Method for preserving microalgae plants

Abstract

The invention relates to the technical field of plant preservation, and discloses a method for preserving microalgae plants. The invention aims to improve the existing microalgae plant preservation method, especially the preservation method for bait microalgae, so as to solve the problems that the existing microalgae preservation time is short, nutritional ingredients are lost after preservation, the activity is reduced and the like. According to the microalgae preservation method, choline chloride is added to maintain the balance of a carbon source and a nitrogen source in a culture medium, nickel salt is added to promote the activity of algae photosynthetic pigments in a stable period, and the purpose of prolonging the preservation time and activity of algae is achieved. The microalgae preserved by the method disclosed by the invention can be preserved for a longer time on the premise of the same activity, and has higher activity on the premise of the same preservation time, and the activity of the microalgae preserved by the method is several times of that of microalgae preserved by a conventional method. The microalgae plant preservation method does not use toxic and expensive reagents, is environment-friendly, and can greatly promote the popularization and application of the microalgae industry.

Description

Technical Field

[0001] The present application relates to the technical field of plant preservation, and in particular to a method for preserving microalgae plants. Background Art

[0002] Microalgae is a lower plant without differentiated organs such as roots, stems, and leaves. Microalgae can synthesize organic matter through photosynthesis using sunlight, carbon dioxide, and water. It is one of the most important primary producers on Earth.

[0003] Microalgae-related products have been widely used in the aquaculture industry, with remarkable effects in improving water quality, regulating aquaculture water environment and being used as biological bait, and have been generally recognized by aquaculture workers. The most widely used microalgae are Chlorophyta and Bacillariophyta.

[0004] In the field of bait microalgae production, due to the mismatch between the peak production season and the peak demand season, the distance between the production site and the final use site, etc., the microalgae need to be directly bottled or concentrated for storage in preparation for use or sale. However, the imperfect microalgae preservation method has hindered the promotion and application of microalgae products.

[0005] In the past, microalgae preservation technology was mainly focused on preservation methods for laboratory microalgae. Such preservation methods have problems such as cumbersome operation and toxic and expensive reagents. For example, the method of using glycerol with a volume coefficient of 10-20% to freeze and preserve microalgae is expensive and may pollute water bodies, so it is not suitable for large-scale application. Another example is the use of DSOM (dimethyl sulfoxide) to preserve microalgae. Dimethyl sulfoxide is biologically toxic and the microalgae needs to be centrifuged and eluted before use, which is also not suitable for large-scale application.

[0006] The existing large-scale preservation method of microalgae is mainly to harvest and concentrate the microalgae and then refrigerate them at low temperature, and preserve them in combination with low light or no light. The microalgae preserved by this method can only be preserved for about 3 months, and as the preservation time increases, the nutrients and activity of the microalgae themselves continue to decrease, causing the utilization value of the microalgae to continue to decline and even have to be discarded, hindering the promotion and application of the microalgae industry. Summary of the invention

[0007] In view of this, the object of the present application is to provide a method for preserving microalgae plants, so that the method can prolong the preservation time of the microalgae and maintain a high activity.

[0008] In order to solve the above technical problems / achieve the above objectives or at least partially solve the above technical problems / achieve the above objectives, as a first aspect of the present application, a method for preserving microalgae plants is provided, comprising: S1. Preliminary cultivation of microalgae in a culture medium under natural conditions; S2. After the microalgae enter the stable culture period, the algae liquid is harvested and stored under low temperature refrigeration combined with low light or no light conditions; Wherein, no more than 1.0 g / L of choline chloride is added to the culture medium of the preliminary culture, and no more than 0.5 mg / g of +2-valent nickel salt is added to the harvested algae liquid; or no more than 0.5 g / g of choline chloride and no more than 0.5 mg / g of +2-valent nickel salt are added to the harvested algae liquid.

[0009] Optionally, the concentration of choline chloride added to the culture medium of the preliminary culture is 0.03-1.0 g / L, and the concentration of +2-valent nickel salt added to the harvested algae liquid is 0.01-0.5 mg / g.

