CN115141755B - Method for separating and culturing aerobiotic microalgae - Google Patents
Method for separating and culturing aerobiotic microalgae Download PDFInfo
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- CN115141755B CN115141755B CN202210991240.7A CN202210991240A CN115141755B CN 115141755 B CN115141755 B CN 115141755B CN 202210991240 A CN202210991240 A CN 202210991240A CN 115141755 B CN115141755 B CN 115141755B
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- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 6
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for separating and culturing aerobiotic microalgae, which is characterized in that a culture solution rich in inorganic nutrient sources and organic carbon sources is prepared, a liquid culture medium and a solid culture medium are obtained based on the culture solution, enrichment culture and expansion culture are carried out by using the liquid culture medium, repeated separation and purification culture is carried out by using the solid culture medium, and the periphery of an algae spot with good characteristic consistency is selected and transferred into the liquid culture medium for proliferation culture, and then pure-seed detection is carried out. The invention can better separate the object of the gas-borne microalgae from the natural environment, effectively promote the growth of the gas-borne microalgae, reduce the influence of bacteria and fungi, and effectively separate and purify the gas-borne microalgae.
Description
Technical Field
The invention relates to the technical field of algae culture, in particular to a separation culture method of aerobiotic microalgae.
Background
Microalgae generally refer to microalgae that need to be identified under a microscope to form, and can be classified into aquatic microalgae, aerogenic microalgae and terrestrial microalgae according to the growth environment. The aerobiotic microalgae generally refers to algae which are directly attached to objects such as rocks, trees, concrete and the like without soil in the growth environment of the microalgae and are exposed to the air for growth and propagation.
The aerobiotic microalgae is an autotrophic plant with wide distribution, rich nutrition and high photosynthetic availability. Compared with conventional aquatic microalgae, the aerobiotic microalgae can adapt to environmental changes faster and can grow in more extreme environments; secondly, the aerobiotic microalgae is usually attached to the surface of rock or concrete for growth, so that the aerobiotic microalgae is utilized for restoring the environment of the bare rock slope, and has a very good application prospect. In addition, the aerobiotic microalgae have more carotenoid, astaxanthin and the like compared with other growth-type microalgae, and have stronger capability of generating substances such as polysaccharide, protein, pigment and the like by cell metabolism, so that the aerobiotic microalgae have good development prospect in the fields of food, medicine, genetic engineering, liquid fuel and the like.
Algae growing in natural ecosystems often form complex symbiotic systems with various microorganisms, and aerobiotic microalgae in natural environments grow on surfaces of rocks, bark, buildings and the like and also form bacterial-algal symbiotic systems with bacteria in the environments. Therefore, to enhance the application and research of the aerobiotic microalgae, the separation and purification of the algae species are required, and the required target microalgae are separated from the zoobiotic system by a certain method from the mixed biological community in the natural environment, which is an indispensable technology for the research of the aerobiotic microalgae. At present, the isolated culture of the aerobiotic microalgae is more difficult than that of the aquatic microalgae due to the special growth characteristics of the aerobiotic microalgae. In the prior art, no method for separating and purifying the aerobiotic microalgae exists, and compared with the culture of the aquatic microalgae, the method for separating and purifying the aerobiotic microalgae and the research on the culture are relatively deficient.
Therefore, how to provide a way for separating and culturing the aerobiotic microalgae, so that the aerobiotic microalgae target object which can be better used for research and application can be better separated and cultured from the natural environment, and the method is a problem to be considered and solved by the person skilled in the art.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to solve the technical problems that: how to provide a method for separating and culturing gas-borne microalgae, which can separate and culture gas-borne microalgae targets better from natural environment, so that the method can be used for researching and applying gas-borne microalgae better.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for separating and culturing the aerobiotic microalgae is characterized by preparing a culture solution rich in inorganic nutrient source and organic carbon source, obtaining a liquid culture medium and a solid culture medium based on the culture solution, carrying out enrichment culture and expansion culture by using the liquid culture medium, carrying out repeated separation and purification culture by using the solid culture medium, selecting the periphery of the algae spots with good characteristic consistency, transferring the periphery to the liquid culture medium for proliferation culture, and then carrying out pure strain detection.
