step 1: Cultivating microalgae under dark heterotrophic culture conditions to obtain multinucleated cells Step 2: Cultivating the resulting multinucleated cells under autotrophic or mixotrophic culture conditions.

[0038] Process 1 In step 1, microalgae are cultured under dark heterotrophic culture conditions to obtain multinucleated cells. In dark conditions, light can be blocked so that the microalgae cannot use it to perform photosynthesis.

[0039] The culture medium may be modified Bristol medium (MBM).

[0040] Examples of heterotrophic culture conditions include a glucose concentration of 1 to 50 g / L, preferably 10 to 20 g / L, as a nutrient source, at a temperature of 10 to 40° C., preferably 20 to 30° C., for example, for 24 to 480 hours.

[0041] The resulting multinucleated cells can be subsequently used in step 2.

[0042] Process 2 In step 2, the resulting multinucleated cells are cultured under autotrophic or mixotrophic culture conditions.

[0043] The medium used for culturing in step 2 includes modified Bristol medium (MBM).

[0044] The autotrophic culture conditions in step 2 utilize photosynthesis, with a light intensity of 30 to 1000 μmol m -2 s -1 , preferably 100 to 300 μmol m -2 s -1 The incubation conditions include a temperature of 10 to 40° C., preferably 20 to 30° C., for example, for 24 to 480 hours.

[0045] In the mixotrophic culture conditions in step 2, autotrophic and heterotrophic culture are combined, so the light intensity is 30 to 1000 μmol m -2 s -1 , preferably 100 to 300 μmol m -2 s -1 The culture can be carried out at a glucose concentration of 1 to 50 g / L, preferably 10 to 20 g / L, and at a temperature of 10 to 40° C., preferably 20 to 30° C., for example, for 24 to 480 hours.

[0046] In step 2, the proliferation rate of the multinucleated cells obtained in step 1 is accelerated. For example, it is accelerated by 1.5 to 2.4 times compared to normal culture.

[0047] In the present invention, the method may further comprise, prior to the above step 1, a step of culturing microalgae under mixotrophic culture conditions or autotrophic culture conditions (stock culture step).

[0048] The mixotrophic or autotrophic culture conditions in the stock culture step can be the same as the culture conditions in step 1 or 2.

[0049] In addition, in the stock culture step, in addition to the above-mentioned mixotrophic culture conditions or autotrophic culture conditions alone, mixotrophic culture and heterotrophic culture conditions may be combined. As the culture conditions for mixotrophic culture and heterotrophic culture in the stock culture step, the conditions of step 1 and step 2, respectively, can be used.

[0050] For example, as a stock culture step, microalgae may be cultured under autotrophic culture conditions and subsequently cultured under heterotrophic culture conditions.

[0051] In this way, in the cultivation of microalgae, multinucleated cells are obtained by pre-culturing, and the subsequent cultivation method of microalgae using the multinucleated cell group advantageously promotes the proliferation of the microalgae.

[0052] In the present invention, the microalgae used in the pre-culture (step 1) can be an algae stock. The algae stock is maintained, for example, in modified Bristol medium (MBM) and has a concentration of 30 to 200 (for example, 60 μmol) mol m -2 s -1 The mixture can be prepared by irradiating the mixture at a light intensity of 12 hours light and 12 hours dark at 10 to 40°C (for example, 25°C).

[0053] The microalgae thus obtained are capable of producing intracellular compounds such as lipids and starch, as well as carotenoids, of which astaxanthin is a typical example. EXAMPLES

[0054] The present invention will be described below with reference to specific embodiments, but it will be understood that the present invention is not limited to these embodiments, and that various changes and modifications therein can be made by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

[0055] [Example 1] (Materials and Experimental Methods) Bacterial strains and culture conditions The green microalga Chromochloris zofingiensis (Donz) Fucikova & L.A. Lewis ATCC 30412 was obtained from the American Type Culture Collection (ATCC, Rockville, USA). Algal stocks were maintained in modified Bristol medium (MBM)

[17] in 250 mL Erlenmeyer flasks at a concentration of 60 μmol m -2 s -1 The plants were exposed to light at 25°C with a light intensity of 12 hours light and 12 hours dark.

[0056] Experimental design To evaluate the effect of pre-culture conditions on the biomass production phase, cells from the three nutrient modes (as pre-cultures) were inoculated into separate cultures in three more nutrient modes (biomass production phase), resulting in a total of nine experimental conditions. As pre-cultures, a series of first pre-culture conditions (in 500 mL Erlenmeyer flasks with a working volume of 300 mL) followed by a series of second pre-culture conditions (in 1 L medium bottles with 700 mL of fresh medium) were performed to adapt / acclimate the cells to the same conditions.

[0057] Preculture Cells from the stock culture were inoculated into the first preculture, where algal cells were grown with three different nutrient modes (Table 1) and continuously aerated (0.2 vvm) in MBM at 25°C for 6 days. A series of successive precultures was performed, where cells from the first preculture were inoculated into the second preculture with the same nutrient mode for the next 6 days. Sampling was performed aseptically every 24 hours.

[0058] [Table 1]

[0059] Biomass Production Process Cells from the second preculture were collected, washed, and resuspended at an optical density of 0.1. Then, batch cultures for the three nutritional modes (autotrophic, heterotrophic, and mixotrophic) at the biomass production stage were performed in 1 L medium bottles with an effective culture volume of 700 mL. Continuous aeration (0.2 vvm) and agitation (260 rpm) were performed in MBM until stationary phase was reached. Light was applied continuously (24 h) for autotrophic and mixotrophic conditions, while the heterotrophic condition was kept in complete darkness. Details of light intensity and glucose concentration are shown in Table 1. Sampling was performed aseptically every 24 h, and all experiments were performed in triplicate.