[0010] Optionally, the concentration of choline chloride added to the harvested algae liquid is 0.03-1.0 g / g, and the concentration of +2-valent nickel salt added is 0.01-0.5 mg / g.

[0011] Optionally, the +2-valent nickel salt includes any one of an organic acid nickel salt and an inorganic acid nickel salt; further optionally, the +2-valent nickel salt includes any one of nickel acetate, nickel nitrate, nickel sulfate, and ammonium nickel sulfate.

[0012] Optionally, the method further comprises performing a secondary culture after adding nickel salt to the harvested algae liquid, wherein the conditions of the secondary culture include: culturing under the original previous culture environment conditions, or culturing under the environment conditions of 20-28°C and a light intensity of 5000-8000 lux.

[0013] As a second aspect of the present application, a method for preserving microalgae plants is provided, comprising: S1. Preliminary cultivation of microalgae in a culture medium under natural conditions; S2. After the microalgae enter the stable culture period, the algae liquid is harvested, and the algae liquid is stored under low temperature refrigeration combined with low light or no light conditions; Wherein, no more than 1.0 g / L of choline chloride and no more than 1.0 mg / L of +2-valent nickel salt are added to the culture medium of the preliminary culture.

[0014] Optionally, the concentration of choline chloride added to the culture medium in the preliminary culture is 0.03-1.0 g / L, and the concentration of +2-valent nickel salt added is 0.01-1.0 mg / L.

[0015] Optionally, the +2-valent nickel salt includes any one of an organic acid nickel salt and an inorganic acid nickel salt; further optionally, the +2-valent nickel salt includes any one of nickel acetate, nickel nitrate, nickel sulfate, and ammonium nickel sulfate.

[0016] The present application aims to improve the existing preservation methods of microalgae plants, especially the preservation methods for bait microalgae, in order to solve the problems of short preservation time of existing microalgae, loss of nutrients and decreased activity after preservation. The present application achieves the purpose of extending the preservation time and activity of algae by adding choline chloride to the microalgae preservation method to maintain the balance of carbon source and nitrogen source in the culture medium, and adding nickel salts to promote the activity of photosynthetic pigments of algae in the stable period. Compared with the conventional low-light or no-light preservation method combined with low-temperature refrigeration, the microalgae preserved by the method of the present application can be preserved for a longer time under the premise of the same activity, and have higher activity under the premise of the same preservation time, and the activity is several times that of microalgae preserved by conventional methods. The preservation method of microalgae plants in the present application does not use toxic and expensive reagents, is environmentally friendly, and can greatly promote the promotion and application of the microalgae industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown is a schematic diagram of the process of preserving microalgae in the present application. DETAILED DESCRIPTION

[0018] The present application discloses a method for preserving microalgae plants. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of this application.

[0019] It should be noted that, in this document, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise one..." do not exclude the existence of other identical elements in the process, method, article or device including the elements. At the same time, the embodiments in this application and the features in the embodiments may be combined with each other without conflict.

[0020] The present application has found in the research that in the conventional preservation of microalgae, the reproduction rate of microalgae is still too high in the early stage of preservation, which causes the concentration of carbon source and nitrogen source in the culture solution to drop sharply, causing the activity of photosynthetic pigments of microalgae to drop and even leading to acute death of microalgae. In order to prolong the preservation of microalgae, the concentration of effective nitrogen source and carbon source in the culture solution should be maintained while reducing the reproduction rate of microalgae, and substances that maintain the activity of its photosynthetic pigments should be provided.

[0021] Choline chloride is a feed additive that promotes digestion. Its molecular formula is C5H 14 ClNO has the potential to be a carbon and nitrogen source for microalgae. According to the research of this application, it can be used as a slow-release nutrient salt for microalgae to provide nitrogen and carbon sources for microalgae, and maintain the carbon and nitrogen source content in the culture medium at a more appropriate concentration. At the same time, this application selects nickel acetate, nickel nitrate, nickel sulfate and other nickel salts to add, which can induce microalgae to adjust their own metabolic mechanism after the growth of microalgae enters the stable period, and increase the amount of carbon and nitrogen sources used for Q A The energy, quantum yield and exciton ratio of electron transfer then promote the activity of algae photosynthetic pigments in the stable period, thereby extending the shelf life and activity of the algae.

