CN111254081A - Large-scale seed preservation and propagation method for pure benthic diatoms - Google Patents
Large-scale seed preservation and propagation method for pure benthic diatoms Download PDFInfo
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Abstract
The invention discloses a large-scale seed preservation and propagation method for pure benthic diatoms, which comprises the following steps: (1) placing the attachment medium in a culture bottle, sterilizing the culture bottle and the attachment medium, cleaning the culture bottle and the attachment medium after sterilization, and then adding a base solution into the culture bottle; (2) adding nutrient solution into the basic solution, and shaking uniformly to obtain culture solution; (3) adding a pure benthic diatom liquid obtained by separation and purification into the culture solution; (4) performing amplification culture to obtain a pure high-concentration algae solution, repeating the step (1) and the step (2) to obtain a culture solution, dividing the pure high-concentration algae solution into multiple parts, wherein each part accounts for 20-30% of the pure high-concentration algae solution, and pouring each part of the pure high-concentration algae solution into one part of the culture solution; (5) and (5) repeating the step (4) to gradually obtain the algae liquid containing a large amount of pure benthic diatoms. The invention can realize large-scale seed preservation and propagation of pure-breed algae liquid and solve the problem that the existing pure-breed benthic diatom can only preserve the seed on a small scale.
Description
Technical Field
The invention relates to the technical field of precious marine product seedling production, in particular to a large-scale seed preservation and propagation method for pure benthic diatoms.
Background
The benthic diatom is high-quality bait for artificial breeding of various aquatic animals such as sea urchins, sea cucumbers, abalones and the like, and the sufficient supply of the benthic diatom can obviously improve the survival rate and the growth speed of the seedlings of the aquatic animals. At present, most seedling raising enterprises adopt the existing method for cultivating benthic diatom feed, namely firstly, seawater containing benthic diatoms is obtained by washing the surfaces of objects such as seaweeds in a sea area, then transparent sheet-shaped PVC corrugated plates (the thickness is 1mm, 33cm multiplied by 40cm) are placed in the benthic diatom seawater to complete inoculation, and the benthic diatoms inoculated on the corrugated plates can be used for collecting seedlings after tens of days of cultivation. Some enterprises also store benthic diatoms cultured on corrugated plates in the last year until the next year, wash down as algae seeds, and inoculate the filtered benthic diatoms to the corrugated plates for reuse. The two methods have the advantages that a large amount of benthic diatoms can be rapidly obtained and used in the large-scale production of rare sea product seedlings, and the defects that the species and the composition of diatoms are uncertain and the quality of diatoms cannot be guaranteed.
The single benthic diatom is easy to obtain, the screened excellent single benthic diatom with different characteristics is expanded and cultured on a large scale, then different single benthic diatoms are reasonably matched according to requirements, and the excellent mixed algae is cultured on a large scale, so that the method has great practical significance for the production of rare sea product seedlings. At present, the single benthic diatom seed preservation method mainly comprises three methods of solid culture medium preservation, liquid culture medium preservation and immobilized preservation. The solid culture medium is prepared by inoculating benthic diatom to agar culture medium containing nutritive salt, and the liquid culture medium is prepared by inoculating benthic diatom to the bottom or inner wall of culture container containing liquid culture medium, and the immobilization preservation mainly comprises preparing colloidal beads from sodium alginate, and allowing benthic diatom to adhere to the colloidal beads for growth. However, these methods are only suitable for small-scale seed preservation, and cannot be used for large-scale seed preservation and large-scale propagation in seed production, so that they cannot be applied in actual production, and a technical method for large-scale single seed preservation and propagation of benthic diatoms is urgently needed in the production of rare sea product seeds.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a large-scale seed preservation and propagation method for pure benthic diatoms, which can realize large-scale seed preservation and propagation of pure benthic diatom liquid, solves the problem that the existing pure benthic diatoms can only preserve seeds in a small scale and cannot be used for large-scale seed preservation and propagation in seedling production, and is convenient to popularize and apply in actual production.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for large-scale seed preservation and propagation of pure benthic diatom, which comprises the following steps:
(1) placing the attachment medium in a culture bottle, sterilizing the culture bottle and the attachment medium, cleaning the culture bottle and the attachment medium after sterilization, and then adding a base solution into the culture bottle;
(2) adding nutrient solution into the basic solution, and shaking uniformly to obtain culture solution;
(3) adding a pure benthic diatom liquid obtained by separation and purification into the culture solution, wherein the separation and purification technology adopts a micro-straw separation method;
(4) performing amplification culture, namely shaking a culture bottle every day during the culture period for 15-20 days to obtain pure benthic diatoms, shaking the bottle violently to obtain pure high-concentration algae liquid, repeating the step (1) and the step (2) to obtain culture liquid, dividing the pure high-concentration algae liquid into multiple parts, wherein each part accounts for 20% -30% of the pure high-concentration algae liquid, and pouring each part of the pure high-concentration algae liquid into one part of the culture liquid;
(5) and (5) repeating the step (4) to gradually obtain the algae liquid containing a large amount of pure benthic diatoms.
