Double-network immobilization carrier of coral symbiotic zooxanthellae and preparation method of double-network immobilization carrier

Abstract

The invention relates to a double-network immobilization carrier of coral symbiotic zooxanthellae and a preparation method, and belongs to the technical field of marine organisms, the carrier is specially designed for zooxanthellae so as to improve the biological activity of zooxanthellae and increase the reproductive capacity of zooxanthellae. The carrier prepared by the method not only can ensure rapid propagation of zooxanthellae in a marine environment and establishment of an efficient symbiotic relationship with madrepore, but also can provide required moisture and nutrition for growth of zooxanthellae. A good affinity effect can be formed between the carrier and zooxanthellae, and the carrier has high biocompatibility. And secondly, the carrier also has excellent tolerance, can keep stability in a severe marine environment, and resists the influence of stormy waves and salinity change. In addition, the carrier not only can protect the zooxanthellae and promote the growth of the zooxanthellae, but also can enhance the resilience and sustainability of the coral reef ecological system.

Description

Technical Field

[0001] The invention belongs to the field of marine biotechnology, and in particular relates to a double-network structure microbial carrier for fixing coral-symbiotic zooxanthellae and a preparation method thereof. Background Art

[0002] Zooxanthellae provide corals with essential organic carbon compounds, such as glucose, glycerol, and amino acids, through photosynthesis. According to statistics, zooxanthellae can meet up to 90% of the energy needs of coral reefs. Fast-growing reef-building corals require a large amount of oxygen, part of which comes from seawater and the other part is provided by zooxanthellae. Zooxanthellae not only provide oxygen and nutrients for corals, but are also closely related to the formation of the calcareous skeleton of coral polyps. The large amount of carbon dioxide (CO2) produced by coral polyps during metabolism will hinder the growth of the skeleton, and zooxanthellae can quickly absorb this CO2 and promote the formation of the coral skeleton. In addition, zooxanthellae also release CO2 during metabolism, and this CO2 is in the form of carbonate (CO3 2- ) and exists in the form of calcium ions (Ca 2+ ) to form the skeleton of corals. Without the presence of zooxanthellae, even if corals can obtain nutrients through other means to survive, their growth will be seriously affected. Zooxanthellae play a vital role in the health and stability of coral reefs and are an indispensable part of coral reef ecosystems. If the zooxanthellae leave or die, the coral reef will lose its color and gradually die. This phenomenon is called bleaching. When environmental pollution and eutrophication of coral reef sediments directly lead to a decrease in the number of zooxanthellae, structural collapse, loss of biodiversity, coral bleaching and death, and loss of ecosystem service functions. Therefore, various marine countries, governments and scientific research departments are extremely concerned about the ecological restoration of coral reef ecosystems. Zooxanthellae inorganic phosphorus transcriptome sequencing revealed differentially expressed genes, allowing planktonic zooxanthellae to work synergistically with symbiotic zooxanthellae to maintain the health of coral communities. Immobilization technology can improve the symbiotic interaction between corals and their symbiotic zooxanthellae by artificially strengthening zooxanthellae and their nutrients, enhance the tolerance of corals to thermal bleaching, and promote the growth of corals.

[0003] The present invention develops an immobilized carrier material with strong biocompatibility with zooxanthellae, which can protect the zooxanthellae from rapid reproduction in the ocean, establish a symbiotic relationship with reef-building corals at a high speed and high efficiency, increase the zooxanthellae content in the corals, and reduce coral bleaching and death.

[0004] Since its advent, immobilized microbial technology has shown broad development prospects. Immobilized microbial technology is an important way to increase the content of zooxanthellae and reduce coral bleaching. At present, there is no microbial restoration technology specifically for zooxanthellae for the restoration of coral reef ecosystems. If free zooxanthellae are directly released into the marine environment, they will be difficult to withstand the impact of waves, resulting in a large loss of zooxanthellae, and will not be able to effectively solve the specific problems faced by coral reef ecosystems in specific sea areas. However, by immobilizing microorganisms on carriers, their anti-interference ability and tolerance can be significantly enhanced. The immobilization technology of zooxanthellae therefore has significant technical advantages and broad application prospects. It not only provides an innovative strategy for the restoration and maintenance of coral reef ecosystems, but also helps to enhance the adaptability of coral reefs to environmental pressures and promote their long-term health and stable development. Summary of the invention

[0005] In view of the defects of the prior art, the present invention aims to provide a novel microorganism immobilization carrier and a preparation method thereof, which is specially designed for zooxanthellae to improve the biological activity and growth and reproduction capacity of zooxanthellae. The carrier prepared by this method can not only ensure the rapid reproduction of zooxanthellae in the marine environment and establish an efficient symbiotic relationship with stony corals, but also provide the required moisture and nutrients for the growth of zooxanthellae.

