CN113462578A

CN113462578A - Microalgae culture medium and culture method

Info

Publication number: CN113462578A
Application number: CN202110990433.6A
Authority: CN
Inventors: 黄成潭; 潘军; 叶蕾; 黄敏
Original assignee: Hainan Green Algae World Biotechnology Co ltd
Current assignee: Hainan Green Algae World Biotechnology Co ltd
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2021-10-01

Abstract

The invention relates to the technical field of microalgae culture, in particular to a microalgae culture medium and a culture method. The culture medium comprises sodium nitrate, ammonium bicarbonate, sodium dihydrogen phosphate dihydrate, trihydrate and potassium dihydrogen phosphate, Tween 80, NaCl, trace elements and vitamins. The culture medium provides proper nutrient components for the growth of microalgae, can effectively promote the mitosis of algae cells and accelerate the growth of algae, and is suitable for large-scale industrial culture.

Description

Microalgae culture medium and culture method

Technical Field

The invention relates to the technical field of microalgae culture, in particular to a microalgae culture medium and a culture method.

Background

Microalgae (Microalgae) is a collective term for a population of small (2-30 microns) lower plants capable of photosynthesis, including both eukaryotic and prokaryotic species. The classical classification method based on the characteristics of the subcellular structure, pigment composition and reproduction mode of microalgae is to classify microalgae into various phyla such as cyanobacteria (cyanobacteria), chlorophyta, rhodophyta, chrysophyta and diatom. There are over 6 thousands of species of microalgae currently found worldwide, more than 6 thousands of species recorded, about hundreds of species involved in scientific research, and only a few tens of species applied to production practice. The microalgae is rich in various nutritional ingredients and bioactive substances such as protein, polysaccharide, fat (polyunsaturated fatty acid), pigment and the like, can be widely applied to a plurality of traditional fields such as human health food, aquatic feed, animal feed, water purification, medicine development and the like, and a new field of microalgae biofuel, and has important economic value and social benefit. The application of microalgae bait in aquatic seedling culture adopts microalgae as initial feed of aquatic animal seedlings and nutrition-enriched food of secondary bait organisms, the position of the microalgae bait in the aquatic seedling culture is not replaceable, and the core position of the microalgae bait is expressed as follows: on one hand, the microalgae are direct initial baits for shellfish, prawn larvae and part of fish larvae, and are equivalent to breast milk of infants; on the other hand, microalgae are also necessary food for culturing secondary food organisms for aquaculture such as rotifers, artemia, copepods and cladocerans, and are equivalent to infant food. The common bait microalgae are more than 20 genera and more than 40 species, including diatom, chrysophyceae and chlorella species, and are mainly used for culturing shellfish and shrimp seedlings and culturing freshwater marine zooplankton (rotifer, copepods and the like). The microalgae has rich and comprehensive nutritional components and is a primary food for aquaculture animal seedlings. The analysis of the component content of more than 40 common bait microalgae shows that: 20-40% of protein, 10-20% of fatty acid and 5-12% of carbohydrate. The microalgae protein has high quality, especially the content of essential amino acids is equal to or even better than that of fish meal, for example, the content of aspartic acid and glutamic acid can reach 7.1-12.9%, the content of cysteine, methionine, tryptophan and histidine is 0.4-3.2%, and the content of other amino acids is 3.2-13.5%. Polyunsaturated fatty acids (PUFA) are essential nutrients for the development of the larvae of aquatic animals, while microalgae are the primary producers of PUFA. Many microalgae, especially diatoms and chrysophytes, are rich in eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA). For example, the EPA content in Chaetoceros can reach 20-25% of the total fatty acid content, and the EPA content in Skeletonema also exceeds 20%; the content of DHA in isochrysis galbana can reach more than 10% of the total fatty acid content, the pavlova contains rich EPA (> 15%) and DHA (10%), and the content of ARA in nannochloropsis and partial diatoms can reach 4% at most.

The microalgae also contains abundant vitamins such as VA, VC, VD2, VD3, VE, VK, VB families (B1, B2, B3, B5, B6 and B12), biotin, beta-carotene, folic acid and the like, is complete in variety and rich in content, and is equivalent to the content in part of human foods and the recommended value of fish feed. Microalgae also contain a variety of microalgal sterols with specific structures, over 10 of which have been found and identified, such as chaetoceros rich in fucosterol, skeletonia, strep, and platymonas rich in campesterol and 24-methylene cholesterol, rhodophyta, and dinoflagellate rich in brassicasterol, and pavlova containing stigmasterol and β -sitosterol. The sterol is an important component of animal cell membranes, and can play important physiological roles of regulating the hormone level in animals, inhibiting the absorption of cholesterol, promoting the catabolism of cholesterol and the like.