[0060] Analysis parameters Cultures were filtered using pre-weighed glass fiber filters (GF / F, Whatman, USA) with a pore size of 0.25 μm. Cells were washed three times with distilled water and dried at 80 °C for 8 h for dry weight measurement. Filtrate samples were stored at -20 °C until analysis, and nutrient concentrations (NO3-N and PO4-P) were determined using an automated nutrient analyzer (FIAlyzer-1000, FIAlab, USA).

[0061] Cell length (μm) was measured by taking pictures using an optical microscope (Axioskop-2 Plus Carl Zeiss, Germany) equipped with a digital camera (EOS Kiss X7i, Canon, Japan). Analysis was then performed using Image J software (National Institutes of Health, USA). To determine the cell size distribution, 30 cells were measured in each sample. For pigment composition, microalgae cells were filtered using a GF / F filter, washed several times with distilled water, and extracted with 5 ml of N,N-dimethylformamide (DMF) at -20 °C for 24 h

[28] . Then, 1 ml of the extracted sample was filtered using a 0.22 μm PTFE filter (Millex-FG, Merck, Japan) before analysis. Measurement and identification of carotenoids in the samples were performed using an LCMS system (ACQUITY UPLC H-Class, Waters, USA). The mobile phase consisted of solvent A (100% acetonitrile), solvent B (100% 2-propanol), and solvent C (10% methanol). At the start of the run, solvent A was 50%, solvent B was 40%, and solvent C was 10%, followed by a linear gradient to 2% solvent A, 40% solvent B, and 58% solvent C. After 12 min, the mixture was switched to 50% solvent A. Chemical standards of astaxanthin, canthaxanthin, lutein, β-carotene, zeaxanthin, violaxanthin, and chlorophyll were purchased from (Danish Hydraulic Institute, Horsholm, Denmark) and analyzed using a reversed-phase column (ACQUITY UPLC BEH C18, 1.7 μm; 2.1 × 50 mm, Waters, USA). Chromatographic peaks of the samples were identified by comparing the absorption and mass spectra with the standard values ​​and then quantified by integrating the peak areas.

[0062] Starch determination was performed using the method of a previous study, but the method was modified before the measurement

[29] . Cells filtered using a GF / F filter were homogenized in 4 ml of 80% ethanol using an ultrasonic homogenizer (UH-50, SMT, Japan). Microalgal cells were then scraped off the filter into a 15 ml centrifuge tube and centrifuged at 4,000 rpm for 10 min. Pigments were extracted three times with 80% ethanol at 68 °C for 15 min, followed by centrifugation at 2,500 rpm for 5 min. The supernatant was removed and the remaining pellet was used for the next step. 30% perchloric acid was added to the pellet to completely hydrolyze starch, and the suspension was stirred at 25 °C for 15 min, then centrifuged to obtain the supernatant. This procedure was repeated three times. The extracts were combined and brought to 10 ml. A 2 ml aliquot of the solubilized starch solution was then reacted with 5 ml concentrated sulfuric acid (98% by weight) and 1 ml phenol (6%, w / v) and then kept at room temperature for 10 min. The absorbance was read using a spectrophotometer (HACH) at 490 nm and the samples were quantified by comparing with a calibration curve using glucose as the standard. The glucose readings were multiplied by 0.9 to obtain the calibration curve for starch measurement

[29] .

[0063] Lipid measurements were performed using the sulfo-phospho-vanillin method

[30] . Phosphovanillin reagent was first prepared by dissolving 0.6 g vanillin in 10 ml absolute ethanol and 90 ml deionized water and stirring continuously. Then, 400 ml concentrated phosphoric acid was added to the mixture and the reagent was stored in the dark until use. The reagent was freshly prepared immediately before every experimental run to ensure its high activity. Cells filtered using GF / F filters were treated with 2 ml concentrated sulfuric acid, heated at 100 °C for 10 min, and then cooled in an ice bath for 5 min. Microalgal cells were centrifuged at 4000 rpm for 5 min, and 1 ml of the supernatant was transferred to a glass vial. Then, 2.5 ml of sulfo-phospho-vanillin was added to the vial and incubated at 37 °C for 15 min. The absorbance was read at 530 nm using a spectrophotometer (HACH) and samples were quantified by comparison with a calibration curve using commercial rapeseed oil (200 mg) in 100 ml of chloroform as the standard.

[0064] All measurements were performed in triplicate and data are expressed as mean ± standard deviation (SD). The significance of the results was calculated by one-way analysis of variance followed by Tukey-Kramer multiple comparison test at P < 0.05.

[0065] (Results and Discussion) Growth of Chromochloris zofingiensis among different nutritional modes in preculture For successive precultures to the same mode (i.e., autotrophic, mixotrophic, or heterotrophic), different growth curves were observed (Figure 1). Compared with the growth curve of the first preculture, the mixotrophic preculture showed higher growth than the autotrophic and heterotrophic precultures. The specific growth rates of the mixotrophic, autotrophic, and heterotrophic first precultures were 0.867 ± 0.067 d, respectively. -1 , 0.586±0.069, 0.387±0.040d -1 The high specific growth rate of the mixotrophic culture was probably due to the synergistic relationship between photosynthesis and carbon assimilation. It has been known for some time that in mixotrophic cultures, microalgae can fix inorganic carbon by photosynthesis (known as photoautotrophy) and simultaneously carbon assimilation (known as heterotrophy) [5]. Thus, in this study, the presence of light and glucose increased the energy conversion up to two-fold in the mixotrophic culture. However, the autotrophic culture was entirely dependent on light for energy conversion. Also, the specific growth rate of the heterotrophic culture was not as high as that of the mixotrophic culture. This is probably due to the absence of light and the use of glucose as the only carbon source for growth.