The method specifically comprises the following steps:
a, preparing a culture solution for standby, wherein the culture solution comprises inorganic nutrient source components and organic carbon source components, and the inorganic nutrient source components comprise ionic sodium, potassium, magnesium, calcium, iron, boron, manganese, zinc, copper and cobalt elements;
b, enrichment culture, namely collecting the gas-borne microalgae of a target to be cultured, cleaning the collected gas-borne microalgae sample by using sterile water to obtain a gas-borne microalgae solution, and adding a culture solution with a small proportion (which is less than the adding proportion in the step c and is usually multiple times of the microalgae solution) for proliferation culture to obtain the microalgae solution after proliferation of the gas-borne microalgae;
c, carrying out expanded culture on the aerobiotic microalgae, taking part of the enriched culture algae liquid, adding a large proportion of culture liquid for proliferation culture, and obtaining the algae liquid with high concentration;
d, inoculating and culturing the aerobiotic microalgae, adding agar into the culture solution prepared in the step a to prepare a solid culture medium, diluting the algae solution obtained in the step c at least once by using sterile water, then coating and inoculating the algae solution onto the surface of the solid culture medium, and carrying out proliferation culture to obtain macroscopic aerobiotic microalgae communities on the solid culture medium;
e, purifying and culturing the aerobiotic microalgae, selecting part of the periphery of the spots with the characteristic consistency from the aerobiotic microalgae community obtained in the step d, diluting with sterile water, streaking and inoculating to the surface of a new solid culture medium, and performing at least one repeated proliferation culture; selecting part of the periphery of the algae spots with good feature consistency from the aerobiotic microalgae community obtained by repeated multiplication culture, and transferring the selected part of the periphery of the algae spots into a new vessel filled with liquid culture solution for multiplication culture;
more specifically, the e-step repeated proliferation culture may specifically be: and d, selecting part of the periphery of the algae spots with the characteristic consistency from the aerobiotic microalgae community obtained in the step d, diluting with sterile water, scribing on the surface of a new solid culture medium, transferring the periphery of the algae spots with the good characteristic consistency to a new conical flask filled with liquid culture solution for proliferation culture after the algae spots grow out, diluting the culture solution with sterile water after the microalgae are fully proliferated in the culture solution, scribing on the surface of the new solid culture medium, and repeating the steps for at least three times.
f, checking the pure strain, sucking the algae liquid obtained in the step e, and separating and culturing the aerogenic microalgae with the content larger than a preset value through naked eye observation and/or microscopic observation, so that the culture is qualified.
In this way, the method adopts the nutrient solution which is rich in a plurality of metal elements and trace elements, so that the method can be widely applied to the culture of different kinds of aerobiotic microalgae, and can promote the rapid growth and propagation of the aerobiotic microalgae. Through repeated separation, dilution and multiplication culture in various modes, the growth of adverse microorganism components such as bacteria in the culture solution is inhibited, so that the aerobiotic microalgae components are rapidly proliferated, and finally, pure aerobiotic microalgae can be rapidly and efficiently obtained. The method adopts liquid culture medium enrichment and expansion culture at the early stage, adopts solid medium to repeatedly streak, separate and purify culture at the later stage, and finally adopts liquid culture medium to proliferate, thereby being more beneficial to removing non-target algae and fungi and obtaining pure-bred target aerobiotic microalgae.
Further, in the step a, part of culture solution is adopted for concentration to obtain a feed supplement for standby; and e, adding part of feed after each dilution, and controlling the total nitrogen content in the culture system to be not less than 200mg/L.
Thus, the constant nutrition condition after dilution is better ensured, and the proliferation culture of the target object is facilitated. In the implementation, the total nitrogen concentration is measured by periodically performing alkaline potassium persulfate digestion ultraviolet spectrophotometry, so that the nutrient concentration in the culture medium can be effectively stabilized by determining the feed addition amount by measuring the nitrogen source content in the culture medium. The specific assay procedure is prior art and is not described in detail herein.
Further, in each step, in proliferation culture, at an illumination intensity of 9000-10000lux, light-dark ratio=16: 8, the culture is realized in an illumination incubator at the temperature of 25-28 ℃, ultraviolet light is used for irradiating for 1h every 12h in the culture process, and mixed sterile air with 3-5% carbon dioxide content is introduced as respiratory gas (aeration treatment can be directly carried out when a liquid culture medium is used).
Thus, the proliferation culture effect can be better ensured.
Further, in the preparation process of the culture solution, the additive components comprise sodium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, EDTA (ethylene diamine tetraacetic acid), magnesium sulfate heptahydrate, calcium chloride dihydrate, ferric ammonium citrate, sodium carbonate, boric acid, manganese chloride monohydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, cobalt nitrate hexahydrate and anhydrous glucose.
The formula is prepared aiming at the culture and propagation of the aerobiotic microalgae, the culture solution contains sufficient various nutritional ingredients and trace elements which are reasonably matched, and all substances are easily dissolved in water and generate ionic mixtures, so that the growth and propagation requirements of the aerobiotic microalgae of various different categories can be met, the practicability is improved, and all the components are safe and harmless; the anhydrous glucose is used as an organic carbon source, so that the carbon source requirement of algae cultivation can be well met; the ethylenediamine tetraacetic acid serving as the chelating agent is favorable for absorbing and utilizing various metal ion elements serving as nutrient sources by the aerogenic microalgae, and greatly improves the proliferation speed of the aerogenic microalgae.
Further, the concentration of each component in the culture solution is 1.5g/L, 0.04g/L of monopotassium phosphate, 0.088g/L of dipotassium phosphate, 0.001g/L of EDTA0.075 g/L of magnesium sulfate heptahydrate, 0.036g/L of calcium chloride dihydrate, 0.0006g/L of ferric ammonium citrate, 0.02g/L of sodium carbonate, 0.00286g/L of boric acid, 0.00181g/L of manganese chloride monohydrate, 0.000222g/L of zinc sulfate heptahydrate, 0.000079g/L of copper sulfate pentahydrate, 0.00039g/L of sodium molybdate dihydrate, 0.000049g/L of cobalt nitrate hexahydrate and 0.01g/L of anhydrous glucose.