The microalgae contains abundant and balanced nutrients (protein, fatty acid, carbohydrate) and various bioactive substances (PUFA, vitamins and sterol), and can meet the nutritional requirements of normal growth and development of aquaculture animals at the seedling stage. Numerous researches and applications prove that in the breeding of prawns (such as Japanese prawns, litopenaeus vannamei, Chinese prawns and south-water prawns), shellfishes (oysters, scallops and abalones), sea cucumbers and the like, bait microalgae are reasonably utilized to improve the survival rate of breeding, ensure the normal metamorphosis and development of seedlings, improve various characteristic indexes such as growth speed, body length, body weight and the like, improve immunity and the like. Besides being used as direct opening bait, the microalgae has another important function of being used for feeding secondary baits such as rotifers, artemia, copepods, cladocerans and the like, can obviously enhance the content of PUFA and various vitamins contained in the organisms of the secondary baits, and further meets the requirement of aquatic animal larvae on high-quality secondary baits. In the seedling production, the nutrition of the microalgae is the direct action of the microalgae, and the indirect action of the microalgae is mainly reflected in the influences on the aspects of water quality, water transmittance, algal facies-bacterial facies in water and the like. The microalgae is put into the seedling water body, oxygen can be generated by utilizing the photosynthesis of the microalgae, carbon dioxide, nitrogen and phosphorus elements discharged by seedlings are absorbed, and the balance of CO2-HCO 3-in the water body is controlled to achieve the effect of stabilizing the pH. Researches prove that the total number of bacteria in the seedling culture water body can be reduced by various microalgae, and meanwhile, the microalgae can play a role in stabilizing the bacterial phase (the structure and the number of the bacteria) in the water body, so that the influence of the concentration of organic matters and antibiotics is reduced. Aiming at the needs of aquaculture industry, corresponding products are published abroad, different types of microalgae concentrated solution and microalgae dry powder are prepared into products with high protein type, high PUFA type and the like by compounding single algae or multiple algae, and the products are respectively suitable for raising the seedlings of prawns and shellfishes and enriching the nutrition of rotifers and artemia.

Most aquatic seedling raising enterprises in China are provided with microalgae culture facilities in seedling raising production, and can produce various kinds of microalgae for baits. However, general seedling farms generally lack corresponding professional technical strength, can only utilize respective algae ponds and natural water bodies for extensive culture, and are respectively positive in bait microalgae germplasm, production technology and application method, so that the microalgae germplasm is disordered, unstable in supply, unbalanced in nutrient content, low in bait titer, and lack of multi-variety intensive production application technology; meanwhile, the method is limited by the limitations of microalgae high-density culture, harvesting technology and concentrated solution preservation technology, and domestic uniform and specialized bait microalgae quality standards and centralized supply points are almost not available.

Due to untimely supply and frequent disjunction with the seedling growing progress, the supply quantity and the requirements of various varieties of fresh and live baits in the seedling growing are difficult to meet, so that the seedling growing survival rate is low, the disease resistance of seedlings is poor, the seedling growing cost is high, and the later-period cultivation is very unfavorable. Therefore, it is urgent to develop high-density microalgae culture technology, keep-alive harvesting and concentrating technology, and concentrated solution preservation technology, and construct standardized bait microalgae production technology system and product quality standard.

The aquaculture scale of China is world-first, and the demand for bait microalgae is huge. Calculated by Muller-Feuga of French scholars, the weight of microalgae biomass required by each million sea water fish, bivalve shellfish and prawn seedlings is respectively 60 kg, 14 kg and 0.65 kg. According to the method, based on the seedling feeding amount of the three aquaculture animals in 2010, 16000 tons of dry microalgae are needed, the current commercial microalgae yield in China is less than 10000 tons, and the commercial bait microalgae yield is very low. Therefore, the market prospect is very wide, and the economic benefit and the social benefit are very obvious.

Disclosure of Invention

In view of the above, the present invention provides a microalgae culture medium and a culture method thereof. The culture medium can effectively promote mitosis of algae cells, accelerate the growth speed of algae, can produce 10 tons of fresh and alive microalgae with high concentration and high activity every day, and is suitable for large-scale industrial culture.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a microalgae culture medium, which comprises the following components:

75-100g/LNaNO₃、13-15g/LNH₄HCO₃、5-10g/LNaH₂PO₄·2H₂O、3-5g/LK₂HPO₄·3H₂O、1-2g/L KH₂PO₄0.05-0.1 v/v% Tween 80, 25-30g/L NaCl, 100-.

Wherein the vitamins consist of vitamin B12, biotin and vitamin B1.

The trace elements are Na₂EDTA、FeCl₃.6H₂O、CuSO₄·5H₂O、ZnSO₄·7H₂O、CoCl₂·6H₂O、MnCl₂·4H₂O and Na₂MoO₄·2H₂And (C) O.

In some embodiments, the microalgae of the invention are cultured and consist of:

the culture medium for microalgae is added with biotin (vitamin H) with a specific concentration, provides extra amino acid nutrients for the microalgae, can effectively promote mitosis of cells and accelerate growth of algae. Meanwhile, in order to maintain the pH value of the microalgae culture environment at 9-10, the pH fluctuation is effectively controlled within a small range by introducing a phosphate buffer system into the formula. Tween 80 is added into the culture medium, so that the permeability of cell membranes can be promoted, and the absorption of nutrients by cells can be accelerated. The combined action of various nutrients is favorable for promoting the mitosis of algae cells and accelerating the growth speed of algae.