[0022] Based on the above-mentioned inventive principle, the present application has two implementation methods. One method is to add choline chloride to the culture medium in the early stage of microalgae cultivation, and to add nickel salt to the algae liquid after harvest; or both choline chloride and nickel salt are added to the algae liquid after harvest; and then the cultured algae liquid is stored according to conventional low-temperature refrigeration combined with low-light or no-light storage conditions; the other method is to add choline chloride and nickel salt to the culture medium in the early stage of microalgae cultivation, and directly harvest them after they grow to a stable period, and store them according to conventional low-temperature refrigeration combined with low-light or no-light storage conditions.

[0023] In the first aspect of the present application, for a first implementation, the present application proposes a method for preserving microalgae plants, comprising: S1. Preliminary cultivation of microalgae in a culture medium under natural conditions; S2. After the microalgae enter the stable culture period, the algae liquid is harvested and stored under low temperature refrigeration combined with low light or no light conditions; Wherein, no more than 1.0 g / L of choline chloride is added to the culture medium of the preliminary culture, and no more than 0.5 mg / g of +2-valent nickel salt is added to the harvested algae liquid (based on the dry weight of microalgae in the algae liquid); or no more than 0.5 g / g of choline chloride and no more than 0.5 mg / g of +2-valent nickel salt are added to the harvested algae liquid (based on the dry weight of microalgae in the algae liquid).

[0024] In certain embodiments of the present application, the microalgae include but are not limited to algae of Chlorophyta and Bacillariophyta, wherein these algae can be freshwater algae or seawater algae. In other embodiments of the present application, the microalgae are selected from Chlorella vulgaris (Chlorophyta), Nannochloropsis chlororaphis (Chlorophyta), Thalassiosira wissenii (Bacillariophyta), and Chaetoceros muehlenii (Bacillariophyta).

[0025] In certain embodiments of the present application, the culture medium is selected according to the type of microalgae, for example, BG11 medium is selected for freshwater Chlorophyta microalgae, BG11 medium without Ca and Mg is selected for marine Chlorophyta microalgae, and F / 2 medium is selected for Bacillariophyta microalgae.

[0026] In some other embodiments of the present application, the BG11 medium composition includes 1500 g of sodium nitrate, 40.00 g of potassium dihydrogen phosphate, 75.00 g of magnesium sulfate heptahydrate, 36.00 g of calcium chloride dihydrate, 6.00 g of citric acid, 6.00 g of ammonium ferric citrate, 1.00 g of disodium ethylenediammonium tetraacetate, 20.00 g of sodium carbonate, 2.86 g of boric acid, 1.86 g of manganese chloride tetrahydrate, and 0.22 g of zinc sulfate heptahydrate per 1000 L of medium. g, 0.39 g sodium molybdate dihydrate, 0.08 g copper sulfate pentahydrate, and 0.05 g cobalt nitrate hexahydrate; the BG11 culture medium without Ca and Mg comprises 80 g urea, 10 g potassium dihydrogen phosphate, 7.00 g ferrous sulfate, 1.00 g manganese chloride tetrahydrate, 0.05 g copper sulfate pentahydrate, 0.05 g cobalt chloride hexahydrate, and 0.39 g sodium molybdate dihydrate per 1000 L culture medium; the F / 2 culture medium comprises 120 g sodium nitrate, 10 g sodium dihydrogen phosphate, 6.3 g ferrous chloride hexahydrate, 0.02 g copper sulfate pentahydrate, 0.044 g zinc sulfate heptahydrate, 0.02 g cobalt chloride hexahydrate, 0.36 g manganese chloride tetrahydrate, 0.1 g nickel sulfate, and 0.012 g sodium molybdate dihydrate per 1000 L culture medium.

[0027] In certain embodiments of the present application, the initial density of the microalgae is (0.1-10)×10 5 cells / mL; the initial density of microalgae in Chlorophyta is preferably (5-10)×10 5 cells / mL, the initial density of microalgae in the diatom phylum is preferably (0.1-1)×10 5 cells / mL.

