CN115786126A - Practical chlorella culture medium and preparation method thereof - Google Patents
Practical chlorella culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a practical chlorella culture medium, which is characterized in that anhydrous sodium acetate, soil leachate, a pineapple honey leaf extract, a maple leaf extract, a macroelement water-soluble fertilizer and a microelement water-soluble fertilizer are mixed to prepare the chlorella culture medium. The chlorella culture medium disclosed by the invention is rich in various nutritional ingredients and growth factors, can ensure the rapid growth requirement of chlorella, has a strong inhibiting effect on bacteria and parasites, can effectively reduce the pollution of infectious microbes and parasites in the chlorella culture process, and has an obvious effect.
Description
Technical Field
The invention relates to the field of chlorella culture media, in particular to a practical chlorella culture medium and a preparation method thereof.
Background
When the aquaculture intensifies, the residual baits and the excrement of the cultured organisms pollute the water body in the culture process, and the culture water body is eutrophicated. Chlorella (Chlorella sp.) is a photoautotrophic organism that utilizes CO under light conditions 2 As a carbon source, absorbing and utilizing the culture wastewaterThe dissolved nutrient salt in the algae culture medium is used for product synthesis and self growth, and synthesizing substances such as protein, polysaccharide, grease and the like in algae cells, and releasing oxygen for utilization by culture organisms. The chlorella is rich in nutrition and is a high-quality biological bait for cultured animals, the chlorella is applied to biological purification treatment of culture water, the method has the advantages of simple and convenient operation, high pollutant removal efficiency and the like, can effectively and safely improve culture water environment, reduce disease occurrence, improve the quality of cultured aquatic animals, and has positive ecological environment significance.
Disclosure of Invention
In view of the above, the present invention is to provide a method for preparing a practical chlorella culture medium, which solves the above problems.
The technical scheme of the invention is realized as follows:
a practical chlorella culture medium comprises the following components per liter: 0.5-1.0g of macroelement water-soluble fertilizer, 0.25-0.5g of trace element water-soluble fertilizer, 0.26-0.5g of anhydrous sodium acetate, 1-2mL of soil leaching solution, 1-2mL of Artocarpus heterophyllus leaf extract, 1-2mL of Acer buergerianum leaf extract and the balance of water.
Further, the preparation method of the soil leachate comprises the following steps: weighing sodium bicarbonate, dissolving in water to obtain sodium bicarbonate water, adding soil into sodium bicarbonate water, stirring for 1-3min, soaking for 15-25min, filtering with 8 layers of gauze, and collecting supernatant to obtain soil leachate.
Further, the preparation method of the Artocarpus heterophyllus leaf extract comprises the following steps: cleaning folium Artocarpi Heterophylli, sun drying, pulverizing, sieving with 80-100 mesh sieve to obtain folium Artocarpi Heterophylli powder, mixing folium Artocarpi Heterophylli powder and ethanol solution, sealing, soaking for 45-50 hr, stirring once every 8 hr, filtering with 600 mesh gauze, standing for 1.5-2.5 hr, and collecting supernatant to obtain folium Artocarpi Heterophylli extract.
Further, the preparation method of the acer truncatum leaf extract comprises the following steps: picking the leaf of the daphniphyllum calycinum, cleaning, drying in the sun, crushing, sieving with a 80-100 mesh sieve to obtain powder of the leaf of the daphniphyllum calycinum, mixing the powder of the leaf of the daphniphyllum calycinum with an ethanol solution, soaking in a sealed condition 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 extract of the leaf of the daphniphyllum calycinum.
Further, the chlorella includes chlorella pyrenoidosa and chlorella vulgaris.
Further, the preparation method comprises the following steps:
(1) Boiling water, and cooling to room temperature;
(2) Measuring water with the total consumption of 1/2, adding the macroelement water-soluble fertilizer, the microelement water-soluble fertilizer and the anhydrous sodium acetate into the water, and stirring and mixing to prepare a mixed solution 1;
(3) Adding the soil leachate, the Artocarpus heterophyllus leaf extract and the Acer nikoense leaf extract into the mixed solution 1, and supplementing water to obtain the practical chlorella culture medium.
Further, the mass volume ratio of the baking soda to the water is 4.0-4.5g, and the mass ratio of the baking soda to the soil is 4.5-5.5.
Furthermore, the mass ratio of the Artocarpus heterophyllus leaf powder to the ethanol solution is 1-17, and the mass concentration of the ethanol solution is 60-80%.
Furthermore, the mass ratio of the daphniphyllum calycinum leaf powder to the ethanol solution is 1.
