Chlorella desert culture medium as well as use method and application thereof

Abstract

The invention provides a desert chlorella culture medium as well as a use method and application thereof, and belongs to the technical field of desert chlorella culture. Each liter of the culture medium for the chlorella desert comprises the following components: 90 to 100 milligrams of NaNO3, 20 to 30 milligrams of KH2PO4, 20 to 30 milligrams of K2HPO4, 2 to 3 milligrams of MgSO4. 7H2O, 7 to 8 milligrams of CaCl2. 2H2O, 2 to 3 milligrams of NaCl, 4 to 6 milligrams of Na2EDTA. 2H2O and 0 to 5 milligrams of KOH, and the optimal pH value of the culture medium is 6.5 to 7.5. The growth speed of the desert chlorella in the desert chlorella culture medium is obviously higher than that of other culture media, and the culture medium and the culture method are simple, convenient and easy to operate, green and environmentally friendly, can be beneficial to later-stage large-scale culture of the desert chlorella, and are of great significance to preparation of chlorella related products.

Description

Technical Field

[0001] The invention belongs to the technical field of desert algae cultivation, and in particular relates to a desert chlorella culture medium and a use method and application thereof. Background Art

[0002] Algae are green autotrophic organisms with various forms, usually with high photosynthetic efficiency, short growth cycle and fast growth. Algae are widely distributed on the earth and can grow almost anywhere with light and moisture, even in deserts. Microalgae are a type of autotrophic plants with rich nutrition and high photosynthetic utilization. The polysaccharides, proteins, pigments, etc. produced by cell metabolism make them have good development prospects in the fields of food, medicine, genetic engineering, liquid fuel, etc. Desert algae are part of microalgae, and as pioneer colonization organisms, desert algae can grow and reproduce in harsh conditions (such as drought, ultraviolet radiation, nutrient deficiency, etc.), and influence and change the environment through their own activities. They have obvious effects in fixing desert soil, retaining water, and crusting and fixing sand. Therefore, the research and development and utilization of desert algae are similar to those of seaweed, but also have their own special features.

[0003] Chlorella is a single-cell plant with many species and a diameter of 3-8 microns. Chlorella contains high nutrients, including 50%-60% protein, 10%-30% fat, and a variety of vitamins. Chlorella can not only be used as an excellent bait for aquatic animals, but also absorb nitrogen, phosphorus and other elements in the water, reduce the eutrophication level of the water body, and purify the water quality. The content of glycoprotein and polysaccharide, the biologically active substances of Chlorella, is quite high. Therefore, the cultivation of Chlorella has a broad prospect. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a desert chlorella culture medium and its use method and application. The desert chlorella culture medium of the present invention contains: NaNO3 90-100 mg, KH2PO4 20-30 mg, K2HPO4 20-30 mg, MgSO4·7H2O 2-3 mg, CaCl2·2H2O 7-8 mg, NaCl 2-3 mg, Na2EDTA·2H2O 4-6 mg, KOH 0-5 mg per liter, and the optimum pH value is 6.5-7.5. The growth rate of desert chlorella in the desert chlorella culture medium is significantly faster than that of other culture media, and the culture medium and culture method are simple and easy to operate, green and environmentally friendly, and can be beneficial to the later large-scale culture of desert chlorella, which is of great significance to the preparation of chlorella-related products.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention provides a desert chlorella culture medium. The desert chlorella culture medium contains 90-100 mg of NaNO3, 20-30 mg of KH2PO4, 20-30 mg of K2HPO4, 2-3 mg of MgSO4·7H2O, 7-8 mg of CaCl2·2H2O, 2-3 mg of NaCl, 4-6 mg of Na2EDTA·2H2O and 0-5 mg of KOH per liter, and the optimum pH value is 6.5-7.5.

[0007] Preferably, each liter of the desert chlorella culture medium contains: NaNO3 95 mg, KH2PO4 25 mg, K2HPO4 25 mg, MgSO4·7H2O 2.5 mg, CaCl2·2H2O 7.5 mg, NaCl 2.5 mg, Na2EDTA·2H2O 5 mg, and the optimal pH value is 6.8.

[0008] Preferably, each liter of the desert chlorella culture medium contains: NaNO3 95 mg, KH2PO4 25 mg, K2HPO4 25 mg, MgSO4·7H2O 2.5 mg, CaCl2·2H2O 7.5 mg, NaCl 2.5 mg, Na2EDTA·2H2O 5 mg, KOH 5 mg, and the optimal pH value is 7.

[0009] The present invention also provides a method for culturing Chlorella vulgaris, wherein the method is carried out using the Chlorella vulgaris culture medium.

