A kind of culture method of nitrogen-fixing Anabaena

Abstract

The present invention discloses a method for culturing nitrogen-fixing Anabaena, which comprises the following steps: preparing a culture medium and culturing under light; sterilizing leucine and arginine at high temperature separately and adding them to the sterilized basal culture medium; sterilizing zeatin by membrane filtration and adding it to the sterilized basal culture medium; inoculating nitrogen-fixing Anabaena seed liquid; culturing under light and shaking for 7 to 9 days, thereby obtaining a microalgae culture solution having an algae cell density of 3.12×10 7 ~5.35×10 7 The present invention can make nitrogen-fixing Anabaena grow rapidly, increase the number of algal cells, shorten the culture time, and reduce the production cost. The nitrogen-fixing Anabaena culture solution can be used as a microbial fertilizer.

Description

Technical Field

[0001] The invention relates to a cultivation method of nitrogen-fixing Anabaena, and belongs to the field of microalgae biotechnology. Background Art

[0002] Microalgae are a type of photosynthetic microorganism with strong carbon and nitrogen fixation capabilities, the ability to produce active ingredients such as exopolysaccharides and plant hormones, and ease of cultivation. Microalgae culture solutions are used in agricultural production to improve soil physical and chemical properties, enhance soil fertility, promote crop growth, improve crop resistance, and increase crop yield and quality. Anabaena azotica is a nitrogen-fixing microalgae from the phylum Cyanobacteria. Its long cultivation cycle, low algal cell biomass, and high production costs have become bottlenecks in its agricultural application.

[0003] Patent CN106591138A discloses a culture medium for Chlorella pyrenoidosa, the ingredients of which are: KNO3, KH2PO4, FeSO4, MnSO4, MgSO4, EDTA-2Na, VB1, VB 12 , plant growth regulators GA3, glycine, and proline. Patent document CN107287125 B discloses a method for culturing Chlorella pyrenoidosa. This method adds fermentation broth of inactivated Rhodobacter sp. and Rhodopseudomonas sp. bacteria to the culture medium, and also adds growth regulators indoleacetic acid and vitamin B2 to the culture medium. This method is beneficial for increasing the growth rate of Chlorella pyrenoidosa, increasing cell density, and promoting oil accumulation. The algal cell density reached 10.11×10 7 pc / mL, but this method primarily targets oil accumulation in algal cells. Patent document CN111349565 A discloses a low-cost, high-biomass, and high-protein cultivation method for Chlorella pyrenoidosa. This method involves saccharifying a recombinant strain of the microalgae with straw, using the straw saccharification liquid as a low-cost, high-quality carbon source, and cultivating Chlorella pyrenoidosa in a mixotrophic manner. This method involves two fermentation processes, resulting in complex operations, a long process flow, and significant investment in facilities and equipment. Furthermore, the raw straw is difficult to store and supply year-round. Patent document CN 112457994 A discloses a method for promoting the growth of Chlorella pyrenoidosa using volatile fatty acids. This method promotes microalgae growth by adding a mixture of volatile fatty acids (acetic acid, propionic acid, and butyric acid) to the culture medium, thereby increasing microalgae oil production. However, the mixed volatile fatty acids required for this method are difficult to supply cheaply and stably. There have been no reports on the cultivation of nitrogen-fixing Anabaena for the production of microbial fertilizers. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a method for culturing nitrogen-fixing Anabaena, which can promote the growth of nitrogen-fixing Anabaena, increase the biomass of algal cells, shorten the cultivation time and reduce production costs.

[0005] There are many types of microalgae, each with its own distinct growth and nutritional characteristics. During microalgae cultivation, both the algae species and environmental factors significantly impact the microalgae cultivation results, with environmental factors having a greater impact, such as nutrient elements, light, temperature, pH, and dissolved oxygen. The present invention provides a method for cultivating nitrogen-fixing Anabaena that can promote the growth of nitrogen-fixing Anabaena, increase algal cell biomass, shorten cultivation time, and reduce production costs. The technical solution employed is:

[0006] A method for culturing nitrogen-fixing Anabaena, comprising preparing a microalgae culture medium and light culturing;

[0007] The preparation of the microalgae culture medium comprises the following specific steps:

[0008] a. Prepare basal medium: The basal medium consists of 1.5 g / L yeast extract powder, 1.5 g / L sodium nitrate, 0.06 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 0.05 g / L ferrous sulfate, 0.036 g / L calcium sulfate, 0.006 g / L ammonium citrate, 0.001 g / L EDTA-2Na, 0.02 g / L sodium carbonate, and 1 mL / L trace element solution.