[0066] When Chromochloris zofingiensis-cells were cultured in two successive precultures (first and second), similar cell growth curves were observed in autotrophic and mixotrophic cultures (Fig. 1a, b). However, the second heterotrophic preculture showed reduced growth compared to the first preculture, with a growth rate of 0.239 ± 0.029 d. -1 This was 0.387±0.040 d after the initial preculture.-1 The growth rate in the second preculture was lower than that in the first preculture. Although the growth rate decreased by nearly 40% in the second preculture, the cells appeared to remain intact. Microscopic observation of fixed samples from the last day of the second preculture revealed that there was no significant difference in the percentage of damaged cells between the heterotrophic (80.87 ± 3.36%) and autotrophic (83.57 ± 3.36%) second precultures. Thus, the lower growth rate in the heterotrophic second preculture was due only to reduced cell division and not to reduced cell viability.

[0067] Autotrophically and mixotrophically precultured cells showed similar growth curves in the first and second precultures, indicating that the cells may have adapted to the same environmental conditions. However, heterotrophically precultured cells showed a lower specific growth rate in the second preculture and the subsequent biomass production stage (Example 3), indicating that the cells still continued to respond to environmental changes. However, when the second heterotrophic preculture was inoculated into the biomass production stage, the cell size remained relatively stable (Example 3). Since the most significant change in cell state in the heterotrophic culture was the increase in cell size, in this example, the second heterotrophic preculture is considered to be valid as the inoculum for the biomass production stage.

[0068] [Example 2] Growth of C. zofingiensis under different nutritional modes during biomass production Comparison of overall growth between different nutritional modes Similar to the preculture, the order of specific growth rate and biomass productivity during the biomass production stage changed from high to low under mixotrophic, autotrophic, and heterotrophic conditions. On average, mixotrophic cultures were significantly higher than autotrophic (0.578 ± 0.306 d -1 ) and heterotrophic (0.447 ± 0.070 d -1 ) mode, the highest maximum specific growth rate (0.868 ± 0.281d -1 ) (Table 2). Similarly, the maximum biomass productivity was 0.448 ± 0.363 g L -1 d -1) and autotrophic (0.065 ± 0.053 g L -1 d -1 ) than the biomass production stage, mixotrophy (1.28 ± 0.033 g L -1 d -1 ) stage (Fig. 2a, b; Table 2). Previous studies have shown that C. zofingiensis can simultaneously utilize both light and carbon for growth [1, 5].

[0069] [Table 2]

[0070] A detailed mechanism of mixotrophic growth promotion in C. zofingiensis has been reported recently [5]. In addition to the simultaneous progression of photosynthesis and glucose assimilation, synergistic cooperation between chloroplasts (photosynthesis), cytosol, and mitochondria (glucose metabolism) was proposed to explain the increased productivity [5]. Chloroplasts and mitochondria contain carbon and energy transporters, such as triosephosphate / phosphate translocator, glucose / phosphate transporter, and ATP / ADP carrier, on their membranes, which allows them to cooperate through the cytosol [5,31-33]. Genes involved in the transport of ATP between mitochondria and cytosol (NTT) and between cytosol and chloroplasts (CAAC) were shown to be upregulated, suggesting the transport of ATP from mitochondria to chloroplasts [5]. This supply of ATP and organic compounds helps to reduce the need for CO2 fixation by RuBisCO, the rate-limiting step of photosynthesis. The enhanced ATP utilization for organic carbon metabolism in chloroplasts further reduces non-photochemical quenching (NPQ) and photorespiration, where light energy is dissipated as heat and chemical energy, leading to more efficient energy utilization for cell growth. Therefore, the synergistic relationship between photosynthesis and carbon assimilation may lead to increased biomass productivity in mixotrophic cultures of C. zofingiensis.

[0071] However, previous studies reported that the addition of organic carbon led to a decrease in photosynthetic activity in mixotrophic cultures. For example, the addition of acetate to irradiated Chlamydomonas cultures significantly reduced inorganic carbon assimilation and oxygen evolution

[34] . In this example, similarly, a lower chlorophyll content was observed during the mixotrophic biomass production phase (Table 2), which likely reduced photosynthetic activity. Nevertheless, due to both organic carbon assimilation and the above-mentioned synergistic cooperation between chloroplasts and mitochondria, the overall biomass productivity of mixotrophic cultures was higher than autotrophic and heterotrophic conditions, despite the reduced photosynthetic activity.

[0072] In this example, the biomass productivity and yield of the mixotrophic culture was higher than the sum of the autotrophic and heterotrophic cultures combined (Table 2), which is similar to the results of previous studies [1,5]. This study showed higher biomass productivity values ​​than many other batch mixotrophic culture studies using Chromochloris zofingiensis (Table 3), which ranged from 0.59 to 0.72 g L -1 d -1 [4, 35, 36]. However, compared to the present example, two previous studies reported higher mixotrophic biomass productivity in batch mixotrophic cultures, ranging from 2.5 to 2.63 g L−1. -1 d -1 [1,5] (Table 3). The difference in productivity may be due to differences in (1) initial cell density and (2) seeding conditions. Most of the previous studies used initial cell densities between 0.5 and 0.6 g L -1 lower than that, which has been adopted in high productivity studies [1,5]. Also, preparation of the inoculum under heterotrophic conditions may enhance growth, as further explained in Example 3, which is the influence of pre-culture conditions.

[0073] [Table 3] A: Autotrophy; M :mixotrophy; H :Heterotrophic;ND:No data available a: Cultures were inoculated with seeds at a volume to volume ratio of 10%.