Thus, the collocation effect of each nutrient substance can be optimized and kept harmless.
Further, rose oil was added to the culture medium at a concentration of 0.01 ml/L.
The rose oil, namely the rose essential oil, belongs to pure natural plant essential oil, has mild sex condition and a certain sterilization effect, and by adding the components in a proper proportion, the terpene compounds in the rose oil can be utilized to effectively inhibit bacterial growth in the early stage of cultivation, but the growth of the aerobiotic microalgae is not influenced, and the proliferation and purification of the aerobiotic microalgae are facilitated. In the later stage, various lipid and alcohol components in the rose oil can be gradually decomposed and used as a nutrient source for absorbing and utilizing the raw microalgae, so that the defect of insufficient carbon source in the later stage of culture is overcome.
Further, when preparing the culture medium, the pH was adjusted to 7-8 by adding each component and then using 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide solution.
The pH value is more favorable for propagation of aerobiotic microalgae.
Further, in the preparation process of the culture solution, the preparation solution is adopted to soak attachments (usually attached rocks) of the collected gas-borne microalgae, and the culture solution is soaked for more than 2 days at normal temperature (usually 25-28 ℃).
Therefore, the method is beneficial to dissolving the components on the surface of the attachment of the gas-borne microalgae into the culture solution, greatly increasing the affinity of the gas-borne microalgae to the aquatic environment of the culture solution, promoting the gas-borne microalgae to realize the primary proliferation and cultivation effect and the growth rate improvement in the water environment of the culture solution, and also facilitating the culture solution to select the gas-borne microalgae which are correspondingly matched for proliferation and cultivation so as to finish enrichment cultivation. Especially when cultivating the gas-borne microalgae and being used for carrying out bare rock slope environment restoration, the gas-borne microalgae collected in the environment to be restored is usually selected for cultivation, so that the culture solution obtained by soaking the attachments of the collected gas-borne microalgae is used for proliferation cultivation in a combined mode, the cultivation is facilitated, the selection of stronger species which are more suitable for survival of the environment to be restored is facilitated, and the subsequent use effect in the process of restoring the bare slope is better improved. More specifically, for example, when there are strong species and weak species in the collected aerobiotic microalgae for cultivation, because the air environments of the two are the same, the reason why the strong species obtain growth advantages will generally be partly because the microalgae of the type and the attached substances are more matched in composition and are more suitable for their growth; therefore, after the culture solution is treated by the method of soaking the attachments of the collected gas-borne microalgae by the modulating solution, the water environment of the culture solution obviously improves the affinity to the strong species of microalgae, is more beneficial to the proliferation and cultivation of the strong species, is finally beneficial to the cultivation, selection and purification of the strong species of microalgae, and is naturally beneficial to the restoration and use of the exposed side slope.
In the step a, a gas-borne microalgae culture solution with a unique preparation mode (which is beneficial to culturing gas-borne microalgae for repairing bare rock environment) is provided, wherein the nutrient components are reasonably matched, anhydrous glucose is used as an organic carbon source, rose oil is added, the characteristic that terpenoid in the rose oil can effectively inhibit bacterial growth is utilized, and finally the culture solution is used for soaking the rock on which the gas-borne microalgae grows in the natural environment, so that a novel culture medium is obtained, and a proper carbon source and nitrogen source are provided for the growth of the gas-borne microalgae. Compared with the culture medium used for culturing general microalgae, the culture medium has the advantages of effectively promoting the growth of the aerobiotic microalgae and having strong antibacterial effect, thereby being beneficial to the separation and purification of the aerobiotic microalgae.
In the step b, the cleaning is carried out by adopting 100Hz ultrasonic oscillation for 30min, so that the aerogenic microalgae and attachments are fully separated and enter into sterile water, the obtained aerogenic microalgae solution is uniformly shaken and kept stand (about 10 min) for precipitation, and then the supernatant is taken out for proliferation culture for 3 days. Therefore, the collected gas-borne microalgae and attachments thereof can enter the cleaning water better by adopting an ultrasonic oscillation mode, so that the content of the microalgae is improved, and meanwhile, the substance components of part of attachments are dissolved in the cleaning water, so that the affinity of the water body cultivation environment to the gas-borne microalgae can be improved, and the proliferation cultivation of the gas-borne microalgae in the water body environment is facilitated.
Further, in the step c, the culture solution with the volume ratio of 20 times is adopted for proliferation culture for 14 days; ensure the expansion culture effect. In the implementation, a 250ml wide-mouth conical flask can be used as a culture container, 95ml of the culture solution in the step a is added, and then 5ml of the aerobiotic microalgae enrichment solution is inoculated for proliferation culture.
Further, in the step d, a solid medium was prepared according to 15g/L of added agar. Ensuring the effect of the solid culture medium.
In the step d, during dilution, 4ml of sterile water is added into each of a plurality of sterilized sample tubes, 1ml of the algae liquid after the expansion culture is firstly taken and added into a first sample tube for shaking uniformly; then, 1ml of algae liquid is taken from the first sample tube, added into the second sample tube and uniformly shaken, and subjected to gradient dilution in sequence; 1ml of the algae liquid was spread on a solid medium from a sample tube with three dilution concentrations, and proliferation culture was performed for 7 days. Therefore, the dilution of other impurity algae or fungi can be well completed, and the purification and proliferation of target algae are facilitated.