[0009] b. High temperature sterilization: sterilize the basal culture medium at high temperature and then cool it to room temperature;

[0010] c. Adding leucine and arginine to the basal medium: under sterile conditions, leucine solution and arginine solution were added to the basal medium to obtain an amino acid medium, and the leucine concentration in the amino acid medium was 3.0 to 5.0 mg / L and the arginine concentration was 1.0 to 3.0 mg / L;

[0011] d. Adding zeatin to the basal medium containing leucine and arginine: Under sterile conditions, the zeatin solution was added to the basal medium containing leucine and arginine, and the zeatin concentration in the medium was 0.02 to 0.05 mg / L to obtain a microalgae culture medium;

[0012] The light culture step comprises: inoculating the Anabaena seed solution into the microalgae culture medium at a 10% inoculum amount, and shaking culturing the solution in a light incubator under a light intensity of 7200 Lux for 7 to 9 days to obtain the nitrogen-fixing Anabaena culture solution;

[0013] The preparation method of the nitrogen-fixing Anabaena seed solution is as follows: 2 to 3 rings of nitrogen-fixing Anabaena slant culture are inoculated into a sterilized basal culture medium, and the culture is shaken in a light incubator under a light intensity of 2400 Lux for 3 days to obtain the nitrogen-fixing Anabaena seed solution.

[0014] A further improvement of the technical solution of the present invention is that the formula of the trace element solution in step a includes: 1.4 g / L boric acid, 1.5 g / L manganese chloride, 0.15 g / L zinc sulfate, 0.25 g / L sodium molybdate, 0.05 g / L copper sulfate, and 0.03 g / L cobalt chloride, with a pH of 7.

[0015] A further improvement of the technical solution of the present invention is that the temperature of the high-temperature sterilization in step b is 121° C. and the sterilization time is 20 to 30 minutes.

[0016] A further improvement of the technical solution of the present invention is that: in step c, the concentration of the leucine solution and the arginine solution added is 5.0 g / L, and they are sterilized at a high temperature of 121° C. for 15 to 20 minutes, and then added to the basal culture medium after cooling to room temperature.

[0017] A further improvement of the technical solution of the present invention is that the concentration of the zeatin solution added in step d is 1.0 mg / L, and it is sterilized by filtration using a 0.22 μm organic membrane before addition.

[0018] A further improvement of the technical solution of the present invention is that during the preparation of the nitrogen-fixing Anabaena culture solution and the nitrogen-fixing Anabaena seed solution, the temperature in the light incubator is 28° C. and the rotation speed is 160 r / min.

[0019] A further improvement of the technical solution of the present invention is that the algae cell density in the nitrogen-fixing Anabaena culture solution is 3.12×10 7 ~5.35×10 7 pieces / mL.

[0020] Due to the adoption of the above technical solution, the technical advancements achieved by the present invention are:

[0021] The cultivation method of the present invention enables the rapid growth of nitrogen-fixing Anabaena. Under normal conditions, the cultivation time of nitrogen-fixing Anabaena is 12 to 15 days, and the algal cell density is 1.12×10 7 ~2.51×10 7 / mL, while the cultivation time of the nitrogen-fixing Anabaena of the present invention is 7 to 8 days, and the cell density of the algae is 3.12×10 7 ~5.35×10 7 cells / mL, significantly increasing the number of algal cells, shortening the culture time and reducing production costs.