[0006] The carrier has many advantages: first, the carrier can form a good affinity with the zooxanthellae and has strong biocompatibility. Second, the carrier also has excellent tolerance and can maintain stability in harsh marine environments and resist the impact of wind and waves and salinity changes. In addition, the carrier can not only protect the zooxanthellae and promote their growth, but also enhance the resilience and sustainability of the coral reef ecosystem.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A double-network microbial carrier of coral-symbiotic zooxanthellae, characterized in that the carrier comprises a first layer of intermediate zooxanthellae three-dimensional network structure carrier, Alginate-CHO solution, coral bones and chitosan.

[0009] The first-layer intermediate zooxanthellae three-dimensional network structure carrier has the following components and the weight ratio of each component is as follows:

[0010]

[0011] in:

[0012] The culture solution containing zooxanthellae Cladocopium goreaui has the following components and the weight ratio of each component is as follows:

[0013]

[0014]

[0015] The Alginate-CHO solution has the following components and the weight ratio of each component is as follows:

[0016]

[0017] The weight ratio of each component of the double network microbial carrier of coral symbiotic zooxanthellae is as follows:

[0018] 10 to 30 copies of the first intermediate zooxanthellae three-dimensional network structure carrier,

[0019] Alginate-CHO solution 0.5-0.9 parts,

[0020] 0.5-1 part of chitosan.

[0021] A method for preparing a double-network microbial carrier of coral-symbiotic zooxanthellae comprises the following steps:

[0022] (1) Preparation of culture medium containing zooxanthellae Cladocopium goreaui

[0023] Take 10 to 20 pieces of coral bones, wash them thoroughly, dry them in a drying oven for 1 hour, and then sieve them through 10-mesh and 20-mesh sieves respectively after crushing to select granular coral bones larger than 10 mesh and smaller than 20 mesh.

[0024] Take 1-3 portions of modified L1 medium, sterilize at 121°C for 15 minutes, cool to room temperature, add 2-10 portions of filtered natural seawater, add 0.5-1.5 portions of glucose, add processed coral bone particles, stir for 2 minutes, and then slowly add 1-5 portions of zooxanthellae Cladocopium goreaui in an environment of 16.72-28.40°C. Use a photoperiod of 12 hours of light and 12 hours of darkness to simulate natural conditions, with a light intensity of 90 μE / m 2 / s. Keep the experimental environment ventilated and culture for 1 to 2 days.

[0025] (2) Preparation of the first intermediate zooxanthellae three-dimensional network structure carrier

[0026] A. Preparation of sodium alginate solution

[0027] 0.4-1.2 parts of sodium alginate (SA) were added to a reaction bottle, mixed with filtered natural seawater, placed in a 40°C constant temperature water bath shaker and heated until dissolved to form a 0.5%-1.0% SA solution, and cooled to room temperature.

[0028] B. Preparation of ethylene glycol diglycidyl ether solution

[0029] Add 0.8-1 parts of ethylene glycol diglycidyl ether (EGDE) to a reaction bottle, mix with filtered natural seawater, place in a 40°C constant temperature water bath shaker and mix at 120 rpm for 20 minutes to form an EGDE solution with a concentration of 0.8% to 1.0%.

[0030] C. Dissolve some coral bones

[0031] Acid-base neutralization reaction: Weigh 1 to 3 parts of coral bone, wash thoroughly, dry in a drying oven for 1 hour, crush and add to a reaction bottle; take 2 to 6 parts of citric acid to prepare a 0.1% to 0.4% citric acid solution, add it to the reaction bottle containing the coral bone powder, place it on a 40°C constant temperature water bath shaker and mix at a rate of 120 rpm for 30 to 60 minutes.

