Method for culturing Chlorella with straw waste liquid produced based on purified cellulose

Abstract

This solution provides a method for culturing Chlorella vulgaris using straw waste liquid produced from purified cellulose, including the steps of: S1: Using a chemical method to purify cellulose from straw to produce straw waste liquid, where the straw waste liquid includes lignin, hemicellulose, and cellulose decomposition products; S2: Diluting the straw waste liquid with water to obtain a culture solution; S3: Culturing and domesticating Chlorella vulgaris using the culture solution, where the domesticated Chlorella vulgaris is domesticated to obtain nutrients in the nutrient solution under acidic conditions, and culturing the domesticated Chlorella vulgaris using the straw waste liquid produced after purifying cellulose from straw, and the culture of Chlorella vulgaris can be satisfied without additional addition of other inorganic nutrients.

Description

Technical Field

[0001] This solution relates to the field of straw waste liquid recycling, and particularly to a method for culturing Chlorella vulgaris with straw waste liquid produced by purifying cellulose. Background Art

[0002] Straw refers to the stem part remaining after harvesting the seeds of crops. Common straws include those of crops such as wheat, corn, rice, sorghum, cotton, and rapeseed. In the past, these straws were mainly disposed of by burning, compressing, returning to the field, etc. However, these methods can cause environmental and climate changes and may also affect human health. In recent years, with the improvement of environmental protection awareness and the progress of technology, the comprehensive utilization of straw has received increasing attention. Using chemical methods to treat straw has the advantages of improving resource utilization rate, producing high-value-added products, reducing environmental pollution, and improving energy efficiency. For example, by chemically treating straw, cellulose can be purified from the straw. At this time, the main purpose of chemical treatment is to remove lignin and hemicellulose from the straw to obtain pure cellulose. However, some by-products and waste liquids may be generated in this process. If these waste liquids are not properly treated, they may pollute the environment. However, some components in these waste liquids, such as organic matter, nitrogen, phosphorus, etc., are actually essential for the growth of Chlorella vulgaris.

[0003] Chlorella vulgaris ( Chlorella ) is a single-celled freshwater green alga belonging to the Chlorophyta, Chlorophyceae, Chlorellales, and Chlorellaceae. It is usually spherical or oval in shape, with a diameter between 2 - 10 μm, having a hard cell wall and abundant chloroplasts, and can carry out photosynthesis efficiently. Chlorella vulgaris is widely distributed in nature, especially growing vigorously in eutrophic waters.

[0004] In order to reduce the production cost of Chlorella, various methods have been tried to culture Chlorella using straw. For example, the invention patent application 201610976321.4 discloses "a method for culturing microalgae using straw fiber hydrolysis solution and its application". This invention uses straw as the raw material, treats the straw by mechanical method, alkali method or ammonium sulfite method to remove lignin, and prepares cellulose hydrolysis solution after adding cellulase; the invention patent application 201710248657.3 discloses "a method for culturing heterotrophic Chlorella using straw". This invention uses physical methods such as steam explosion method or ball milling method to crush the straw, and uses cellulase and hemicellulose for enzymatic hydrolysis to produce sugar, which is used as the carbon source for heterotrophic culture of Chlorella; the invention patent application 201811569914.4 "a method for culturing Chlorella pyrenoidosa with high biomass and high protein content" uses Clostridium thermocellum to anaerobically ferment the pretreated straw to saccharify the straw and obtain a saccharified solution mainly composed of glucose. On this basis, by adding essential inorganic nutrients, it is used as the heterotrophic culture medium for Chlorella. However, all the methods mentioned above still need to add essential inorganic nutrients for Chlorella, and there are relatively high production costs for enzymatic hydrolysis of straw and using strains to ferment straw.

[0005] In other words, there are currently technical solutions for culturing Chlorella using straw waste liquid. However, most of the solutions directly cultivate Chlorella with the waste liquid generated by chemical or physical treatment of straw, which can only provide the carbon source required for the growth of Chlorella, and other nutrients required for the growth of Chlorella need to be added additionally, resulting in an increase in the cultivation cost of Chlorella and also failing to fully utilize the straw waste liquid. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for culturing Chlorella using straw waste liquid generated by purifying cellulose, culturing domesticated Chlorella with the straw waste liquid generated after purifying cellulose from straw, and without the need to add other inorganic nutrients additionally to meet the cultivation of Chlorella.