[0022] The leucine and arginine of the present invention have a very obvious growth-promoting effect on nitrogen-fixing Anabaena, and have a synergistic effect; zeatin also has a good growth-promoting effect on nitrogen-fixing Anabaena, and when leucine, arginine and zeatin are used together, they have a synergistic effect on the growth of nitrogen-fixing Anabaena, and the growth-promoting effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of forchlorfenuron concentration on the growth of Anabaena;

[0024] Figure 2 This is a graph showing the effect of zeatin concentration on the growth of Anabaena according to the present invention;

[0025] Figure 3 This is a graph showing the effect of gibberellin concentration on the growth of Anabaena;

[0026] Figure 4 This is a graph showing the effect of diethylaminoethyl hexanoate concentration on the growth of Anabaena;

[0027] Figure 5 This is a graph showing the effect of the concentration of sodium 5-nitroguaiacol on the growth of Anabaena;

[0028] Figure 6 This is a graph showing the effect of IAA concentration on the growth of Anabaena according to the present invention;

[0029] Figure 7 This is a graph showing the effect of NAA concentration on the growth of Anabaena according to the present invention;

[0030] Figure 8 This is a graph showing the effect of adding amino acids and zeatin simultaneously on the growth of Anabaena according to the present invention. DETAILED DESCRIPTION

[0031] The present invention is described in further detail below in conjunction with the embodiments:

[0032] (1) Preparation:

[0033] Algae species:

[0034] The microalgae cultured in the present invention is nitrogen-fixing Anabaena azotica FACHB-119, which was purchased from the freshwater algae seed bank of the Chinese Academy of Sciences.

[0035] Basal culture medium:

[0036] The basal medium consists of 1.5 g / L yeast extract, 1.5 g / L sodium nitrate, 0.06 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, 0.05 g / L ferrous sulfate, 0.036 g / L calcium sulfate, 0.006 g / L ammonium citrate, 0.001 g / L EDTA-2Na, 0.02 g / L sodium carbonate, and 1 mL / L trace element solution.

[0037] The formula of the trace element solution includes: 1.4 g / L boric acid, 1.5 g / L manganese chloride, 0.15 g / L zinc sulfate, 0.25 g / L sodium molybdate, 0.05 g / L copper sulfate, 0.03 g / L cobalt chloride, pH = 7.

[0038] Preparation of nitrogen-fixing Anabaena seed solution:

[0039] 2-3 rings of nitrogen-fixing Anabaena slant culture were inoculated into the sterilized basal culture medium and cultured in a light incubator under the conditions of temperature of 28°C, rotation speed of 160 r / min and light intensity of 2400 Lux for 3 days to obtain nitrogen-fixing Anabaena seed solution.

[0040] (2) Analytical methods:

[0041] The growth of nitrogen-fixing Anabaena was determined by turbidimetry, and the OD value (OD 680 );

[0042] Algal cell number: Counting method was adopted by microscopic observation.

[0043] (3) Experimental methods:

[0044] Experiment 1: Effects of amino acid type and sterilization method

[0045] Six amino acids (arginine, tyrosine, proline, isoleucine, glycine, and glutamic acid) were prepared into amino acid stock solutions at a concentration of 500 mg / L and added to the basal culture medium. Two sterilization methods were used for adding the amino acid stock solutions to the basal culture medium: separate sterilization, where the six amino acid stock solutions and the basal culture medium were sterilized at high temperature (121°C for 20 minutes) and then added to the basal culture medium to obtain the amino acid culture medium. A mixed sterilization method was used, where the six amino acid stock solutions were first added to the basal culture medium and then sterilized at high temperature (121°C for 20 minutes) to obtain the amino acid culture medium. The concentrations of arginine, tyrosine, proline, isoleucine, glycine, and glutamic acid in the six amino acid culture media were all 5.0 mg / L. A basal culture medium without amino acids was used as a control.

[0046] The Anabaena seed solution was inoculated into the above amino acid culture medium at a 10% inoculum amount, and cultured in a light incubator under a light intensity of 7200 Lux for 7 days with shaking. The results are shown in Table 1.

[0047] Table 1 Effects of amino acid types and sterilization methods on growth (OD 680 )

[0048] control group Arg Gly Glu Pro Leu Tyr Sterilize separately 1.36 1.75 1.45 1.43 1.50 1.96 1.52 Mixed sterilization 1.36 0.59 0.70 1.44 1.57 1.40 1.58

[0049] As shown in Table 1, after 7 days of light culture, the six sterilized amino acids, with the exception of leucine and arginine, showed significant growth-promoting effects compared to the control group. Leucine and arginine increased biomass by 44.1% and 28.7%, respectively, compared to the control group. The mixed sterilized amino acids showed no significant growth-promoting effect, and arginine and glycine even had an inhibitory effect.

