CN113502226A - Culture medium for separating, purifying and amplifying culture of plateau microalgae - Google Patents

Culture medium for separating, purifying and amplifying culture of plateau microalgae Download PDF

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CN113502226A
CN113502226A CN202111011381.XA CN202111011381A CN113502226A CN 113502226 A CN113502226 A CN 113502226A CN 202111011381 A CN202111011381 A CN 202111011381A CN 113502226 A CN113502226 A CN 113502226A
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culture medium
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microalgae
plateau
vitamin
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布多
王金虎
李静
杨俊�
杜梅
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Tibet University
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Abstract

The invention discloses a culture medium for separating, purifying and amplifying culture of plateau microalgae, belonging to the technical field of microalgae biology, and the culture medium comprises: ca (NO)3)2·4H2O、CaCl2·2H2O、NaNO3、KNO3、K2HPO4、MgSO4·7H2O, citric acid, ferric ammonium citrate, EDTA-2Na, Na2CO3Beta-sodium glycerophosphate, vitamin B12, vitamin H, vitamin B1, trace metal solution, 4-hydroxyethyl piperazine ethanesulfonic acid and Na2SiO3·9H2O and 40-60mL/L of soil extract; the invention adopts a method of filtering first, then pre-culturing and then diluting and separating, has simple equipment, simple and convenient operation and small workload, and is particularly suitable for the preliminary separation of the water sample collected from the natural water area. The invention has the characteristics of strong pertinence, short culture period and large culture biomass, andis not easy to be interfered by bacteria, and the sampling operation is simple and convenient.

Description

Culture medium for separating, purifying and amplifying culture of plateau microalgae
Technical Field
The invention relates to the technical field of microalgae biology, in particular to a culture medium for separating, purifying and amplifying culture of plateau microalgae.
Background
Microalgae is an autotrophic microorganism capable of photosynthesis, and has the characteristics of short growth cycle, high photosynthetic efficiency, abundant types of cell metabolites and the like. Meanwhile, the main components of the microalgae cells are active substances with application values, such as polysaccharide, protein, pigment, fatty acid and the like. Therefore, the microalgae has good development prospects in the fields of food, medicine, genetic engineering, liquid fuel and the like. Pure culture of microalgae is essential for research on nutrition, physiology, biochemistry and the like of marine microalgae, but during the culture process of microalgae, the microalgae is usually polluted by other microorganisms, so that the growth speed and the culture density of the microalgae are influenced. Sterile pure algae is the basis for deeply developing the research of algae physiology and genetics, so that pure culture and preservation are the basic and critical links of the application of the pure algae, and are the essential steps for researching the physiology, biochemistry, nutritional value, pharmacology, toxicology and the like of the pure algae.
At present, there are several technical methods for microalgae separation: a micro pipette (capillary tube) separation method, a water droplet separation method, a dilution separation method, and an agar plate method, i.e., a solid medium separation method. These methods all have some disadvantages: the micro straw (capillary) separation method is generally only suitable for separating larger algae, and requires high technical skill level for operators and sufficient care and patience; the water drop separation method is simple and convenient to operate, but is more suitable for separating dominant species appearing in the pre-culture and algae in the culture solution polluted by a small amount of organisms; the dilution separation method is simple and easy to operate, but has certain blindness; the water drop separation method and the dilution method really take a long time to separate and purify. Therefore, by adopting the prior technical methods, a considerable part of microalgae germplasm resources cannot be obtained, and a plurality of high-quality algae species cannot be known.
Disclosure of Invention
The invention aims to provide a culture medium for separating, purifying and amplifying culture of plateau microalgae, which is used for solving the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a culture medium for separating, purifying and amplifying culture of plateau microalgae, which comprises the following components:
Ca(NO3)2·4H2O 0.1-0.2g/L，CaCl2·2H2O 4-8mg/L，NaNO3 1-2g/L，KNO3 0.05-0.15g/L，K2HPO4 0.03-0.05g/L，MgSO4·7H244-52mg/L of O, 6-10mg/L of citric acid, 6-10mg/L of ferric ammonium citrate, 1-1.4mg/L of EDTA-2Na and Na2CO30.01-0.03g/L, 20-30mg/L of beta-sodium glycerophosphate, 120.05-0.15 mu g/L of vitamin B, 0.05-0.15 mu g/L of vitamin H, 18-12 mu g/L of vitamin B, 8-12mL/L of trace metal solution, 0.4-0.6g/L of 4-hydroxyethyl piperazine ethanesulfonic acid and Na2SiO3·9H20.1-0.2g/L of O and 40-60mL/L of soil extract.