[0007] To achieve the above purpose, the present solution provides a method for culturing Chlorella using straw waste liquid generated by purifying cellulose, including the following steps:

[0008] S1: Use chemical method to purify cellulose from straw to generate straw waste liquid, where the straw waste liquid includes lignin, hemicellulose and cellulose decomposition products;

[0009] S2: Dilute the straw waste liquid with water to obtain a culture solution;

[0010] S3: Use the culture solution to culture domesticated Chlorella, where the domesticated Chlorella is domesticated to obtain nutrients in the nutrient solution under acidic conditions.

[0011] This solution uses a chemical method to treat straw to purify cellulose in the straw, and at the same time directly uses the straw waste liquid generated from the purified cellulose to cultivate Chlorella without adding additional inorganic nutrient components. Since the straw waste liquid in this solution includes lignin, hemicellulose, and cellulose decomposition products, sugars such as glucose, xylose, and arabinose in the straw waste liquid can be used as organic substances to provide a carbon source for heterotrophic cultivation of Chlorella. Cellulose provides glucose, while hemicellulose provides xylose and arabinose. The nitrogen and phosphorus elements contained in the straw waste liquid serve as raw materials for the photosynthesis of Chlorella to provide autotrophic cultivation. Therefore, the straw waste liquid contains sufficient carbon source and nitrogen and phosphorus elements necessary for the growth of Chlorella.

[0012] In step S1, the production of straw waste liquid by purifying cellulose from straw using a chemical method includes the steps of: crushing the straw into straw powder, mixing the straw powder with sodium hydroxide and hydrogen peroxide for a period of time to obtain a first mixed solution, adding a sulfuric acid solution to the first mixed solution and mixing for a period of time to obtain a second mixed solution, and filtering the cellulose in the second mixed solution to obtain the straw waste liquid.

[0013] In some embodiments, the straw powder is mixed with 2% sodium hydroxide and 0.6% hydrogen peroxide for a period of time to obtain a first mixed solution, and then 1.4% sulfuric acid solution is added to the first mixed solution and mixed for a period of time to obtain a second mixed solution. The cellulose in the second mixed solution is filtered using a 10 μm filter membrane to obtain the straw waste liquid.

[0014] Since this solution uses the straw waste liquid generated from purifying cellulose from straw as the culture solution, although the cellulose decomposition products in the straw waste liquid can provide (heterotrophic: glucose) nutrition for Chlorella, the acidic substances in the cellulose decomposition products will also have an obvious inhibitory effect on Chlorella. The acidic substances include, but are not limited to, phenolic acids, ferulic acid, p - coumaric acid, levulinic acid, etc. The research team of this application has overcome the technical prejudice that the acidic substances in the straw waste liquid inhibit Chlorella. By domesticating Chlorella, domesticated Chlorella that can obtain the nutrients in the nutrient solution even in an acidic environment is obtained, eliminating the adverse effects of these cellulose decomposition products that inhibit the growth of Chlorella on the growth of Chlorella, and thus enabling this part of the domesticated Chlorella to make full use of the nutrients in the straw waste liquid.

[0015] In step S2, the present solution dilutes the straw waste liquid with water to obtain a culture solution. The purpose is to reduce the concentration of organic acids and other harmful substances in the waste liquid, thereby reducing their inhibitory effect on the growth of Chlorella. Although the domesticated Chlorella in the present solution can obtain nutrients under acidic conditions, if the concentration of acid substances in the straw waste liquid is too high, these substances may still have an adverse effect on the growth of microorganisms such as Chlorella at high concentrations. Therefore, the present solution can reduce the concentration of these substances by diluting with water to make it harmless or low-harmful to Chlorella. In some embodiments, the volume ratio of water to straw waste liquid is 2:1.

[0016] In addition, in some embodiments, step S2 further includes the steps of: diluting the straw waste liquid with water to obtain a dilution, adjusting the pH value of the dilution to weakly acidic with an alkaline solution and sterilizing it at a high temperature to obtain a culture solution.

[0017] The present solution can use NaOH as the alkaline solution to adjust the pH value of the dilution to 6.2 - 6.3. The purpose is to create an environment suitable for the growth of domesticated Chlorella.

[0018] In addition, the present solution can perform sterilization at a high temperature above 100 °C for 30 - 40 min to kill other microorganisms that may exist in the culture solution, prevent these microorganisms from competing for nutrients with Chlorella during the cultivation process, and even secrete harmful substances to inhibit the growth of Chlorella. By sterilization, it can ensure that the nutrients in the culture solution are mainly absorbed and utilized by Chlorella, and at the same time avoid the risk of foreign microorganism contamination.