[0074] The maximum heterotrophic biomass productivity in this example was 0.448±0.363 g L -1 d -1 , which is the highest productivity reported in previous studies, at 7.03 g L -1 d -1 [2]. The large difference in biomass productivity may be due to the difference in feeding conditions, where the present study performed a one-off batch culture, whereas the other study performed fed-batch culture. The present study showed a higher biomass productivity under mixotrophic conditions than under heterotrophic conditions, with a higher biomass productivity of 7.03 g L−1 when fed-batch mixotrophic culture was performed. -1 d -1 Thus, further studies on fed-batch mixotrophic cultures with high initial cell densities should be confirmed if mixotrophic conditions are superior to heterotrophic conditions in high-density cultures with limited light penetration.

[0075] [Example 3] Effect of pre-culture nutrition mode on biomass production stage The effect of preculture nutrition mode on biomass productivity was different between dark (heterotrophic) and illuminated (autotrophic and mixotrophic) conditions. During the heterotrophic biomass production phase in the dark, the lowest growth rate was observed in heterotrophic preculture cells. When cells were transferred from the stock culture to the first preculture, the specific growth rate was 0.387 ± 0.040 d -1 and decreased after the second pre-incubation (0.239 ± 0.029 d -1 ; calculated from Fig. 1). However, autotrophically and mixotrophically pre-cultured cells showed significantly higher oxidative stress than heterotrophically pre-cultured cells (0.364 ± 0.008 d -1 , 0.034±0.062gL -1 d -1 ) in the heterotrophic biomass production phase compared to the control (0.504 ± 0.022 and 0.437 ± 0.015 d -1), and higher biomass productivity (0.714 ± 0.284 and 0.596 ± 0.108 g L -1 d -1 ) were observed. These results suggest that the growth rate and productivity of Chromochloris zofingiensis declines when light is absent for a long period of time. The highest productivity of Chromochloris zofingiensis was observed under heterotrophic conditions (7.03 g L−1). -1 d -1 [2]), and since many studies have focused on heterotrophic cultivation of Chromochloris zofingiensis [2,11,17,22,37,38], the low heterotrophic productivity was rather surprising. All the above studies showed that the heterotrophic productivity was low under low light conditions (20–90 μmol m -2 s -1 It should be noted that the seed culture was maintained in the presence of glucose, which probably allows for high biomass productivity. Productivity may decrease if heterotrophic culture is continued for more than one batch. Light illumination therefore appears to be an essential part of cell growth before the microalgae are transferred to the dark culture mode. Similar observations were reported in the cyanobacterium (Synechocystis sp.) strain PCC6803, where periodic light pulses were required for continued cell growth under dark heterotrophic culture

[39] . The inventors suggested that light acts as an environmental signal to regulate heterotrophic metabolism, cell cycle, or cell division. It has also been shown that in Chromochloris zofingiensis, cell density is significantly reduced under complete darkness compared to illuminated conditions with glucose supply [8]. Thus, light may be required to stimulate heterotrophic growth, especially for cell division.

[0076] The fact that cells irradiated during preculture (autotrophic and mixotrophic precultures) showed higher growth in the next heterotrophic culture suggests that the physiological characteristics of those cells remained in the latter culture. This need for light indicates that gene expression or intracellular compounds remain in the next stage. It is generally known that light can induce morphological changes and gene expression responses and alter intracellular components

[40] . Intracellular compounds produced during photosynthetic stimulation may be necessary for growth.

[0077] In contrast to dark conditions, under illumination conditions (autotrophic and mixotrophic biomass production stages), heterotrophically precultured cells showed higher specific growth rates and biomass productivity than autotrophically and mixotrophically precultured cells (Fig. 2a, b). During the autotrophic biomass production stage, heterotrophic precultured cells had approximately 2.2-2.4 times higher maximum specific growth rates compared to autotrophic and mixotrophic precultured cells (n=3, p<0.05; Table 2). Similarly, during the mixotrophic biomass production stage, heterotrophic precultured cells showed 1.6-1.8 times higher maximum specific growth rates compared to autotrophic and mixotrophic precultured cells (n=3, p<0.05). Interestingly, previous studies with high mixotrophic productivity also employed dark heterotrophic conditions for inoculum activation [1,5] (Table 3). Preparing inoculum under dark heterotrophic conditions for illumination cultivation could improve productivity. In fact, the maximum specific growth rate (1.19 ± 0.044 d -1 ) was obtained during the mixotrophic biomass production phase using heterotrophically pre-cultured cells (Table 2). This value was significantly higher than that of the mixotrophically pre-cultured conditions (0.057d -1 ; [4]), and the value of another study in which dark conditions were used as pre-culture conditions (1.27d -1 [5]). Two possible mechanisms could explain the high growth rate of heterotrophically precultured cells: (1) accumulation of growth-promoting factors and (2) a synergistic relationship between photosynthesis and carbon assimilation.

[0078] Heterotrophically precultured cells may have accumulated growth-promoting factors, which may help to increase the growth rate during the autotrophic and mixotrophic biomass production stages. The accumulated growth-promoting factors may have been of at least two types; 1) nuclei, and (2) intracellular compounds, such as lipids and starches. Depending on the culture conditions, Chromochloris zofingiensis divides cells in a binary (two daughter cells) or multiple division (4–64 daughter cells) division pattern

[39] . The characteristics of multiple division are: cells grow to a size of about 15 μm compared to 3–5 μm for normal cells, and each cell has multiple nuclei within it, allowing rapid proliferation. Heterotrophic cells in this study also showed large cell size. The cell size of heterotrophic cells was significantly larger (13.37 ± 1.19 μm, respectively) than mixotrophic (9.21 ± 1.02 μm) and autotrophic (5.72 ± 1.25 μm) cells, regardless of the preculture condition (n = 9, p < 0.05; Figure 3). This suggests that heterotrophic cells may be in a state of multiple divisions and ready to divide. An increase in cell size was observed when autotrophic and mixotrophic cells were transitioned to heterotrophic conditions, suggesting an accumulation of nuclei. The cell size of autotrophically and mixotrophically precultured cells increased from day 0 (5.33 ± 1.61 and 6.93 ± 2.00 μm, respectively) to the end (13.67 ± 2.76 and 14.07 ± 5.72 μm, respectively), while that of heterotrophically precultured cells did not change much (Figure 3c).