The culture medium contains N, P, CO3 2-The nutrient components required by the growth of the microalgae, such as Si, Fe, Ca, Mg, trace elements, vitamins and the like, are very important for the normal growth of the microalgae, particularly for the synthesis of cell walls.
Further, the pH of the medium is 6.8 to 7.2.
Further, the pH of the medium is adjusted with 0.01mol/L hydrochloric acid or sodium hydroxide solution.
Further, the trace metal solution comprises the following components:
H3BO3 2.9g/L，MnCl2·4H2O 1.9g/L，ZnSO4·7H2O 0.2g/L，Na2MoO4·2H2O 0.4g/L，CuSO4·5H2O 0.08g/L，Co(NO3)2·6H2O 0.05g/L，ZnCl2·7H2O 0.005g/L，FeCl3·6H2O 0.1g/L，CoCl2·6H2O 0.002g/L。
further, the preparation method of the soil extracting solution comprises the following steps:
mixing the soil without fertilizer with water, heating, cooling, precipitating, filtering, sterilizing the supernatant, and storing.
Further, the culture medium also comprises 25-30g/L nutrient agar culture medium for preparing solid culture medium.
The nutrient agar culture medium is purchased from the market and comprises the following components: 10g of peptone, 3g of beef extract, 5g of sodium chloride and 15-20g of agar.
The invention also provides application of the culture medium for the separation, purification and amplification culture of the plateau microalgae in the separation, purification and amplification of the plateau microalgae.
The invention also provides a method for separating, purifying and amplifying the plateau microalgae, which comprises the step of culturing a plateau water sample by adopting the culture medium.
The invention discloses the following technical effects:
the invention adopts a method of filtering first, then pre-culturing and then diluting and separating, has simple equipment, simple and convenient operation and small workload, is particularly suitable for the preliminary separation of water samples collected from natural waters, and can accelerate the growth of microalgae, clean the growth of fungi and facilitate the operation of transferring and separating by adopting the culture medium.
The invention has the characteristics of strong pertinence, short culture period and large culture biomass, is not easy to be interfered by bacteria, and is simple and convenient to sample and operate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a photograph of a Microcyclus species of Diatoma of a plateau isolated and purified according to the present invention;
FIG. 2 is a photograph of a Microcyclus species of Diatom of the plateau isolated and purified according to the present invention;
FIG. 3 is a picture of the end-stage logarithmic growth of the isolated and purified Cyclotella tenera of the present invention.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
In the following examples, the preparation of the soil extract includes the following steps:
putting 200g of unfertilized soil into a triangular flask, adding 1000mL of distilled water, mixing, sealing the opening of the flask by using a breathable plug, heating in boiling water in a water bath for 3h, cooling, precipitating for 24h, continuously carrying out the process for three times, then filtering, taking supernatant, sterilizing in an autoclave, and storing in a refrigerator at 4 ℃ for later use.
In the embodiment of the invention, the adopted soil is the garden soil which is collected from the university of Tibet and is not fertilized, and meanwhile, the inventor compares the garden soil of a plurality of places to prove that the soil effect of each place is similar, so that the invention does not limit the soil source of the soil extract and only needs the soil which is not fertilized.
The soil extract of the garden soil is subjected to component detection, and the garden soil extract is found to be rich in nutrient components and mainly comprises macrominerals and micronutrients. The major minerals mainly comprise N, P, K, Ca, Mg, and SO4 2-Etc., the micronutrients include: fe. Mn, Cu, Zn, B and the like, and trace minerals such as Co, Cr, Ga, Ge, Mo, Ni, Pb, Sn, V, W and the like.
In the following examples, trace metal solutions, include the following components:
H3BO3 2.9g/L，MnCl2·4H2O 1.9g/L，ZnSO4·7H2O 0.2g/L，Na2MoO4·2H2O 0.4g/L，CuSO4·5H2O 0.08g/L，Co(NO3)2·6H2O 0.05g/L，ZnCl2·7H2O 0.005g/L，FeCl3·6H2O 0.1g/L，CoCl2·6H2O 0.002g/L。
example 1
A culture medium for separating, purifying and amplifying culture of plateau microalgae comprises the following components:
Ca(NO3)2·4H2O 0.15g/L，CaCl2·2H2O 6mg/L，NaNO3 1.5g/L，KNO3 0.1g/L，K2HPO40.04g/L，MgSO4·7H248mg/L of O, 8mg/L of citric acid, 8mg/L of ferric ammonium citrate, 1.2mg/L of EDTA-2Na and Na2CO30.02g/L, 25mg/L of beta-sodium glycerophosphate, 120.1 mu g/L of vitamin B, 0.1 mu g/L of vitamin H, 110 mu g/L of vitamin B, 10mL/L of trace metal solution, 0.5g/L of 4-hydroxyethyl piperazine ethanesulfonic acid, Na2SiO3·9H2O0.15 g/L and soil extract 50 mL/L.