In some embodiments, the culture medium of the present invention further comprises agar, i.e., a plating medium of the present invention.

In some embodiments, the concentration of agar in the microalgal medium is 1.7-2 g/L. In some embodiments, the medium is 1.7 g/L.

The invention also provides a culture method of the microalgae, which comprises the following steps:

step 1, streaking microalgae liquid through a plate culture medium, and separating single algae colonies;

step 2, inoculating the single algae colony into a liquid culture medium, and culturing for 2-3 days under 3000-;

and step 3: inoculating the culture solution and the liquid culture medium in the step 2 into the liquid culture medium according to the ratio of 1:3-1:10, placing the liquid culture medium in an indoor window along 0.5-1 meter for natural light culture on the first day, carrying out 3000-;

and 4, step 4: inoculating the culture solution obtained in the step (3) into a liquid culture medium, and carrying out step-by-step enlarged culture;

the plate culture in the step 1 is the microalgae culture medium added with agar, and the liquid culture medium in the steps 2-4 is the microalgae culture medium not added with agar.

In the present invention, the ratio of the inoculation in step 3 may be specifically 1:3, 1:5 or 1: 10.

In the invention, the step-by-step enlarged culture comprises 250ml triangular flask culture, 2.5L triangular flask culture, 5L oil drum culture, 20L oil drum culture, 100L reactor culture and 400L upright column culture.

Specifically, the culture conditions of the 250ml triangular flask culture are as follows: culturing for 24h under full illumination with light intensity of 3000-;

the culture conditions of the 2.5L triangular flask culture are as follows: culturing for 24h in full light, with light intensity of 5000-;

the culture conditions of the 5L oil drum culture and the 20L oil drum culture are as follows: culturing for 24h under full illumination with light intensity of 6000-;

the culture conditions of the 100L reactor culture are as follows: culturing for 48-72h at 25-32 ℃ under full illumination with the illumination intensity of 5000-;

the culture conditions of the 400L upright column culture are as follows: culturing for 48-72h at 25-32 ℃ under natural illumination with the illumination intensity of 5000-;

the microalgae culture medium provided by the invention provides proper nutrient components for the growth of microalgae, can effectively promote the mitosis of algae cells and accelerate the growth speed of algae, and is suitable for large-scale industrial culture. The culture method provided by the invention is expanded from the algae seeds to the upright column production step by step, and covers the whole process of industrial culture of the microalgae. 10 tons of fresh and alive microalgae with high concentration and high activity can be produced every day, and the microalgae can be cultured according to the culture method of the invention, and the production scale of the microalgae can reach 200 t/year.

Drawings

FIG. 1 is a schematic drawing of a plating streaking method;

FIG. 2 shows a schematic representation of the coated plate and the algae seed separation after streaking of the plate.

Detailed Description

The invention provides a microalgae culture medium and a culture method. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated below with reference to the following examples:

example 1

1. Flat coating

1.1 culture Medium, preparation

1) Nutrient solution mother liquor formula (1L)

TABLE 1

Name of material	Molecular formula	Rank of	Weight (g)
				Sodium nitrate	NaNO₃	AR	75
Ammonium bicarbonate	NH₄HCO₃	AR	13
				Sodium dihydrogen phosphate dihydrate	NaH₂PO₄·2H₂O	AR	5
Dipotassium hydrogen phosphate trihydrate	K₂HPO₄·3H₂O	AR	3
				Potassium dihydrogen phosphate	KH₂PO₄	AR	1.2
Tween 80	Tween-80	AR	0.5ml

Vitamin mother liquor formula (100 ml):

TABLE 2

Sterilizing the nutrient solution mother liquor at 121 deg.C for 20min, cooling, and adding 1ml vitamin mother liquor (0.22 membrane filtration sterilization of the mother liquor).

2) Microelement solution mother liquor formula (1L)

TABLE 3

Dissolving 17g of agar in 1L of purified water, adding 25g of NaCl, heating to dissolve, placing in a sterilization pot, sterilizing at 121 ℃ for 30 min. And (3) sucking 1ml of nutrient solution mother liquor and 1ml of trace element solution on a super clean workbench by using a liquid transfer device when the nutrient solution mother liquor and the trace element solution are hot after the sterilization is finished, adding the nutrient solution mother liquor and the trace element solution into the agar solution, shaking up, sealing, and cooling. (nutrient solution mother liquor and microelement solution need to be sterilized in advance at 121 ℃ for 30min)

1.2 sterile preparation

1) A cotton ball is taken by tweezers to wipe the surface of the super clean bench (a square area with the same width as the shoulder, which is an operation area). The waste cotton balls were placed in a beaker.

2) The alcohol lamp is placed at the center of the operation area, a sterile (121 ℃, 30min sterilization) flat plate is placed on the left side, an inoculating ring, a coating rod and a pipette are placed on the right side, and a lighter is placed outside the operation area on the right side.

3) And opening the ultra-clean bench for ultraviolet irradiation, closing the glass cover and the ventilation system, and performing ultraviolet irradiation for 30 min.