[0019] In step S3, the present solution uses domesticated Chlorella that can obtain nutrients in the nutrient solution under acidic conditions, and cultivates the domesticated Chlorella with the culture solution to realize the resource utilization of the nutrients in the straw waste liquid after purifying cellulose.

[0020] In some embodiments, the domesticated Chlorella is inoculated into the culture solution at an inoculation amount of 5 - 10% by volume for mixotrophic cultivation. The conditions for mixotrophic cultivation are a temperature of 20 - 25 °C, a rotation speed of 80 - 120 rpm / min, and a light intensity of 40 - 60 μmolm -3 s -1 , and the cultivation time is 5 - 8 days. The cultivation temperature of the present solution is controlled at 20 - 25 °C to provide suitable thermodynamic conditions, which is helpful for the metabolism and cell division of Chlorella; the cultivation rotation speed is controlled at 80 - 120 rpm / min to provide sufficient oxygen and help maintain the uniform distribution of nutrients, prevent the deposition of algal cells at the bottom of the container, and can also promote the exchange of carbon dioxide; the light intensity is controlled at 40 - 60 μmolm -3 s -1, to ensure that the domesticated Chlorella vulgaris can effectively carry out photosynthesis without suffering from photoinhibition due to excessive light intensity; the cultivation time is controlled within 5 to 8 days to allow the domesticated Chlorella vulgaris to complete several growth cycles. In other words, the cultivation conditions for mixotrophic cultivation in this solution are designed to create an environment that is conducive to the growth of Chlorella vulgaris and enables it to adapt to and utilize the nutrients in the culture solution. In this way, the effective utilization of nutrients in the culture solution by Chlorella vulgaris can be achieved, and at the same time, it helps to reduce the environmental impact of straw waste liquid discharge. In addition, such cultivation conditions also help to increase the yield of Chlorella vulgaris, providing a basis for subsequent applications (such as biofuel production, wastewater treatment, etc.).

[0021] In some preferred embodiments, the conditions for mixotrophic cultivation are a temperature of 23°C, a rotation speed of 100 rpm / min, and a light intensity of 50 μmolm -3 s -1 , and the cultivation time is 7 days.

[0022] It should be specifically noted that no additional nutrients are added during the entire mixotrophic cultivation process of domesticating Chlorella vulgaris. During the cultivation process, the cell concentration, QY, and pigment content of the domesticated Chlorella vulgaris are detected every day to ensure that the Chlorella vulgaris is in a normal growth state.

[0023] This solution uses a multi-stage domestication method to domesticate wild Chlorella vulgaris to obtain domesticated Chlorella vulgaris that can obtain nutrients in the nutrient solution under acidic conditions. In step S3, this solution uses a domestication medium with different concentration ratios of N8 medium and straw waste liquid to multi-stage domesticate wild Chlorella vulgaris. The concentration of straw waste liquid in the domestication medium increases with the increase in the number of domestication times. The straw waste liquid is the waste liquid produced by chemically purifying cellulose from straw, and the straw waste liquid includes lignin, hemicellulose, and cellulose decomposition products.

[0024] In some embodiments, this solution prepares a primary domestication medium with a volume ratio of N8 medium to straw waste liquid of 2:1, a first-stage domestication medium with a volume ratio of N8 medium to straw waste liquid of 1:1, a second-stage domestication medium with a volume ratio of N8 medium to straw waste liquid of 1:2, and a third-stage domestication medium with a volume ratio of water to straw waste liquid of 2:1. The wild Chlorella vulgaris is sequentially domesticated by the primary domestication medium, the first-stage domestication medium, the second-stage domestication medium, and the third-stage domestication medium to obtain the finally surviving Chlorella vulgaris as the domesticated Chlorella vulgaris.

[0025] The composition of the N8 medium in this solution is as follows: potassium nitrate 1.0 g / L, potassium dihydrogen phosphate 0.74 g / L, disodium hydrogen phosphate dihydrate 0.26 g / L, calcium chloride dihydrate 0.013 g / L, Fe EDTA 0.01 g / L, magnesium sulfate heptahydrate 0.05 g / L, and trace elements 1.0 mL. The components in the trace elements are aluminum sulfate hydrate 3.58 g / L, manganese chloride tetrahydrate 12.98 g / L, copper sulfate pentahydrate 1.83 g / L, and zinc sulfate heptahydrate 3.2 g / L.

