A practical chlorella culture medium and preparation method thereof

Abstract

The present invention provides a practical method for preparing a chlorella culture medium, wherein anhydrous sodium acetate, soil leaching solution, jackfruit leaf extract, oxalis leaf extract, macronutrient water-soluble fertilizer and tracenutrient water-soluble fertilizer are mixed to prepare the chlorella culture medium. The chlorella culture medium of the present invention is rich in various nutrients and growth factors, can ensure the rapid growth requirements of chlorella, has a strong inhibitory effect on bacteria and parasites, can effectively reduce the contamination of miscellaneous bacteria and parasites during the chlorella culture process, and has obvious effects.

Description

Technical Field

[0001] The invention relates to the field of chlorella culture medium, and in particular to a practical chlorella culture medium and a preparation method thereof. Background Art

[0002] As aquaculture develops intensively, the residual bait and excrement of farmed organisms in the breeding process pollute the water body and cause eutrophication of the breeding water body. Chlorella sp. is a type of photoautotrophic organism that uses CO2 as a carbon source under light conditions, absorbs and utilizes dissolved nutrients in aquaculture wastewater for product synthesis and self-growth, synthesizes proteins, polysaccharides, oils and other substances inside algae cells, and releases oxygen for use by farmed organisms. Chlorella sp. is rich in nutrients and is a high-quality biological feed for farmed animals. Applying Chlorella sp. to the biological purification of aquaculture water has the advantages of simple operation and high pollutant removal efficiency. It can effectively and safely improve the aquaculture water environment, reduce the occurrence of diseases, and improve the quality of farmed aquatic animals, which has positive ecological and environmental significance. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a practical method for preparing a Chlorella culture medium to solve the above problems.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A practical chlorella culture medium, each liter of the culture medium comprises the following components: 0.5-1.0 g of a macroelement water-soluble fertilizer, 0.25-0.5 g of a trace element water-soluble fertilizer, 0.26-0.5 g of anhydrous sodium acetate, 1-2 mL of soil leaching solution, 1-2 mL of a jackfruit leaf extract, 1-2 mL of a maple leaf extract and the remainder of water.

[0006] Furthermore, the soil leachate preparation method is: weigh baking soda, dissolve it in water to prepare baking soda water, take soil and add it to the baking soda water, stir for 1-3 minutes, soak for 15-25 minutes, filter with 8 layers of gauze, take the supernatant, and prepare the soil leachate.

[0007] Furthermore, the preparation method of the jackfruit leaf extract is: picking jackfruit leaves, washing and drying them, crushing them, passing them through an 80-100 mesh sieve to obtain jackfruit leaf powder, mixing the jackfruit leaf powder and an ethanol solution, sealing and soaking them for 45-50 hours, stirring them once every 8 hours, filtering them with a 600-mesh gauze after soaking and extraction, standing them for 1.5-2.5 hours, taking the supernatant, and obtaining the jackfruit leaf extract.

[0008] Furthermore, the preparation method of the maple leaf extract is: picking maple leaves, washing and drying them, crushing them, passing them through an 80-100 mesh sieve to obtain maple leaf powder, mixing the maple leaf powder with an ethanol solution, sealing and soaking them for 45-50 hours, stirring them once every 8 hours, filtering them with 600 mesh gauze after soaking and extraction, standing them for 1.5-2.5 hours, taking the supernatant, and obtaining the maple leaf extract.

[0009] Furthermore, the Chlorella includes Chlorella pyrenoidosa and Chlorella vulgaris.

[0010] Furthermore, the preparation method comprises the following steps:

[0011] (1) Boil water and cool to room temperature for later use;

[0012] (2) taking 1 / 2 of the total amount of water, adding a macro-element water-soluble fertilizer, a trace element water-soluble fertilizer, and anhydrous sodium acetate into the water, stirring and mixing, and preparing a mixed solution 1;

[0013] (3) Add soil extract, jackfruit leaf extract and safflower leaf extract to the mixed solution 1, and add water to prepare a practical Chlorella culture medium.

[0014] Furthermore, the mass volume ratio of the baking soda to water is 4.0-4.5 g:1 L, and the mass ratio of the baking soda water to soil is 4.5-5.5:1.

[0015] Furthermore, the mass ratio of the jackfruit leaf powder to the ethanol solution is 1:17-23, and the mass concentration of the ethanol solution is 60-80%.