[0079] Another possible growth-promoting factor is the accumulation of intracellular compounds. From previous literature, it is known that Chromochloris zofingiensis cells accumulate several intracellular compounds, such as lipids and starch [8, 40]. Utilization of these intracellular compounds for cell proliferation may have occurred, resulting in a higher proliferation rate when exposed to light. For example, Chen et al. [8] showed that a sharp decrease in lipid yield coincided with cell proliferation upon illumination compared to dark conditions. In this study, heterotrophic cells (0.171 ± 0.024 mg / ml) were found to accumulate more than 1000 mg / ml. -1 DW) and mixotrophic cells (0.181 ± 0.051 mg-1 DW had more autotrophic cells (0.063±0.017mg -1 DW: higher starch content than in the control (Table 2). With regard to lipid accumulation, a higher lipid content was observed in heterotrophic culture (13.56 ± 1.98%) than in autotrophic (8.83 ± 4.18%) and mixotrophic cultures (9.21 ± 0.72%). The conserved intracellular compounds may have functioned as either a carbon source or a growth promoting factor to achieve a high specific growth rate.

[0080] However, mixotrophic cells did not show growth-promoting functions compared to heterotrophic cells, although here, both cell size, lipid, and starch contents of heterotrophic and mixotrophic cells were almost similar. The specific reason for the differential enhancement effect is not clear in the current experiment. However, in contrast to the mixotrophic condition where cell division was not limited, the long-term growth restriction in the second heterotrophic preculture may have induced the accumulation of nuclei without cell division. In addition, the enhanced accumulation of growth-promoting intracellular compounds in the heterotrophic culture compared to the mixotrophic preculture may have been a potential cause of the difference.

[0081] Another possible mechanism for the higher growth rate of heterotrophic cells could be the synergistic relationship between photosynthesis and glucose metabolism occurring in chloroplasts, cytosol, and mitochondria. Although such synergistic relationships have been found to occur in mixotrophic conditions (see Example 2; [5]), a similar cooperative cooperation may have occurred in the transition of heterotrophic cells to autotrophic and mixotrophic biomass production stages. As mentioned above, cells precultured in heterotrophic conditions accumulated starch and possibly lipids in them. When these cells were transferred to illuminated autotrophic and mixotrophic conditions, they may have exhibited a synergistic relationship between photosynthesis, glycolysis, and the citric acid cycle by utilizing the accumulated carbon sources to provide intermediate organic matter and ATP to the chloroplasts.

[0082] Further analyses, such as nuclear staining with SYBR®Gold, transmission electron microscopy, comprehensive analysis of cellular compounds, metabolomics, and genomics, may shed light on the mechanisms of growth-promoting factors in heterotrophically pre-cultured cells. The elucidation of growth-promoting factors may also be beneficial for the mass production of Chromochloris zofingiensis and other inoculants. (R) indicates a registered trademark.

[0083] For example, as shown in Figure 7, the increased growth rate of heterotrophically precultured cells may be due to multiple divisions by the multinucleated Chromochloris zofingiensis. In fact, it has been observed by microscopic observation that the heterotrophically precultured strain forms multinucleated cells, which become mononucleated when shifted to autotrophy (Figure 5). In our study, the heterotrophically precultured cells had many more nuclei than the autotrophically precultured cells (Figure 7). These multinucleated cells showed a rapid decrease in the average number of nuclei from 7.19 to 1.26 from day 0 to day 2, and the decrease in the number of nuclei was consistent with rapid cell proliferation. This is probably due to the rapid division (multiple divisions) of the multinucleated cells when the culture was shifted to an autotrophic state. On the other hand, it was observed that the high cell proliferation of the heterotrophically precultured cells continued even after the number of nuclei was reduced in the same way as the autotrophically precultured cells. The reason for the overall growth improvement may not only be the multiple divisions, but also the utilization of growth promoters stored during the heterotrophic period.

[0084] Our research has revealed that heterotrophic culture in the dark contributes to the generation of multinucleated cells (Figure 6). When autotrophically pre-cultured cells were transferred to heterotrophic culture, the average number of nuclei increased from day 0 to day 2 (Figure 8). This indicates that when Chromochloris zofingiensis is cultured in the dark, cell division is suppressed and only nuclear division continues.

[0085] In this example, a clear effect of preculture nutrition mode in the later stage was observed, indicating the importance of optimization in successful mass cultivation. It was shown that heterotrophic biomass production requires light in the preculture to maintain high growth rate and biomass productivity. However, heterotrophically precultured Chromochloris zofingiensis cells could accumulate growth-promoting factors such as nuclear and intracellular compounds. This could lead to higher growth and biomass yield when grown in the presence of light (mixotrophic and autotrophic biomass production stages). Therefore, heterotrophic cultivation in the preculture stage to induce growth-promoting compounds, and mixotrophic cultivation in the later stage are proposed as good choices to increase biomass productivity.

[0086] Pigment content measurement Chlorophyll a content was significantly higher during the autotrophic biomass production stage (7.54 ± 3.62 mg / L). -1 ) but not mixed nutrition (1.84±0.121mgg -1 ) and heterotrophic (1.25 ± 0.511 mgg -1 ) Biomass production stage Floor was significantly higher than that of the control group (n=3, p<0.05 )( Table 2). Similarly, the total carotenoids achieved here were significantly higher in the autotrophic biomass production stage than in the mixotrophic and heterotrophic biomass production stages (n=3, p<0.05) (Figure 4). The high carotenoid content in the autotrophic condition was due to the effect of light, since light upregulates carotenoid biosynthesis genes in Chromochloris zofingiensis [2]. However, the mixotrophic condition showed a lower carotenoid content here, since it was performed in the presence of light and glucose. The effect of light transmittance could be one of the reasons, since the high cell density in the mixotrophic biomass production stage resulted in self-shading and a decrease in the carotenoid content.