[0032] D. Preparation of the first intermediate zooxanthellae three-dimensional network structure carrier mixed solution

[0033] The culture solution containing zooxanthellae Cladocopium goreaui prepared in (1) is added to a synthesis reactor, followed by slowly adding 2 to 6 parts of filtered natural seawater, slowly adding 1 to 5 parts of glucose as a suspending agent, slowly adding a prepared 0.8% to 1.0% EGDE solution as a cross-linking agent, slowly adding a solution after acid-base neutralization reaction as a calcium ion source, and finally adding a prepared 0.5% to 1.0% SA solution as an embedding agent. The synthesis reactor is placed in a 30°C constant temperature water bath shaker and mixed at a speed of 60 rpm for 20 minutes to form a first layer of intermediate zooxanthellae three-dimensional network structure carrier mixed solution that embeds the zooxanthellae and suspends the coral bone particle matrix.

[0034] (3) Preparation of Alginate-CHO Solution

[0035] A. Preparation of sodium alginate solution

[0036] Add 1 to 5 parts of sodium alginate (SA) to a reaction bottle, mix with filtered natural seawater, place in a 40°C constant temperature water bath shaker and heat until it dissolves to form a 0.5% to 1.0% SA solution, and cool to room temperature.

[0037] B. Oxidation reaction

[0038] At room temperature and in the dark, 0.2-1 part of sodium periodate (NaIO4) is slowly added to a 0.5%-1.0% sodium alginate solution. Sodium periodate acts as an oxidant to oxidize the hydroxyl group (-OH) in the sodium alginate into an aldehyde group (-CHO).

[0039] C. Reaction termination

[0040] After adding sodium periodate for oxidation for 24 hours, 0.1 to 0.5 parts of ethanol were added dropwise as a reducing agent to terminate the oxidation reaction, and the reaction was stirred for 15 minutes to terminate the oxidation reaction.

[0041] D. Purification

[0042] Place the reaction solution in a dialysis bag, immerse the dialysis bag in water, and make sure that the dialysis bag is completely submerged and let stand for 1 to 3 days. Then wash with a large amount of deionized water.

[0043] E. Drying

[0044] The washed Alginate-CHO is freeze-dried at -40°C for 1 to 3 days to obtain Alginate-CHO powder.

[0045] F. Adjust the concentration

[0046] The obtained Alginate-CHO powder was redissolved in water to adjust the concentration to 5% to 9%.

[0047] (4) Preparation of dual-network microbial vectors for coral symbiotic zooxanthellae

[0048] In the synthesis kettle, take 10 to 30 parts of the prepared first-layer intermediate zooxanthellae three-dimensional network structure carrier mixed solution and add it to the 5% to 9% concentration Alginate-CHO solution, then add 0.5 to 1 part of chitosan, and place the synthesis kettle in a 30°C constant temperature water bath shaker and mix at 60 rpm for 20 minutes.

[0049] The synthesis kettle was then placed in a vacuum drying oven and dried at a constant temperature of 30°C for 12 hours to obtain a double-network microbial carrier of coral symbiotic zooxanthellae.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The preparation of the double-network immobilized zooxanthellae carrier of the present invention has achieved technical innovation. It is the first time that biotechnology is introduced to repair the coral reef ecosystem and improve the coral growth environment. The prepared double three-dimensional network structure carrier has better mechanical properties and stability than the single network structure. In addition, it shows good biocompatibility with zooxanthellae and can be used in complex seawater environments.

[0052] First, the coral bones used in the present invention are mainly composed of calcium carbonate (CaCO3). 2+ The porous structure of coral bones provides a large surface area, allowing more Ca 2+ It can react with sodium alginate. The reaction formula of sodium alginate and coral skeleton is:

[0053] (C6H7NaO6) n +CaCO3→(C6H7COO)2Ca+CO2.

[0054] However, due to the Ca 2+ The diffusion rate into the sodium alginate solution is slow, and the Ca content in the gel solution needs to be increased. 2 + The concentration is used to increase the diffusion rate, thereby accelerating the formation of gel. Using coral bones and citric acid, the reaction equation is: CaCO3+2C6H8O7→Ca(C6H7O7)2+CO2+H2O. This enhanced gel layer can better fix zooxanthellae, improve their survival rate under environmental pressure, and provide a more stable carrier. However, when Ca 2+ When the source concentration is too high, the flexibility of the formed gel will decrease and it will be easy to break. Therefore, it is necessary to control the reaction time of the coral bone and citric acid. It is appropriate to immerse for about 1 to 2 hours. The reaction time can be appropriately shortened when heated.