[0016] Furthermore, the mass ratio of the ox-ear maple leaf powder to the ethanol solution is 1:18-22, and the mass concentration of the ethanol solution is 60-80%.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The practical chlorella culture medium of the present invention has comprehensive nutrition, a large amount of water-soluble fertilizer is a general-purpose horticultural plant compound fertilizer rich in nitrogen, phosphorus, and potassium, a trace element water-soluble fertilizer is rich in nitrate nitrogen, calcium, magnesium, boron, zinc, iron, manganese and other elements, anhydrous sodium acetate provides a carbon source, and the soil leaching solution contains relatively rich minerals, trace elements, etc., which can promote the rapid growth of chlorella, and the ingredients are simple and easy to obtain, which is suitable for a large number of breeders in tropical areas. In the present invention, it is found that adding jackfruit leaf extract and cow ear maple leaf extract on this basis has a strong inhibitory effect on bacteria and parasites, can effectively reduce the contamination of miscellaneous bacteria and parasites during the cultivation of chlorella, maintain the pH value of the water body, can provide nutrients to promote the growth of chlorella, and can also improve the immunity of farmed animals. Jackfruit leaf and cow ear maple leaf extracts are common ingredients in tropical areas, with low cost, and a large number of breeders are capable of preparing and using them. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Example 2 Chlorella cultivation effect diagram

[0020] Figure 2 Example 5: Chlorella cultivation effect diagram

[0021] Figure 3 Comparative Example 2: Chlorella cultivation effect diagram

[0022] Figure 4 Comparative Example 4: Chlorella Cultivation Effect Diagram DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0026] The macronutrient water-soluble fertilizer used in the embodiment of the present invention is Stanley brand home gardening fertilizer, and the total nutrient (N+P2O5+K2O) is ≥60%.

[0027] The trace element water-soluble fertilizer used in the embodiment of the present invention is Green Shield brand all-water-soluble trace element fertilizer, with calcium ≥13%, magnesium ≥6.0%, and boron + zinc + iron + manganese + copper + molybdenum ≥0.4%.

[0028] The soil leachate preparation method is as follows: weigh 4.2 g of baking soda, dissolve it in 1 L of tap water, take 200 g of soil and add it to the baking soda water, stir for 2 minutes, soak for 20 minutes, filter with 8 layers of gauze, take the supernatant, and obtain the soil leachate.

[0029] The preparation method of the jackfruit leaf extract comprises the following steps: picking jackfruit leaves, washing, drying, crushing, and passing through a 100-mesh sieve to obtain jackfruit leaf powder; mixing the jackfruit leaf powder with an ethanol solution, wherein the mass ratio of the jackfruit leaf powder to the ethanol solution is 1:20, and the mass concentration of the ethanol solution is 70%; sealing and soaking for 48 hours, stirring once every 8 hours, filtering with a 600-mesh gauze after soaking and extraction, standing for 2 hours, and taking the supernatant to obtain the jackfruit leaf extract.

[0030] The preparation method of the maple leaf extract comprises the following steps: picking maple leaves, washing, drying, crushing, and passing through a 100-mesh sieve to obtain maple leaf powder; mixing the maple leaf powder with an ethanol solution, wherein the mass ratio of the maple leaf powder to the ethanol solution is 1:20, and the mass concentration of the ethanol solution is 70%; sealing and soaking for 48 hours, stirring once every 8 hours, filtering with a 600-mesh gauze after soaking and extraction, standing for 2 hours, and taking a supernatant to obtain the maple leaf extract.

[0031] Example 1

[0032] Weigh the following components: 0.5 g of macro-element water-soluble fertilizer, 0.25 g of trace element water-soluble fertilizer, 0.25 g of anhydrous sodium acetate, and 1.0 mL of soil extract.

[0033] Example 2

[0034] Weigh the following components: 1.0 g of macro-element water-soluble fertilizer, 0.5 g of trace element water-soluble fertilizer, 0.5 g of anhydrous sodium acetate, and 2.0 mL of soil extract.

[0035] The preparation method of the culture medium of Example 1-2 is:

[0036] (1) Boil water and cool to room temperature for later use;

[0037] (2) Take 500 ml of water, weigh out macro-element water-soluble fertilizer, trace element water-soluble fertilizer, and anhydrous sodium acetate in turn and add them into the water. Stir each reagent thoroughly to dissolve. Measure the soil extract and add it into the water. Stir evenly. Add water to 1 L. Filter the insoluble solids in the solution with a 600-mesh sieve to prepare the Chlorella culture medium.

[0038] Example 3

[0039] The preparation method of the oxalis powder comprises the following steps: harvesting 5-6 month old oxalis fresh fruits, cutting them in half, drying them in the sun, crushing them, and passing them through a 80-100 mesh sieve to obtain the oxalis powder.