The pH is adjusted to 7.0 with 0.01mol/L hydrochloric acid or sodium hydroxide solution.
The preparation method of the liquid culture medium comprises the following steps: weighing the medicines in the proportion, adding the medicines into a 1000mL conical flask, adding water to a constant volume, oscillating until the medicines are completely dissolved, adjusting the pH to 7.0 by using 1M HCl or NaOH, covering the opening of the conical flask with a rubber plug, filtering in a clean bench by using a 0.22 mu M sterilization membrane, and filtering for later use.
The preparation method of the solid culture medium comprises the following steps: adding 28g of nutrient agar culture medium into the prepared liquid culture medium, heating to 55 ℃ in a water bath kettle, and shaking to completely dissolve. Then, the mixture was filtered through a 0.22 μm sterile filter in a clean bench and was used after filtration. And after the culture medium is cooled to 38 ℃, pouring the culture medium into a sterile culture dish, and continuously cooling to obtain the solid culture medium.
Example 2
A culture medium for separating, purifying and amplifying culture of plateau microalgae comprises the following components:
Ca(NO3)2·4H2O 0.2g/L，CaCl2·2H2O 4mg/L，NaNO3 2g/L，KNO3 0.05g/L，K2HPO40.05g/L，MgSO4·7H2o44 mg/L, citric acid 10mg/L, ferric ammonium citrate 6mg/L, EDTA-2Na 1.4mg/L, Na2CO30.01g/L, 30mg/L of beta-sodium glycerophosphate, 120.05 mu g/L of vitamin B, 0.15 mu g/L of vitamin H, 18 mu g/L of vitamin B, 12mL/L of trace metal solution, 0.4g/L of 4-hydroxyethyl piperazine ethanesulfonic acid, Na2SiO3·9H2O0.2 g/L and soil extract 40 mL/L.
The preparation method of the liquid culture medium comprises the following steps: weighing the medicines in the proportion, adding the medicines into a 1000mL conical flask, adding water to a constant volume, oscillating until the medicines are completely dissolved, adjusting the pH to 6.8 by using 1M HCl or NaOH, covering the opening of the conical flask with a rubber plug, filtering by using a 0.22 mu M sterilizing membrane in a clean bench, and filtering for later use.
The preparation method of the solid culture medium comprises the following steps: adding nutrient agar culture medium 30g into the prepared liquid culture medium, heating to 50 deg.C with water bath kettle, and shaking to dissolve completely. Then, the mixture was filtered through a 0.22 μm sterile filter in a clean bench and was used after filtration. And after the culture medium is cooled to 40 ℃, pouring the culture medium into a sterile culture dish, and continuously cooling to obtain the solid culture medium.
Example 3
A culture medium for separating, purifying and amplifying culture of plateau microalgae comprises the following components:
Ca(NO3)2·4H2O 0.1g/L，CaCl2·2H2O 8mg/L，NaNO3 1g/L，KNO3 0.15g/L，K2HPO40.03g/L，MgSO4·7H2o52 mg/L, citric acid 6mg/L, ferric ammonium citrate 10mg/L, EDTA-2Na 1mg/L, Na2CO30.03g/L, 20mg/L of beta-sodium glycerophosphate, 120.15 mu g/L of vitamin B, 0.05 mu g/L of vitamin H, 112 mu g/L of vitamin B, 8mL/L of trace metal solution, 0.6g/L of 4-hydroxyethyl piperazine ethanesulfonic acid, Na2SiO3·9H2O0.1 g/L and soil extract 60 mL/L.
The preparation method of the liquid culture medium comprises the following steps: weighing the medicines in the proportion, adding the medicines into a 1000mL conical flask, adding water to a constant volume, oscillating until the medicines are completely dissolved, adjusting the pH to 7.2 by using 1M HCl or NaOH, covering the opening of the conical flask with a rubber plug, filtering by using a 0.22 mu M sterilizing membrane in a clean bench, and filtering for later use.
The preparation method of the solid culture medium comprises the following steps: adding 25g of nutrient agar culture medium into the prepared liquid culture medium, heating to 60 ℃ in a water bath kettle, and shaking to completely dissolve. Then, the mixture was filtered through a 0.22 μm sterile filter in a clean bench and was used after filtration. And after the culture medium is cooled to 35 ℃, pouring the culture medium into a sterile culture dish, and continuously cooling to obtain the solid culture medium.