[0055] Using EGDE as a crosslinker, its epoxy group (C2H4O) reacts with the carboxylic acid group (-COOH) of sodium alginate to form an ester bond. Under gentle stirring and appropriate temperature, the epoxy group of EGDE opens and reacts with the carboxylic acid group to form a crosslinked network. In this process, the long-chain molecules of sodium alginate are connected by EGDE to form a three-dimensional structure, thereby solidifying sodium alginate into a gel. This reaction is combined with the reaction of calcium ions with sodium alginate to obtain a three-dimensional mesh gel with significantly improved stability and mechanical properties.

[0056] Sodium alginate-COOH+EGDE→Sodium alginate-COO-CH2CH2O-CH2CH2-Sodium alginate

[0057] On this basis, Alginate-CHO is used to react with chitosan. Alginate-CHO and chitosan can react with Schiff base to generate a three-dimensional network structure gel with strong biocompatibility, which interacts with the first layer structure to produce a double network structure microbial carrier with good biocompatibility, stability and mechanical properties, which can cope with the complex environment of seawater. The Schiff base reaction is a chemical reaction in which the carbonyl group (C=O) of an aldehyde or ketone reacts with an amino group (-NH2) to form a Schiff base. In this case, the aldehyde group (-CHO) of Alginate-CHO reacts with the amino group of chitosan. This cross-linking reaction leads to the formation of a three-dimensional network structure, which can absorb water and swell in water to form a hydrogel.

[0058] The following is a simplified reaction:

[0059]

[0060] In addition, part of the glucose added during the preparation process serves as a nutrient for zooxanthellae, and the other part serves as a suspending agent, which slows down the sedimentation rate of coral bone particles that have not reacted with citric acid and dissolved, facilitates their suspension, provides attachment points for zooxanthellae, and enhances the activity and stability of zooxanthellae in the carrier.

[0061] After testing, the concentration of microorganisms embedded in the developed dual-network structure immobilized zooxanthellae carrier was between 5.4×10 7 to 2.8×10 8 The carrier exhibits strong environmental tolerance, with a mechanical strength of 75 to 86 mN, and can immobilize zooxanthellae in a seawater environment, a temperature of 10 to 35°C, and a pH of 6.4 to 9.0, with an efficiency of 82.15% to 95.43%. In addition, the relative biological activity of the immobilized zooxanthellae is about 60% higher than that of the free state. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0063] Figure 1 is a flow chart of the preparation method of the present invention;

[0064] Figure 2 This is a real picture of coral bones in an embodiment of the present invention;

[0065] Figure 3 is an electron microscope scanning effect diagram of the immobilized zooxanthellae in an embodiment of the present invention;

[0066] Figure 4 is an activity curve diagram of the immobilized zooxanthellae and the control sample (free zooxanthellae) at different time periods at 26° C. in the embodiment of the present invention;

[0067] Figure 5 is a SEM scanning photo of the immobilized zooxanthellae in the embodiment of the present invention;

[0068] Figure 6 This is a schematic diagram of the preparation of Alginate-CHO. DETAILED DESCRIPTION

[0069] The technical solution of the present invention is further specifically described below in conjunction with examples.

[0070] Example 1

[0071] like Figure 1 As shown, in this embodiment, a method for preparing a double-network microbial carrier of coral-symbiotic zooxanthellae comprises the following steps:

[0072] (1) Preparation of culture medium containing zooxanthellae Cladocopium goreaui

[0073] Take 10g of coral bones, wash them thoroughly, dry them in a drying oven for 1h, and then sieve them through 10-mesh and 20-mesh sieves after crushing to select granular coral bones with a size larger than 10 mesh and a size smaller than 20 mesh.