[0040] The preparation method of jackfruit leaf powder comprises the following steps: washing and drying jackfruit leaves, crushing the leaves, and passing the leaves through a 80-100 mesh sieve to obtain the jackfruit leaf powder.

[0041] Weigh the following components: 1.0 g of macro-element water-soluble fertilizer, 0.5 g of trace element water-soluble fertilizer, 0.5 g of anhydrous sodium acetate, 2.0 mL of soil extract, 1.0 g of jackfruit leaf powder, and 1.0 g of Acer truncatum powder.

[0042] The method for preparing the culture medium is as follows:

[0043] (1) Boil water and cool to room temperature for later use;

[0044] (2) Take 500 ml of water, weigh out macro-element water-soluble fertilizer, trace element water-soluble fertilizer, anhydrous sodium acetate, jackfruit leaf powder, and truncatum powder, and add them to the water. Stir each reagent thoroughly to dissolve it. Filter the insoluble solids in the solution with a 600-mesh sieve. Take out the soil extract and add it to the water. Stir evenly and add water to 1 L. This is how to prepare the Chlorella culture medium.

[0045] Example 4

[0046] Weigh the following components: 1.0 g of macro-element water-soluble fertilizer, 0.5 g of trace element water-soluble fertilizer, 0.5 g of anhydrous sodium acetate, 2.0 mL of soil extract, 1.0 mL of jackfruit leaf extract, and 1.0 mL of calamus leaf extract.

[0047] Example 5

[0048] Weigh the following components: 1.0 g of macro-element water-soluble fertilizer, 0.5 g of trace element water-soluble fertilizer, 0.5 g of anhydrous sodium acetate, 2.0 mL of soil extract, 2.0 mL of jackfruit leaf extract, and 2.0 mL of calamus leaf extract.

[0049] Example 4-5 Preparation method of culture medium:

[0050] (1) Boil water and cool to room temperature for later use;

[0051] (2) Take 500 ml of water, weigh out macro-element water-soluble fertilizer, trace element water-soluble fertilizer, and anhydrous sodium acetate in turn and add them into the water. Stir each reagent thoroughly to dissolve it. Filter the insoluble solids in the solution with a 600-mesh sieve. Measure out soil extract, jackfruit leaf extract, and yew leaf extract and add them into the water. Stir evenly and add water to 1 L to prepare the Chlorella culture medium.

[0052] Comparative Example 1

[0053] A commonly used Chlorella culture medium (Aquatic No. 6), each liter of culture medium includes the following components: NaNO3 (sodium nitrate) 0.25g, K2HPO4·3H2O (dipotassium hydrogen phosphate trihydrate) 0.075g, MgSO4·7H2O (magnesium sulfate heptahydrate) 0.08g, CaCl2·2H2O (calcium chloride dihydrate) 0.05g, KH2PO4 (potassium dihydrogen phosphate) 0.2g, NaCl (sodium chloride) 0.025g, FeCl3·6H2O (ferric chloride hexahydrate) 0.005g, and soil extract 0.5mL.

[0054] The culture medium was prepared by

[0055] (1) Boil water and cool to room temperature for later use;

[0056] (2) Take 500 mL of water, weigh the above culture medium components in turn, add them into the water and stir to dissolve; add soil extract, add water to 1 L, mix well, and prepare Chlorella culture medium.

[0057] Comparative Example 2

[0058] A commonly used Chlorella culture medium (BG11), the following components per liter of culture medium: NaNO3 (sodium nitrate) 0.15g, K2HPO4·3H2O (potassium hydrogen phosphate trihydrate) 0.04g, MgSO4·7H2O (magnesium sulfate heptahydrate) 0.075g, CaCl2·2H2O (calcium chloride dihydrate) 0.036g, citric acid monohydrate 0.006g, ammonium ferric citrate 0.006g, EDTA-Na2 0.001g, Na2CO3 (sodium carbonate) 0.02g, A5 (trace elements) 1mL.

[0059] The formula of A5 (trace elements) is as follows. Each liter of A5 includes the following components: 2.86g H3BO3 (boric acid), 1.81g MnCl2·4H2O (manganese chloride tetrahydrate), 0.22g ZnSO4·7H2O (zinc sulfate heptahydrate), 0.39g Na2MoO4·2H2O (sodium molybdate dihydrate), 0.079g CuSO4·5H2O (copper sulfate pentahydrate), and 0.05g Co(NO3)2·6H2O (cobalt nitrate hexahydrate).

[0060] The method for preparing the culture medium comprises the following steps:

[0061] (1) Boil water and cool to room temperature for later use;

[0062] (2) Prepare A5 (trace elements) seed solution: Take 500 mL of water and add the weighed components in sequence. Stir each reagent thoroughly to dissolve and add water to 1 L.

