Method for promoting rapid heterotrophic growth of microalgae cells

Abstract

The invention provides a method for promoting rapid heterotrophic growth of microalgae cells. Comprising the following steps: S1, purification: separating and purifying microalgae cells by a plate streaking method to obtain a single algal colony; a nitrogen source, a carbon source and a growth stimulant are added into a culture medium, the single algae colony in the step S1 is inoculated for culture, and the growth stimulant comprises one or two of a yeast extract and a soy sauce residue extract. The growth stimulant can promote rapid heterotrophic growth of chlorella pyrenoidosa, shorten the culture period and improve the conversion rate of glucose to microalgae biomass.

Description

Technical Field

[0001] The invention relates to the technical field of unicellular algae cultivation, and in particular to a method for promoting rapid heterotrophic growth of microalgae cells. Background Art

[0002] Microalgae cells can accumulate a large number of high value-added products such as oils, proteins, polysaccharides, pigments and polyunsaturated fatty acids, and have broad application prospects in many industries such as energy, food, feed, bait, health products and cosmetics. However, the low biomass concentration and low target product yield of microalgae during photoautotrophic growth have led to limited production capacity and high prices of microalgae-related products, which seriously restricts the practical application of microalgae in the above industries. Compared with the photoautotrophic mode, microalgae grow faster and have higher biomass concentration during heterotrophic culture, and the entire culture process is time-saving and efficient, which has greater potential for industrial production. At the same time, the study also found that using heterotrophic high-density cultured microalgae cells as algae species for photoautotrophy can promote rapid cell growth, efficient CO2 fixation, and is more conducive to cell accumulation of target products. Therefore, the heterotrophic mode is an effective means to achieve efficient production of microalgae-derived target products and large-scale preparation of high-quality algae species.

[0003] However, the heterotrophic culture model also has many disadvantages. First, compared with bacteria or yeast, the cycle of heterotrophic culture of microalgae is still relatively long (about one week), which not only increases the production cost, but also increases the risk of contamination and failure of culture. Secondly, when microalgae are heterotrophic, they use high-cost organic carbon sources as the only carbon source and energy source, which makes the heterotrophic model face severe challenges in terms of economy. To this end, adopting reasonable culture methods and strategies to promote the rapid heterotrophic growth of microalgae cells, shorten the culture cycle, and improve the conversion rate of organic carbon sources to microalgae biomass is one of the important countermeasures to solve the above two problems.

[0004] The main factors affecting the heterotrophic growth of microalgae include environmental factors (temperature, pH, dissolved oxygen and salinity, etc.) and nutritional factors (carbon source, nitrogen source, C / N ratio, phosphorus concentration, etc.). In addition, some vitamins or amino acids can also significantly affect the heterotrophic growth of microalgae. For example, a certain concentration of vitamin B1 and B12 can significantly accelerate the heterotrophic growth rate of Poterioochromonas malhamensis, while a certain concentration of amino acids can promote the rapid heterotrophic growth of Chlorella sp. Since the types and concentrations of vitamins and amino acids that affect the heterotrophic growth characteristics of different algae strains are diverse, it is time-consuming and laborious to screen these substances and their concentrations separately.

[0005] Chlorella pyrenoidosa is a unicellular microalgae of the genus Chlorella in the Chlorophyta. It grows fast and has a variety of nutritional modes. It can be photoautotrophic, heterotrophic or mixotrophic using organic carbon sources. In addition, algal cells can accumulate a large number of high-value-added products such as protein and lutein. It has been approved as a new food raw material in my country. At present, the heterotrophic model has been successfully applied to the high-density cultivation of multiple strains of Chlorella, including Chlorella pyrenoidosa. It was found that there are large strain differences in the optimal nitrogen source and C / N ratio for heterotrophic growth of Chlorella, and the optimal culture temperature is usually around 30°C, and the optimal pH is about 6.5. In addition, there is still an obvious lag period in the growth process of algal cells (especially in the shake bottle stage), the culture cycle is long, and the conversion rate of glucose to microalgae biomass is generally less than 50%, and the culture efficiency is still low. For example, in CN113913298A, yeast extract and its analogs, peptone and its analogs are used as carbon sources or nitrogen sources to provide nutritional factors for the heterotrophic growth of microalgae. Not only is the utilization rate of organic matter low and needs to be further improved, but the growth efficiency is also low, the growth cycle is long, and the production cost is high. In the non-patent document "Feasibility Study on Protein Accumulation in Cultivation of Chlorella with Soy Sauce Residue Hydrolyzate", soy sauce residue hydrolyzate is used as a nutritional factor for the heterotrophic growth of Chlorella, but there is also a problem of low utilization rate of organic matter and needs to be further improved. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for promoting the rapid heterotrophic growth of microalgae cells, which solves the problems of long microalgae cell culture cycle, less than 50% conversion rate of glucose to microalgae biomass and low culture efficiency in the prior art.