[0026] Specifically, mix the N8 medium and straw waste liquid in a volume ratio of 2:1, adjust the pH value to 6.2 - 6.3 with NaOH, and then add agar powder in a mass ratio of 15%. Sterilize at high temperature to obtain the primary acclimation medium. Similarly, mix the N8 medium and straw waste liquid in a volume ratio of 1:1, adjust the pH value to 6.2 - 6.3 with NaOH, and then add agar powder in a mass ratio of 15%. Sterilize at high temperature to obtain the first-level acclimation medium. Mix the N8 medium and straw waste liquid in a volume ratio of 1:2, adjust the pH value to 6.2 - 6.3 with NaOH, and then add agar powder in a mass ratio of 15%. Sterilize at high temperature to obtain the second-level acclimation medium. Mix water and straw waste liquid in a volume ratio, adjust the pH value to 6.2 - 6.3 with NaOH, and then add agar powder in a mass ratio of 15%. Sterilize at high temperature to obtain the third-level acclimation medium.

[0027] In a specific embodiment, the condition for sterilization at high temperature is to sterilize at a high temperature above 100°C for 30 - 40 min. Preferably, the condition for sterilization at high temperature is to sterilize at 121°C for 30 min.

[0028] The process of hierarchical acclimation is as follows:

[0029] Prepare a Chlorella vulgaris culture solution from wild Chlorella vulgaris. Dilute the Chlorella vulgaris culture solution and spread it on the primary acclimation medium for acclimation culture for 20 - 40 days. Take the surviving single colonies and place the single colonies in a 24-cell medium for culture for 5 - 9 days, and conduct primary screening according to the OD 750 value change;

[0030] Spread the algal species that survived the primary screening on the first-level acclimation medium for acclimation culture for 20 - 40 days. Take the surviving single colonies and place the single colonies in a 24-cell medium for culture for 5 - 9 days, and conduct screening according to the OD 750 value change;

[0031] The screened surviving algal species are spread on the secondary acclimation medium for acclimation culture for 20 - 40 days. Take the surviving single algal colonies and place them in a 24 - well medium for culture for 25 - 35 days, and screen according to the change of OD 750 value;

[0032] The screened surviving algal species are spread on the tertiary acclimation medium for acclimation culture for 10 - 20 days to obtain the finally surviving algal species as the acclimated Chlorella vulgaris.

[0033] In some embodiments, the conditions for acclimation culture are set with the temperature at 20 - 25 °C and the light intensity at 40 - 60 μmolm -3 s -1 . Preferably, the acclimation culture conditions are set with the temperature at 20 °C and the light intensity at 50 μmolm -3 s -1 .

[0034] In some embodiments, the acclimation culture is carried out for 30 days in the primary acclimation medium, the first - stage acclimation medium, and the secondary acclimation medium, and for 15 days in the tertiary acclimation medium.

[0035] In some embodiments, the 24 - well medium is N8 medium, and 2 - 4 ml of N8 medium is added to each well. Preferably, 3 ml of N8 medium is added to each well in the 24 - well medium and cultured for 7 days.

[0036] Preferably, the Chlorella vulgaris culture solution is diluted to an OD of 0.1 and then spread on the primary acclimation medium.

[0037] This solution aims to domesticate wild Chlorella vulgaris through a multi - stage domestication method to obtain domesticated Chlorella vulgaris that can grow rapidly using the culture solution. Therefore, in the initial stage of domestication, this solution uses N8 medium to dilute the straw waste liquid and gradually increases the proportion of the straw waste liquid, so that after finally using water to dilute the straw waste liquid, such domesticated Chlorella vulgaris can still grow rapidly in the straw waste liquid.

[0038] Compared with the prior art, the present technical solution has the following characteristics and beneficial effects: Different from the traditional process of directly using the waste liquid from straw treatment and additionally adding elements such as nitrogen and phosphorus to cultivate Chlorella, this solution uses the straw waste liquid after purifying cellulose from straw as the culture medium to cultivate Chlorella. Due to the nutrient components contained in the straw waste liquid after purifying cellulose, such as organic matter, nitrogen, phosphorus, and potassium, etc., it can enable Chlorella to carry out heterotrophic and autotrophic processes simultaneously in the straw waste liquid. Therefore, by combining the straw waste liquid produced by chemically treating straw to purify cellulose with the cultivation of domesticated Chlorella, an environmentally friendly and economical ecosystem can be constructed, which not only solves the environmental pollution problem of straw waste liquid treatment but also provides a nutrient source for the growth of Chlorella. The harvest of Chlorella, in turn, provides high-value product raw materials for industries such as biofuels, biofertilizers, and health foods. This comprehensive utilization method embodies the concept of circular economy, that is, by innovative technologies, waste is transformed into valuable resources, promoting the high-value utilization of agricultural waste, and at the same time, it also promotes the development of sustainable agriculture and clean energy. In this way, the waste liquid that might originally become an environmental burden can be transformed into a beneficial resource that promotes ecological balance and economic development. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 FIG. is a schematic diagram of the process of wild domestication of Chlorella according to an embodiment of the present invention.