[0028] In certain embodiments of the present application, the concentration of choline chloride added to the culture medium of the initial culture is 0.03-1.0 g / L, and the concentration of +2-valent nickel salt added to the harvested algae liquid is 0.01-0.5 mg / g; in other embodiments of the present application, the concentration of choline chloride added to the culture medium of the initial culture is 0.1-0.5 g / L, for example, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, 0.5 g / L, etc., and the harvested The concentration of +2-valent nickel salt added to the algae liquid is 0.02~0.3 mg / g, for example, 0.02 mg / g, 0.03 mg / g, 0.04 mg / g, 0.05 mg / g, 0.06 mg / g, 0.07 mg / g, 0.08 mg / g, 0.09 mg / g, 0.1 mg / g, 0.12 mg / g, 0.14 mg / g, 0.16 mg / g, 0.18 mg / g, 0.2 mg / g, 0.22 mg / g, 0.24 mg / g, 0.26 mg / g, 0.28 mg / g, 0.30 mg / g, etc.

[0029] In certain embodiments of the present application, the concentration of choline chloride added to the harvested algae liquid is 0.03-1.0 g / g, and the concentration of +2-valent nickel salt added is 0.01-0.5 mg / g; in other embodiments of the present application, the concentration of choline chloride added to the harvested algae liquid is 0.03-0.5 g / g, for example, 0.03 g / g, 0.05 g / g, 0.08 g / g, 0.1 g / g, 0.15 g / g, 0.2 g / g, 0.25 g / g, 0.3 g / g, 0.35 g / g, 0.4 g / g, 0.45 g / g, 0.5 g / g g, etc., and the concentration of added +2-valent nickel salt is 0.02~0.3 mg / g, for example 0.02 mg / g, 0.03 mg / g, 0.04 mg / g, 0.05 mg / g, 0.06 mg / g, 0.07 mg / g, 0.08 mg / g, 0.09 mg / g, 0.1 mg / g, 0.12 mg / g, 0.14 mg / g, 0.16 mg / g, 0.18 mg / g, 0.2 mg / g, 0.22 mg / g, 0.24 mg / g, 0.26 mg / g, 0.28 mg / g, 0.30 mg / g, etc.

[0030] In certain embodiments of the present application, the +2-valent nickel salt includes any one of an organic acid nickel salt and an inorganic acid nickel salt; in other embodiments of the present application, the +2-valent nickel salt includes any one of nickel acetate, nickel nitrate, nickel sulfate, and nickel ammonium sulfate. In other embodiments of the present application, the concentration of each nickel salt added to the secondary culture of the algae liquid can be referenced as follows: nickel acetate tetrahydrate 0.02~0.1mg / g, nickel nitrate 0.03~0.12mg / g, nickel sulfate 0.02~0.10mg / g, nickel ammonium sulfate 0.04~0.18mg / g.

[0031] In certain embodiments of the present application, in order to accelerate the absorption of nickel salts by microalgae into the algae, promote the Q A Subsequent electron transfer also includes secondary culture after adding nickel salt to the harvested algae liquid, and the conditions of the secondary culture include: culture under the original previous culture environment conditions, or culture under the environment conditions of 20~28℃ and light intensity of 5000~8000lux, and the secondary culture time is 12h±5h; in some other embodiments of the present application, the pH value of the algae liquid is adjusted to weak alkalinity before the secondary culture, for example, the pH value is 7.5~8.5, which can be adjusted with sodium hydroxide or glacial acetic acid.

[0032] In certain embodiments of the present application, the low temperature refrigeration condition is generally 2-6°C, and the low light condition is generally 800-1500 lux, 24h light; in addition, before low temperature refrigeration combined with low light or no light storage, the pH value of the algae liquid is adjusted to a weak alkaline, for example, a pH value of 7.5-8.5, which can be adjusted with sodium hydroxide or glacial acetic acid. In other embodiments of the present application, when low temperature refrigeration combined with low light or no light storage is performed, the algae liquid is evenly dispersed every 1-5 days, and the uniform dispersion process can be appropriately shaken according to the algae liquid container used, such as a shake bottle operation.