Further, the method comprises the step g: and d, coating the algae liquid obtained in the step f on a solid culture medium for bacterial cultivation, and culturing for 2d in a light incubator at 25-28 ℃, wherein if no bacteria grow, the pure seed separation is successful.
Therefore, the separated pure-breed aerobiotic microalgae can be better ensured to contain no or little bacterial components, and is more beneficial to being used for repairing the bare rock slope.
Further, all the culture products were sterilized at high temperature and high pressure (121 ℃,20 min) or ultraviolet (30 min). Avoiding the influence of new bacteria and fungi introduced in the cultivation process on the cultivation effect.
Compared with the technology for separating microalgae by using a general solid culture medium, the method has the beneficial effects that: (1) The method solves the problem that the separation and purification of the general inorganic solid culture medium is not ideal, the gas-borne microalgae are very suitable for rapid growth on the solid culture medium, and through experiments, dominant microalgae communities can be formed in a short time, namely, the method can form the gas-borne microalgae communities meeting the separation requirement before mold and bacteria form colonies on the culture medium, so that the method has good separation and purification effects on the gas-borne microalgae, and is very suitable for the purification and separation of the gas-borne microalgae. Can also be combined with the physical and chemical mutagenesis of the aerogenic microalgae gene work to realize the rapid acquisition of satisfactory improved algae species. (2) The invention can realize the growth of short-term large biomass of the aerobiotic microalgae, and can form large aerobiotic microalgae communities in 7 days when streaking growth is carried out, thus being very beneficial to subsequent large-scale liquid culture, and pure aerobiotic microalgae with higher concentration can be obtained in about 10 days, while other solid culture mediums can grow slowly, even can not grow, have long separation period and are easy to be infected by mould. The method has the advantages of good effect of separating and purifying the gas-borne microalgae, low workload, and easy operation, and overcomes the defect of complex procedure of separating the gas-borne microalgae by the prior general solid culture medium.
In conclusion, the method can separate the object of the aerobiotic microalgae better from the natural environment, can effectively promote the growth of the aerobiotic microalgae, and reduce the influence of bacteria and fungi, thereby effectively separating and purifying the aerobiotic microalgae.
Drawings
FIG. 1 is a photograph showing the result of the culture in test example 1.
FIG. 2 is a photograph showing the result of the culture in comparative example 1.
FIG. 3 is a photograph showing the result of streaking inoculation in test example 2.
FIG. 4 is a photograph showing the result of the streaking inoculation in comparative example 2.
FIG. 5 is a photograph showing the result of the plating-type inoculation in test example 2.
FIG. 6 is a photograph showing the result of the plating-type inoculation in comparative example 2.
FIG. 7 is a photograph showing the result of microscopic examination of the streak inoculation in test example 2.
FIG. 8 is a photograph showing the result of microscopic examination of the streak inoculation in comparative example 2.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments and test comparative examples.
The specific embodiment is as follows:
a method for separating and culturing the aerobiotic microalgae includes such steps as preparing the culture liquid rich in inorganic nutritive source and organic carbon source, preparing liquid culture medium and solid culture medium, enriching and amplifying, culturing, repeatedly separating and purifying, choosing the periphery of the spots, culturing, and detecting pure strain.
The method specifically comprises the following steps:
a, preparing a culture solution for standby, wherein the culture solution comprises inorganic nutrient source components and organic carbon source components, and the inorganic nutrient source components comprise ionic sodium, potassium, magnesium, calcium, iron, boron, manganese, zinc, copper and cobalt elements;
wherein, in the preparation process of the culture solution, the additive components comprise sodium nitrate, monopotassium phosphate, dipotassium phosphate, EDTA (ethylene diamine tetraacetic acid), magnesium sulfate heptahydrate, calcium chloride dihydrate, ferric ammonium citrate, sodium carbonate, boric acid, manganese chloride monohydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, cobalt nitrate hexahydrate and anhydrous glucose.
The formula is prepared aiming at the culture and propagation of the aerobiotic microalgae, the culture solution contains sufficient various nutritional ingredients and trace elements which are reasonably matched, and all substances are easily dissolved in water and generate ionic mixtures, so that the growth and propagation requirements of the aerobiotic microalgae of various different categories can be met, the practicability is improved, and all the components are safe and harmless; the anhydrous glucose is used as an organic carbon source, so that the carbon source requirement of algae cultivation can be well met; the ethylenediamine tetraacetic acid serving as the chelating agent is favorable for absorbing and utilizing various metal ion elements serving as nutrient sources by the aerogenic microalgae, and greatly improves the proliferation speed of the aerogenic microalgae.
Wherein the content concentration of each component in the culture solution is 1.5g/L, 0.04g/L of monopotassium phosphate, 0.088g/L of dipotassium phosphate, 0.001g/L of EDTA0.075 g/L of magnesium sulfate heptahydrate, 0.036g/L of calcium chloride dihydrate, 0.0006g/L of ferric ammonium citrate, 0.02g/L of sodium carbonate, 0.00286g/L of boric acid, 0.00181g/L of manganese chloride monohydrate, 0.000222g/L of zinc sulfate heptahydrate, 0.000079g/L of copper sulfate pentahydrate, 0.00039g/L of sodium molybdate dihydrate, 0.000049g/L of cobalt nitrate hexahydrate and 0.01g/L of anhydrous glucose. Thus, the collocation effect of each nutrient substance can be optimized and kept harmless.