[0050] Experiment 2: Study on the synergistic effect of arginine and leucine

[0051] The basal medium, leucine stock solution, and arginine stock solution were sterilized separately at high temperature (121°C for 20 min). Then, under aseptic conditions, the leucine stock solution, arginine stock solution, and leucine stock solution and arginine stock solution were added to the basal medium, respectively, or simultaneously and sequentially, to obtain a medium containing 5.0 mg / L leucine (Leu), 5.0 mg / L arginine (Arg), and 3.0 mg / L leucine + 2.0 mg / L arginine (Leu + Arg). A basal medium without amino acid addition served as a control.

[0052] The Anabaena seed solution was inoculated into the above amino acid culture medium at a 10% inoculum size, and cultured in a light incubator under a light intensity of 7200 Lux for 7 days with shaking. The results are shown in Table 2.

[0053] Table 2 Effects of arginine and leucine on growth (OD 680 )

[0054] control group Leu Arg Leu+Arg Separate sterilization 1.42 2.01 1.81 2.32

[0055] As shown in Table 2, when both leucine and arginine were added to the basal medium, Anabaena grew better than when either arginine or leucine was added alone, indicating a synergistic effect. When both leucine and arginine were added, the biomass of Anabaena increased by 57% compared to the control.

[0056] Experiment 3: Effects of biological growth regulators on the growth of Anabaena

[0057] Seven plant growth regulator stock solutions were prepared respectively; the plant growth regulator stock solutions were sterilized by filtration using a 0.22 μm organic membrane; a control without plant growth regulator was used, and three concentrations of each plant growth regulator were set: high, medium, and low.

[0058] (1) Forchlorfenuron

[0059] The mother solution of chlorfenuron was added to the basal culture medium, and its concentration was adjusted to 0.0 mg / L (control), 2.0 mg / L (low concentration), 5.0 mg / L (medium concentration), and 10 mg / L (high concentration). The seed solution of Anabaena was inoculated into the above chlorfenuron culture medium at a 10% inoculum amount, and the culture was shaken in a light incubator under a light intensity of 7200 Lux for 7 days. The results are shown in FIG. Figure 1 and Table 3.

[0060] Table 3 Effects of forchlorfenuron and its concentration on the growth of Anabaena (OD 680 )

[0061] comparison Low concentration Medium concentration High concentration forchlorfenuron 1.54 1.72 1.23 0.89

[0062] Figure 1 Table 3 shows the effects of different forchlorfenuron concentrations on the biomass of Anabaena. When the forchlorfenuron concentration was 2 mg / L, the biomass of Anabaena was higher than that of the control, indicating that forchlorfenuron at this concentration promoted the growth of Anabaena; the biomass increased by 11.7%. When the forchlorfenuron concentrations were 5 mg / L and 10 mg / L, the biomass of Anabaena was lower than that of the control, indicating that forchlorfenuron at these concentrations inhibited the growth of Anabaena.

[0063] (2) Zeatin

[0064] Zeatin was added to the basal culture medium at concentrations of 0.0 mg / L (control), 0.05 mg / L (low concentration), 0.2 mg / L (medium concentration), and 2.0 mg / L (high concentration). Anabaena seed solution was inoculated into the zeatin culture medium at a 10% inoculum rate and cultured in a light incubator under a light intensity of 7200 Lux for 7 days. The results are shown in FIG. Figure 2 and Table 4.

[0065] Table 4 Effects of zeatin and its concentration on the growth of Anabaena (OD 680 )

[0066] control group Low concentration Medium concentration High concentration Zeatin 1.54 2.36 0.15 0.13

[0067] Figure 2Table 4 shows the effects of different zeatin concentrations on Anabaena biomass. When the zeatin concentration was 0.05 mg / L, the biomass of Anabaena was higher than the control, indicating that zeatin at this concentration promoted the growth of Anabaena; the biomass increased by 53.2%. When the zeatin concentrations were 0.2 mg / L and 2.0 mg / L, the biomass of Anabaena was significantly lower than the control, indicating that zeatin at these concentrations inhibited the growth of Anabaena.