Compared with the prior art, the invention has the beneficial effects that:
the practical chlorella culture medium disclosed by the invention is comprehensive in nutrition, the macroelement water-soluble fertilizer is a general horticultural plant compound fertilizer rich in nitrogen, phosphorus and potassium elements, the microelement water-soluble fertilizer is rich in nitrate nitrogen, calcium, magnesium, boron, zinc, iron, manganese and other elements, the anhydrous sodium acetate provides a carbon source, and the soil leachate contains rich mineral substances, trace elements and the like, so that the chlorella can be promoted to grow quickly, the components are simple and easy to obtain, and the practical chlorella culture medium is suitable for vast farmers in tropical regions. The invention discovers that the addition of the Artocarpus heterophyllus leaf extract and the Acer nikoense leaf extract has strong inhibition effect on bacteria and parasites, can effectively reduce the pollution of mixed bacteria and parasites in the chlorella culture process, maintains the pH value of a water body, can provide nutrients to promote the growth of the chlorella and can also improve the immunity of cultured animals. The extracts of the leaves of Artocarpus heterophyllus and the leaves of Acer nikoense are common food materials in tropical regions, the cost is low, and the broad masses of farmers can prepare and use the extracts.
Drawings
FIG. 1 example 2 Chlorella culture Effect graph
FIG. 2 example 5 Chlorella culture Effect graph
FIG. 3 is a graph showing the effect of culturing chlorella in comparative example 2
FIG. 4 graph showing the culture effect of Chlorella of comparative example 4
Detailed Description
In order that the technical contents of the invention may be better understood, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
The macroelement water-soluble fertilizer used in the embodiment of the invention is Strobile household gardening fertilizer with total nutrient (N + P) 2 O 5 +K 2 O)≥60％。
The trace element water-soluble fertilizer used in the embodiment of the invention is a green shield full water-soluble trace element fertilizer, calcium is more than or equal to 13%, magnesium is more than or equal to 6.0%, and boron, zinc, iron, manganese, copper and molybdenum are more than or equal to 0.4%.
The preparation method of the soil leaching solution comprises the following steps: weighing 4.2g of baking soda, dissolving in 1L of tap water, adding 200g of soil into the baking soda water, stirring for 2min, soaking for 20min, filtering with 8 layers of gauze, and collecting supernatant to obtain soil leachate.
The preparation method of the Artocarpus heterophyllus leaf extract comprises the following steps: cleaning and drying the seeds of Artocarpus heterophyllus in the sun, crushing, sieving with a 100-mesh sieve to obtain Artocarpus heterophyllus powder, mixing the Artocarpus heterophyllus powder with an ethanol solution, wherein the mass ratio of the Artocarpus heterophyllus powder to the ethanol solution is 1.
The preparation method of the daphniphyllum calycinum leaf extract comprises the following steps: the method comprises the steps of picking the daphniphyllum calycinum leaves, cleaning, drying in the sun, crushing, screening by a 100-mesh screen to obtain daphniphyllum calycinum leaf powder, mixing the daphniphyllum calycinum leaf powder with an ethanol solution, wherein the mass ratio of the daphniphyllum calycinum leaf powder to the ethanol solution is 1.
Example 1
The following components were weighed: 0.5g of macroelement water-soluble fertilizer, 0.25g of trace element water-soluble fertilizer, 0.25g of anhydrous sodium acetate and 1.0mL of soil leaching solution.
Example 2
The following components were weighed: 1.0g of macroelement water-soluble fertilizer, 0.5g of trace element water-soluble fertilizer, 0.5g of anhydrous sodium acetate and 2.0mL of soil leachate.
Example 1-2 the preparation method of the culture medium was:
(1) Boiling water, and cooling to room temperature;
(2) Weighing 500ml of water, sequentially weighing a macroelement water-soluble fertilizer, a microelement water-soluble fertilizer and anhydrous sodium acetate, adding into the water, adding each reagent, fully stirring and dissolving, weighing a soil leaching solution, adding into the water, uniformly stirring, supplementing water to 1L, and filtering insoluble solids in the solution by using a 600-mesh screen to obtain the chlorella culture medium.
Example 3
The preparation method of the auricularia auricula-judae powder comprises the steps of collecting 5-6 months old fresh auricularia auricula-judae leaves, cutting the fresh fruits into half, drying in the sun, crushing, and sieving by a 80-100 mesh sieve to obtain the auricularia auricula-judae powder.
The Artocarpus heterophyllus leaf powder is prepared by cleaning Artocarpus heterophyllus leaf, sun drying, pulverizing, and sieving with 80-100 mesh sieve.