The culture solution is also added with rose oil with the content of 0.01 ml/L. The rose oil, namely the rose essential oil, belongs to pure natural plant essential oil, has mild sex condition and a certain sterilization effect, and by adding the components in a proper proportion, the terpene compounds in the rose oil can be utilized to effectively inhibit bacterial growth in the early stage of cultivation, but the growth of the aerobiotic microalgae is not influenced, and the proliferation and purification of the aerobiotic microalgae are facilitated. In the later stage, various lipid and alcohol components in the rose oil can be gradually decomposed and used as a nutrient source for absorbing and utilizing the raw microalgae, so that the defect of insufficient carbon source in the later stage of culture is overcome.
When preparing the culture medium, each component was added, and then the pH was adjusted to about 7.5 with 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide solution. The pH value is more favorable for propagation of aerobiotic microalgae.
In the preparation process of the culture solution, the preparation solution is adopted to soak attachments (usually attached rocks) of the collected aerobiotic microalgae for more than 2 days at normal temperature (usually 25-28 ℃).
Therefore, the method is beneficial to dissolving the components on the surface of the attachment of the gas-borne microalgae into the culture solution, greatly increasing the affinity of the gas-borne microalgae to the aquatic environment of the culture solution, promoting the gas-borne microalgae to realize the primary proliferation and cultivation effect and the growth rate improvement in the water environment of the culture solution, and also facilitating the culture solution to select the gas-borne microalgae which are correspondingly matched for proliferation and cultivation so as to finish enrichment cultivation. Especially when cultivating the gas-borne microalgae and being used for carrying out bare rock slope environment restoration, the gas-borne microalgae collected in the environment to be restored is usually selected for cultivation, so that the culture solution obtained by soaking the attachments of the collected gas-borne microalgae is used for proliferation cultivation in a combined mode, the cultivation is facilitated, the selection of stronger species which are more suitable for survival of the environment to be restored is facilitated, and the subsequent use effect in the process of restoring the bare slope is better improved. More specifically, for example, when there are strong species and weak species in the collected aerobiotic microalgae for cultivation, because the air environments of the two are the same, the reason why the strong species obtain growth advantages will generally be partly because the microalgae of the type and the attached substances are more matched in composition and are more suitable for their growth; therefore, after the culture solution is treated by the method of soaking the attachments of the collected gas-borne microalgae by the modulating solution, the water environment of the culture solution obviously improves the affinity to the strong species of microalgae, is more beneficial to the proliferation and cultivation of the strong species, is finally beneficial to the cultivation, selection and purification of the strong species of microalgae, and is naturally beneficial to the restoration and use of the exposed side slope.
In the step a, the gas-borne microalgae culture solution with a unique preparation mode is provided, wherein all nutrient components are reasonably matched, anhydrous glucose is used as an organic carbon source, rose oil is added, the characteristic that terpenoid in the rose oil can effectively inhibit bacterial growth is utilized, and finally the gas-borne microalgae are soaked in the culture solution to adhere to and grow on rocks in a natural environment, so that a novel culture medium is obtained, and a proper carbon source and a proper nitrogen source are provided for the growth of the gas-borne microalgae. Compared with the culture medium used for culturing general microalgae, the culture medium has the advantages of effectively promoting the growth of the aerobiotic microalgae and having strong antibacterial effect, thereby being beneficial to the separation and purification of the aerobiotic microalgae.
b, enrichment culture, namely collecting the gas-borne microalgae of a target to be cultured, cleaning the collected gas-borne microalgae sample by using sterile water to obtain a gas-borne microalgae solution, and adding a culture solution with a small proportion (which is less than the adding proportion in the step c and is usually multiple times of the microalgae solution) for proliferation culture to obtain the microalgae solution after proliferation of the gas-borne microalgae;
specifically, in the step b, 100Hz ultrasonic oscillation is adopted for 30min during cleaning, so that the aerobiotic microalgae and attachments are fully separated and enter sterile water, the obtained aerobiotic microalgae solution is uniformly shaken and kept stand (about 10 min) for precipitation, and then the supernatant is taken out for proliferation culture for 3 days. Therefore, the collected gas-borne microalgae and attachments thereof can enter the cleaning water better by adopting an ultrasonic oscillation mode, so that the content of the microalgae is improved, and meanwhile, the substance components of part of attachments are dissolved in the cleaning water, so that the affinity of the water body cultivation environment to the gas-borne microalgae can be improved, and the proliferation cultivation of the gas-borne microalgae in the water body environment is facilitated.
c, carrying out expanded culture on the aerobiotic microalgae, taking part of the enriched culture algae liquid, adding a large proportion of culture liquid for proliferation culture, and obtaining the algae liquid with high concentration;
specifically, in the step c, the culture solution with the volume ratio of 20 times is adopted for proliferation culture for 14 days; ensure the expansion culture effect. In the implementation, a 250ml wide-mouth conical flask can be used as a culture container, 95ml of the culture solution in the step a is added, and then 5ml of the aerobiotic microalgae enrichment solution is inoculated for proliferation culture.