[0074] Take 100 ml of modified L1 medium, autoclave at 121°C for 15 minutes, cool to room temperature, add 200 ml of filtered natural seawater, add 10 g of glucose, add the processed coral bone particles, stir for 2 minutes, and then slowly add 1 to 5 portions of zooxanthellae Cladocopium goreaui in a 26°C environment. Use a photoperiod of 12 hours of light and 12 hours of darkness to simulate natural conditions, with a light intensity of 90 μE / m 2 / s. Keep the experimental environment ventilated and culture for 1 day.

[0075] (5) Preparation of the first intermediate zooxanthellae three-dimensional network structure carrier

[0076] A. Preparation of sodium alginate solution

[0077] 0.75 g of sodium alginate (SA) was added to a reaction bottle, mixed with 100 ml of filtered natural seawater, and placed in a 40° C. constant temperature water bath shaker to heat until it dissolved to form a 0.75% SA solution, which was then cooled to room temperature.

[0078] B. Preparation of ethylene glycol diglycidyl ether solution

[0079] 0.9 g of ethylene glycol diglycidyl ether (EGDE) was added to the reaction bottle, mixed with 100 ml of filtered natural seawater, placed in a 40° C. constant temperature water bath shaker and mixed at 120 rpm for 20 min to form an EGDE solution with a concentration of 0.9%.

[0080] E. Dissolve some coral bones

[0081] Acid-base neutralization reaction: Weigh 10g of coral bone, wash it thoroughly, dry it in a drying oven for 1h, crush it, and add it to a reaction bottle; take 20g of citric acid and add water to prepare a 0.25% citric acid solution, add it to the reaction bottle containing the coral bone powder, place it in a 40℃ constant temperature water bath shaker and mix it at a rate of 120rpm for 30min.

[0082] F. Preparation of the first intermediate zooxanthellae three-dimensional network structure carrier mixed solution

[0083] The culture solution of zooxanthellae Cladocopium goreaui prepared in (1) was added to the synthesis kettle, followed by slowly adding 400 ml of filtered natural seawater, slowly adding 3 g of glucose as a suspending agent, slowly adding a prepared 0.9% EGDE solution as a cross-linking agent, slowly adding a mixed solution after acid-base neutralization reaction as a calcium ion source, and finally adding a prepared 0.75% SA solution as an embedding agent. The synthesis kettle was placed in a 30°C constant temperature water bath shaker and mixed at a speed of 60 rpm for 20 minutes to form a first layer of intermediate zooxanthellae three-dimensional network structure carrier mixed solution that embeds the zooxanthellae and suspends the coral bone particle matrix.

[0084] (6) Preparation of Alginate-CHO Solution

[0085] A. Preparation of sodium alginate solution

[0086] 5 g of sodium alginate (SA) was added to a reaction bottle, mixed with 100 ml of filtered natural seawater, placed in a 40° C. constant temperature water bath shaker and heated until it dissolved to form a 0.5% SA solution, and cooled to room temperature.

[0087] G. Oxidation reaction

[0088] At room temperature and in the dark, 0.6 g of sodium periodate (NaIO4) was slowly added to a 0.5% sodium alginate solution. Sodium periodate, as an oxidant, oxidized the hydroxyl group (-OH) in the sodium alginate into an aldehyde group (-CHO).

[0089] H. Reaction Termination

[0090] After adding sodium periodate for oxidation for 24 h, 3 ml of ethanol was added dropwise as a reducing agent to terminate the oxidation reaction, and the reaction was stirred for 15 min to terminate the oxidation reaction.

[0091] I. Purification

[0092] The reaction solution was placed in a dialysis bag, which was immersed in water to ensure that the dialysis bag was completely submerged and allowed to stand for 1 day. Then, a large amount of deionized water was used for washing.

[0093] J. Drying

[0094] The washed Alginate-CHO was freeze-dried at -40°C for 1 day to obtain Alginate-CHO powder.

[0095] K. Adjust the concentration

[0096] The obtained Alginate-CHO powder was redissolved in water to adjust the concentration to 6%.

[0097] (7) Preparation of dual-network microbial vectors for coral symbiotic zooxanthellae

[0098] In the synthesis kettle, take 30 ml of the prepared first-layer intermediate zooxanthellae three-dimensional network structure carrier mixture and add it to the 6% concentration of Alginate-CHO solution, then add 0.8 g of chitosan, place the synthesis kettle in a 30°C constant temperature water bath shaker and mix at 60 rpm for 20 minutes.