[0010] Preferably, the method comprises using the desert chlorella culture medium for seed liquid culture and expansion culture; the process of the seed liquid culture is: inoculating algae into the desert chlorella culture medium, culturing under the conditions of light intensity of 4500-5500 Lux, temperature of 26-28 ° C, light-dark ratio of 12:12, and vibration speed of 110-130 rpm, and waiting for the desert chlorella to grow to the logarithmic growth phase to obtain seed liquid; the process of the expansion culture is: inoculating the seed liquid into the expansion culture medium at a volume ratio of 8-12%, culturing it to a concentration of 5×10 7 -1×10 8 Pieces / ml.

[0011] Preferably, the culture conditions of the seed solution are: light intensity 5000 Lux, temperature 27° C., light-dark ratio 12:12, and vibration speed 120 rpm.

[0012] Preferably, the inoculation ratio of the seed solution in the expanded culture is 10%.

[0013] Preferably, the illumination intensity of the expanded culture is 5000 Lux and the temperature is 29°C.

[0014] The present invention also provides an application of the desert chlorella obtained by culturing using the method in the preparation of chlorella-related products.

[0015] Preferably, the Chlorella-related product is specifically a fertilizer containing the Desert Chlorella.

[0016] Compared with the prior art, the present invention has the following technical effects:

[0017] The growth rate of desert chlorella in the desert chlorella culture medium of the present invention is significantly faster than that in other culture media, and the culture medium and the culture method are simple and easy to operate, green and environmentally friendly, which can be beneficial to the later large-scale culture of desert chlorella and is of great significance to the preparation of chlorella-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the OD value of the two cultures in Example 3 of the present invention;

[0019] Figure 2 It is the pH value of the two cultures in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention are within the scope of the present invention. In the examples of the present invention, the equipment, raw materials, etc. used are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.

[0021] The desert chlorella used in the present invention is Chlorella sorokiniana.

[0022] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0023] Example 1

[0024] A method for cultivating desert chlorella, comprising the following steps:

[0025] (1) Inoculate algae into a flask with 150 ml of culture medium and culture it in an incubator under the conditions of 5000 Lux light intensity, 27°C temperature, 12:12 light-dark ratio, and 120 rpm vibration speed. After 3-4 days, the liquid in the flask gradually turns green. When the algae grow to the logarithmic growth phase, the seed solution is obtained.

[0026] (2) The seed solution obtained in step (1) was inoculated into the expansion medium at a volume ratio of 10%, and cultured under a light intensity of 4000 Lux, a temperature of 26° C., shaking 2-3 times / day, and ventilation until the concentration reached 5×10 7 Pieces / ml.

[0027] The culture medium formula used for mother culture and large-scale culture is: NaNO395mg / L, KH2PO425mg / L, K2HPO425mg / L, MgSO4·7H2O 2.5mg / L, CaCl2·2H2O 7.5mg / L, NaCl 2.5mg / L, Na2EDTA·2H2O5mg / L, and the optimal pH is 6.8.

[0028] Determination of various indicators: OD values ​​were measured starting from the first day of inoculation. The method was as follows: 200 μL of algae solution was placed in a 96-well plate, three replicates were made, and OD values ​​were measured using an ELISA reader. PH values ​​were measured every two days.

[0029] Example 2

[0030] (1) Inoculate algae into a flask with 150 ml of culture medium and culture it in an incubator under the conditions of 5000 Lux light intensity, 27°C temperature, 12:12 light-dark ratio, and 120 rpm vibration speed. After 3-4 days, the liquid in the flask gradually turns green. When the algae grow to the logarithmic growth phase, the seed solution is obtained.

[0031] (2) The seed solution obtained in step (1) was inoculated into the expansion medium at a volume ratio of 10%, and cultured under a light intensity of 5000 Lux, a temperature of 29° C., shaking 2-3 times / day, and ventilation until the concentration reached 1×10 8 Pieces / ml.

[0032] The culture medium formula used for mother culture and large-scale culture is: NaNO395mg / L, KH2PO425mg / L, K2HPO425mg / L, MgSO4·7H2O 2.5mg / L, CaCl2·2H2O 7.5mg / L, NaCl 2.5mg / L, Na2EDTA·2H2O5mg / L, KOH 5mg / L, and the optimal pH value is 7.

[0033] Determination of various indicators: OD values ​​were measured starting from the first day of inoculation. The method was as follows: 200 μL of algae solution was placed in a 96-well plate, three replicates were made, and OD values ​​were measured using an ELISA reader. PH values ​​were measured every two days.