1.3 plate inverting operation

1) And (4) turning off the ultra-clean bench ultraviolet lamp, turning on the ultra-clean bench for ventilation, and turning on the glass cover for 20cm in height. Exhausting for 5 min.

2) The slightly cooled agar medium was sprayed with 75% alcohol onto the surface of the flask and placed in a clean bench.

3) And (3) lighting an alcohol lamp, placing the sterilized culture dish on a table top beside the flame, holding the conical flask filled with the culture medium by the right hand, opening the sealing film by the left hand, and holding the conical flask by the right hand to enable the bottle mouth to rapidly pass through the flame.

4) The culture dish is opened by the left hand to form a gap slightly larger than the bottle mouth, the culture medium (about 10-20 ml) in the triangular flask is poured into the culture dish by the right hand, the culture dish is immediately covered by the left hand, and the culture dish is gently shaken up.

1.4 Flat scribing operations

1) Placing the inoculating loop on flame for firing until the inoculating loop is red; cooling the inoculating loop beside the flame, and opening a test tube cover of the algae liquid to be inoculated;

2) and (3) passing the opening of the test tube through flame, extending the cooled inoculating loop into the algae liquid, and dipping a loop of algae liquid.

3) The left hand opens the dish cover beside the alcohol burner to make the opening angle less than 45 degrees, the right hand rapidly stretches the inoculating loop stained with the algae seeds into the flat plate, draws three to five parallel lines, and covers the dish cover. Care was taken not to lacerate the medium. The scribe pattern is shown in figure 1.

4) And finally, putting the bottom of the dish held by the left hand back into the dish cover, sealing the edge by using a sealing film, and burning residual algae liquid on the inoculating ring by using the outer flame of an alcohol lamp.

5) The streaked plates were placed in a culture rack (2000-.

1.5 Flat coating operation

1) The algae species to be coated were diluted 1:1, 1:10, 1:100, 1:1000 with sterile glass tubes.

2) And (4) sucking the diluted algae liquid by using a sterile pipette with the right hand, opening a plate cover beside an alcohol lamp with the left hand to enable the opening angle to be smaller than 45 degrees, and dropping a drop of algae liquid in the middle of the plate.

3) Burning the coating rod by an alcohol lamp, and uniformly coating the algae liquid on the flat plate after cooling. The bottom of the dish held by the left hand is put back into the dish cover, the edge is sealed by a sealing film, and the residual algae liquid on the coating rod is burnt out by the outer flame of the alcohol lamp.

4) Placing the streaked plate on a light culture frame for inverted culture for 5-7 days.

5) And (3) sample reserving and culturing: the plate or test tube of the source algae needs to be cultured continuously for later retrospective inspection, and the algae can not be discarded until the batch of algae is normally produced (production is finished).

FIG. 2 is a schematic diagram of the coated plate and the algae seeds after streaking of the plate.

2 test tube culture

2.1 preparation of microalgae culture Medium

Adding 25g NaCl into 1L of purified water, sterilizing at 121 deg.C for 30 min. And (3) sucking 1ml of nutrient solution mother liquor on a super clean workbench by using a liquid transfer machine when the mother liquor is hot after the sterilization is finished, adding 1ml of trace element solution into the culture medium, shaking up, sealing, and cooling. (nutrient solution mother liquor and microelement solution need to be sterilized in advance at 121 ℃ for 30min)

2.2 Disinfection

1) Placing the sterilized test tube, test tube cover, sterilized culture medium, triangular flask, seed picking needle (or inoculating loop), and test tube rack into a super clean bench, and ultraviolet sterilizing for 30 min.

2) Before the air conditioner is used, the ultraviolet lamp is turned off, the fluorescent lamp is turned on, the windshield in front of the super clean bench is opened to be about 25-30cm high, the fan switch is pressed to start the fan, and the air speed is set to be medium.

2) Spraying 75% alcohol on the flat plate to be sampled and the hand, sterilizing, and placing into a clean bench.

2.3 inoculation procedure

1) Igniting the alcohol lamp, epitaxially burning the culture medium bottle cap by the flame of the alcohol lamp, opening the cap beside the alcohol lamp, and epitaxially burning the culture medium bottle mouth and the bottle cap by the flame of the alcohol lamp.

2) Opening the sealing film of the triangular flask beside the alcohol lamp, burning the opening of the triangular flask by the flame extension of the alcohol lamp, and then pouring the culture medium into the triangular flask.

3) The culture medium in the triangular flask is uniformly subpackaged into the test tubes, so that each test tube has the same volume and is placed on the test tube rack in a staggered manner.

4) And (3) starting a flat plate to be sampled beside the alcohol lamp, burning and disinfecting the inoculating loop by using outer flame of the alcohol lamp, and picking proper single algae to fall into the test tube after cooling.

(how to judge whether the temperature of the inoculating loop is proper: the inoculating loop can be tried on the edge of the plate, if the agar is melted, the inoculating loop is not cooled, and the inoculating loop is tried again after a little time)

5) Quickly burning the test tube opening and the test tube cover, closing the test tube cover, winding and sealing by using a breath sealing film, and labeling.