Weighing the following components: 1.0g of macroelement water-soluble fertilizer, 0.5g of trace element water-soluble fertilizer, 0.5g of anhydrous sodium acetate, 2.0mL of soil leaching solution, 1.0g of pineapple honey leaf powder and 1.0g of daphniphyllum calycinum powder.
The preparation method of the culture medium comprises the following steps:
(1) Boiling water, and cooling to room temperature;
(2) Taking 500ml of water, sequentially weighing a major-element water-soluble fertilizer, a trace-element water-soluble fertilizer, anhydrous sodium acetate, pineapple leaf powder and daphniphyllum calycinum powder, adding into the water, fully stirring and dissolving each reagent, filtering insoluble solids in the solution by using a 600-mesh screen, weighing a soil leaching solution, adding into the water, uniformly stirring, and supplementing water to 1L to obtain the chlorella culture medium.
Example 4
Weighing the following components: 1.0g of macroelement water-soluble fertilizer, 0.5g of microelement water-soluble fertilizer, 0.5g of anhydrous sodium acetate, 2.0mL of soil leachate, 1.0mL of pineapple honey leaf extract and 1.0mL of auricularia auricula-judae leaf extract.
Example 5
Weighing the following components: 1.0g of macroelement water-soluble fertilizer, 0.5g of microelement water-soluble fertilizer, 0.5g of anhydrous sodium acetate, 2.0mL of soil leachate, 2.0mL of pineapple honey leaf extract and 2.0mL of auricularia auricula-judae leaf extract.
Examples 4-5 preparation of the culture Medium:
(1) Boiling water, and cooling to room temperature;
(2) Weighing 500ml of water, sequentially weighing a macroelement water-soluble fertilizer, a microelement water-soluble fertilizer and anhydrous sodium acetate, adding into the water, adding each reagent, fully stirring and dissolving, filtering insoluble solids in the solution by using a 600-mesh screen, weighing a soil leaching solution, a Artocarpus heterophyllus leaf extract and a Acer nikoense leaf extract, adding into the water, uniformly stirring, and supplementing water to 1L to obtain the chlorella culture medium.
Comparative example 1
A commonly used chlorella culture medium (aquatics No. 6) comprising the following components per liter of culture medium: naNO 3 (sodium nitrate) 0.25g 2 HPO 4 ·3H 2 O (dipotassium hydrogenphosphate trihydrate) 0.075g 4 ·7H 2 O (magnesium sulfate heptahydrate) 0.08g 2 ·2H 2 0.05g of O (calcium chloride dihydrate) 2 PO 4 0.2g of potassium dihydrogen phosphate, 0.025g of NaCl (sodium chloride) 3 ·6H 2 0.005g of O (ferric trichloride hexahydrate) and 0.5mL of soil leaching solution.
The preparation method of the culture medium comprises
(1) Boiling water, and cooling to room temperature;
(2) Taking 500mL of water, sequentially weighing the culture medium components, adding the culture medium components into the water, and stirring for dissolving; adding soil leachate, supplementing water to 1L, and mixing to obtain Chlorella culture medium.
Comparative example 2
A commonly used Chlorella medium (BG 11) contains the following components per liter of medium: naNO 3 0.15g of (sodium nitrate) 2 HPO 4 ·3H 2 O (dipotassium hydrogenphosphate trihydrate) 0.04g, mgSO 4 ·7H 2 0.075g of O (magnesium sulfate heptahydrate) 2 ·2H 2 0.036g of O (calcium chloride dihydrate), 0.006g of citric acid monohydrate, 0.006g of ferric ammonium citrate, EDTA-Na 2 0.001g，Na 2 CO 3 (sodium carbonate) 0.02g, A5 (trace element) 1mL.
Wherein the formula of A5 (trace elements) is as follows, and each liter of A5 comprises the following components: h 3 BO 3 (boric acid) 2.86g, mnCl 2 ·4H 2 O (manganese chloride tetrahydrate) 1.81g 4 ·7H 2 0.22g of O (zinc sulfate heptahydrate) 2 MoO 4 ·2H 2 0.39g of O (sodium molybdate dihydrate) 4 ·5H 2 0.079g of O (copper sulfate pentahydrate) (NO) 3 ) 2 ·6H 2 0.05g of O (cobalt nitrate hexahydrate).