Comparative example 1
The difference from example 1 is that no soil extract was added to the medium components.
Experimental example 1 separation, screening and identification of plateau microalgae
The media prepared in example 1 and comparative example 1 were used, respectively, and the samples were filtered, pre-cultured, and then diluted and separated by plate coating.
(1) By filtering before preculture
And under the condition that the purification workbench is opened, sequentially pouring the collected water samples into a Buchner funnel, filtering under the suction filtration of a water ring vacuum pump, and selecting a 0.45-micron water system filter membrane as the filter paper.
Filtering with a total volume of 100L water sample for three times, replacing a new filter membrane each time, and putting the replaced filter membrane into a liquid culture medium prepared in advance for culture. Three times of filtration can obtain 3 parallel groups of culture solution. The bottom mud collected from the water sample barrel does not need to be filtered, and about 30g of bottom mud in each group is independently put into a liquid culture medium for culture, and three groups are needed in parallel. The top of the conical flask is sealed by a breathable sealing film to prevent external bacteria from entering.
Shaking regularly 3 times daily in an illumination incubator (light-to-dark ratio 12 h: 12h, illumination intensity 2000Lx) at 25 + -1 deg.C, and randomly placing each sample position to allow uniform illumination. Culturing for 7-15 days until the whole solution system is yellow green or green, and performing microscopic examination and dilution to separate algae strains.
(2) Method for separating algae strain with high biomass by pre-culture solution dilution separation method and solid medium coating method
Fully and uniformly mixing the pre-cultured microalgae culture solution samples, adding 0.5mL of the uniformly mixed sample into a test tube containing 4.5mL of sterile water in an ultra-clean workbench, uniformly mixing, sequentially carrying out gradient dilution by 10-100 times, carrying out microscopic examination while diluting, stopping dilution until each drop of the sample in a visual field only contains 1 cell, and starting tapping culture. Then, 200. mu.L of the suspension was applied to a solid medium from a test tube of the corresponding concentration, and the suspension was irradiated with light at a temperature of 25. + -. 1 ℃ and a light intensity of 2000 Lx. Inoculating the separated and purified algae strain into 200mL liquid culture medium, culturing in a light incubator with the illumination intensity of 2000Lx and the temperature of 25 +/-1 ℃ (the light-dark ratio is 12 h: 12h), shaking slightly three times daily, culturing for 7-15 days, performing microscopic examination after the color of algae is found in a test tube, and if single species is detected by microscopic examination, successfully separating, as shown in figures 1 and 2, the figure is the separated and purified chlorella of the plateau diatom phylum, and at this time, the purposes of separation and purification are achieved, and the expanded culture can be performed.
The test results are shown in table 1, and it can be seen from table 1 that the culture period of comparative example 1 without adding the soil extract is increased, the growth of algae is slow, white algae solids appear in the solution, and the culture effect of microalgae is affected.
TABLE 1
Figure BDA0003239095780000091
Experimental example 2 laboratory purification culture of microalgae
The solid medium prepared in example 2 was used.
The inoculating loop is dipped with algae liquid inoculum (inoculum preparation: fresh algae liquid at the late logarithmic growth stage of about l0 days in culture is centrifuged for 5-10min at 5000rpm, the supernatant is discarded, the culture liquid is washed once, and the culture liquid is suspended for inoculation (the experimental operation is carried out under aseptic condition), and the dense streaking is carried out.
Placing at 25 +/-1 ℃,2000Lx illumination intensity, light-dark ratio of 12 h: culturing for 12h in a light incubator for 7-15 days, transferring single algae colony in a culture dish by using a sterile inoculating ring under a clean workbench, then performing microscopic examination under a microscope, if single algae is present, indicating that the separation method is reliable, inoculating the separated and purified algae strain into 200mL of liquid culture medium, culturing in a light incubator with the light intensity of 2000Lx and the temperature of 25 +/-1 ℃ (the light-dark ratio is 12 h: 12h), slightly shaking for three times every day, culturing for 7-15 days, performing microscopic examination after the fact that the test tube has algae color, and if single algae is detected, indicating that the separation is successful.
Experimental example 3 laboratory microalgae amplification culture
The liquid medium prepared in example 1 was used.
(1) The microalgae with better growth are picked by a sterilized inoculating loop and dropped into a centrifuge tube filled with lmL water for injection and sterilized, and the mixture is shaken by an oscillator.
(2) And (3) transferring the microalgae which are blown and beaten uniformly into a liquid culture medium filled with 50mL, culturing for 20-30 days at 25 +/-1 ℃ under illumination of 2000Lx, slightly shaking for three times every day, and transferring again, wherein the growth state of the microalgae is good, and the biomass is obviously visible.