[0007] In one aspect, the present invention provides a method for promoting the rapid heterotrophic growth of microalgae cells, wherein a growth stimulant is added to a culture medium, and the growth stimulant comprises one or two of yeast extract and soy sauce residue extract.

[0008] Furthermore, the method comprises the following steps:

[0009] S1 purification: The microalgae cells were separated and purified by the plate streak method to obtain single algae colonies;

[0010] S2 culture: Add nitrogen source, carbon source and growth stimulant to the culture medium, and inoculate the single algae colony in step S1 for culture.

[0011] Furthermore, the final concentration of the yeast extract is 1-10 g / L, preferably 1-5 g / L.

[0012] Furthermore, the preparation method of the soy sauce residue extract comprises dissolving the soy sauce residue in water and filtering to obtain the soy sauce residue.

[0013] Furthermore, the final concentration of the soy sauce residue extract is 4.0-5.0 g / L, preferably 4.5 g / L.

[0014] A final concentration that is too high will inhibit the growth of Chlorella vulgaris, which may be due to the high salt content of the high-concentration soy sauce residue extract causing salt stress to the algae cells; a concentration that is too low will not promote the growth of the algae cells.

[0015] Furthermore, the microalgae cells include Chlorella cells, Scenedesmus cells, Haematococcus pluvialis cells, preferably Chlorella pyrenoidosa cells.

[0016] Furthermore, the culture medium includes one of Endo culture medium, BG-11 culture medium, BBM culture medium and C culture medium.

[0017] Furthermore, the nitrogen source is one or both of urea and ammonium acetate; the carbon source includes one or more of glucose, sucrose and acetate.

[0018] Preferably, the C / N ratio of the carbon source and the nitrogen source is 18-36, preferably the C / N ratio is 24.

[0019] Furthermore, the culture time is 2-5 days.

[0020] In another aspect, the present invention provides a culture medium for promoting rapid heterotrophic growth of microalgae cells, the culture medium comprising a nitrogen source, a carbon source and a growth stimulant.

[0021] Wherein, the nitrogen source includes one or two of urea and ammonium acetate; the carbon source includes one or more of glucose, sucrose, and acetate; the growth stimulant includes one or two of yeast extract and soy sauce residue extract; the culture medium includes one of Endo culture medium, BG-11 culture medium, BBM culture medium, and C culture medium;

[0022] Preferably, the final concentration of the yeast extract is 1-10 g / L, preferably 1-5 g / L;

[0023] Preferably, the final concentration of the soy sauce residue extract is 4.0-5.0 g / L, preferably 4.5 g / L;

[0024] Preferably, the C / N ratio of the carbon source and the nitrogen source is 18-36, preferably the C / N ratio is 24.

[0025] Autotrophy: also known as photosynthetic autotrophy (Photoautotrophy), refers to the microalgae using light energy for photosynthesis, assimilating CO2 (inorganic carbon) and water into organic matter (glucose), this nutritional lifestyle of obtaining energy.

[0026] Mixotrophy: The nutritional mode in which microalgae utilizes both CO2 and organic carbon for growth and metabolism in the presence of light is called mixotrophy.

[0027] Heterotrophy: refers to adding organic carbon source or organic nitrogen source to autotrophic culture medium and carrying out aerobic fermentation growth under dark conditions, eliminating the effect of light on cell growth, which is called heterotrophy.

[0028] Culture medium: essentially refers to culture medium without added induction factors and / or production of induction factors, such as BBM medium, BG11 medium, TAP medium, M11 medium, C medium, Endo medium, etc.