[0068] (3) Gibberellic acid

[0069] Gibberellic acid was added to the basal culture medium, and its concentration was adjusted to 0.0 mg / L (control), 0.05 mg / L (low concentration), 0.5 mg / L (medium concentration), and 5.0 mg / L (high concentration). The Anabaena seed liquid was inoculated into the above-mentioned gibberellin culture medium at a 10% inoculum amount, and cultured in a light incubator with shaking under a light intensity of 7200 Lux for 7 days. The results are shown in FIG. Figure 3 and Table 5.

[0070] Table 5 Effect of gibberellin concentration on the growth of Anabaena (OD 680 )

[0071] comparison Low concentration Medium concentration High concentration Gibberellic acid 1.54 1.93 1.93 1.61

[0072] Figure 3 Table 5 shows the effects of different gibberellin concentrations on the biomass of Anabaena. When the gibberellin concentrations were 0.05 mg / L, 0.5 mg / L, and 5.0 mg / L, the biomass of Anabaena was higher than that of the control, indicating that gibberellin at this concentration can promote the growth of Anabaena; the biomass increased by 25.1%, 25%, and 4.7%, respectively.

[0073] (4) Aminoethyl ester

[0074] The plant hormone diethylaminoethyl was added to the basal culture medium, and its concentration was adjusted to 0.0 mg / L (control), 10.0 mg / L (low concentration), 30.0 mg / L (medium concentration), and 50.0 mg / L (high concentration). The seed liquid of Anabaena was inoculated into the above diethylaminoethyl culture medium at a 10% inoculum amount, and the culture was shaken in a light incubator under a light intensity of 7200 Lux for 7 days. The results are shown in FIG. Figure 4 and Table 6.

[0075] Table 6 Effect of aminoethyl hexanoate concentration on the growth of Anabaena (OD 680 )

[0076] comparison Low concentration Medium concentration High concentration Aminoethyl hexanoate 1.54 1.72 1.58 1.35

[0077] Figure 4Table 6 shows the effects of different diethylaminoethyl hexanoate concentrations on the biomass of Anabaena. When diethylaminoethyl hexanoate concentrations were 10 mg / L and 30 mg / L, the biomass of Anabaena was higher than that of the control, indicating that diethylaminoethyl hexanoate at these concentrations promoted the growth of Anabaena; the biomass increased by 11.8% and 2.5%, respectively. When the concentration of diethylaminoethyl hexanoate was 50 mg / L, the biomass of Anabaena was lower than that of the control, indicating that diethylaminoethyl hexanoate at this concentration inhibited the growth of Anabaena.

[0078] (5) 5-nitroguaiacol sodium

[0079] The plant hormone sodium 5-nitroguaiacol was added to the basal culture medium, and its concentration was adjusted to 0.0 mg / L (control), 0.5 mg / L (low concentration), 2.0 mg / L (medium concentration), and 5.0 mg / L (high concentration). The Anabaena seed liquid was inoculated into the above sodium 5-nitroguaiacol culture medium at a 10% inoculum amount, and the culture was shaken in a light incubator under a light intensity of 7200 Lux for 7 days. The results are shown in FIG. Figure 5 and Table 7.

[0080] Table 7 Effect of sodium 5-nitroguaiacol on the growth of Anabaena (OD 680 )

[0081] comparison Low concentration Medium concentration High concentration 5-Nitroguaiacol sodium 1.54 1.75 1.96 2.01

[0082] Figure 5 Table 7 shows the effect of different sodium 5-nitroguaiacol concentrations on the biomass of Anabaena. When the concentration of sodium 5-nitroguaiacol is 0.5 mg / L, 2.0 mg / L, and 5.0 mg / L, the biomass of Anabaena is higher than that of the control, indicating that sodium 5-nitroguaiacol can promote the growth of Anabaena at these three concentrations; among them, the biomass is the highest when the concentration is 5.0 mg / L, which is 30.7% higher than that of the control.

[0083] (6) IAA (indoleacetic acid)

[0084] To study the effect of IAA on the growth of Anabaena, the plant hormone IAA was added to the optimized culture medium at concentrations of 0.0 mg / L (control), 0.05 mg / L (low concentration), 0.5 mg / L (medium concentration), and 5.0 mg / L (high concentration). Anabaena seed liquid was inoculated into the above IAA culture medium at a 10% inoculum size and cultured in a light incubator under a light intensity of 7200 Lux for 7 days. The results are shown in Table 1. Figure 6 and Table 8.