Preferably, in the step (1), the attachment base includes a hollow transparent tubular structure, inner protrusions are respectively arranged on inner walls of two ends of the transparent tubular structure, and a movable transparent round ball is arranged in the transparent tubular structure between the two inner protrusions.
Preferably, the tubular structure is a transparent PET or PVC hard transparent round tube with the thickness of 2mm, the height of 30mm, the outer diameter of 10mm and the inner diameter of 6 mm.
Preferably, the inner bulge is 2mm away from the end part of the transparent circular tube, and the inner bulge protrudes 2mm from the inner wall of the tubular structure.
Preferably, the round ball is a hard transparent PET or PVC round ball, and the diameter of the round ball is 5 mm.
Preferably, the culture flask is an erlenmeyer flask, and the volume of the erlenmeyer flask is 5L.
Preferably, in the step (1), the disinfection is performed by pouring a disinfection solvent into the culture flask and soaking and disinfecting the culture flask in the chlorine dioxide solution of 100mg/L for 15-25 minutes without passing through the attachment medium.
Preferably, the volume of the chlorine dioxide solution is 75-85% of the volume of the culture flask.
Preferably, in the step (1), the attachment medium and the inner wall of the culture flask are washed 3-4 times by using seawater which is boiled for 4-6min and cooled to below 40 ℃.
Preferably, in step (1), the base liquid is seawater boiled for 4-6min and cooled to 15-25 ℃.
Preferably, in the step (2), the volume ratio of the nutrient solution to the base solution is (2-3) mL: 1L; the nutrient solution comprises a first nutrient solution and a second nutrient solution in a volume ratio of 1:1, and the two nutrient solutions can prevent sodium metasilicate nonahydrate and disodium ethylene diamine tetraacetate from directly forming a complex compound to influence the using effect of the nutrient salt. The formula of the nutrient solution is beneficial to the differentiation and growth of diatom, and when the nutrient solution is generally used, the first nutrient solution is poured into the base solution and mixed uniformly, and then the second nutrient solution is added and mixed uniformly, so that the risk that sodium silicate nonahydrate and disodium ethylene diamine tetraacetate directly form a complex is reduced.
The first nutrient solution comprises urea, monopotassium phosphate, ferric ammonium citrate, ethylene diamine tetraacetic acid and purified water, wherein the solid-to-liquid ratio of the urea, the monopotassium phosphate, the ferric ammonium citrate, the ethylene diamine tetraacetic acid and the purified water is (10-13), (3-5), (1), (9-11): (0.7-0.8) L;
the second nutrient solution comprises the following components in a solid-to-liquid ratio of (12-18) g: 1L of sodium silicate nonahydrate and purified water.
Preferably, in step (4), the flask is shaken not less than 2 times a day within 1 to 5 days of the culture, and after 5 days, the flask is shaken vigorously 1 time every 2 days except that the flask is shaken not less than 2 times a day.
Through shaking the culture bottle, make mutual striking, friction between adhesion base outer wall, mutual striking, friction between adhesion base outer wall and the culture bottle inner wall to and mutual striking, friction between pellet in the adhesion base and the adhesion base inner wall, benthic diatom on most adhesion base inner and outer wall gets into the algae liquid of formation higher concentration in the basic solution.
Preferably, in the step (4), before bottle sealing and culture, the volume of the base solution is determined, so that the volume of the solution accounts for 75-85% of the volume of the culture bottle.
Preferably, the concentration of the algae cells is greater than or equal to 5000 cells/mL after the volume is determined by the base solution.
In the step (4), the bottle sealing is performed by sterilizing quantitative filter paper. The quantitative filter paper was autoclaved at 121 ℃ for 20 min.
Preferably, in step (4), the culture conditions are such that the culture is performed under the illumination of 1000-.
In a second aspect of the present invention, there is provided a pure benthic diatom produced by the above method.
In a third aspect of the present invention, there is provided the use of the above pure benthic diatoms for the preparation of a composite benthic diatom algae solution of a specific species of algae.