[0040] Figure 2 FIG. is a graph showing the change in the cell concentration of Chlorella according to Embodiment 1 of the present solution.

[0041] Figure 3 FIG. is a graph showing the change in the QY value of Chlorella according to Embodiment 1 of the present solution.

[0042] Figure 4 FIG. is a graph showing the change in the pigment situation of Chlorella according to Embodiment 1 of the present solution.

[0043] Figure 5 FIG. is a graph showing the change in the cell concentration of Chlorella according to Embodiment 2 of the present solution.

[0044] Figure 6 FIG. is a graph showing the change in the QY value of Chlorella according to Embodiment 2 of the present solution.

[0045] Figure 7 FIG. is a graph showing the change in the pigment situation of Chlorella according to Embodiment 2 of the present solution.

[0046] Figure 8 FIG. is a graph showing the change in the cell concentration of Chlorella according to Comparative Example 1 of the present solution.

[0047] Figure 9It is the change curve graph of the QY value of Chlorella in Comparative Example 1 according to this solution.

[0048] Figure 10 It is the change curve graph of the pigment situation of Chlorella in Comparative Example 1 according to this solution.

[0049] Figure 11 It is the change curve graph of the cell concentration of Chlorella in Comparative Example 2 according to this solution.

[0050] Figure 12 It is the change curve graph of the QY value of Chlorella in Comparative Example 2 according to this solution.

[0051] Figure 13 It is the change curve graph of the pigment situation of Chlorella in Comparative Example 2 according to this solution. Detailed implementation manners

[0052] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art belong to the scope protected by the present invention.

[0053] It can be understood that the term "one" should be understood as "at least one" or "one or more". That is, in one embodiment, the number of an element can be one, while in other embodiments, the number of the element can be multiple. The term "one" cannot be understood as a limitation on the number.

[0054] I. Raw material preparation:

[0055] Obtaining of straw waste liquid: Weigh 15 g of sieved corn straw powder and place it in a blue-capped bottle. Add 200 mL of a mixed solution containing 2% sodium hydroxide and 0.6% hydrogen peroxide thereto, and then place it on a magnetic stirrer at 70 °C and ≥630 r / min for reaction for 2 h. After the reaction is completed, filter to obtain a filter residue. Add 100 mL of pure water and 0.5 mL of glacial acetic acid to the filter residue, filter to obtain a filter residue, and wash it three times (100 mL / time) and discard the supernatant to obtain straw waste liquid.

[0056] Obtaining of N8 medium: The specific formula of N8 medium is as follows: potassium nitrate 1.0 g / L, potassium dihydrogen phosphate 0.74 g / L, disodium hydrogen phosphate dihydrate 0.26 g / L, calcium chloride dihydrate 0.013 g / L, Fe EDTA 0.01 g / L, magnesium sulfate heptahydrate 0.05 g / L, trace elements 1.0 mL (the specific formula of trace elements is: aluminum sulfate octadecahydrate 3.58 g / L, manganese chloride tetrahydrate 12.98 g / L, copper sulfate pentahydrate 1.83 g / L, zinc sulfate heptahydrate 3.2 g / L).

[0057] 24-cell medium: N8 medium, add 3 ml of N8 medium to each well.

[0058] Chlorella culture solution: The Chlorella culture solution is purchased from ccap company.