[0087] Herein, it can be noticed that despite the low carotenoid content in the heterotrophic and mixotrophic pre-culture stages, there was a comparable carotenoid content in the autotrophic biomass production stage (Figure 4a). This indicates that autotrophic cells are not the only option for high carotenoid production. The results obtained here suggest that the nutritional mode of the pre-culture stage does not significantly affect the carotenoid content in the later stages.

[0088] [Example 4] Combination of nutritional modes In this example, the highest biomass productivity was observed in mixotrophic mode, especially when the culture was inoculated with heterotrophic cells (Fig. 2b). This result suggests that the combination of heterotrophic preculture and mixotrophic biomass production steps is a good strategy to improve the biomass productivity of Chromochloris zofingiensis. However, the heterotrophic culture faced a decrease in growth rate when the cells were cultured heterotrophically for a long period of time (i.e., two or three consecutive heterotrophic cultures) (Figs. 1c and 2b). This result indicates that the inoculum for heterotrophic culture should be from either autotrophic or mixotrophic culture. However, for stock culture, mixotrophic mode is not suitable because glucose may cause contamination during storage, and the consumption and depletion of glucose during the stock step may also make it difficult to maintain good cell condition.

[0089] Also, the heterotrophically pre-cultured cells showed a lower growth rate during the heterotrophic biomass production stage. Therefore, our experimental results suggest that the optimal combination of nutritional modes is as follows; - Autotrophic culture for stock (to induce growth factors for subsequent heterotrophic cultures), - Heterotrophic culture as a preculture (to increase the number of multinucleated cells and at the same time accumulate intracellular growth-promoting compounds such as starch and lipids), and - Mixotrophic or autotrophic cultivation during the biomass production stage (to maximize biomass productivity).

[0090] In the present invention, for example, the 7.03 g L -1 d -1 , or 5.8 gL according to Sun et al., 2019[9]. -1 d -1 Although the highest biomass productivity in all existing studies on Chromochloris zofingiensis has not been reached, optimization of the reactor conditions in fed-batch culture may achieve similar or even higher productivity since the culture faced nutrient and glucose limitations. The increased biomass productivity may further improve the overall astaxanthin productivity.

[0091] conclusion The inventors showed that heterotrophically grown pre-cultured Chromochloris zofingiensis cells could accumulate intracellular compounds, which could enhance the growth rate and productivity by light irradiation. It was also observed that light is required for the preceding culture stages, such as stock and pre-culture, to successfully grow Chromochloris zofingiensis in heterotrophic mode. Furthermore, the inventors revealed that the nutritional mode of the preceding culture stage does not significantly affect the carotenoid content of the later stages. Our experimental results indicate that the combination of appropriate nutritional modes in the stock, pre-culture, and biomass production stages may be a feasible method to enhance the biomass productivity of Chromochloris zofingiensis. Also, the optimization of conditions could enhance the productivity of astaxanthin. [Industrial Applicability]

[0092] In microalgae, particularly microalgae such as those of the genus Chromochloris, a dramatic increase in growth rate can be achieved by changing the nutritional form according to the culture stage, making it possible to easily culture the microalgae in large quantities, which can ultimately lead to the production of astaxanthin.

[0093] References [1] Z. Zhang, D. Sun, T. Wu, Y. Li, Y. Lee, J. Liu, F. Chen, The synergistic energy and carbon metabolism under mixotrophic cultivation reveals the coordination between photosynthesis and aerobic respiration in Chlorella zofingiensis, Algal Res. 25 (2017) 109-116. https: / / doi.org / 10.1016 / j.algal.2017.05.007. [2] Z. Zhang, J.J. Huang, D. Sun, Y. Lee, F. Chen, Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP), Bioresour. Technol. 224 (2017) 515-522. https: / / doi.org / 10.1016 / j.biortech.2016.10.081. [3] J. Liu, Z. Sun, H. Gerken, Z. Liu, Y. Jiang, F. Chen, Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: Biology and industrial potential, Mar. Drugs. 12 (2014) 3487-3515. https: / / doi.org / 10.3390 / md12063487. [4] J. hui Chen, L. Liu, D. Wei, Enhanced production of astaxanthin by Chromochloris zofingiensis in a microplate-based culture system under high light irradiation, Bioresour. Technol. 245 (2017) 518-529. https: / / doi.org / 10.1016 / j.biortech.2017.08.102. [5] Z. Zhang, D. Sun, K.W. Cheng, F. Chen, Investigation of carbon and energy metabolic mechanism of mixotrophy in Chromochloris zofingiensis, Biotechnol. Biofuels. 14 (2021) 1-16. https: / / doi.org / 10.1186 / s13068-021-01890-5. [6] S.N. Ani Azaman, N. Nagao, F.M. Yusoff, S.W. Tan, S.K. Yeap, A comparison of the morphological and biochemical characteristics of Chlorella sorokiniana and Chlorella zofingiensis cultured under photoautotrophic and mixotrophic conditions, PeerJ. 2017 (2017). https: / / doi.org / 10.7717 / peerj.3473. [7] Y. Li, J. Huang, G. Sandmann, F. Chen, High-light and sodium chloride stress differentially regulate the biosynthesis of astaxanthin in Chlorella zofingiensis (Chlorophyceae), J. Phycol. 45 (2009) 635-641. https: / / doi.org / 10.1111 / j.1529-8817.2009.00689.x. [8] T. Chen, J. Liu, B. Guo, X. Ma, P. Sun, B. Liu, F. Chen, Light attenuates lipid accumulation while enhancing cell proliferation and starch synthesis in the glucose-fed oleaginous microalga Chlorella zofingiensis, Sci. Rep. 5 (2015) 1-10. https: / / doi.org / 10.1038 / srep14936. [9] Z. Sun, Y. Zhang, L.P. Sun, J. Liu, Light elicits astaxanthin biosynthesis and accumulation in the fermented ultrahigh-density Chlorella zofinginesis, J. Agric. Food Chem. 67 (2019) 5579-5586. https: / / doi.org / 10.1021 / acs.jafc.9b01176.