The invention has the beneficial effects that:
1. the method can realize large-scale seed preservation and propagation of the pure benthic diatom liquid, solves the problem that the existing pure benthic diatom can only preserve seeds on a small scale and cannot be used for large-scale seed preservation and propagation in seedling production, and is convenient to popularize and apply in actual production.
2. According to the invention, the attachment medium is placed in the culture bottle, so that the growth area of benthic diatoms in unit space can be increased, and the benthic diatoms comprise the inner and outer walls of the attachment medium and the surfaces of the small balls, so that the yield of algae seeds is effectively improved; by shaking the culture bottle, the outer walls of the attachment medium can be impacted and rubbed with each other, the outer walls of the attachment medium and the inner walls of the culture bottle can be impacted and rubbed with each other, and the small balls in the attachment medium and the inner walls of the attachment medium can be impacted and rubbed with each other, so that the benthic diatoms fall off from the attachment medium and the culture bottle into the culture solution in the culture bottle.
3. The method can produce the pure algae liquid in a large scale, match different kinds of pure algae liquids according to a proportion, can prepare the high-quality composite algae liquid, can directly replace the algae liquid obtained by scouring the seaweeds to be used for large-scale inoculation in seedling production, and can provide high-quality and nutritional benthic diatom mixed bait for the cultured organisms.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, the current single benthic diatoms are mainly preserved in a solid medium, a liquid medium and an immobilized medium, wherein the solid medium is preserved by inoculating benthic diatoms on an agar medium containing nutrient salts, the liquid medium is preserved by inoculating benthic diatoms on the bottom or inner wall of a culture container containing the liquid medium, and the immobilized preservation is mainly realized by preparing colloidal beads from sodium alginate so that the benthic diatoms can grow on the colloidal beads. These methods are only suitable for small-scale seed preservation and cannot be used for large-scale seed preservation and large-scale expanding culture in seedling production.
Based on the method, the attaching medium is placed in the culture bottle by adopting the method for large-scale seed preservation and propagation of the pure benthic diatoms, so that the growth area of the benthic diatoms in unit space is increased, and the formula of the optimized nutrient solution is matched, so that the stable and rapid culture of the pure benthic diatoms is realized, and the large-scale seed preservation and propagation of the pure benthic diatom solution is ensured.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Examples
(1) The method comprises the following steps of placing an attachment base in a 5L conical bottle in a scattered mode, enabling the upper surface of the scattered attachment base to be approximately at the 3L scale mark of the conical bottle, wherein the attachment base comprises a transparent PET or PVC hard circular tube, the thickness of the transparent PET or PVC hard circular tube is 2mm, the height of the transparent PET or PVC hard circular tube is 30mm, the outer diameter of the transparent PET or PVC hard circular tube is 10mm, the inner diameter of the transparent PET or PVC hard transparent circular tube is 6mm, inner protrusions are arranged on the inner walls of two ends of the hard circular tube respectively, the distance between the inner protrusions and the end portion of the circular tube is 2mm, the inner protrusions protrude out of the inner wall of a tubular structure;
pouring 100mg/L chlorine dioxide solution into the culture bottle and immersing and disinfecting the culture bottle and the attachment medium for 15-25 minutes, wherein the volume of the chlorine dioxide solution accounts for 80% of the volume of the culture bottle, and disinfecting the culture bottle and the attachment medium;
after disinfection, cleaning the attachment medium and the inner wall of the culture bottle for 3-4 times by using seawater which is boiled for 4-6min and cooled to below 40 ℃, and then adding 3L of base liquid into the culture bottle, wherein the base liquid is seawater which is boiled for 4-6min and cooled to 15-25 ℃;
(2) adding nutrient solution into the basic solution, and shaking uniformly to prepare the culture solution, wherein the volume ratio of the nutrient solution to the basic solution is 2 mL: 1L; the nutrient solution comprises a first nutrient solution and a second nutrient solution in a volume ratio of 1:1, and when the nutrient solution is used, the first nutrient solution is poured into the base solution and mixed uniformly, and then the second nutrient solution is added and mixed uniformly;
the first nutrient solution comprises urea, monopotassium phosphate, ferric ammonium citrate, disodium ethylene diamine tetraacetate and purified water, wherein the solid-to-liquid ratio of the urea to the monopotassium phosphate to the ferric ammonium citrate to the disodium ethylene diamine tetraacetate to the purified water is 12g:4g:1g:10 g: 0.75L;
the second nutrient solution comprises the following components in a solid-to-liquid ratio of 15 g: 1L of sodium silicate nonahydrate and purified water;