(3) 50mL of the cultured algal solution was added to 250mL of a liquid medium and cultured (in a ratio of 1: 5) with gentle shaking three times a day.
(4) Normal culture conditions: 25 +/-1 ℃,200 r/min, 3000Lx illumination intensity, light-dark ratio of 12 h: 12h, the culture period is 45 days, then an ultraviolet spectrophotometer is used for detecting whether the absorbance, namely the optical density value, corresponding to the characteristic absorption wavelength of the microalgae reaches 0.7-0.8, if the absorbance does not reach the value, then inoculation is carried out, the microalgae liquid is centrifuged for 5-10min at 5000rpm, the microalgae at the bottom of the centrifuge tube is taken out and combined, then the microalgae liquid is put into a liquid culture medium with the same volume before centrifugation for continuous culture, the culture period is 45 days until the biomass optical density value of the microalgae reaches 0.7-0.8 (the absorbance corresponding to the characteristic absorption wavelength of the microalgae) at the last stage of growth, and then the microalgae is separated and used for other subsequent experimental purposes.
Experimental example 4 microalgae Biomass amplification protocol
Open raceway pond: the organic glass material is 20cm high, 20cm wide and 40cm long, the culture solution is 5-10 cm deep, the stirring speed is 300-500 rpm, the suitable biomass range is 400-500 mg/L of dry weight, namely the optical density OD of the algae solution at the late logarithmic growth stage4200.7-0.8, and the pH is controlled by sodium bicarbonate, but the sodium ion is increased by introducing CO2To solve the problem.
The Cyclotella tenera separated and purified in test example 1 was inoculated into an open raceway pond, cultured for 45 days, and then OD was measured by an ultraviolet spectrophotometer420That is, whether the optical density value reaches 0.7-0.8, if notAnd then inoculating the absorbance value, centrifuging the algae solution for 5-10min at 5000rpm, taking out and combining the microalgae at the bottom of the centrifugal tube, putting the combined microalgae into a liquid culture medium with the same volume before centrifugation, and continuously culturing for 45 days until the biomass of the microalgae reaches the final growth stage, wherein the biomass is 0D as shown in figure 3420＝0.793。
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (8)

1. A culture medium for separating, purifying and amplifying culture of plateau microalgae is characterized by comprising the following components:
Ca(NO3)2·4H2O 0.1-0.2g/L，CaCl2·2H2O 4-8mg/L，NaNO3 1-2g/L，KNO30.05-0.15g/L，K2HPO4 0.03-0.05g/L，MgSO4·7H244-52mg/L of O, 6-10mg/L of citric acid, 6-10mg/L of ferric ammonium citrate, 1-1.4mg/L of EDTA-2Na and Na2CO30.01-0.03g/L, 20-30mg/L of beta-sodium glycerophosphate, 120.05-0.15 mu g/L of vitamin B, 0.05-0.15 mu g/L of vitamin H, 18-12 mu g/L of vitamin B, 8-12mL/L of trace metal solution, 0.4-0.6g/L of 4-hydroxyethyl piperazine ethanesulfonic acid and Na2SiO3·9H20.1-0.2g/L of O and 40-60mL/L of soil extract.
2. The culture medium according to claim 1, wherein the pH of the culture medium is 6.8 to 7.2.
3. The culture medium according to claim 2, wherein the pH of the culture medium is adjusted with 0.01mol/L hydrochloric acid or sodium hydroxide solution.
4. The culture medium according to claim 1, wherein the trace metal solution comprises the following components:
H3BO3 2.9g/L，MnCl2·4H2O 1.9g/L，ZnSO4·7H2O 0.2g/L，Na2MoO4·2H2O0.4g/L，CuSO4·5H2O 0.08g/L，Co(NO3)2·6H2O 0.05g/L，ZnCl2·7H2O0.005g/L，FeCl3·6H2O 0.1g/L，CoCl2·6H2O 0.002g/L。
5. the culture medium according to claim 1, wherein the preparation method of the soil extract comprises the following steps:
mixing the soil without fertilizer with water, heating, cooling, precipitating, filtering, sterilizing the supernatant, and storing.
6. The culture medium of claim 1, further comprising 25-30g/L nutrient agar medium.
7. Use of the culture medium for plateau microalgae isolation, purification and amplification culture as claimed in any one of claims 1-6 in plateau microalgae isolation, purification and amplification.
8. A method for separating, purifying and amplifying plateau microalgae, which is characterized by comprising the step of culturing a plateau water sample by using the culture medium of any one of claims 1 to 6.
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