[0029] The technical principle of the present invention is: due to the various nutritional modes of microalgae (autotrophy, heterotrophy, mixotrophy), microalgae have evolved a variety of metabolic strategies, which have different effects on the growth characteristics, biochemical components and carbon metabolic pathways of microalgae. The prior art includes a lot of research on the use of different nutritional modes and culture techniques to promote the heterotrophic growth of microalgae cells. For example, in CN113913298A, yeast extract and its analogs are added to the basal culture medium as a carbon source to convert the nutritional mode of microalgae from photoautotrophy to heterotrophy or mixotrophy, which can promote the growth of algae cells, increase cell density and increase biomass. However, there are relatively few documents on how to promote the growth of microalgae cells, improve the utilization rate of organic carbon sources and heterotrophic metabolic mechanisms under heterotrophic mode. The inventors found that when conducting heterotrophic culture of microalgae, compared with adding a carbon source and a nitrogen source alone, or adding yeast extract or soy sauce extract alone, adding an appropriate amount of carbon source and a nitrogen source to the basal culture medium and then adding a small amount of yeast extract or soy sauce residue extract as a growth stimulant can improve the utilization efficiency of the organic carbon source, significantly promote cell growth, shorten the culture cycle, and help to further reduce the production cost of heterotrophic culture of microalgae.

[0030] This may be due to the changes in the growth and metabolic pathways of algae cells such as carbon metabolism and nitrogen metabolism under different nutritional modes, which changes the carbon source utilization rate. Specifically, yeast extract is rich in proteins, peptides, amino acids, vitamins and trace elements, and is a common nutrient source for cultivating microorganisms. Soy sauce residue, as the main by-product in the production process of soy sauce, is cheap and easy to obtain (700-800 yuan per ton), and its extract also contains a variety of substances such as protein, fat, carbohydrates, amino acids, soy isoflavones and trace elements. Studies have shown that its hydrolyzate can be used as a culture medium for the growth of microalgae. Therefore, these two substances, as nutrient-rich, comprehensive and cost-controlled nutrient sources, can provide a variety of potential essential substances for the growth and division of microalgae, and both have the potential to become stimulants for the heterotrophic growth of microalgae, improve the utilization efficiency of organic carbon sources, promote the rapid division and proliferation of microalgae cells, further improve quality and efficiency, and reduce the cost of heterotrophic mode production of microalgae biomass.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention provides a method for promoting the rapid heterotrophic growth of Chlorella pyrenoidosa, shortening the culture cycle, and improving the conversion rate of glucose to microalgae biomass by using yeast extract and soy sauce residue extract as stimulants. Compared with the control group without the addition of stimulants, the method can increase the average biomass yield of Chlorella pyrenoidosa by about 30%, shorten the growth cycle by 1 day, and increase the conversion rate of glucose to microalgae biomass by about 10%, which can significantly improve the quality and efficiency. In addition, the carbon source and nitrogen source, yeast extract or soy sauce residue extract have a synergistic effect.

[0033] (2) The method provided by the present invention is easy to control, simple and efficient, provides a basis for high-density and large-scale cultivation of economical microalgae, and is easy to apply to industrial production.

[0034] (3) The present invention can achieve significant effects by adding 1g / L yeast extract or 4.5g / L soy sauce residue extract. These additives are small in amount and low in cost. Not only will they not increase production costs, but they will reduce the final production costs due to improved quality and efficiency, which has significant economic benefits and provides an efficient and economical technical solution for heterotrophic production of microalgae-related products and preparation of a large number of high-quality algae species. In addition, the method can also be applied to promote the rapid heterotrophic growth of other microalgae, and has strong scalability. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The streaking culture result of Chlorella pyrenoidosa in Example 1 of the present invention is shown in FIG. 1 , wherein A is the streaking culture result of Chlorella pyrenoidosa in Example 1 of the present invention ... - Single algae colony obtained by streaking on the plate, B is EG + Pure algae strains were obtained by streaking on the plates.

[0036] Figure 2 This is a diagram showing the effects of different nitrogen sources (A) and C / N ratios (B) on the heterotrophic growth of Chlorella pyrenoidosa in Example 1 of the present invention.