The preparation method of the culture medium comprises the following steps:
(1) Boiling water, and cooling to room temperature;
(2) Preparing A5 (trace element) seed liquid: taking 500mL of water, sequentially adding the weighed formula components, fully stirring and dissolving each time one reagent is added, and supplementing water to 1L;
(3) Taking 500mL of water, sequentially weighing the culture medium components, adding the culture medium components into the water, and stirring for dissolving; adding A5 (trace element) seed liquid, supplementing water to 1L, and mixing to obtain the chlorella culture medium.
Comparative example 3
A multivitamin stock solution, per 100 mL: vitamin B 1 0.1g, nicotinic acid 0.1g and biotin 1.5mg.
A trace element stock solution, per 100 mL: 0.004g of copper nitrate heptahydrate, 0.024g of zinc sulfate heptahydrate, 0.075g of sodium molybdate dihydrate and the balance of water.
The following media components were weighed: NH 4 0.25g of Cl (ammonium chloride), 0.2g of sodium acetate, mgSO 4 ·7H 2 0.02g of O (magnesium sulfate heptahydrate) 2 PO 4 (monopotassium phosphate) 0.175g, K 2 HPO 4 ·3H 2 0.075g of O (dipotassium hydrogenphosphate trihydrate) 2 ·2H 2 0.01g of O (calcium chloride dihydrate), 1ml of compound vitamin stock solution, 20ml of trace element stock solution and the balance of water.
The preparation method of the culture medium comprises the following steps:
(1) Boiling water, and cooling to room temperature;
(2) Preparing a composite vitamin stock solution: taking 50mL of water, sequentially adding the weighed formula components, fully stirring and dissolving each reagent, and supplementing water to 100mL;
(3) Preparing a trace element stock solution: taking 50mL of water, sequentially adding the weighed formula components, fully stirring and dissolving each time one reagent is added, and supplementing the water to 100mL;
(4) Taking 500mL of water, sequentially weighing the culture medium components, adding the culture medium components into the water, and stirring for dissolving; adding composite vitamin stock solution and microelement stock solution, supplementing water to 1L, and mixing to obtain culture medium.
Comparative example 4
The preparation method of the water extract of the Artocarpus heterophyllus leaves comprises the following steps: the method comprises the steps of picking up the seeds of the Artocarpus heterophyllus, cleaning, drying in the sun, crushing, screening by a 100-mesh screen to obtain Artocarpus heterophyllus leaf powder, mixing the Artocarpus heterophyllus leaf powder with sterile water, wherein the mass ratio of the Artocarpus heterophyllus leaf powder to the sterile water is 1.
The preparation method of the daphniphyllum calycinum leaf extract comprises the following steps: the method comprises the steps of picking the daphniphyllum calycinum leaves, cleaning, drying in the sun, crushing, screening by a 100-mesh screen to obtain daphniphyllum calycinum leaf powder, mixing the daphniphyllum calycinum leaf powder with sterile water, wherein the mass ratio of the daphniphyllum calycinum leaf powder to the sterile water is 1.
Weighing the following components: 1.0g of macroelement water-soluble fertilizer, 0.5g of trace element water-soluble fertilizer, 2.0mL of soil leaching solution, 1.0g of aqueous extract of Artocarpus heterophyllus leaves and 1.0g of aqueous extract of Acer palmatum leaves.
The preparation method of the culture medium comprises the following steps:
(1) Boiling water, and cooling to room temperature;
(2) Taking 500ml of water, sequentially weighing a major element water-soluble fertilizer, a trace element water-soluble fertilizer, anhydrous sodium acetate, a ludwigia octovalvis leaf water extract and a daphniphyllum leaf water extract, adding each reagent into the water, fully stirring and dissolving, filtering insoluble solids in the solution by using a 600-mesh screen, weighing a soil leaching solution, adding the soil leaching solution into the water, uniformly stirring, and supplementing water to 1L to obtain the chlorella culture medium.
Test example 1
The culture effect of Chlorella was compared with the culture media prepared in examples 1 to 5 and comparative examples 1 to 4, and the Chlorella used in the experiment was Chlorella (Chlorella sp.) selected by the inventors from the culture water environment in Hainan province. Preparing 2L of culture medium according to a proportion, putting the culture medium into a 5L triangular flask, arranging three parallel culture media for each culture medium, respectively inoculating 20% of chlorella algae activated in a laboratory, culturing under the outdoor natural illumination condition, shaking the chlorella 3 times in the morning, the noon and the evening every day, respectively sampling the chlorella in the culture solution and the 24 th, 48 th, 96 th and 144 th hours of the culture after the inoculation and the culture, and calculating the number of the chlorella in the culture solution and detecting the pH value of the culture solution by using a blood counting chamber, wherein the results are shown in tables 1 and 2.