[0059] Obtaining of domesticated Chlorella: (1) Preparation of primary domestication medium: Take 200 mL of N8 medium and 100 mL of straw waste liquid, dilute the straw waste liquid with N8 medium at a ratio of 2:1, adjust the pH value to 6.2 with NaOH, add agar powder at a mass ratio of 15%, and sterilize at 121 °C for 30 min to obtain a solid medium; (2) Primary domestication of Chlorella: Dilute the Chlorella culture solution to OD of 0.1 and coat it on the above domestication medium for domestication culture; set the temperature at 20 °C and the light intensity at 50 μmolm -3 s -1 , the culture time is 30 days, and 2358 single colonies are obtained. Place the single colonies in 24-cell medium for culturing for 7 days, and conduct primary screening according to the change of OD 750 value; (3) Preparation of first-stage domestication medium: Take 100 mL of N8 medium and 100 mL of straw waste liquid, dilute the straw waste liquid with N8 medium at a ratio of 1:1, adjust the pH value to 6.2 with NaOH, add agar powder at a mass ratio of 15%, and sterilize at 121 °C for 30 min to obtain a solid medium; (4) First-stage domestication of Chlorella: Further coat the 137 algal strains screened in (2) on the solid medium obtained in (3), set the temperature at 20 °C and the light intensity at 50 μmolm -3 s -1, The cultivation time was 30 days, and 4158 single colonies of algae were obtained, which were further screened according to the screening method in (2); (5) Preparation of the secondary domestication medium: Take 100 mL of N8 medium and 200 mL of straw waste liquid, dilute the straw waste liquid with N8 medium at a ratio of 1:2, adjust the pH value to 6.2 with NaOH, add agar powder at a mass ratio of 15%, and sterilize at 121 °C for 30 min to obtain a solid medium; (6) Secondary domestication of Chlorella: Further inoculate the 570 strains of algal species obtained by screening in (4) into the solid medium obtained in (5), set the temperature at 20 °C, and the light intensity at 50 μmolm -3 s -1 , The cultivation time was 30 days, and 3150 single colonies of algae were obtained, which were further screened according to the screening method in (2); (7) Preparation of the tertiary domestication medium: Take 200 mL of natural water and 100 mL of straw waste liquid, dilute the straw waste liquid with tap water at a ratio of 2:1, adjust the pH value to 6.2 with NaOH, add agar powder at a mass ratio of 15%, and sterilize at 121 °C for 30 min to obtain a solid medium; (8) Final domestication of Chlorella: Further inoculate the 769 strains of algal species obtained by screening in (6) into the solid medium obtained in (7), set the temperature at 20 °C, and the light intensity at 50 μmolm -3 s -1 , The cultivation time was 15 days, and the final algal species were obtained for liquid culture, regarding the hierarchical domestication of wild Chlorella.

[0060] II. Example design:

[0061] Example 1:

[0062] Take 20 mL of natural water and 10 mL of straw waste liquid, dilute the straw waste liquid with tap water at a ratio of 2:1, adjust the pH value to 6.2 with NaOH, and sterilize at 121 °C for 30 min to obtain a culture solution. According to an inoculation amount of 5% by volume, inoculate the domesticated Chlorella into the culture solution for mixotrophic culture, and set the culture conditions as follows: temperature 25 °C, rotation speed 100 rpm / min, light intensity 50 μmolm -3 s -1 , The cultivation time was 7 days, and no additional nutrients were added during the cultivation process.

[0063] Take 30 mL of N8 medium, and cultivate it under the same conditions as in Example 1. During the cultivation process, the cell concentration, QY, and pigment content of Chlorella were detected every day to ensure that Chlorella was in a normal growth state. The change curve of the cell concentration of the observed Chlorella is as Figure 2 shown, the change curve of the QY value of Chlorella is as Figure 3 shown, and the change curve of the pigment content of Chlorella is as Figure 4As shown, where N8 represents N8 medium, and H2O:CSW represents the diluted straw waste liquid of this scheme. Figure 4 In (a), it represents the change in the content of lutein a. Figure 4 In (b), it represents the change in the content of lutein b. Figure 4 In (c), it represents the change in the content of carotenoids. It can be seen that the domesticated Chlorella vulgaris in this scheme can grow well in the straw waste liquid designed in this scheme.

[0064] Example 2:

[0065] Take 20 mL of natural water and 10 mL of straw waste liquid. Dilute the straw waste liquid with tap water according to a ratio of 2:1, adjust the pH value to 6.2 with NaOH, and sterilize it at 121 °C for 30 min to obtain a culture solution. According to an inoculation amount of 10% by volume, inoculate the domesticated Chlorella vulgaris into the culture solution for mixotrophic culture. Set the culture conditions as: temperature 25 °C, rotation speed 100 rpm / min, light intensity 50 μmolm -3 s -1 , and the culture time is 7 days. No additional nutrients are added during the culture process.

[0066] Take 30 mL of N8 medium and culture it under the same conditions as in Example 1. During the culture process, detect the cell concentration, QY, and pigment content of Chlorella vulgaris every day to ensure that Chlorella vulgaris is in a normal growth state. The change curve graph of the cell concentration of the observed Chlorella vulgaris is as Figure 5 shown, the change curve graph of the QY value of Chlorella vulgaris is as Figure 6 shown, and the change curve graph of the pigment content of Chlorella vulgaris is as Figure 7 shown, where N8 represents N8 medium, and H2O:CSW represents the diluted straw waste liquid of this scheme. Figure 7 In (a), it represents the change in the content of lutein a. Figure 7 In (b), it represents the change in the content of lutein b. Figure 7 In (c), it represents the change in the content of carotenoids. It can be seen that the domesticated Chlorella vulgaris in this scheme can grow well in the straw waste liquid designed in this scheme.