[0034] Example 3

[0035] The method in Example 2 was used as the experimental group N, and the method using the M1 culture medium was used as the control group M1. The culture method of the desert chlorella and the OD and pH value measurement methods were the same as in Example 2. The M1 culture medium formula was: NHCI 450 mg / L, K2HPO4 25 mg / L, MgSO4·7H2O 2.5 mg / L, CaCl2·2H2O 7.5 mg / L, Na2EDTA·2H2O 1 mg / L, Na2CO3 20 mg / L, FeCI3·H2O 36 mg / L, and the pH value was the natural pH value. The experimental results of M1 and N are shown in Tables 1 to 2 and Figure 1-2 shown.

[0036] Table 1 OD value measurement results

[0037] M1 / OD value N / OD value Day 1 0.06 0.0698 Day 2 0.1878 0.2366 Day 3 0.6628 1.6036 Day 4 0.8252 1.9642 Day 5 0.8499 1.9761 Day 6 0.9638 1.9812 Day 7 0.922 2.0804 Day 8 0.9528 2.2965 Day 9 0.9729 2.2779 Day 10 1.1794 2.4147 Day 11 1.2934 2.532 Day 12 1.2052 2.528

[0038] Table 2 pH value measurement results

[0039] M1 / pH N / pH Day 1 6.8 7 Day 3 7.3 7.3 Day 5 8.3 8 Day 7 9 9 Day 9 9.5 9.3 Day 11 9.3 9

[0040] From Table 1 to Table 2 and Figure 1-2 It can be seen from the experimental data that the use of the culture medium of the present invention can make Chlorella proliferate faster than the use of M1 culture medium, and the maximum concentration that Chlorella can reach is also higher than that of M1 culture medium. And the color of the algae liquid becomes darker and the cell density increases from the third day of culture using the culture medium of the present invention. The pH value of the culture medium of the present invention also increases with the culture time, but the increase rate is slower than that of M1 culture medium. It can be seen that the culture medium provided by the present invention has obvious advantages.

[0041] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A culture medium for Chlorella vulgaris, characterized in that: Each liter of the desert chlorella culture medium contains: NaNO3 90-100 mg, KH2PO4 20-30 mg, K2HPO4 20-30 mg, MgSO4·7H2O 2-3 mg, CaCl2·2H2O 7-8 mg, NaCl 2-3 mg, Na2EDTA·2H2O 4-6 mg, KOH 0-5 mg, and the optimum pH value is 6.5-7.

5.

2. The desert Chlorella culture medium according to claim 1, characterized in that Each liter of the desert chlorella culture medium contains: NaNO395 mg, KH2PO425 mg, K2HPO425 mg, MgSO4·7H2O 2.5 mg, CaCl2·2H2O7.5 mg, NaCl 2.5 mg, Na2EDTA·2H2O 5 mg, and the optimum pH value is 6.

8.

3. The culture medium of desert Chlorella according to claim 1, characterized in that Each liter of the desert chlorella culture medium contains: NaNO395 mg, KH2PO425 mg, K2HPO425 mg, MgSO4·7H2O 2.5 mg, CaCl2·2H2O7.5 mg, NaCl 2.5 mg, Na2EDTA·2H2O 5 mg, KOH 5 mg, and the optimal pH value is 7.

4. A method for cultivating desert chlorella, characterized in that: The method is carried out using the desert Chlorella culture medium according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The method comprises using the desert chlorella culture medium to carry out seed liquid culture and expansion culture; the process of the seed liquid culture is: inoculating algae seeds into the desert chlorella culture medium, culturing under the conditions of light intensity of 4500-5500 Lux, temperature of 26-28° C., light-dark ratio of 12:12, and vibration speed of 110-130 rpm, and waiting for the desert chlorella to grow to the logarithmic growth phase to obtain seed liquid; the process of the expansion culture is: inoculating the seed liquid into the expansion culture medium at a volume ratio of 8-12%, culturing it to a concentration of 5×10 7 -1×10 8 Pieces / ml.

6. The method according to claim 5, characterized in that The culture conditions of the seed solution are: light intensity 5000 Lux, temperature 27° C., light-dark ratio 12:12, and vibration speed 120 rpm.

7. The method according to claim 6, characterized in that The inoculation ratio of the seed liquid in the expanded culture is 10%.

8. The method according to claim 7, characterized in that The illumination intensity of the expanded culture is 5000 Lux and the temperature is 29° C.

9. Use of the desert chlorella obtained by culturing using the method described in any one of claims 4 to 8 in the preparation of chlorella-related products.

10. The use according to claim 9, characterized in that: The Chlorella-related product is specifically a fertilizer containing the Desert Chlorella.

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