[0085] Table 8 Effect of IAA concentration on the growth of Anabaena (OD 680 )

[0086] comparison Low concentration Medium concentration High concentration IAA 1.54 1.34 1.81 1.39

[0087] Figure 6 Table 8 shows the effects of different IAA concentrations on Anabaena biomass. At an IAA concentration of 0.5 mg / L, the biomass of Anabaena was higher than the control, indicating that IAA at this concentration promoted the growth of Anabaena; the biomass increased by 17.5%. At IAA concentrations of 0.05 mg / L and 5.0 mg / L, the biomass of Anabaena was lower than the control, indicating that IAA at these concentrations inhibited the growth of Anabaena.

[0088] (7) NAA (naphthaleneacetic acid)

[0089] To study the effect of NAA on the growth of Anabaena, the plant hormone NAA was added to the optimized culture medium at concentrations of 0.0 mg / L (control), 0.1 mg / L (low concentration), 0.5 mg / L (medium concentration), and 2.0 mg / L (high concentration). Anabaena seed liquid was inoculated into the above NAA culture medium at a 10% inoculum and cultured in a light incubator under a light intensity of 7200 Lux for 7 days. The results are shown in Table 1. Figure 7 and Table 9.

[0090] Table 9 Effect of NAA concentration on the growth of Anabaena (OD 680 )

[0091] control group Low concentration Medium concentration High concentration NAA 1.54 1.83 1.59 2.19

[0092] Figure 7 Table 9 shows the effects of different NAA sodium concentrations on the biomass of Anabaena. When the NAA concentrations were 0.1 mg / L, 0.5 mg / L, and 2.0 mg / L, the biomass of Anabaena was higher than that of the control, indicating that NAA could promote the growth of Anabaena at all three concentrations. Among them, the biomass was the highest when the concentration was 2.0 mg / L, which was 42.3% higher than that of the control.

[0093] In summary, after 7 days of light culture, different plant hormones at different concentrations had different effects on the growth of Anabaena. All promoted the growth of Anabaena, and the order of promotion was: zeatin (0.05 mg / L) > naphthaleneacetic acid (2.0 mg / L) > sodium 5-nitroguaiacol (5.0 mg / L) > gibberellin (0.05 mg / L) > indoleacetic acid (0.5 mg / L) > diethylaminoethyl hexanoate (10 mg / L) > forchlorfenuron (2 mg / L). Zeatin (0.05 mg / L) had the greatest promoting effect, increasing growth by 53% compared to the control group. Naphthaleneacetic acid (2.0 mg / L) increased growth by 42% compared to the control group.

[0094] Experiment 4: Study on the synergistic effect of amino acids and zeatin

[0095] The basal culture medium and arginine mother solution (500 mg / L) and leucine mother solution (500 mg / L) were sterilized at high temperature (121°C for 20 min) and then cooled to room temperature. The leucine mother solution and arginine mother solution were then added to the basal culture medium to obtain a culture medium containing 3.0 mg / L leucine + 2.0 mg / L arginine. 1.0 mg / L zeatin was then added to the above culture medium (sterilized by filtration with a 0.22 μm organic membrane before addition) to obtain a zeatin concentration of 0.03 mg / L to obtain a leucine + arginine + zeatin culture medium. Anabaena seed liquid was inoculated into the microalgae culture medium at a 10% inoculum size and cultured in a light incubator under a light intensity of 7200 Lux with shaking. Samples were taken at regular intervals. The test results are shown in FIG. Figure 8 .

[0096] The basal culture medium and arginine mother solution (500 mg / L) and leucine mother solution (500 mg / L) were sterilized at high temperature (121°C for 20 min) and then cooled to room temperature. Then, leucine mother solution and arginine mother solution were added to the basal culture medium to obtain a culture medium containing 3.0 mg / L leucine and 2.0 mg / L arginine. The Anabaena seed solution was inoculated into the microalgae culture medium at a 10% inoculum, and the culture was shaken in a light incubator under a light intensity of 7200 Lux. Samples were taken at regular intervals. The test results are shown in FIG. Figure 8 .