[0067] Comparative Example 1:

[0068] Take 30 mL of straw waste liquid, adjust the pH value to 6.2 with NaOH, and sterilize it at 121 °C for 30 min to obtain a culture solution. According to an inoculation amount of 5% by volume, inoculate the domesticated Chlorella vulgaris into the culture solution for mixotrophic culture. Set the culture conditions as: temperature 25 °C, rotation speed 100 rpm / min, light intensity 50 μmolm -3 s -1, the cultivation time was 7 days, and no additional nutrients were added during the cultivation process. The cell concentration, QY, and pigment content of Chlorella vulgaris were detected every day during the cultivation process to ensure that Chlorella vulgaris was in a normal growth state.

[0069] Take 30 mL of N8 medium and cultivate it under the same conditions as in Comparative Example 1. During the cultivation process, the cell concentration, QY, and pigment content of Chlorella vulgaris were detected every day to ensure that Chlorella vulgaris was in a normal growth state. The change curve of the cell concentration of the observed Chlorella vulgaris is as Figure 8 shown, and the change curve of the QY value of Chlorella vulgaris is as Figure 9 shown, and the change curve of the pigment content of Chlorella vulgaris is as Figure 10 shown, where N8 represents the N8 medium and CSE represents the straw waste liquid of this scheme. Figure 10 In (a), it represents the change in the content of lutein a. Figure 10 In (b) of it, it represents the change in the content of lutein b. Figure 10 In (c) of it, it represents the change in the content of carotenoids. It can be seen that the wild Chlorella vulgaris of this scheme cannot grow in the straw waste liquid.

[0070] Comparative Example 2:

[0071] Take 20 mL of natural water and 10 mL of straw waste liquid, dilute the straw waste liquid with tap water according to a ratio of 2:1, adjust the pH value to 6.2 with NaOH, and sterilize it at 121 °C for 30 min to obtain a culture solution. According to an inoculation amount of 5% by volume, inoculate wild Chlorella vulgaris into the culture solution for mixotrophic cultivation. Set the cultivation conditions as: temperature 25 °C, rotation speed 100 rpm / min, light intensity 50 μmolm -3 s -1 , the cultivation time was 7 days, and no additional nutrients were added during the cultivation process. The cell concentration, QY, and pigment content of Chlorella vulgaris were detected every day during the cultivation process to ensure that Chlorella vulgaris was in a normal growth state.

[0072] Take 30 mL of N8 medium and cultivate it under the same conditions as in Comparative Example 2. During the cultivation process, the cell concentration, QY, and pigment content of Chlorella vulgaris were detected every day to ensure that Chlorella vulgaris was in a normal growth state. The change curve of the cell concentration of the observed Chlorella vulgaris is as Figure 11 shown, and the change curve of the QY value of Chlorella vulgaris is as Figure 12 shown, and the change curve of the pigment content of Chlorella vulgaris is as Figure 13 shown, where N8 represents the N8 medium and ALE represents the diluted straw waste liquid of this scheme. Figure 13 In (a), it represents the change in the content of lutein a. Figure 13 In (b) of it, it represents the change in the content of lutein b. Figure 13Among them, (c) represents the change in the content of carotenoids. It can be seen that the wild Chlorella vulgaris in this solution cannot grow in the straw waste liquid.

[0073] From the test results, it can be seen that the growth rate of the domesticated Chlorella vulgaris under the straw waste liquid culture conditions (Example 1 and Example 2) is significantly faster than that under the N8 medium conditions, and the cell concentration has also increased significantly. This means that in the same volume of culture medium, the biomass of Chlorella vulgaris is larger. At the same time, the culture medium is only composed of tap water and waste liquid, and the raw material cost is extremely low, which is of great significance for improving production efficiency and reducing costs. More importantly, the pigment content and QY value of Chlorella vulgaris have also increased, which is usually related to the enhancement of photosynthesis, indicating that the light energy conversion efficiency of Chlorella vulgaris under this culture condition is higher.

[0074] The present invention is not limited to the above best implementation mode. Any person can obtain other various forms of products under the inspiration of the present invention. However, no matter what changes are made in its shape or structure, as long as it has the same or similar technical solutions as this application, it falls within the protection scope of the present invention.