6 after sampling, covering a flat plate cover, sealing by using a breathing sealing film, and marking.

7) And filling in batch production records.

8) And (3) sample reserving and culturing: the algae seed plate after being sampled is not discarded, the plate is covered by flame for two or three times to sterilize, the plate is closed, a sealing film is used for sealing, and the plate is placed on a light culture frame for continuous culture so as to facilitate the retrospective examination at the later period. The algae species can not be discarded until the batch of algae species is normally produced (production is finished).

2.4 culture conditions

The inoculated test tube liquid is placed in a light culture rack (3000 plus 6000lux) in a clean area for culture, and is ventilated for 2-3 times every day by hands or a vortex oscillator, the inclination angle of the test tube is noticed in the ventilation process, and the liquid in the test tube is prevented from contacting the opening of the test tube.

3. 250ml triangular flask culture

3.1 Disinfection

1) Placing 250ml triangular flask sterilized at 121 deg.C, a 1L triangular flask, and high temperature sterilized culture medium (prepared with culture medium 2.1) into a super clean bench, and ultraviolet sterilizing for 30 min.

3) Spraying 75% alcohol to test tube and hand, sterilizing, and loading into clean bench.

3.2 inoculation procedure

2) Opening a 1L triangular flask sealing film beside the alcohol lamp, burning the triangular flask mouth by the flame extension of the alcohol lamp, and then pouring the culture medium into the triangular flask.

3) The rubber band of a 250ml triangular flask is removed beside the alcohol lamp, one corner of the sealing film is clamped by the thumb and the forefinger of the left hand, the other three fingers hold the other side of the shake flask, the 1L triangular flask is held by the right hand, the mouth of the flask is burned by the outer flame of the alcohol lamp, and then the culture medium is slowly poured into the 250ml triangular flask.

4) Quickly closing the sealing film of the 250ml triangular flask, and pushing aside for standby.

5) According to the third and fourth points, the culture medium is completely subpackaged, and the liquid level of the culture medium in each 250ml triangular flask is ensured to be consistent.

6) The tubes to be inoculated were checked for turbidity and algae phase difference group was removed.

7) Randomly extracting the test tubes qualified by visual inspection (the extraction ratio is 1:5, namely 6 test tubes are selected for microscopic inspection for 30 test tubes), quickly burning the tube opening by flame, opening the tube cover, sucking 1ml of algae liquid by using a sterile pipettor and a sterile gun head, putting the algae liquid into a 2ml centrifuge tube, and conveying the algae liquid from a super clean bench for microscopic inspection to ensure sterility and no insects.

8) The test tube mouth is burnt fast to flame, opens the test tube lid, presss from both sides the one corner of 250ml triangular flask sealing membrane with left hand thumb and forefinger, and the other three fingers hold the opposite side of shake flask, and the test tube is held to the right hand, and the test tube mouth is burnt fast to the outer flame of alcohol burner, waits to cool, then pours the algae kind in the test tube into 250ml triangular flask, and every three test tubes merge into a triangular flask.

9) And (3) after inoculation is finished, quickly closing the sealing film of the 250ml triangular flask, checking whether the sealing film is complete, and replacing a new sealing film if the sealing film is broken. When the sealing film is replaced, the new sealing film needs to be sterilized by passing flame for 2-3 times. The triangular bottle is sealed and then is fastened by 2 rubber bands.

10) And (3) sample reserving and culturing: when the test tube is poured, a small amount of algae liquid is left at the bottom of the test tube, a fresh culture medium is added for sample culture, after the culture medium is added, the flame burns the mouth of the test tube and the test tube cover, the test tube cover is closed, the sealing film is used for sealing, and then the test tube is placed on the test tube rack for continuous culture until the batch of algae seeds are normally produced (production is completed), so that the algae seeds can be discarded.

11) Each triangular bottle needs to be labeled and forms a one-to-one corresponding relation with the connected test tubes.

3.3 culture conditions

Culturing in indoor window (0.5-1 m) in the first day (natural light to make the strain adapt to new culture system), culturing in full light culture (3000 plus 6000lux) in the second day, and culturing in light shaking table in the third day. Ventilating for 2-3 times daily, and avoiding liquid in the shake flask contacting the bottle mouth.

42.5L Erlenmeyer flask culture

4.1 Disinfection

1) And (3) sufficiently brushing a 2.5L triangular bottle by using a test tube brush, sealing the bottle by using a silica gel plug, sealing tin foil paper, and placing the bottle in an oven for dry heat sterilization at 160-180 ℃ for 2 hours.

2) Filling tap water into a water bucket for killing, adding 25g/L NaCl, 1ml/L nutrient solution mother liquor, 1ml/L trace element solution and 0.5ml/L sodium hypochlorite solution according to the actual volume, fully and uniformly stirring by using a stirring rod, putting a plastic water ladle, closing the bucket cover, and killing overnight.