[0033] In the second aspect of the present application, for the second implementation, the present application proposes a method for preserving microalgae plants, comprising: S1. Preliminary cultivation of microalgae in a culture medium under natural conditions; S2. After the microalgae enter the stable culture period, the algae liquid is harvested, and the algae liquid is stored under low temperature refrigeration combined with low light or no light conditions; Wherein, no more than 1.0 g / L of choline chloride and no more than 1.0 mg / L of +2-valent nickel salt are added to the culture medium of the preliminary culture.

[0034] In certain embodiments of the present application, the types of microalgae, culture medium types, low-temperature refrigeration combined with low light or no light conditions used in the second implementation method can refer to the relevant records in the first aspect of the present application.

[0035] In certain embodiments of the present application, the concentration of choline chloride added to the medium of the preliminary culture is 0.03-1.0 g / L, and the concentration of the +2-valent nickel salt added is 0.01-1.0 mg / L. In other embodiments of the present application, in other embodiments of the present application, the concentration of choline chloride added to the medium of the preliminary culture is 0.1-0.5 g / L, such as 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, 0.5 g / L, etc., and the concentration of the +2-valent nickel salt added is 0.04-0.5 mg / L, such as 0.04 mg / L, 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08mg / L, 0.1mg / L, 0.12mg / L, 0.14mg / L, 0.16mg / L, 0.18mg / L, 0.2mg / L, 0.22mg / L, 0.24mg / L, 0.26mg / L, 0.28mg / L, 0.3mg / L, 0.32mg / L, 0.34mg / L, 0.36mg / L, 0.38mg / L, 0.4mg / L, 0.42mg / L, 0.44mg / L, 0.46mg / L, 0.48mg / L, 0.5mg / L, etc.

[0036] In certain embodiments of the present application, the +2-valent nickel salt includes any one of an organic acid nickel salt and an inorganic acid nickel salt; in other embodiments of the present application, the +2-valent nickel salt includes any one of nickel acetate, nickel nitrate, nickel sulfate, and nickel ammonium sulfate. In other embodiments of the present application, the concentration of each nickel salt added to the culture medium in the early stage of the culture can be referenced as follows: nickel acetate tetrahydrate 0.05~0.2mg / L, nickel nitrate 0.06~0.22mg / L, nickel sulfate 0.04~0.18mg / L, nickel ammonium sulfate 0.08~0.34mg / L.

[0037] In each group of comparative experiments provided in this application, unless otherwise specified, other experimental conditions, materials, etc. are kept consistent except for the differences indicated in each group, so as to provide comparability. In addition, all materials used in this application can be purchased through commercial channels.

[0038] The following is a further description of a method for preserving microalgae plants provided in the present application.

[0039] Example 1: Freshwater Chlorella vulgaris ( Chlorella vulgaris ) In a 1-ton pipeline photobioreactor located outdoors, 1 ton of tap water was added, aerated for 12 h, 1500 g of sodium nitrate, 200 g of choline chloride, and 40.00 g of potassium dihydrogen phosphate were added, mixed, 75.00 g of magnesium sulfate heptahydrate and 36.00 g of calcium chloride dihydrate were added, mixed, 6.00 g of citric acid, 6.00 g of ammonium ferric citrate, 1.00 g of disodium ethylenediammonium tetraacetate, 20.00 g of sodium carbonate, 2.86 g of boric acid, 1.86 g of manganese chloride tetrahydrate, 0.22 g of zinc sulfate heptahydrate, 0.39 g of sodium molybdate dihydrate, 0.08 g of copper sulfate pentahydrate, and 0.05 g of cobalt nitrate hexahydrate were added, mixed, and the Chlorella algae solution was added, mixed, and the initial Chlorella density was adjusted to 10×10 5 cells / ml, cultured outdoors under normal day-night cycle and light intensity.

[0040] After 10 days of cultivation, Chlorella entered the stable cultivation period with a density of 410×10 5 cells / ml, concentrated, harvested and bottled, dry weight of 133g / L, bottle volume of 5L, 0.06g of nickel nitrate added to each bottle, air-conditioned in the transplant room to maintain an environment of 26℃, and cultured for 12h under a light intensity of 8000lux.