[10] M.S. Roth, S.D. Gallaher, D.J. Westcott, M. Iwai, K.B. Louie, M. Mueller, A. Walter, F. Foflonker, B.P. Bowen, N.N. Ataii, J. Song, J.H. Chen, C.E. Blaby-Haas, C. Larabell, M. Auer, T.R. Northen, S.S. Merchant, K.K. Niyogia, Regulation of oxygenic photosynthesis during trophic transitions in the green alga Chromochloris zofingiensis, Plant Cell. 31 (2019) 579-601. https: / / doi.org / 10.1105 / tpc.18.00742.

[11] N. Sun, Y. Wang, Y.T. Li, J.C. Huang, F. Chen, Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta), Process Biochem. 43 (2008) 1288-1292. https: / / doi.org / 10.1016 / j.procbio.2008.07.014.

[12] M.N. Metsoviti, G. Papapolymerou, I.T. Karapanagiotidis, N. Katsoulas, Effect of light intensity and quality on growth rate and composition of Chlorella vulgaris, Plants. 9 (2020) 1-17. https: / / doi.org / 10.3390 / plants9010031.

[13] M. Benedetti, V. Vecchi, S. Barera, L. Dall’Osto, Biomass from microalgae: The potential of domestication towards sustainable biofactories, Microb. Cell Fact. 17 (2018) 1-18. https: / / doi.org / 10.1186 / s12934-018-1019-3.

[14] M. Rise, E. Cohen, M. Vishkautsan, M. Cojocaru, H.E. Gottlieb, S. (Malis) Arad, Accumulation of secondary carotenoids in Chlorella zofingiensis, J. Plant Physiol. 144 (1994) 287-292. https: / / doi.org / 10.1016 / S0176-1617(11)81189-2.

[15] J. Fan, Y. Cui, J. Huang, W. Wang, W. Yin, Z. Hu, Y. Li, Suppression subtractive hybridization reveals transcript profiling of Chlorella under heterotrophy to photoautotrophy transition, PLoS One. 7 (2012). https: / / doi.org / 10.1371 / journal.pone.0050414.

[16] X.F. Shen, Q.W. Qin, S.K. Yan, J. Le Huang, K. Liu, S.B. Zhou, Biodiesel production from Chlorella vulgaris under nitrogen starvation in autotrophic, heterotrophic, and mixotrophic cultures, J. Appl. Phycol. 31 (2019) 1589-1596. https: / / doi.org / 10.1007 / s10811-019-01765-1.

[17] P.F. Ip, F. Chen, Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark, Process Biochem. 40 (2005) 733-738. https: / / doi.org / 10.1016 / j.procbio.2004.01.039.

[18] J. Zhan, J. Rong, Q. Wang, Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass / bioenergy production, and bioremediation, advances and prospect, Int. J. Hydrogen Energy. 42 (2017) 8505-8517. https: / / doi.org / 10.1016 / j.ijhydene.2016.12.021.

[19] C. Yang, Q. Hua, K. Shimizu, Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic / dark-heterotrophic conditions, Biochem. Eng. J. 6 (2000) 87-102. https: / / doi.org / 10.1016 / S1369-703X(00)00080-2.

[20] Y. Imaizumi, N. Nagao, F.M. Yusoff, S. Taguchi, T. Toda, Estimation of optimum specific light intensity per cell on a high-cell-density continuous culture of Chlorella zofingiensis not limited by nutrients or CO2, Bioresour. Technol. 162 (2014) 53-59. https: / / doi.org / 10.1016 / j.biortech.2014.03.123.

[21] Y. Imaizumi, N. Nagao, F.M. Yusoff, N. Kurosawa, N. Kawasaki, T. Toda, Lumostatic operation controlled by the optimum light intensity per dry weight for the effective production of Chlorella zofingiensis in the high cell density continuous culture, Algal Res. 20 (2016) 110-117. https: / / doi.org / 10.1016 / j.algal.2016.09.015.

[22] Y. Wang, J. Peng, Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta), World J. Microbiol. Biotechnol. 24 (2008) 1915-1922. https: / / doi.org / 10.1007 / s11274-008-9692-8.

[23] E. Bar, M. Rise, M. Vishkautsan, S. (Malis) Arad, Pigment and structural changes in Chlorella zofingiensis upon light and nitrogen stress, J. Plant Physiol. 146 (1995) 527-534. https: / / doi.org / 10.1016 / S0176-1617(11)82019-5.

[24] G. Minyuk, R. Sidorov, A. Solovchenko, Effect of nitrogen source on the growth, lipid, and valuable carotenoid production in the green microalga Chromochloris zofingiensis, J. Appl. Phycol. 32 (2020) 923-935. https: / / doi.org / 10.1007 / s10811-020-02060-0.

[25] Y. Kou, M. Liu, P. Sun, Z. Dong, J. Liu, High light boosts salinity stress-induced biosynthesis of astaxanthin and lipids in the green alga Chromochloris zofingiensis, Algal Res. 50 (2020) 101976. https: / / doi.org / 10.1016 / j.algal.2020.101976.