Claims

1. A method for culturing Chlorella based on straw waste liquid produced by purifying cellulose, characterized in that: The following steps are involved: S1: Purifying cellulose from straw by chemical method to produce straw waste liquid, wherein the straw waste liquid includes lignin, hemicellulose and cellulose decomposition products; Wherein step S1 comprises: purifying cellulose from straw by chemical method to produce straw waste liquid comprises the steps of: crushing the straw into straw powder, mixing the straw powder with sodium hydroxide and hydrogen peroxide for a period of time to obtain a first mixed liquid, adding sulfuric acid solution to the first mixed liquid and mixing for a period of time to obtain a second mixed liquid, filtering cellulose in the second mixed liquid to obtain straw waste liquid; S2: adding water to dilute the straw waste liquid to obtain the culture solution; S3: Using culture medium to cultivate domesticated Chlorella, using a multi-stage domestication method to domesticate wild Chlorella to obtain domesticated Chlorella that can obtain nutrients in the nutrient solution under acidic conditions, wherein domestication medium containing N8 culture medium and straw waste liquid in different concentration ratios is used to domesticate wild Chlorella in multiple stages, and the concentration of straw waste liquid in the domestication medium increases with the increase in the number of domestication times.

2. The method for culturing Chlorella based on straw waste liquid produced by purifying cellulose according to claim 1, characterized in that: Step S2 includes the steps of: adding water to dilute the straw waste liquid to obtain a dilution liquid, adjusting the pH value of the dilution liquid to weak acidity using an alkaline solution, and sterilizing at high temperature to obtain a culture solution.

3. The method for culturing Chlorella based on straw waste liquid produced by purifying cellulose according to claim 2, characterized in that: NaOH was used as an alkaline solution to adjust the pH value of the diluent to 6.2-6.3, and a high temperature of more than 100°C was used for sterilization for 30-40 minutes.

4. The method for culturing Chlorella based on straw waste liquid produced by purifying cellulose according to claim 1, characterized in that: The culture solution was obtained by diluting the straw waste liquid with a volume ratio of water to the straw waste liquid of 2:

1.

5. The method for culturing Chlorella based on straw waste liquid produced by purifying cellulose according to claim 1, characterized in that: In step S3, the domesticated Chlorella is inoculated into the culture medium with an inoculation volume fraction of 5-10% for mixotrophic culture, wherein the mixotrophic culture conditions are a temperature of 20-25°C, a rotation speed of 80-120 rpm / min, and a light intensity of 40-60 μmol / m -3 s -1 The culture time is 5 to 8 days.

6. The method for culturing Chlorella based on straw waste liquid produced by purifying cellulose according to claim 1, characterized in that: A primary acclimation medium is prepared with a volume ratio of N8 medium and straw waste liquid of 2:1, a first-level acclimation medium is prepared with a volume ratio of N8 medium and straw waste liquid of 1:1, a second-level acclimation medium is prepared with a volume ratio of N8 medium and straw waste liquid of 1:2, and a third-level acclimation medium is prepared with a volume ratio of water and straw waste liquid of 2:1, wherein the straw waste liquid is waste liquid produced by chemically purifying cellulose from straw, and the straw waste liquid includes lignin, hemicellulose and cellulose decomposition products. The primary acclimation medium, the first-level acclimation medium, the second-level acclimation medium and the third-level acclimation medium are used in sequence to acclimate wild Chlorella to obtain the surviving Chlorella as the acclimated Chlorella.

7. The method for culturing Chlorella based on straw waste liquid produced by purifying cellulose according to claim 6, characterized in that: Wild chlorella was prepared into chlorella culture solution, and the chlorella culture solution was diluted and applied to the primary acclimation medium for 20 to 40 days of acclimation culture. The surviving single algae colony was taken and placed in N8 medium for 5 to 9 days. The OD value was calculated. 750 The algae species that survived the initial screening were spread in the primary acclimation medium for 20 to 40 days, and the surviving single algae colony was taken and placed in N8 medium for 5 to 9 days. 750 The surviving algae species were spread on the secondary acclimation medium for 20 to 40 days, and the surviving single algae colonies were taken and placed in N8 medium for 25 to 35 days. 750 The surviving algae species are screened according to the change of the value; the surviving algae species are spread on the third-level domestication medium for domestication culture for 10 to 20 days, and the final surviving algae species are obtained as the domesticated Chlorella.

8. The method for culturing Chlorella based on straw waste liquid produced by purifying cellulose according to claim 1, characterized in that: The conditions for acclimatization and culture were set at a temperature of 20-25°C and a light intensity of 40-60 μmol / m -3 s -1 .

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