3) Neutralizing the water with sodium thiosulfate, adding the sodium thiosulfate to the sodium hypochlorite solution to obtain a solution with the same effective chlorine content (for example, adding 40ml of sodium hypochlorite with the effective chlorine content of 10% for killing, then neutralizing with 4g of sodium thiosulfate), and detecting with a residual chlorine tester to ensure that the water does not contain residual chlorine.

4.2 inoculation procedure

1) Closing the air conditioner of the room, and spraying the room with the benzalkonium bromide solution for disinfection;

2) the vaccinee should wear the lab coat, wear the mask and gloves, and sterilize the hands and table top with 75% alcohol solution.

3) An alcohol lamp is lighted on the desk, the barrel cover of the disinfected water barrel is opened, 2.5L of triangular flask tinfoil paper and a silica gel plug are opened on the edge of the alcohol lamp by the left hand, the silica gel plug is placed on the desk (on the edge of the alcohol lamp) in an inverted mode, and the right hand grabs a water ladle in the water barrel to scoop 1.5L of water into the triangular flask. And (4) putting the water ladle back to the water bucket, plugging the silica gel plug on the flame for two or three times, sealing the triangular bottle mouth, and sealing by using the tin foil paper.

4) And (3) subpackaging all 2.5L triangular flasks to be inoculated with the culture medium, sealing, and spraying the culture medium to the surrounding environment by using a Xinjieer solution.

5) Opening the triangular flask of the algae seed solution beside the alcohol lamp, igniting the bottle mouth by flame, opening the tinfoil paper and the silica gel plug of the 2.5L triangular flask, inoculating the algae seed solution into the 2.5L triangular flask, and reserving a small part of the algae seed solution for sample reserving culture.

6) Burning the triangular bottle mouth by an alcohol lamp, plugging the silica gel plug on flame for two or three times, sealing the triangular bottle mouth, sealing by using tin foil paper, sticking a label, and indicating the inoculation date, the algal species batch number and the inoculator.

7) And (3) sample reserving and culturing: adding fresh culture medium into the reserved triangular flask of the algae liquid, killing the triangular flask mouth by flame, sealing by using an original sealing film, and placing in a proper environment for continuous culture. The algae species can not be discarded until the batch of algae species is normally produced (production is finished).

4.3 aerated culture

1) Transferring the 2.5L triangular flask to an algae seed expanding culture chamber, continuously performing aeration culture, lifting the air stone and the air pipe, boiling, sterilizing, and placing in an oven for drying.

2) Before the air pipe is connected with the air stone, the mask is worn, the hands and the surrounding environment of the air pipe opening are sprayed with 75% alcohol, the air stone and the air pipe which are fully dried are taken out from the sterile bag, and the air stone and the air pipe are quickly connected.

3) And opening a silica gel plug of the 2.5L triangular flask, putting air stones into the culture solution, penetrating the silica gel plug at the upper end to be connected to the air port, quickly closing the silica gel plug, and covering the air port at the top of the silica gel plug by using tinfoil paper.

4) The air valve is opened to make the air in the bottle uniformly escape.

5) The ratio of the algae solution in the same batch is 1:10 proportion, randomly sampling and performing microscopic examination, and if the infected bacteria or the infected insects are found, discarding. (if the algae liquid is obviously turbid and yellow and the algae bodies are more sunk, microscopic examination is required)

4.4 culture conditions

24h full light irradiation, light intensity of 5000-.

55L oil drum culture

5.1 Disinfection

1) Detecting whether the 5L oil drum is dry and clean;

2) 5L oil drums and drum covers are put into a first dressing room in a clean area one night in advance, and ultraviolet disinfection (disinfection at regular time of one night and two nights, 00 per night: 00-02:00 is the UV on time). When in sterilization, the front of the bucket opening is required to be upward and is completely opened.

3) The next day, spray both hands with 75% alcohol, get into the clean first dressing room of district, close 5L oil drums with the bung. And then taken out of the denuded zone.

4) Preparing a funnel and multiple 500 mesh gauze, placing in a sterilization bag, and sterilizing at 121 deg.C for 30 min.

The same water for cultivation was used for sterilization as 4.1.

5.2 inoculation procedure

3) The alcohol lamp is lighted on the desk, the barrel cover of the disinfected and killed water barrel is opened, the barrel cover of the oil barrel is opened on the edge of the alcohol lamp, the water barrel is placed on the desk (on the edge of the alcohol lamp) in an inverted mode, the sterilization bag is opened, the funnel is taken out and placed on the opening of the oil barrel, then the water ladle in the water barrel is grabbed, and 3.5L of water is scooped into the oil barrel. The water ladle is put back into the water bucket, the bucket cover is quickly put on the flame for two or three times, and the oil bucket is covered.

4) And (3) filling the required oil receiving barrel with the culture medium with the same volume according to the operation, and spraying the culture medium to the surrounding environment by using the Xinjieer solution again.