[0037] Figure 3 Graph showing the effects of different concentrations of yeast extract on the heterotrophic growth (A) and glucose consumption rate (B) of Chlorella pyrenoidosa in Example 2 of the present invention.

[0038] Figure 4 The figures are the effects of different concentrations of soy sauce residue extract on the heterotrophic growth (A) and glucose consumption rate (B) of Chlorella pyrenoidosa in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0040] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods. The algae species required for the present invention were purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0041] The soy sauce residue in the invention is the residue after soy sauce is made by fermenting, drying, crushing and other process technologies using soybean, wheat, peanut or flour as raw materials, and the moisture content is less than 15% and the protein content is greater than 10%.

[0042] The soy sauce residue extract / extract in the present invention refers to the soy sauce residue water extract or water extract. For example, 100g of dry soy sauce residue is dissolved in 1000mL ddH2O. After sufficient dissolution, the extract is filtered through a 0.22μm membrane to obtain an extract. Analysis of the extract shows that 18.0g / L of soy sauce residue extract is dissolved in each liter of ddH2O.

[0043] Example 1 Cultivation of Chlorella pyrenoidosa

[0044] 1. Aseptic purification of Chlorella pyrenoidosa: First, prepare two solid plate Endo medium (formula see Table 1), the first one is Endo medium without glucose (Endo-glucose - :EG - ), and the second was Endo medium supplemented with 30 g / L glucose (Endo-glucose + :EG +), add 1L of deionized water and 20g of agar to the above ingredients according to the formula, and sterilize them in an autoclave at 121℃ for 20min. The culture medium containing antibiotics needs to be sterilized and cooled to below 60℃. Add 20mg / L ampicillin and 10mg / L kanamycin that have been filtered and sterilized in a clean bench. Pour the culture medium into a plate to prepare a solid culture medium containing antibiotics. Then, use an inoculation loop to pick a ring of protein core algae liquid and streak it on the EG containing antibiotics. - The culture was carried out on a plate in a light incubator at 25°C and a light intensity of about 40 μmol m -2 s -1 , light-dark cycle 14h:10h. After single algae appeared after about 7 days of cultivation, continue to streak the single algae colony on EG containing antibiotics - Plates were cultured and then incubated for about 5 times in EG - After streaking on the plate, sterile single algae colonies can be obtained ( Figure 1 A) Finally, EG - A single algae colony on the plate was streaked onto EG containing antibiotics. + On the plate, after about 3 times in EG + After streaking on the plate, pure algae strains can be obtained ( Figure 1 B).

[0045] Table 1. Endo medium formula

[0046]

[0047] The above ingredients were added to 1 L of deionized water (ddH2O), the pH was adjusted to 6.5 with 3 M NaOH and 1 M HCl, and then sterilized at 121°C in an autoclave for 20 min.

[0048] 2. Screening of the best nitrogen source for heterotrophic growth of Chlorella pyrenoidosa

[0049] After obtaining the pure algae strain, first use an inoculation loop to pick up 3 single algae colonies in the clean bench and transfer them to a conical flask (100 mL) containing 50 mL of sterile Endo liquid culture medium. Cultivate in a shaker in the dark at a temperature of 30°C and a shaker speed of 240 rpm. After 6-7 days of cultivation, use them as algae seeds.

[0050] Then, Endo medium containing different nitrogen sources was prepared, the medium had a fixed glucose concentration of 30 g / L, a C / N ratio of 18, and five nitrogen sources, namely KNO3, NaNO3, NH4Cl, urea (CO(NH2)2) and CH3COONH4, with corresponding concentrations of 4.81, 4.05, 2.549, 1.46 and 4.05 g / L, respectively. These five nitrogen sources were added to Endo medium without glucose and sterilized at high temperature. Glucose was prepared into a solution (concentration of 1.5 g / mL ddH2O) and then sterilized at high temperature separately, cooled to room temperature, and then added to Endo medium for mixing and use.

[0051] Finally, the above cultured algae were transferred into 250 mL flasks containing 100 mL Endo liquid culture medium with different nitrogen sources using a sterile pipette, with an inoculation volume of 5% (v / v), and cultured in a shaker in the dark at a temperature of 30°C and a shaker speed of 240 rpm. The culture period was 6 days, and the biomass concentration was measured after the culture was completed.