[26] D. Pelah, A. Sintov, E. Cohen, The effect of salt stress on the production of canthaxanthin and astaxanthin by Chlorella zofingiensis grown under limited light intensity, World J. Microbiol. Biotechnol. 20 (2004) 483-486. https: / / doi.org / 10.1023 / B:WIBI.0000040398.93103.21.

[27] Y. Ye, J.C. Huang, Defining the biosynthesis of ketocarotenoids in Chromochloris zofingiensis, Plant Divers. 42 (2020) 61-66. https: / / doi.org / 10.1016 / j.pld.2019.11.001.

[28] K. Furuya, M. Hayashi, Y. Yabushita, HPLC determination of phytoplankton pigments using N,N-dimethylformamide, J. Oceanogr. 54 (1998) 199-203. https: / / doi.org / 10.1007 / BF02751695.

[29] I. Branyikova, B. Marsalkova, J. Doucha, T. Branyik, K. Bisova, V. Zachleder, M. Vitova, Microalgae-novel highly efficient starch producers, Biotechnol. Bioeng. 108 (2011) 766-776. https: / / doi.org / 10.1002 / bit.23016.

[30] S.K. Mishra, W.I. Suh, W. Farooq, M. Moon, A. Shrivastav, M.S. Park, J.W. Yang, Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method, Bioresour. Technol. 155 (2014) 330-333. https: / / doi.org / 10.1016 / j.biortech.2013.12.077.

[31] U.-I. Flu, R.E. Ha, F. Ludewig, M. Gierth, The role of transporters in supplying energy to plant plastids, J. Exp. Bot. 62 (2011) 2381-2392. https: / / doi.org / 10.1093 / jxb / erq361.

[32] Y.M. Dyo, S. Purton, The algal chloroplast as a synthetic biology platform for production of therapeutic proteins, Microbiology. 164 (2018) 113-121. https: / / doi.org / 10.1099 / mic.0.000599.

[33] I. Haferkamp, J.H.P. Hackstein, F.G.J. Voncken, G. Schmit, J. Tjaden, Functional integration of mitochondrial and hydrogenosomal ADP / ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids, Eur. J. Biochem. 269 (2002) 3172-3181. https: / / doi.org / 10.1046 / j.1432-1033.2002.02991.x.

[34] P.B. Heifetz, B. Forster, C.B. Osmond, L.J. Giles, J.E. Boynton, Effects of acetate on facultative autotrophy in Chlamydomonas reinhardtii assessed by photosynthetic measurements and stable isotope analyses, Plant Physiol. 122 (2000) 1439-1445. https: / / doi.org / 10.1104 / pp.122.4.1439.

[35] S.S. Oncel, E. Imamoglu, E. Gunerken, F.V. Sukan, Comparison of different cultivation modes and light intensities using mono-cultures and co-cultures of Haematococcus pluvialis and Chlorella zofingiensis, (n.d.). https: / / doi.org / 10.1002 / jctb.2532.

[36] Y. Wang, Z. Liu, S. Qin, Effects of iron on fatty acid and astaxanthin accumulation in mixotrophic Chromochloris zofingiensis, Biotechnol. Lett. 35 (2013) 351-357. https: / / doi.org / 10.1007 / s10529-012-1096-z.

[37] C.W. Zhang, F.L. Zhang, S.J. Dong, Y.Y. He, X.M. Xu, J. Peng, J.P. Yuan, The discrepancy of fatty acid composition of astaxanthin, esters and total fatty acids in photoautotrophic and heterotrophic Chlorella zofingiensis, JAOCS, J. Am. Oil Chem. Soc. 96 (2019) 891-901. https: / / doi.org / 10.1002 / aocs.12230.

[38] J. Liu, J. Huang, Z. Sun, Y. Zhong, Y. Jiang, F. Chen, Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: Assessment of algal oils for biodiesel production, Bioresour. Technol. 102 (2011) 106-110. https: / / doi.org / 10.1016 / j.biortech.2010.06.017.

[39] S.L. Anderson’, L. Mcintosh13, Light-activated heterotrophic growth of the Cyanobacterium synechocystis sp. strain PCC 6803: a blue-light-requiring process, 173 (n.d.). https: / / journals.asm.org / journal / jb (accessed July 22, 2021).

[40] J. Fan, J. Huang, Y. Li, F. Han, J. Wang, X. Li, W. Wang, S. Li, Sequential heterotrophy-dilution-photoinduction cultivation for efficient microalgal biomass and lipid production, Bioresour. Technol. 112 (2012) 206-211. https: / / doi.org / 10.1016 / j.biortech.2012.02.046.

Claims

1. A method for culturing microalgae, comprising the following steps 1 and 2: Step 1: Cultivating microalgae under dark heterotrophic culture conditions to obtain multinucleated cells Step 2: culturing the obtained multinucleated cells under autotrophic or mixotrophic culture conditions; wherein the microalgae is Chromochloris zofingiensis.

2. 2. The method of claim 1, further comprising culturing the microalgae under mixotrophic or autotrophic culture conditions prior to step 1.

3. The method according to claim 1 or 2, wherein in step 2, the obtained multinucleated cells are cultured under autotrophic culture conditions.

4. The method according to claim 1 or 2, wherein in step 2, the obtained multinucleated cells are cultured under mixotrophic culture conditions.

5. The method of claim 1 , wherein steps 1 and 2 are repeated.

6. A method for accelerating the growth rate of microalgae, comprising the steps of: Step 1: Cultivating microalgae under dark heterotrophic culture conditions to obtain multinucleated cells; Step 2: culturing the multinucleated cells under autotrophic or mixotrophic culture conditions; wherein the microalgae is Chromochloris zofingiensis.

7. 7. The method of claim 6, further comprising culturing the microalgae under mixotrophic or autotrophic culture conditions prior to step 1.

Images