5) Placing the algae seeds to be inoculated with 2.5L in sequence beside an alcohol burner, opening an oil barrel cover, putting a funnel, putting a 500-mesh filter screen in the funnel, opening a bottle of 2.5L algae seeds, slightly burning a bottle opening, and pouring the algae seeds into a 5L oil barrel. (if the filter screen is not smooth, the new filter screen can be replaced in time)

6) And (3) sample reserving and culturing: every bottle of 2.5L algae seeds is subpackaged into 2 oil barrels of 5L. A part of the algal species is reserved and transferred to a sterilized 250ml triangular flask, and fresh culture medium is added at a ratio of 1:1 for continuous reserved culture.

7) After the split charging is finished, the barrel cover is quickly put on flame for two or three times, the barrel opening is sealed, the label is pasted, the inoculation date, the algae seed batch number and the inoculation person are marked, and then the barrel cover is transferred to an algae seed expanding culture room.

5.3 aeration culture

1) Transferring 5L oil barrel to algae seed expanding culture chamber, performing aeration culture, boiling in advance for killing air stone and air pipe matched with the oil barrel cover, and oven drying.

3) Opening the barrel cover of the oil barrel, putting air stones into the culture solution, connecting the upper end of the oil barrel with an air valve on the barrel cover, closing the barrel cover, connecting the air valve on the upper end of the barrel cover and the air pipe opening by using a sterilized air pipe, and opening the air valve to enable the air stones in the barrel to uniformly discharge air.

4) The culture conditions are as follows: 24h full light irradiation, light intensity 6000-.

The ratio of the algae solution inoculated in the same batch to the algae solution was 1:10 proportion, randomly sampling and performing microscopic examination, and if the infected bacteria or the infected insects are found, discarding. (if the algae liquid is obviously turbid and yellow and the algae bodies are more sunk, microscopic examination is required)

5.4 culture conditions

Light supplement is carried out in the whole process (6000-.

620L oil drum culture

6.1 Disinfection

The disinfection method is the same as the step 5.1.

6.2 inoculation procedure

2) the vaccinee should wear the lab coat, wear the mask and gloves, and sterilize the hands and ground with 75% alcohol solution.

3) Opening the sterilized cover of the bucket, opening the cover of the oil bucket, standing upside down on a table (on the edge of an alcohol burner), opening the sterilization bag, taking out the funnel, placing the funnel on the opening of the oil bucket, grabbing a water ladle in the bucket, and scooping 14L of culture medium into the oil bucket. The water ladle is put back into the water bucket, the bucket cover is quickly put on the flame for two or three times, and the oil bucket is covered.

5) Placing 5L oil drum algae seeds to be received beside an alcohol burner in sequence, opening an oil drum cover, placing a funnel, placing a 500-mesh filter screen in the funnel, opening a 5L bucket of algae seeds, slightly firing a bottle mouth, and pouring the algae seeds into a 20L oil drum. (if the filter screen is not smooth, the new filter screen can be replaced in time)

6) A bucket 5L oil drum algae kind connects to in 1 20L oil drums.

7) And (3) sample reserving and culturing: a portion of the algal species at the bottom was removed and transferred to a 250ml Erlenmeyer flask (sterilized at 121 ℃) and fresh medium was added at 1:1 for further culture.

After inoculation is finished, the barrel cover is quickly placed on flame for two or three times, the barrel opening is sealed, a label is pasted, the inoculation date, the algal species batch number and the inoculation person are marked, and then the barrel cover is transferred to an algal species expanding culture room.

6.3 aeration culture

The method is the same as the step 5.3.

6.4 culture conditions

Light supplement is carried out in the whole process (6000-.

Inoculation culture of 7100L reactor

7.1 Disinfection

1) Weigh 2kg of crude salt and pour into a salt dissolving basin. Boiling 3L of boiled water, pouring into a salt dissolving basin to dissolve the crude salt. Placing funnel and 400 mesh sieve on 5L blue cap bottle, filtering the dissolved crude salt into blue cap bottle, and sterilizing in sterilizing pot at 121 deg.C for 30 min.

2) The 100L reactor was filled with water (80L), and the hot crude salt sterilized at 121 ℃ was poured into the reactor and 40ml of sodium hypochlorite solution was added, and the water pump was started to circulate for 5 min.

3) And (5) closing the water pump, and killing the water body for more than 4 hours.

4) Neutralization was carried out with sodium thiosulfate in the same amount as 4.1. Meanwhile, an air pump and a water pump of the reactor are opened, and aeration is fully performed.

5) 80ml of nutrient solution mother liquor and trace element solution (which need to be sterilized at 121 ℃ in advance) are respectively measured on a super-clean workbench by using a sterile measuring cylinder and are quickly added into a reactor.

7.2 inoculation procedure

1) Closing the room air conditioner, and spraying a new benzalkonium bromide solution to the periphery of the reactor for disinfection;

2) the vaccinee should wear a lab coat, wear a mask and gloves, and disinfect his hands with a 75% alcohol solution.

3) Opening the sealing film of the inoculation port, opening the sterilization bag, taking out the funnel, placing the funnel on the inoculation port, and placing a sterilized 500-mesh filter screen.

4) And opening a cover of the algae seed barrel to be connected with the 5L oil drum, and pouring the algae seeds into the reactor through a funnel. (if the filter screen is not smooth, the new filter screen can be replaced in time)

5) Four barrels of 5L oil drum algae are inoculated in 1 reactor. A part of the algal species at the bottom is kept, transferred to a triangular flask (sterilized at 121 ℃) and added with fresh culture medium for continuous culture.