[0052] The method for determining the biomass concentration is as follows: take a certain volume (2 mL) of algae liquid, filter it onto a dried and weighed glass fiber membrane (pore size 0.22 μm, GF / C, Whatman), add 10 mL of deionized water and wash three times, then place it in a 105°C constant temperature oven for 24 hours to dry to constant weight. Biomass concentration DW (g / L) = (W1-W0) / V; wherein W1 is the total weight of the glass fiber membrane and algae after drying (g), W0 is the weight of the dried glass fiber membrane weighed in advance (g), and V is the volume of the algae liquid (L). The measurement results are shown in Table 2. Figure 2 As shown in A.

[0053] Table 2 Effects of different nitrogen sources on the biomass concentration of Chlorella pyrenoidosa

[0054] Type of nitrogen source Biomass concentration g / L <![CDATA[KNO3]]> 10.1 <![CDATA[NaNO3]]> 9.35 <![CDATA[NH4Cl]]> 1.4 <![CDATA[Urea (CO(NH2)2)]]> 11.9 <![CDATA[CH3COONH4]]> 12.9

[0055] The results show that when NH4Cl is used as the nitrogen source, the biomass concentration of Chlorella pyrenoidosa is the lowest, only 1.4g / L; when NaNO3 and KNO3 are used as the nitrogen sources, the biomass concentration of Chlorella pyrenoidosa increases significantly, reaching 9.35 and 10.1g / L respectively; when urea and CH3COONH4 are used as the nitrogen sources, the biomass concentration of Chlorella pyrenoidosa further increases to 11.9 and 12.9g / L respectively. Figure 2A). These results indicate that urea and CH3COONH4 are both suitable as nitrogen sources for the rapid heterotrophic growth and efficient accumulation of biomass of Chlorella pyrenoidosa. Considering that the current market price of urea (about 2,000 yuan / ton) is much lower than that of CH3COONH4 (about 7,000 yuan / ton), the use of urea as a nitrogen source is more conducive to reducing the production cost of heterotrophic microalgae. Therefore, urea was used as a nitrogen source to cultivate Chlorella pyrenoidosa in subsequent studies.

[0056] 3. C / N ratio of heterotrophic growth of Chlorella pyrenoidosa

[0057] Urea was used as the nitrogen source for the heterotrophic growth of Chlorella vulgaris, and Endo liquid culture medium containing different C / N ratios was prepared. The glucose concentration of the culture medium was fixed at 30 g / L, and 7 different C / N ratios (6, 12, 18, 24, 30, 36 and 42, corresponding to urea concentrations of 4.62, 2.22, 1.46, 1.09, 0.72 and 0.62 g / L, respectively) were set. Urea of ​​different concentrations was added to Endo culture medium without glucose and sterilized at high temperature; glucose was prepared into a solution (concentration of 1.5 g / mL ddH2O) and then sterilized at high temperature alone, cooled to room temperature and then added to Endo culture medium.

[0058] Use a sterile pipette to transfer the algae cultured in step 2 to 100 mL of Endo liquid culture medium with different C / N ratios in a triangular flask (250 mL). The inoculation amount is 5% (v / v). The culture is kept in a shaker in the dark at a temperature of 30°C and a shaker speed of 240 rpm. The culture period is 6 days, and the biomass concentration is measured after the culture is completed. The results are as follows Figure 2 As shown in B.

[0059] The results show that when the C / N ratio gradually increases from 6 to 24, the biomass concentration of Chlorella pyrenoidosa increases, reaching the highest at a C / N ratio of 24, which is 13.6 g / L; and when the C / N ratio increases further, the biomass concentration decreases, reaching the lowest at a C / N ratio of 42, which is only 9.4 g / L. This may be due to the continuous decrease in nitrogen concentration, or even lack, which cannot meet the heterotrophic growth of cells. The above results show that 24 is the optimal C / N ratio for heterotrophic growth of Chlorella pyrenoidosa.