d, inoculating and culturing the aerobiotic microalgae, adding agar into the culture solution prepared in the step a to prepare a solid culture medium, diluting the algae solution obtained in the step c at least once by using sterile water, then coating and inoculating the algae solution onto the surface of the solid culture medium, and carrying out proliferation culture to obtain macroscopic aerobiotic microalgae communities on the solid culture medium;
in step d, a solid medium was prepared according to 15g/L of agar added. Ensuring the effect of the solid culture medium.
d, in the dilution process, adding 4ml of sterile water into each of a plurality of sterilized sample tubes, firstly taking 1ml of the amplified culture algae liquid, adding the algae liquid into a first sample tube, and shaking uniformly; then, 1ml of algae liquid is taken from the first sample tube, added into the second sample tube and uniformly shaken, and subjected to gradient dilution in sequence; 1ml of the algae liquid was spread on a solid medium from a sample tube with three dilution concentrations, and proliferation culture was performed for 7 days. Therefore, the dilution of other impurity algae or fungi can be well completed, and the purification and proliferation of target algae are facilitated.
e, purifying and culturing the aerobiotic microalgae, selecting part of the periphery of the spots with the characteristic consistency from the aerobiotic microalgae community obtained in the step d, diluting with sterile water, streaking and inoculating to the surface of a new solid culture medium, and performing at least one repeated proliferation culture; selecting part of the periphery of the algae spots with good feature consistency from the aerobiotic microalgae community obtained by repeated multiplication culture, and transferring the selected part of the periphery of the algae spots into a new vessel filled with liquid culture solution for multiplication culture;
the step e of repeating the proliferation culture is implemented specifically as follows: and d, selecting part of the periphery of the algae spots with the characteristic consistency from the aerobiotic microalgae community obtained in the step d, diluting with sterile water, scribing on the surface of a new solid culture medium, transferring the periphery of the algae spots with the good characteristic consistency to a new conical flask filled with liquid culture solution for proliferation culture after the algae spots grow out, diluting the culture solution with sterile water after the microalgae are fully proliferated in the culture solution, scribing on the surface of the new solid culture medium, and repeating the steps for at least three times.
f, checking the pure strain, absorbing the algae liquid obtained in the step e, and separating and culturing until the content of the gas-borne microalgae is greater than a preset value through naked eye observation and/or microscopic observation, thereby obtaining qualified separation and culture.
In this way, the method adopts the nutrient solution which is rich in a plurality of metal elements and trace elements, so that the method can be widely applied to the culture of different kinds of aerobiotic microalgae, and can promote the rapid growth and propagation of the aerobiotic microalgae. Through repeated separation, dilution and multiplication culture in various modes, the growth of adverse microorganism components such as bacteria in the culture solution is inhibited, so that the aerobiotic microalgae components are rapidly proliferated, and finally, pure aerobiotic microalgae can be rapidly and efficiently obtained. The method adopts liquid culture medium enrichment and expansion culture at the early stage, adopts solid medium to repeatedly streak, separate and purify culture at the later stage, and finally adopts liquid culture medium to proliferate, thereby being more beneficial to removing non-target algae and fungi and obtaining pure-bred target aerobiotic microalgae.
In the embodiment, in the step a, part of culture solution is adopted for concentration to obtain a feed supplement for standby; and e, adding part of feed after each dilution, and controlling the total nitrogen content in the culture system to be not less than 200mg/L.
Thus, the constant nutrition condition after dilution is better ensured, and the proliferation culture of the target object is facilitated. In the implementation, the total nitrogen concentration is measured by periodically performing alkaline potassium persulfate digestion ultraviolet spectrophotometry, so that the nutrient concentration in the culture medium can be effectively stabilized by determining the feed addition amount by measuring the nitrogen source content in the culture medium. The specific assay procedure is prior art and is not described in detail herein.
In each of the above steps, during the proliferation culture, the light-dark ratio=16 at the illumination intensity of 9000 to 10000 lux: 8, the culture is realized in an illumination incubator at the temperature of 25-28 ℃, ultraviolet light is used for irradiating for 1h every 12h in the culture process, and mixed sterile air with 3-5% carbon dioxide content is introduced as respiratory gas (aeration treatment is directly carried out when a liquid culture medium is used).
Thus, the proliferation culture effect can be better ensured.
The present embodiment further includes step g: and d, coating the algae liquid obtained in the step f on a solid culture medium for bacterial cultivation, and culturing for 2d in a light incubator at 25-28 ℃, wherein if no bacteria grow, the pure seed separation is successful.
Therefore, the separated pure-breed aerobiotic microalgae can be better ensured to contain no or little bacterial components, and is more beneficial to being used for repairing the bare rock slope.
In the above examples, all the culture products were sterilized at high temperature under high pressure (121 ℃,20 min) or by ultraviolet rays (> 30 min). Avoiding the influence of new bacteria and fungi introduced in the cultivation process on the cultivation effect.