(3) adding the separated and purified Navicula sp solution into the culture solution;
(4) fixing the volume with the basic liquid to make the liquid volume account for 80% of the volume of the culture bottle, wherein the concentration of the algae cells is greater than or equal to 5000 cells/mL after the volume is fixed;
sealing the bottle through sterilized quantitative filter paper, and culturing, wherein the quantitative filter paper is autoclaved for 20min at 121 ℃;
the culture conditions are that the culture is carried out under the illumination of 1000-3000Lux, the room temperature of 15-25 ℃ and the humidity of 40% -60%; shaking the culture bottle for not less than 2 times every day within 1-5 days of culture, after 5 days, shaking the culture bottle vigorously for 1 time every 2 days except that the culture bottle is shaken for not less than 2 times every day, after culturing for 15-20 days, obtaining pure benthic diatom, shaking the bottle vigorously to obtain high-concentration algae liquid, repeating the steps (1) and (2) to obtain culture liquid, dividing the pure high-concentration algae liquid into multiple parts, wherein each part accounts for 25% of the pure high-concentration algae liquid, and each part of the pure high-concentration algae liquid is poured into one part of culture liquid;
(5) and (5) repeating the step (4) to gradually obtain the algae liquid containing a large amount of pure benthic diatoms.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A large-scale seed preservation and propagation method for pure benthic diatoms is characterized by comprising the following steps:
(1) placing the attachment medium in a culture bottle, sterilizing the culture bottle and the attachment medium, cleaning the culture bottle and the attachment medium after sterilization, and then adding a base solution into the culture bottle;
(2) adding nutrient solution into the basic solution, and shaking uniformly to obtain culture solution;
(3) adding a pure benthic diatom liquid obtained by separation and purification into the culture solution;
(4) performing amplification culture, namely shaking a culture bottle every day during the culture period to obtain pure benthic diatoms after the culture, violently shaking the bottle to obtain pure high-concentration algae liquid, repeating the step (1) and the step (2) to obtain the culture liquid, dividing the pure high-concentration algae liquid into a plurality of parts, wherein each part accounts for 20-30% of the pure high-concentration algae liquid, and pouring each part of the pure high-concentration algae liquid into one part of the culture liquid;
(5) and (5) repeating the step (4) to gradually obtain the algae liquid containing a large amount of pure benthic diatoms.
2. The method of claim 1, wherein: in the step (1), the attachment base comprises a hollow transparent tubular structure, inner bulges are respectively arranged on the inner walls of two ends of the transparent tubular structure, and a movable transparent round ball is arranged in the tubular structure between the two inner bulges.
3. The method of claim 1, wherein: in the step (1), the disinfection is to pour a disinfection solvent into the culture bottle and soak and disinfect the culture bottle without passing through the adhesive medium, wherein the disinfection solvent is 100mg/L chlorine dioxide solution, and the soaking and disinfection are carried out for 15-25 minutes.
4. The method of claim 1, wherein: in the step (1), the basic liquid is seawater which is boiled for 4-6min and cooled to 15-25 ℃.
5. The method of claim 1, wherein: in the step (2), the volume ratio of the nutrient solution to the basic solution is (2-3) mL: 1L; the nutrient solution comprises a first nutrient solution and a second nutrient solution in a volume ratio of 1:1, and when the nutrient solution is used, the first nutrient solution is poured into the base solution and mixed uniformly, and then the second nutrient solution is added and mixed uniformly.
The first nutrient solution comprises urea, monopotassium phosphate, ferric ammonium citrate, ethylene diamine tetraacetic acid and purified water, wherein the solid-to-liquid ratio of the urea, the monopotassium phosphate, the ferric ammonium citrate, the ethylene diamine tetraacetic acid and the purified water is (10-13), (3-5), (1), (9-11): (0.7-0.8) L;
the second nutrient solution comprises the following components in a solid-to-liquid ratio of (12-18) g: 1L of sodium silicate nonahydrate and purified water.
6. The method according to claim 1, wherein in the step (4), the flask is shaken not less than 2 times a day within 1 to 5 days of the culture, and after 5 days, the flask is shaken vigorously 1 time every 2 days except that the flask is shaken not less than 2 times a day.
7. The method according to claim 1, wherein in the step (4), before the amplification culture, the volume of the base solution is determined to be 75-85% of the volume of the culture flask.
8. The method as claimed in claim 1, wherein in the step (4), the culture conditions are such that the culture is carried out under an illumination of 1000-3000Lux, at a room temperature of 15-25 ℃ and at a humidity of 40% -60%.
9. A pure benthic diatom produced by the method of any one of claims 1-8.
10. Use of the pure benthic diatoms of claim 9 for the preparation of a composite benthic diatom broth of a specific species of algae.
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2020
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