[0097] The culture medium without arginine, leucine and zeatin was used as the control. Samples were taken at regular intervals and the test results are shown in Figure 8 .

[0098] from Figure 8 It can be found that compared with the control, the addition of arginine, leucine and zeatin to the culture medium has a great promoting effect on Anabaena. The time for the control to reach the maximum biomass is 14 days, and the OD 680 2.56 (algal cell number 1.23×10 7 The time for Anabaena to reach the maximum biomass when grown in arginine + leucine medium was 10 days, OD 680 was 2.97 (algal cell number 2.63×10 7 / mL), while the time for arginine + leucine + zeatin medium to reach the maximum biomass was 8 days, and OD 680 is 3.25 (the number of algal cells is 3.12×10 7 The experimental results showed that the addition of arginine, leucine and zeatin promoted the growth of algal cells, significantly shortened the culture time and reduced the production cost.

Claims

1. A method for cultivating nitrogen-fixing Anabaena, characterized in that: Including the preparation of microalgae culture medium and light cultivation; The preparation of the microalgae culture medium comprises the following specific steps: a. Prepare basal medium: The basal medium comprises yeast extract powder 1.5 g / L, sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 0.06 g / L, magnesium sulfate 0.1 g / L, ferrous sulfate 0.05 g / L, calcium sulfate 0.036 g / L, ammonium citrate 0.006 g / L, EDTA-2Na 0.001 g / L, sodium carbonate 0.02 g / L, and trace element solution 1 mL / L; the trace element solution comprises boric acid 1.4 g / L, manganese chloride 1.5 g / L, zinc sulfate 0.15 g / L, sodium molybdate 0.25 g / L, copper sulfate 0.05 g / L, and cobalt chloride 0.03 g / L, pH = 7; b. High temperature sterilization: sterilize the basal culture medium at high temperature and then cool it to room temperature; c. Adding leucine and arginine to the basal medium: Under sterile conditions, leucine solution and arginine solution are separately added to the basal medium to obtain an amino acid medium, and the leucine concentration in the amino acid medium is 3.0-5.0 mg / L and the arginine concentration is 1.0-3.0 mg / L; the sterile conditions are sterilization at 121°C for 15-20 minutes, cooling to room temperature, and then adding them to the basal medium; d. Adding zeatin to the basal medium containing leucine and arginine: Under sterile conditions, the zeatin solution was added to the medium containing leucine and arginine, and the zeatin concentration in the medium was 0.02 to 0.05 mg / L to obtain a microalgae culture medium; The light culture step comprises: inoculating the Anabaena seed solution into the microalgae culture medium at a 10% inoculum amount, and shaking culturing the solution in a light incubator under a light intensity of 7200 Lux for 7 to 9 days to obtain the nitrogen-fixing Anabaena culture solution; The preparation method of the nitrogen-fixing Anabaena seed solution is as follows: 2 to 3 rings of nitrogen-fixing Anabaena slant culture are inoculated into a sterilized basal culture medium, and the culture is shaken in a light incubator under a light intensity of 2400 Lux for 3 days to obtain the nitrogen-fixing Anabaena seed solution.

2. The method for cultivating nitrogen-fixing Anabaena according to claim 1, wherein: The temperature of the high temperature sterilization in step b is 121° C., and the sterilization time is 20 to 30 minutes.

3. The method for culturing nitrogen-fixing Anabaena according to claim 1, wherein: In step c, the added concentration of the leucine solution and the arginine solution is both 500 mg / L.

4. The method for culturing nitrogen-fixing Anabaena according to claim 1, wherein: The concentration of the zeatin solution added in step d is 1.0 mg / L, and the solution is sterilized by filtration using a 0.22 μm organic membrane before addition.

5. The method for culturing nitrogen-fixing Anabaena according to claim 1, wherein: During the preparation of the nitrogen-fixing Anabaena culture solution and the nitrogen-fixing Anabaena seed solution, the temperature in the light incubator was 28° C. and the rotation speed was 160 r / min.

6. The method for culturing nitrogen-fixing Anabaena according to claim 1, wherein: The algae cell density in the nitrogen-fixing Anabaena culture solution was 3.12×10 7 ~5.35×10 7 pieces / mL.

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