[0041] After 12 hours, the initial pH was measured to be 8.8. Glacial acetic acid was used to adjust the pH to 7.8 and the bottle was stored in a 4°C cold storage. The light intensity in the cold storage was adjusted to 1200 lux with 24 hours of illumination. The bottle was shaken every 3 days to ensure that the algae liquid was evenly dispersed in the bottle.

[0042] Example 2: Marine Chlorophyta Nannochloropsis sp. In a 1-ton pipeline photobioreactor located outdoors, 1 ton of natural seawater, 80 g of urea, 120 g of choline chloride, and 10 g of potassium dihydrogen phosphate were added and mixed. Then, 7.00 g of ferrous sulfate, 1.00 g of manganese chloride tetrahydrate, 0.05 g of copper sulfate pentahydrate, 0.05 g of cobalt chloride hexahydrate, 0.39 g of sodium molybdate dihydrate, and 0.18 g of nickel acetate were added and mixed. The microgreen chlorophyll algae solution was added and mixed. The initial microgreen chlorophyll density was adjusted to 7 × 10 5 cells / ml, cultured outdoors under normal day-night cycle and light intensity.

[0043] After 8 days of cultivation, Nannochloropsis chlororaphis entered the stable cultivation period with a density of 230×10 5 cells / ml, concentrated, harvested and bottled, initial pH 8.6, adjusted to pH 7.8 with glacial acetic acid, stored in a 4°C cold storage, adjusted light intensity in the cold storage to 1200 lux, 24h lighting, shaken the bottle every 3 days to evenly disperse the algae liquid in the bottle.

[0044] Example 3: Thalassiosira weissflogii from marine diatoms In a 1-ton wide-mouth beef tendon barrel indoors, add 1 ton of natural seawater, 120 g of sodium nitrate, 100 g of choline chloride, and 10 g of sodium dihydrogen phosphate, mix well, add 6.3 g of ferrous chloride hexahydrate, 0.02 g of copper sulfate pentahydrate, 0.044 g of zinc sulfate heptahydrate, 0.02 g of cobalt chloride hexahydrate, 0.36 g of manganese chloride tetrahydrate, 0.1 g of nickel sulfate, and 0.012 g of sodium molybdate dihydrate, mix well, inoculate Thalassiosira wissenii, mix well, and adjust the initial density of Thalassiosira wissenii to 5 × 10 4 cells / ml, adjust the inflation volume to slightly boiling state, adjust the light intensity to 4000 lux, adjust the room temperature to 26℃, and adjust the light for 24 hours.

[0045] After 12 days of cultivation, Thalassiosira wissensis entered the stable cultivation period with a density of 58×10 4 cells / ml, harvest and bottle, initial pH8.2, use glacial acetic acid to adjust pH to 7.8, store in a 4℃ cold storage, adjust the light intensity in the cold storage to 800lux, 24h lighting, shake the bottle once every 3 days to make the algae liquid evenly dispersed in the bottle.

[0046] Example 4: Chaetoceros muelleri In a 1-ton wide-mouthed tendon barrel, add 1 ton of natural seawater, 120 g of sodium nitrate, 10 g of sodium dihydrogen phosphate, mix well, add 6.3 g of ferrous chloride hexahydrate, 0.02 g of copper sulfate pentahydrate, 0.044 g of zinc sulfate heptahydrate, 0.02 g of cobalt chloride hexahydrate, 0.36 g of manganese chloride tetrahydrate, and 0.012 g of sodium molybdate dihydrate, mix well, inoculate Chaetoceros muelleri, mix well, and adjust the initial Chaetoceros muelleri density to 8 × 10 4 cells / ml, adjust the inflation volume to slightly boiling state, adjust the light intensity to 4000 lux, adjust the room temperature to 26℃, and adjust the light for 24 hours.

[0047] After 12 days of cultivation, Chaetoceros muelleri entered the stable period of cultivation, with a density of 207×10 4 cells / ml, harvest and bottle, initial pH8.6, add 0.1mg / g nickel sulfate and 0.1g / g choline chloride, use glacial acetic acid to adjust the pH to 7.8, store in a 4℃ cold storage, adjust the light intensity in the cold storage to 800lux, 24h lighting, shake the bottle once every 3 days to make the algae liquid evenly dispersed in the bottle.