[0099] The synthesis kettle was then placed in a vacuum drying oven and dried at a constant temperature of 30°C for 12 hours to obtain a double-network microbial carrier of coral symbiotic zooxanthellae.

[0100] (5) Detection of the physical and chemical properties of the immobilized carrier

[0101] The physical and chemical properties of the synthetic immobilized microorganism carrier, such as adsorption capacity, specific surface area, mechanical strength, and immobilization efficiency, were tested. The test results are shown in Table 1.

[0102] Table 1 Physical and chemical properties test table of immobilized carrier

[0103] Adsorption capacity Specific surface area Mechanical strength Immobilization efficiency <![CDATA[2.8×10 8 cell / g]]> <![CDATA[1032.43m 2 / g]]> 86mN 95.43％

[0104] After testing, the concentration of microorganisms embedded in the developed dual-network structure immobilized zooxanthellae carrier was between 5.4×10 7 to 2.8×10 8 The carrier exhibits strong environmental tolerance, with a mechanical strength of 75 to 86 mN, and can immobilize zooxanthellae in a seawater environment, a temperature of 10 to 35°C, and a pH of 6.4 to 9.0, with an efficiency of 82.15% to 95.43%. In addition, the relative biological activity of the immobilized zooxanthellae is about 60% higher than that of the free state.

[0105] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A double-network microbial carrier of coral-symbiotic zooxanthellae, characterized in that: The carrier contains a first layer of intermediate zooxanthellae three-dimensional network structure carrier, Alginate-CHO solution and chitosan; The first-layer intermediate zooxanthellae three-dimensional network structure carrier comprises 1 to 5 parts of culture solution containing zooxanthellae Cladocopium goreaui, 0.4 to 1.2 parts of embedding agent, 0.8 to 1 part of cross-linking agent, 1 to 5 parts of suspending agent, and 1 to 3 parts of coral bone, forming a first-layer intermediate zooxanthellae three-dimensional network structure carrier mixed solution that embeds the zooxanthellae and suspends the coral bone particle matrix.

2. The dual network microbial carrier according to claim 1, characterized in that: The culture solution containing zooxanthellae Cladocopium goreaui contains 1 to 3 parts of L1 culture medium, 1 to 5 parts of zooxanthellae Cladocopium goreaui, 0.5 to 1.5 parts of glucose, 2 to 10 parts of filtered seawater and 10 to 20 parts of coral bones.

3. The dual network microbial carrier according to claim 1, characterized in that: The Alginate-CHO solution contains 1 to 5 parts of sodium alginate, 0.2 to 1 part of sodium periodate, and 0.1 to 0.5 parts of ethanol; and 10 to 100 parts of filtered seawater.

4. The dual-network microbial carrier according to claim 1, characterized in that: The carrier contains 10 to 30 parts of a first-layer intermediate zooxanthellae three-dimensional network structure carrier, 0.5 to 0.9 parts of Alginate-CHO solution and 0.5 to 1 part of chitosan.

5. The method for preparing the double-network microbial carrier of coral-symbiotic zooxanthellae according to any one of claims 1 to 4, characterized in that: 10 to 30 parts of the prepared first-layer intermediate zooxanthellae three-dimensional network structure carrier mixed solution are added to a 5% to 9% concentration Alginate-CHO solution, and then 0.5 to 1 part of chitosan is added, mixed, and dried to prepare the solution.

6. The method for preparing the double-network microbial carrier of coral-symbiotic zooxanthellae according to claim 5, wherein the preparation of the first intermediate zooxanthellae three-dimensional network structure carrier mixed solution comprises: A culture solution containing zooxanthellae Cladocopium goreaui is added to a synthesis kettle, followed by slow addition of natural seawater, slow addition of a suspending agent, slow addition of a cross-linking agent, slow addition of a solution after acid-base neutralization reaction as a calcium ion source, and finally addition of an embedding agent, and a constant temperature water bath is performed to form a first layer of intermediate zooxanthellae three-dimensional network structure carrier mixed solution that embeds the zooxanthellae and the suspended coral bone particle matrix.

7. Use of the double-network microbial carrier of coral-symbiotic zooxanthellae according to any one of claims 1 to 4, wherein the carrier is used for fixing the zooxanthellae.

Images