[0063] (3) Take 500 mL of water, weigh the above culture medium components in turn, add them into the water and stir to dissolve; add A5 (trace elements) seed solution, add water to 1 L, mix well, and the Chlorella culture medium is prepared.

[0064] Comparative Example 3

[0065] Multivitamin stock solution, per 100mL: vitamin B1 0.1g, niacin 0.1g, biotin 1.5mg.

[0066] Trace element stock solution, per 100 mL: 0.004 g copper nitrate heptahydrate, 0.024 g zinc sulfate heptahydrate, 0.075 g sodium molybdate dihydrate, and the balance water.

[0067] Weigh the following culture medium components: NH4Cl (ammonium chloride) 0.25 g, sodium acetate 0.2 g, MgSO4·7H2O (magnesium sulfate heptahydrate) 0.02 g, KH2PO4 (potassium dihydrogen phosphate) 0.175 g, K2HPO4·3H2O (dipotassium hydrogen phosphate trihydrate) 0.075 g, CaCl2·2H2O (calcium chloride dihydrate) 0.01 g, multivitamin stock solution 1 ml, trace element stock solution 20 ml, and the balance water.

[0068] The method for preparing the culture medium comprises the following steps:

[0069] (1) Boil water and cool to room temperature for later use;

[0070] (2) Preparation of multivitamin stock solution: Take 50 mL of water and add the weighed formula components in sequence. Stir each reagent thoroughly to dissolve and add water to 100 mL.

[0071] (3) Preparation of trace element stock solution: Take 50 mL of water and add the weighed formula components in sequence. Stir each reagent thoroughly to dissolve and add water to 100 mL.

[0072] (4) Take 500 mL of water, weigh the above culture medium components in order, add them into the water, stir and dissolve; add the multivitamin stock solution and the trace element stock solution, add water to 1 L, mix well, and the culture medium is prepared.

[0073] Comparative Example 4

[0074] The preparation method of the jackfruit leaf water extract comprises the following steps: picking jackfruit leaves, washing, drying, crushing, passing through a 100-mesh sieve to obtain jackfruit leaf powder, mixing the jackfruit leaf powder and sterile water, wherein the mass ratio of the jackfruit leaf powder to the sterile water is 1:20, sealing and soaking for 48 hours, stirring once every 8 hours, filtering with a 600-mesh gauze after soaking and extraction, standing for 2 hours, taking the supernatant, and obtaining the jackfruit leaf water extract.

[0075] The preparation method of the maple leaf extract is as follows: picking maple leaves, washing, drying, crushing, passing through a 100-mesh sieve to obtain maple leaf powder, mixing the maple leaf powder and sterile water, wherein the mass ratio of the maple leaf powder to the sterile water is 1:20, sealing and soaking for 48 hours, stirring once every 8 hours, filtering with 600-mesh gauze after soaking and extraction, standing for 2 hours, taking the supernatant, and obtaining the maple leaf water extract.

[0076] Weigh the following components: 1.0 g of macro-element water-soluble fertilizer, 0.5 g of trace element water-soluble fertilizer, 2.0 mL of soil extract, 1.0 g of jackfruit leaf water extract, and 1.0 g of Acer truncatum leaf water extract.

[0077] The method for preparing the culture medium is as follows:

[0078] (1) Boil water and cool to room temperature for later use;

[0079] (2) Take 500 ml of water, weigh out macro-element water-soluble fertilizer, trace element water-soluble fertilizer, anhydrous sodium acetate, water extract of jackfruit leaves and water extract of maple leaves in turn and add them into the water. Stir each reagent thoroughly to dissolve it. Filter the insoluble solids in the solution with a 600-mesh sieve. Take out the soil extract and add it into the water. Stir evenly and add water to 1 L. This is how to prepare the Chlorella culture medium.

[0080] Test Example 1

[0081] The culture medium prepared in the above-mentioned Examples 1-5 and Comparative Examples 1-4 was used to compare the culture effect of Chlorella. The Chlorella used in the test was the Chlorella (Chlorella sp.) screened and purified by the inventor from the aquaculture water environment in Hainan. 2L of the culture medium was prepared in proportion and placed in a 5L triangular flask. Three parallels were set for each culture medium, and 20% of the laboratory-activated Chlorella species were inoculated respectively. The culture was carried out under outdoor natural light conditions, and the algae were shaken 3 times a day in the morning, noon and evening. Samples were taken at the time of inoculation and at the 24th, 48th, 96th and 144th hours of culture, and the number of Chlorella in the culture solution was calculated using a hemocytometer and the pH value of the culture solution was detected. The results are shown in Tables 1 and 2.