In order to further verify the effect of the present invention, in particular, the effect of the aerobiotic microalgae culture solution prepared in the special manner in the present invention. The applicant has additionally carried out the following comparative tests alone. The comparison test adopts the aerobiotic microalgae culture solution obtained in the specific embodiment and a solid culture medium (obtained by adding agar into the culture solution provided by the method), and the culture solution and the solid culture medium of conventional algae culture are respectively inoculated with the same aerobiotic microalgae for comparison. The following test examples and comparative examples are specifically included.
Test example 1: with the aerobiotic microalgae culture solution and the aerobiotic microalgae strain obtained in the specific embodiment above, the aerobiotic microalgae strain was first inoculated into a 250ml conical flask, and the pH was adjusted to 7.5 using 0.1mol of hydrochloric acid and 0.1mol of sodium hydroxide solution. The inoculated liquid medium was placed and cultured in an illumination incubator (illumination 9000-10000lux, light-dark ratio=16:8, temperature 25-28 ℃) for 7d. And (5) photographing and observing, and detecting to obtain the chlorophyll content and carotenoid content in the algae liquid.
Comparative example 1: the procedure of test example 1 was followed using a conventional microalgae culture solution, followed by inoculation and cultivation for 7 days. And (5) photographing and observing, and detecting to obtain the chlorophyll content and carotenoid content in the algae liquid.
Test example 2: the solid culture medium obtained in the specific embodiment is adopted, and the aerobiotic microalgae species are respectively inoculated on the surface of the solid culture medium in a streaking and coating mode. The inoculated solid medium was left to stand and cultured in an illumination incubator (illumination 9000-10000lux, light-dark ratio=16:8, temperature 25-28 ℃) for 7d. And photographing and observing, and detecting to obtain chlorophyll content and carotenoid content in the algal plaques.
Comparative example 2: a common conventional solid medium was used and inoculated and cultured for 7 days in the same manner as in test example 2. And photographing and observing, and detecting to obtain chlorophyll content and carotenoid content in the algal plaques.
The results of photographing observation and microscopic examination of the above comparative experiments are shown in fig. 1-8, and the algae liquid obtained in test example 1 can be seen in fig. 1-2, so that the algae liquid has more green algae components and the other liquid components have high transparency, which indicates that the other impurity fungus components are less, while the algae liquid obtained in comparative example 1 has more impurities and yellow color, which indicates that the impurity fungus content is greater and the purifying cultivation effect is worse.
As can be seen from FIGS. 3 to 6, in test example 2, the algae liquid inoculated by streaking or coating method produced dark green algae spots after cultivation, and the other yellow components were less, indicating that the microalgae purification cultivation effect was better. In comparative example 2, a large amount of yellow regions were generated, the components of the green regions were significantly smaller, and the microalgae purification and cultivation effects were poor.
As can be seen from the photographs of the microscopic examination results in FIGS. 7 to 8, the aerobiotic microalgae obtained after streaking culture in test example 2 has no obvious other impurities, which indicates that the separation and purification effects are better, while the aerobiotic microalgae obtained after streaking culture in comparative example 2 has a large amount of other impurities and cells, which indicates that the separation and purification effects are worse.
The applicant then carried out the detection of chlorophyll content and carotenoid content using the algae solutions obtained in test example 1 and comparative example 1, and the algae plaques obtained in test example 2 and comparative example 2, respectively. The detection results are shown in Table 1.
Table 1 chlorophyll content of aerogenic microalgae in different test examples and comparative examples.
| Comparative example 1 | Comparative example 2 | Test example 1 | Test example 2 | |
| Chlorophyll a (mg/g) | 3.962 | 3.323 | 5.713 | 7.298 |
| Chlorophyll b (mg/g) | 0.43 | 0.39 | 4.21 | 3.54 |
| Carotenoid (mg/g) | 2.014 | 1.634 | 2.907 | 3.715 |
Therefore, the comparison test results show that the aerobiotic microalgae culture solution can effectively improve the photosynthetic efficiency of the aerobiotic microalgae, accelerate the growth rate of the aerobiotic microalgae and effectively improve the purity of the aerobiotic microalgae.