[0048] Experimental example: After mixing the algae liquid, randomly sample it and drop it on the blood cell counting plate for counting and observation. The algae with intact cell membrane structure and uniform distribution of chromatophores are judged as living algae. The activity of microalgae is calculated based on the density at the initial storage. For example, if the initial density of algae cells is 100×10 4 cells / ml. After a period of storage, the cell membrane structure was intact and the chromatophores were evenly distributed. The cell density was 98×10 4 cells / ml, the activity of the algae solution is 98%.

[0049] Referring to the above activity detection method, the microalgae were stored for different days using the storage methods of Examples 1-4 and their respective corresponding conventional storage methods (i.e., no choline chloride and nickel salt were added in each embodiment, the culture medium was cultured to the stable period, and the low temperature and low light storage method after harvesting and concentration) and the activity was detected. The results are shown in Table 1 below. Table 1

[0050] According to the activity test results in Table 1 above, it can be clearly seen that the addition of choline chloride and nickel salt according to the method for preserving microalgae in the present application can prolong the storage time of the microalgae while ensuring a relatively high activity, which has an extremely significant improvement effect compared to the current large-scale microalgae preservation method. Moreover, the preservation method of the present application is simpler than laboratory preservation technology, does not use toxic and expensive reagents, is environmentally friendly, and is suitable for large-scale microalgae preservation.

[0051] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for preserving microalgae, characterized in that: include: S1. Preliminary cultivation of microalgae in a culture medium under natural conditions; S2. After the microalgae enter the stable culture period, the algae liquid is harvested and stored under low temperature refrigeration combined with low light or no light conditions; Wherein, no more than 1.0 g / L of choline chloride is added to the culture medium of the preliminary culture, and no more than 0.5 mg / g of +2-valent nickel salt is added to the harvested algae liquid; or no more than 0.5 g / g of choline chloride and no more than 0.5 mg / g of +2-valent nickel salt are added to the harvested algae liquid.

2. The method according to claim 1, characterized in that The concentration of choline chloride added to the culture medium of the preliminary culture is 0.03-1.0 g / L, and the concentration of +2-valent nickel salt added to the harvested algae liquid is 0.01-0.5 mg / g.

3. The method according to claim 1, characterized in that The concentration of choline chloride added to the harvested algae liquid is 0.03-1.0 g / g, and the concentration of +2-valent nickel salt added is 0.01-0.5 mg / g.

4. The method according to any one of claims 1 to 3, characterized in that: The +2-valent nickel salt includes any one of an organic acid nickel salt and an inorganic acid nickel salt.

5. The method according to claim 4, characterized in that The +2-valent nickel salt includes any one of nickel acetate, nickel nitrate, nickel sulfate, and nickel ammonium sulfate.

6. The method according to claim 1, characterized in that It also includes secondary culture after adding nickel salt to the harvested algae liquid, and the conditions of the secondary culture include: culture under the original previous culture environment conditions, or culture under the environment conditions of 20~28℃ and light intensity of 5000~8000lux.

7. A method for preserving microalgae plants, characterized in that: include: S1. Preliminary cultivation of microalgae in a culture medium under natural conditions; S2. After the microalgae enter the stable culture period, the algae liquid is harvested, and the algae liquid is stored under low temperature refrigeration combined with low light or no light conditions; Wherein, no more than 1.0 g / L of choline chloride and no more than 1.0 mg / L of +2-valent nickel salt are added to the culture medium of the preliminary culture.

8. The method according to claim 7, characterized in that The concentration of choline chloride added to the culture medium in the preliminary culture is 0.03-1.0 g / L, and the concentration of +2-valent nickel salt added is 0.01-1.0 mg / L.

9. The method according to claim 7 or 8, characterized in that: The +2-valent nickel salt includes any one of an organic acid nickel salt and an inorganic acid nickel salt.

10. The method according to claim 9, characterized in that The +2-valent nickel salt includes any one of nickel acetate, nickel nitrate, nickel sulfate, and nickel ammonium sulfate.

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