TABLE 1 growth of Chlorella vulgaris 1 generation species in different medium formulations
TABLE 2 pH Change of the culture solution
Comparative examples 1 and 2 are media used for large-scale propagation of chlorella, and as shown in tables 1 and 2, comparative examples 1 and 2 are significantly less favorable for growth of chlorella than the examples, the number of chlorella cells is low, and the growth rate of chlorella in comparative example 1 is the slowest.
Comparative example 3 is a modified version of HSM in a common chlorella culture medium without the addition of Artocarpus heterophyllus leaves and Acer nikoense leaves. Comparative example 4 No sodium acetate was added, and the aqueous extracts of Artocarpus heterophyllus leaf and the aqueous extract of Acer nikoense leaf were added, and the addition of sodium acetate was found to be effective in promoting the growth of Chlorella vulgaris.
Examples 3 to 5 and comparative example 4 added with powders or extracts of Artocarpus heterophyllus leaves and Acer niphyllum leaves or fresh Acer niphyllum fruits, the number of chlorella cells was significantly increased, the pH of the culture solution was stable, the change in the culturing process was small, and the number of chlorella cells was the highest in all examples after adding the Artocarpus heterophyllus leaf extract and Acer niphyllum leaf ethanol extract in example 5, compared with examples 1 to 2 and comparative examples 1 to 3 without adding.
In conclusion, in the test examples with the addition of the Artocarpus heterophyllus leaves and the Acer nikoense leaves, no obvious mixed bacteria pollution phenomenon exists, and the results prove that the addition of the Artocarpus heterophyllus leaves and the Acer nikoense leaves in the culture medium can effectively inhibit the growth of the mixed bacteria, and the addition of the Artocarpus heterophyllus leaf extract and the Acer nikoense leaf extract is more beneficial to the growth of chlorella.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (9)
1. A practical chlorella culture medium is characterized in that each liter of culture medium comprises the following components: 0.5-1.0g of macroelement water-soluble fertilizer, 0.25-0.5g of microelement water-soluble fertilizer, 0.26-0.5g of anhydrous sodium acetate, 1-2mL of soil leachate, 1-2mL of Artocarpus heterophyllus leaf extract, 1-2mL of Acer buergerianum leaf extract and the balance of water.
2. The practical chlorella culture medium according to claim 1, wherein the soil leachate is prepared by the following method: weighing sodium bicarbonate, dissolving in water to obtain sodium bicarbonate water, adding soil into sodium bicarbonate water, stirring for 1-3min, soaking for 15-25min, filtering with 8 layers of gauze, and collecting supernatant to obtain soil leachate.
3. The practical chlorella culture medium according to claim 1, wherein the preparation method of the honey bolea leaf extract comprises: cleaning and drying the seeds of Artocarpus heterophyllus in the sun, crushing, sieving with a 80-100 mesh sieve to obtain Artocarpus heterophyllus powder, mixing the Artocarpus heterophyllus powder with an ethanol solution, soaking in a sealed condition 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 Artocarpus heterophyllus leaf extract.
4. The practical chlorella culture medium according to claim 1, wherein the extract of the maple leaves of the bole is prepared by: picking the leaf of the daphniphyllum calycinum, cleaning, drying in the sun, crushing, sieving with a 80-100 mesh sieve to obtain powder of the leaf of the daphniphyllum calycinum, mixing the powder of the leaf of the daphniphyllum calycinum with an ethanol solution, soaking in a sealed condition 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 extract of the leaf of the daphniphyllum calycinum.
5. The practical chlorella culture medium according to claim 1, wherein the chlorella includes chlorella pyrenoidosa and chlorella vulgaris.
6. The practical chlorella culture medium according to claim 1, wherein the preparation method comprises the following steps:
(1) Boiling water, and cooling to room temperature;
(2) Measuring water with the total consumption of 1/2, adding the macroelement water-soluble fertilizer, the trace element water-soluble fertilizer and anhydrous sodium acetate into the water, and stirring and mixing to obtain a mixed solution 1;
(3) Adding the soil leachate, the Artocarpus heterophyllus leaf extract and the Acer nikoense leaf extract into the mixed solution 1, and supplementing water to obtain the practical chlorella culture medium.
7. The practical chlorella culture medium according to claim 2, wherein the mass volume ratio of the baking soda to the water is 4.0-4.5g.
8. The practical chlorella culture medium according to claim 3, wherein the mass ratio of the pineapple honey leaf powder to the ethanol solution is 1.
9. The practical chlorella culture medium according to claim 4, wherein the mass ratio of the maple leaf powder to the ethanol solution is 1.
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