6) After completion, the inoculation port is covered by making a cup shape with tinfoil paper (the cup is not closed, and the unsmooth exhaust is avoided), and the inoculation record is made on the record book.

7) Sampling by a bottom valve, performing microscopic examination, and recording parameters such as density, pH and the like of the cultured initial algae.

8) Samples were taken once a day in the morning and afternoon and the algal growth density and pH changes were recorded.

9) And (3) sample reserving and culturing: before releasing the algae seeds, a part of algae liquid is taken out from a sampling port by using a small sterile triangular flask, and a fresh culture medium is added in an equal volume for sample reserving culture so as to be checked later. Before the sampling port is connected with the algae liquid, a part of the algae liquid is discharged to scrub the sampling valve, and then the algae liquid is connected into the triangular flask.

7.3 culture conditions

Light supplement, ventilation and a boiled water pump are carried out in the whole process, and the temperature is controlled below 32 ℃. If the temperature is too high, the light supplement lamp is turned off, so that the light supplement lamp is turned on after the temperature is reduced.

8400L/column production

8.1 Disinfection

The disinfection is uniformly carried out by adopting bleaching water to disinfect to the main concentration of 500 ppm. For example, one column of 400 liters requires 200mL of bleaching water.

After the bleaching water is added into the water body, the bleaching water is ensured to be sufficiently uniform (aeration is carried out for 10-20 minutes), and then the air is stopped to seal the disinfection container. All the instruments to be used are sterilized. The neutralization was carried out with sodium thiosulfate at a concentration of 50 ppm. For example, 20 g of sodium liu sulphate is needed to be added into 400L of one upright post. After the sodium thiosulfate needs to be fully dissolved by boiled water, the sodium thiosulfate needs to be fully and uniformly added into the water body (aeration), residual chlorine is detected after 20 minutes, and if the residual chlorine still exists, ten percent of the sodium thiosulfate needs to be added for continuous neutralization.

8.3 harvesting

When the algae liquid is discharged, the algae liquid needs to be filtered by 400 meshes (and above), and then the algae liquid is pumped to a centrifugal machine through a water pump. The algae liquid from the centrifuge also needs to be filtered by 400 meshes (and above).

Example 2

The method and the culture medium of example 1 are compared with the culture medium which is commonly available in the market by using the chlorella as the algae species, the growth rate of the microalgae is calculated, the algae species and the culture medium are shown in a table 4, and the algae density result is shown in a table 5. The results are as follows:

TABLE 4

TABLE 5 algal Density results (units ten thousand/ml)

Incubation time	Experimental group	Control group 1	Control group 2	Control group 3
					0h	2.6	2.3	1.9	1.75
12h	5.8	3.8	4.8	2.55
					24h	10.95	7.35	5.5	4.25
36h	15.5	5.9	6.6	5.5
					48h	23.8	10.15	5.25	4.6
60h	27.55	8.5	9.2	4.35
					72h	36.05	10.1	9.6	3.73

The results show that the growth rate of microalgae cultured by the culture medium is more than 3 times that of the control group, and the cell density is higher.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A microalgae culture medium is characterized by comprising the following components:

75-100g/L NaNO₃、13-15g/L NH₄HCO₃、5-10g/L NaH₂PO₄·2H₂O、3-5g/L K₂HPO₄·3H₂O、1-2g/L KH₂PO₄0.05-0.1 v/v% Tween 80, 25-30g/L NaCl, 100-.

2. The microalgal culture medium of claim 1, wherein the vitamins consist of vitamin B12, biotin and vitamin B1.

3. The microalgal culture medium of claim 2, wherein the trace element is Na₂EDTA、FeCl₃.6H₂O、CuSO₄·5H₂O、ZnSO₄·7H₂O、CoCl₂·6H₂O、MnCl₂·4H₂O and Na₂MoO₄·2H₂And (C) O.

4. The culture medium according to claim 1, consisting of:

5. the microalgal culture medium according to any one of claims 1 to 4, further comprising agar.

6. The microalgal medium of claim 5, wherein the concentration of agar in the microalgal medium is 1.7 g/L.

7. A culture method of microalgae is characterized by comprising the following steps:

wherein the plate culture in the step 1 is the microalgae culture medium according to claim 5 or 6, and the liquid culture medium in the steps 2 to 4 is the microalgae culture medium according to any one of claims 1 to 4.

8. The culture method according to claim 7, wherein the stepwise scale-up culture comprises 250ml triangular flask culture, 2.5L triangular flask culture, 5L oil drum culture, 20L oil drum culture, 100L reactor culture, and 400L upright column culture.

9. The culture method according to claim 8, wherein the culture conditions for the 250ml Erlenmeyer flask culture are: culturing for 24h under full illumination with light intensity of 3000-;

the culture conditions of the 400L upright column culture are as follows: culturing at 25-32 deg.C under natural illumination for 48-72h with illumination intensity of 5000-.