[0060] Example 2 Effects of different concentrations of yeast extract on heterotrophic growth of Chlorella pyrenoidosa

[0061] Using urea as nitrogen source and a C / N ratio of 24, yeast extracts of 5 different concentrations (0.5, 1.0, 5.0, 10.0 and 20.0 g / L) were added to Endo medium without glucose for sterilization. Glucose was prepared into a solution (concentration of 1.5 g / mL ddH2O) and then sterilized at high temperature alone. After cooling to room temperature, it was added to Endo and mixed. Endo medium without yeast extract was used as the control group.

[0062] Then, in a clean bench, the cultured algae were transferred to a 250 mL flask containing 100 mL of the Endo liquid medium using a sterile pipette. The inoculation volume was 5% (v / v). The culture was carried out in a shaker in the dark at a temperature of 30°C and a shaker speed of 240 rpm. The culture period was 6 days, and the biomass concentration and the remaining glucose concentration in the algae solution were measured regularly every day. The results are shown in Table 3. Figure 3 shown.

[0063] The method for determining the glucose concentration is as follows: 1 mL of algae solution was added to a 1.5 mL EP tube and centrifuged at 5,000 rpm for 1 min. The glucose concentration (mmol / L) in the supernatant was determined using an Anwen blood glucose meter (Sino Biosensor Co., Ltd., Changsha, Hunan) and matching test strips (the measurement range is between 1.1-33.3 mmol / L. If the concentration is too high, it needs to be diluted with ddH2O). The concentration (g / L) was converted according to the molecular weight of glucose.

[0064] Calculation method of average biomass productivity and conversion rate of glucose to microalgae biomass: Average biomass productivity (Average biomass productivity: ABP) (g / L / d) = (DW1-DW0) / T; Conversion rate of glucose to microalgae biomass (Conversion rate: CR) (%) = (DW1-DW0) / (30-GC1); wherein DW1 is the biomass concentration (g / L) when glucose is consumed or the culture ends, DW0 is the biomass concentration at the initial inoculation (g / L), T is the culture time (d) when glucose is consumed or the culture ends, 30 is the glucose concentration of 30 g / L at the initial inoculation, and GC1 is the glucose concentration in the algae solution (g / L) when glucose is consumed or the culture ends.

[0065] The results show that after adding different concentrations of yeast extract, algal cells can grow rapidly after inoculation, and the biomass concentration after 2 days of culture is higher than that of the control group; after 5 days of culture, the treatment groups with 1.0, 5.0 and 10.0 g / L yeast extract can reach the highest biomass concentration, which are 16.65, 16.95 and 15.65 g / L respectively; while the control group and the treatment groups with 0.5 and 20.0 g / L yeast extract need 6 days of culture to reach the highest biomass concentration, which are 14.90, 14.71 and 15.25 g / L respectively, which are significantly lower than the highest biomass concentration obtained when 1.0 and 5.0 g / L yeast extract are added (p<0.05)( Figure 3 A). The changes in glucose concentration and biomass concentration in the algae liquid showed opposite trends, that is, the higher the biomass concentration, the faster the glucose concentration decreased. When 1.0 and 5.0 g / L yeast extract was added, the glucose in the algae liquid could be consumed after 5 days of cultivation, while the control group and other treatment groups needed 6 days to consume the glucose ( Figure 3 B). In addition, by comparing the average biomass yield and the conversion rate of glucose to microalgae biomass, it was found that when 1.0 and 5.0 g / L yeast extract were added, the average biomass yield of Chlorella pyrenoidosa reached 3.24 and 3.29 g / L / d, respectively, which were more than 33% higher than the control group (2.41 g / L / d), and the conversion rate of glucose to microalgae biomass reached 54.18% and 55.19%, respectively, which were more than 12% higher than the control group (48.33%) (Table 3). These results show that the addition of 1.0 and 5.0 g / L yeast extract can significantly promote the heterotrophic growth of Chlorella pyrenoidosa, shorten the culture cycle, and improve the utilization efficiency of glucose. Considering that there is no significant difference in the effect of adding 1.0 and 5.0 g / L yeast extract on the growth indicators of Chlorella pyrenoidosa (p>0.05), and the addition of 5.0 g / L yeast extract will cause waste of raw materials and increase production costs. Therefore, adding 1.0 g / L yeast extract in future industrial production can meet the purpose of improving the quality and efficiency of heterotrophic growth of microalgae.