[0082] Table 1 Growth of the first generation of Chlorella in different culture medium formulations

[0083]

[0084] Table 2 Changes in pH value of culture medium

[0085]

[0086] Comparative Examples 1 and 2 are commonly used culture media for large-scale expansion of Chlorella. As can be seen from Tables 1 and 2, Comparative Examples 1 and 2 are obviously not conducive to the growth of Chlorella compared with the embodiment, and the number of algal cells is low. The growth rate of Chlorella in Comparative Example 1 is the slowest.

[0087] Comparative Example 3 is a modified version of the commonly used Chlorella medium HSM, without the addition of jackfruit leaves and oxalis leaves. Comparative Example 4 does not add sodium acetate, but adds jackfruit leaf water extract and oxalis leaf water extract. From the test results, adding sodium acetate can effectively promote the growth of Chlorella.

[0088] In Examples 3-5 and Comparative Example 4, powder or extract of jackfruit leaf and Acer truncatum leaf or Acer truncatum fresh fruit was added. Compared with Examples 1-2 and Comparative Examples 1-3 without adding powder or extract, the number of Chlorella cells increased significantly, and the pH of the culture solution was relatively stable and did not change much during the culture process. In Example 5, after adding jackfruit leaf extract and Acer truncatum leaf ethanol extract, the number of Chlorella cells was the highest among all the examples.

[0089] In summary, there was no obvious contamination by foreign bacteria in the test cases with the addition of jackfruit leaves and red maple leaves, which proved that the addition of jackfruit leaves and red maple leaves to the culture medium can effectively inhibit the growth of foreign bacteria, and the addition of jackfruit leaf extract and red maple leaf extract is more conducive to the growth of Chlorella.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A practical Chlorella culture medium, characterized in that: Each liter of culture medium includes the following components: 0.5-1.0 g of macronutrient water-soluble fertilizer, 0.25-0.5 g of trace element water-soluble fertilizer, 0.26-0.5 g of anhydrous sodium acetate, 1-2 mL of soil leaching solution, 1-2 mL of jackfruit leaf extract, 1-2 mL of oxear maple leaf extract and the balance of water; The soil leachate preparation method comprises: weighing baking soda, dissolving it in water to prepare baking soda water, adding soil to the baking soda water, stirring for 1-3 minutes, soaking for 15-25 minutes, filtering with 8 layers of gauze, taking the supernatant to prepare the soil leachate; The preparation method of the jackfruit leaf extract comprises: picking jackfruit leaves, washing, drying, crushing, passing through an 80-100 mesh sieve to obtain jackfruit leaf powder, mixing the jackfruit leaf powder with an ethanol solution, sealing and soaking for 45-50 hours, stirring once every 8 hours, filtering with a 600-mesh gauze after soaking and extraction, standing for 1.5-2.5 hours, taking the supernatant, and obtaining the jackfruit leaf extract; The preparation method of the maple leaf extract comprises: picking maple leaves, washing, drying, crushing, and passing through an 80-100 mesh sieve to obtain maple leaf powder; mixing the maple leaf powder with an ethanol solution, sealing and soaking for 45-50 hours, stirring once every 8 hours, filtering with a 600-mesh gauze after soaking and extraction, standing for 1.5-2.5 hours, and taking the supernatant to obtain the maple leaf extract; The method for preparing the practical Chlorella culture medium comprises the following steps: (1) Boil water and cool to room temperature for later use; (2) taking 1 / 2 of the total amount of water, adding a macro-element water-soluble fertilizer, a trace element water-soluble fertilizer, and anhydrous sodium acetate into the water, stirring and mixing, and preparing a mixed solution 1; (3) Add soil extract, jackfruit leaf extract and safflower leaf extract to the mixed solution 1, and add water to prepare a practical Chlorella culture medium.

2. The practical Chlorella culture medium according to claim 1, characterized in that: In the soil leachate preparation method, the mass volume ratio of the baking soda to water is 4.0-4.5 g:1 L, and the mass ratio of the baking soda water to soil is 4.5-5.5:

1.

3. The practical Chlorella culture medium according to claim 1, characterized in that: In the preparation method of the jackfruit leaf extract, the mass ratio of the jackfruit leaf powder to the ethanol solution is 1:17-23, and the mass concentration of the ethanol solution is 60-80%.

4. The practical Chlorella culture medium according to claim 1, characterized in that: In the method for preparing the oxalis leaf extract, the mass ratio of the oxalis leaf powder to the ethanol solution is 1:18-22, and the mass concentration of the ethanol solution is 60-80%.

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