Claims (6)
1. A method for separating and culturing gas-borne microalgae is characterized in that a culture solution rich in inorganic nutrient sources and organic carbon sources is prepared, a liquid culture medium and a solid culture medium are obtained based on the culture solution, enrichment culture and expansion culture are carried out by using the liquid culture medium, repeated separation and purification culture is carried out by using the solid culture medium, and the periphery of a algae spot with good characteristic consistency is selected and transferred into the liquid culture medium for proliferation culture and then pure strain detection is carried out;
the method specifically comprises the following steps:
a, preparing a culture solution for standby, wherein the culture solution comprises inorganic nutrient source components and organic carbon source components, and the inorganic nutrient source components comprise ionic sodium, potassium, magnesium, calcium, iron, boron, manganese, zinc, copper and cobalt elements;
b, enrichment culture, namely collecting the aerobiotic microalgae of a target to be cultured, cleaning the collected aerobiotic microalgae sample by using sterile water to obtain an aerobiotic microalgae solution, adding a culture solution for proliferation culture, and obtaining an alga solution after proliferation of the aerobiotic microalgae;
c, carrying out expanded culture on the aerobiotic microalgae, taking part of the enriched culture algae liquid, adding a large proportion of culture liquid for proliferation culture, and obtaining the algae liquid with high concentration;
d, inoculating and culturing the aerobiotic microalgae, adding agar into the culture solution prepared in the step a to prepare a solid culture medium, diluting the algae solution obtained in the step c at least once by using sterile water, then coating and inoculating the algae solution onto the surface of the solid culture medium, and carrying out proliferation culture to obtain macroscopic aerobiotic microalgae communities on the solid culture medium;
e, purifying and culturing the aerobiotic microalgae, namely selecting part of the periphery of the aerobiotic microalgae community obtained in the step d, diluting with sterile water, scribing on the surface of a new solid culture medium, transferring part of the periphery of the aerobiotic microalgae with good characteristic consistency into a new conical flask filled with liquid culture solution for proliferation culture after the aerobiotic microalgae grows out of the aerobiotic microalgae community, diluting with sterile water after the microalgae fully proliferates in the culture solution, scribing on the surface of the new solid culture medium, and repeating the steps for at least three times; selecting part of the periphery of the algae spots with good feature consistency from the aerobiotic microalgae community obtained by repeated multiplication culture, and transferring the selected part of the periphery of the algae spots into a new vessel filled with liquid culture solution for multiplication culture;
f, checking the pure strain, absorbing the algae liquid obtained in the step e, and observing the algae liquid by naked eyes and/or a microscope, wherein the content of the microalgae grown in the gas to be separated and cultured is greater than a preset value, and the culture is qualified;
in the preparation process of the culture solution, the additive components comprise sodium nitrate, monopotassium phosphate, dipotassium phosphate, EDTA, magnesium sulfate heptahydrate, calcium chloride dihydrate, ferric ammonium citrate, sodium carbonate, boric acid, manganese chloride monohydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, cobalt nitrate hexahydrate and anhydrous glucose; the concentration of each component in the culture solution is 1.5g/L, the concentration of sodium nitrate is 0.04g/L, the concentration of dipotassium phosphate is 0.088g/L, the concentration of EDTA0.001g/L, the concentration of magnesium sulfate heptahydrate is 0.075g/L, the concentration of calcium chloride dihydrate is 0.036g/L, the concentration of ferric ammonium citrate is 0.0006g/L, the concentration of sodium carbonate is 0.02g/L, the concentration of boric acid is 0.00286g/L, the concentration of manganese chloride monohydrate is 0.00181g/L, the concentration of zinc sulfate heptahydrate is 0.000222g/L, the concentration of copper sulfate pentahydrate is 0.000079g/L, the concentration of sodium molybdate dihydrate is 0.00039g/L, the concentration of cobalt nitrate hexahydrate is 0.000049g/L, and the concentration of anhydrous glucose is 0.01g/L;
in the step a, part of culture solution is adopted for concentration to obtain feed supplement for standby; in the step e, adding partial feed after each dilution, and controlling the total nitrogen content in the culture system to be not less than 200mg/L;
in each step, during proliferation culture, at an illumination intensity of 9000-10000lux, a light-dark ratio=16: 8, the culture is carried out in an illumination incubator at the temperature of 25-28 ℃, ultraviolet light is used for irradiating for 1h every 12h in the culture process, and mixed sterile air with the carbon dioxide content of 3-5% is introduced as breathing gas.
2. The method for isolated culture of aerobiotic microalgae of claim 1 wherein the culture medium is further supplemented with rose oil at a level of 0.01 ml/L.
3. The method for isolated culture of aerobiotic microalgae as claimed in claim 1 characterized in that, when preparing the culture solution, the pH is adjusted to 7-8 by using 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide solution after adding each component.
4. The method for separating and culturing airborne microalgae according to claim 1, wherein the culture solution is prepared by soaking the attachments of the acquired airborne microalgae in the preparation solution at normal temperature for more than 2 days.
5. The method for separating and culturing airborne microalgae according to claim 4, wherein in the step, the steps are carried out by adopting 100Hz ultrasonic oscillation for 30min during cleaning, so that the airborne microalgae and attachments are fully separated and enter sterile water, and the obtained solution of the airborne microalgae is uniformly shaken, kept stand and deposited, and then the supernatant is taken out for proliferation culture for 3 days;
in the step c, the culture solution with the volume ratio of 20 times is adopted for proliferation culture for 14 days;
in the step d, preparing a solid culture medium according to 15g/L of added agar;
d, in the dilution process, adding 4ml of sterile water into each of a plurality of sterilized sample tubes, firstly taking 1ml of the amplified culture algae liquid, adding the algae liquid into a first sample tube, and shaking uniformly; then, 1ml of algae liquid is taken from the first sample tube, added into the second sample tube and uniformly shaken, and subjected to gradient dilution in sequence; 1ml of the algae liquid was spread on a solid medium from a sample tube with three dilution concentrations, and proliferation culture was performed for 7 days.
6. The method for isolated culture of aerobiotic microalgae of claim 1 further comprising step g: and d, coating the algae liquid obtained in the step f on a solid culture medium for bacterial cultivation, and culturing for 2d in a light incubator at 25-28 ℃, wherein if no bacteria grow, the pure seed separation is successful.
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