[0066] Example 3 Effects of different concentrations of soy sauce residue extract on heterotrophic growth of Chlorella pyrenoidosa

[0067] Using urea as a nitrogen source and under the condition of a C / N ratio of 24, first obtain a soy sauce residue extract, that is, dissolve 100g of dry soy sauce residue in 1000mL ddH2O, and after sufficient dissolution, obtain an extract through a 0.22μm membrane filtration. The extract is analyzed and found to contain 18.0g / L of soy sauce residue extract per liter of ddH2O. Then, a stepwise dilution method is used to dilute the soy sauce residue extract to 9.0, 4.5, 2.25 and 1.125g / L respectively by adding ddH2O; Endo culture medium components without glucose are added to these dilutions for sterilization, and glucose is prepared into a solution (concentration of 1.5g / mL ddH2O) and then sterilized at high temperature alone, and then added to these culture media after cooling to room temperature for mixing, and Endo culture medium without soy sauce residue extract is used as a control group;

[0068] Then, in a clean bench, the cultured algae were transferred to a 250 mL flask containing 100 mL of the above culture medium using a sterile pipette. The inoculation volume was 5% (v / v). The culture was carried out in a shaker in the dark at a temperature of 30°C and a shaker speed of 240 rpm. The culture period was 6 days, and the biomass concentration and the remaining glucose concentration in the algae solution were measured regularly every day. The results are shown in Table 3. Figure 4 shown.

[0069] The results show that the addition of different concentrations of soy sauce residue extract has different effects on the heterotrophic growth of protein-nucleated Chlorella. The addition of high concentrations of soy sauce residue extract (9.0 and 18.0 g / L) will inhibit the growth of protein-nucleated Chlorella, and the biomass concentration after 3 days of cultivation is significantly lower than that of the control group (p<0.05). This may be due to the high salt content of high concentrations of soy sauce residue extract, which causes salt stress to algal cells. The addition of low concentrations of soy sauce residue extract (1.125 and 2.25 g / L) has no promoting effect on the growth of algal cells. When 4.5 g / L of soy sauce residue extract is added, algal cells can grow rapidly, and the biomass concentration after 2 days of cultivation is significantly higher than that of the control group (p<0.05). The highest biomass concentration of 16.31 g / L can be reached after 5 days of cultivation. Figure 4 A). The glucose concentration in the algae liquid showed that when 4.5 g / L soy sauce residue extract was added, the algae cells could completely consume glucose after 5 days of culture; while the addition of low concentration soy sauce residue and the control group required 6 days of culture to consume all the glucose; the addition of high concentration soy sauce residue extract inhibited glucose consumption, and there was still glucose in the algae liquid at the end of the culture ( Figure 4B). In addition, by comparing the average biomass yield and the conversion rate of glucose to microalgae biomass, it was found that when 4.5 g / L of soy sauce residue extract was added, the average biomass yield of Chlorella pyrenoidosa reached 3.19 g / L / d, which was more than 30% higher than the control group (2.41 g / L / d), and the conversion rate of glucose to microalgae biomass reached 53.37%, which was more than 10% higher than the control group (48.33%) (Table 3). These results show that the addition of 4.5 g / L of soy sauce residue extract can significantly promote the heterotrophic growth of Chlorella pyrenoidosa, shorten the culture period, and improve the utilization efficiency of glucose.

[0070] Table 3. Effects of different concentrations of yeast extract and soy sauce residue extract added to Endo medium on heterotrophic growth of Chlorella vulgaris

[0071]

[0072] Comparative Example 1

[0073] Similar to Example 2, except that no nitrogen source or carbon source is added.

[0074] After testing, the average biomass yield of Chlorella pyrenoidosa was 0.03 g / L / d, and the conversion rate of organic matter (2 g / L peptone and 1 g / L yeast extract) to microalgae biomass was only 9%, which was significantly lower than that in Example 2.

[0075] Comparative Example 2

[0076] Similar to Example 3, except that no nitrogen source or carbon source is added.

[0077] After testing, the average biomass yield of Chlorella pyrenoidosa was 0.07 g / L / d, and the conversion rate of organic matter (reducing sugar 4.153 g / L) to microalgae biomass was only 12.8%, which was significantly lower than that in Example 3.

[0078] It can be seen that when yeast extract and its analogs or soy sauce residue extract are added alone to Endo medium, the carbon source conversion rate and average material yield are low. When glucose, urea and yeast extract are added to Endo medium, the carbon source conversion rate and average material yield are significantly improved. This is because the microalgae cells in Comparative Examples 1 and 2 mainly use yeast extract or soy sauce residue extract as a growth carbon source or nitrogen source, while in Example 2 and Example 3, yeast extract or soy sauce residue extract interacts with carbon source and nitrogen source as a growth stimulant for algae cells, and jointly promotes the rapid growth of cells by efficiently utilizing organic matter such as glucose, improves the utilization efficiency of organic carbon sources, shortens the culture cycle, reduces production costs, and thus increases economic benefits. For example, adding 30g / L glucose, 1.09g / L urea, and 1g / L yeast extract to Endo medium can increase the average biomass yield of protein core Chlorella by more than 33%, increase the conversion rate of glucose to microalgae biomass by more than 12%, shorten the culture cycle by 1 day, and significantly improve the efficiency of heterotrophic culture. Alternatively, adding 30g / L glucose, 1.09g / L urea, and 4.5g / L soy sauce residue extract to the Endo medium can increase the average biomass yield of Chlorella pyrenoidosa by more than 30%, and increase the conversion rate of glucose to microalgae biomass by more than 10%, significantly improving the heterotrophic culture efficiency. Moreover, after adding a small amount of yeast extract or soy sauce residue extract, the algae cells still grow heterotrophically and do not change the nutritional mode of the microalgae.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for promoting rapid heterotrophic growth of microalgae cells, characterized in that: A growth stimulant is added to the culture medium, wherein the growth stimulant comprises one or both of yeast extract and soy sauce residue extract.

2. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 1, characterized in that: The following steps are involved: S1 purification: The microalgae cells were separated and purified by the plate streak method to obtain single algae colonies; S2 culture: Add nitrogen source, carbon source and growth stimulant to the culture medium, and inoculate the single algae colony in step S1 for culture.

3. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 2, characterized in that: The final concentration of the yeast extract is 1-10 g / L, preferably 1-5 g / L.

4. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 2, characterized in that: The preparation method of the soy sauce residue extract comprises dissolving the soy sauce residue in water and filtering to obtain the soy sauce residue.

5. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 2, characterized in that: The final concentration of the soy sauce residue extract is 4.0-5.0 g / L, preferably 4.5 g / L.

6. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 2, characterized in that: The microalgae cells include Chlorella cells, Scenedesmus cells, Haematococcus pluvialis cells, preferably Chlorella pyrenoidosa cells.

7. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 2, characterized in that: The culture medium includes one of Endo culture medium, BG-11 culture medium, BBM culture medium and C culture medium.

8. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 2, characterized in that: The nitrogen source is one or both of urea and ammonium acetate; the carbon source includes one or more of glucose, sucrose and acetate; Preferably, the C / N ratio of the carbon source and the nitrogen source is 18-36, preferably the C / N ratio is 24.

9. A method for promoting rapid heterotrophic growth of microalgae cells as claimed in claim 2, characterized in that: The culture time is 2-5 days.

10. A culture medium for promoting rapid heterotrophic growth of microalgae cells, characterized in that: The culture medium comprises a nitrogen source, a carbon source and a growth stimulant, Wherein, the nitrogen source includes one or two of urea and ammonium acetate; the carbon source includes one or more of glucose, sucrose, and acetate; the growth stimulant includes one or two of yeast extract and soy sauce extract; the culture medium includes one of Endo culture medium, BG-11 culture medium, BBM culture medium, and C culture medium; Preferably, the final concentration of the yeast extract is 1-10 g / L, preferably 1-5 g / L; Preferably, the final concentration of the soy sauce residue extract is 4.0-5.0 g / L, preferably 4.5 g / L; Preferably, the C / N ratio of the carbon source and the nitrogen source is 18-36, preferably the C / N ratio is 24.

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