Method for promoting propagation of Halasiosira wissii and used microorganism

Abstract

The invention provides a method for promoting propagation of Hilasiosira wiskei and a microorganism used in the method. The microorganism used in the method is flagellimonas sp. FLG-A strain, and the preservation number of the FLG-A strain is CGMCC No.34666. The invention further provides a method for promoting propagation of Hilasiosira wiskei. The flagelmonas FLG-A strain provided by the invention can effectively improve the propagation effect of the sealasiosira wissi, and has good production and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine microalgae cultivation, and in particular relates to a method for promoting the propagation of Thalassiosira wiseri and the microorganisms used therein. Background Art

[0002] Thalassiosira wiseri ( Thalassiossira weissflogii ) is a typical species of planktonic diatoms in the Centromorpha. Due to its small size, short growth cycle, high degree of cell wall silicification, and strong survival, it is widely distributed in various aquatic environments. As a typical microalgae, Thalassiosira wisseni is rich in ω-3 unsaturated fatty acids (EPA and DHA) and fucoxanthin, an important carotenoid widely found in diatoms and brown algae, with antioxidant, anti-inflammatory, and anti-tumor activities. In aquaculture, Thalassiosira wisseni is widely used as a bait, not only promoting the growth and development of aquatic animals and providing essential nutrients, but also enhancing their immunity and antioxidant capacity. Therefore, it is considered a potential high-quality bait.

[0003] Currently, research on the cultivation of bait microalgae such as Thalassiosira wissenii, both domestically and internationally, focuses primarily on regulating environmental factors such as light, salinity, and temperature, as well as optimizing the organic and inorganic nutrients in the culture medium. Despite some progress in these areas, the actual cultivation of Thalassiosira wissenii remains insufficiently dense to meet the feed requirements for large-scale seedling production of aquatic animals.

[0004] In recent years, studies have found that there are complex interactions between algal bacteria and microalgae, which have an important impact on the proliferation of microalgae. For example, Haematococcus pluvialis ( Haematococcus pluvialis ) Cultivable bacteria in the algae genus Methylobacterium ( Methylobacterium sp.) can significantly increase the biomass of Haematococcus pluvialis; the algal bacteria of Chlorella (Rosobacter hengxiongzhi) Dinoroseobacter shiba ) has a promoting effect on the growth of Chlorella and can be used as a biological inoculant to serve the aquaculture industry; Bacillus sulfitans ( Sulfitobacter sp.) can promote the reproduction of diatoms by secreting indoleacetic acid.

[0005] However, the current propagation technology for Thalassiosira wiseri does not consider the impact of phycobiotic bacteria. Therefore, screening phycobiotic bacteria that can promote the growth of Thalassiosira wiseri has important research significance and application prospects. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for promoting the propagation of Thalassiosira wiseri and the microorganisms used therefor, thereby overcoming the deficiencies of the prior art.

[0007] The present invention first provides a flagellate monocytogenes (Flagellimonas Bacillus sp. FLG-A strain, the 16S rDNA sequence of which is SEQ ID NO: 1, and which was deposited on May 23, 2025 at the China General Microbiological Culture Collection Center, located at No. 1, Huayuancun, Beijing, China, and assigned accession number CGMCC No. 34666.

[0008] The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. Flagellimonas The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. Flagellimonas The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. Flagellimonas The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect.

[0009] The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. Flagellimonas The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect.

[0010] The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. Flagellimonas The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. Flagellimonas The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. Figure 2 The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect. DETAILED DESCRIPTION

[0012] The provided Bacillus sp. FLG-A strain can effectively improve the expansion effect of A. cartanica, and has good production application prospect.

[0013] Example 1: Screening of symbiotic strain The symbiotic strain is isolated and screened from the culture solution of Stephanodinium williamsi in January 2024. The strain isolation method is as follows: the culture solution of Stephanodinium williamsi is gradient diluted, and after dilution, it is coated on 2216E solid culture medium, and the culture condition is 28 DEG C, dark, and inverted cultivation for about 1 week, so that different types, shapes and sizes of single colony colonies can be obtained.

[0014] The single colony colonies with high isolation degree, clear boundary and various shapes are picked and transferred to new 2216E solid culture medium, and the purified strain is obtained through multiple bacterial three-zone streaking method. The purified strain is inoculated into 2216E liquid culture medium and placed in a shaking incubator for culture, and the culture condition is 28 DEG C, dark, and the rotation speed is 200 r·min -1 , and after 12 hours, logarithmic phase bacterial solution can be obtained, and glycerol is added and stored in a-80 DEG C refrigerator.

[0015] Stephanodinium williamsi and the interstitial bacteria isolated are respectively cultured to the exponential growth phase. Different interstitial bacteria are added to the culture solution of Stephanodinium williamsi at a ratio of 1:200 of the cell density of the algae and bacteria, and three parallel groups are set for each group, and sterile seawater medium is used as a control group. The cell concentration of Stephanodinium williamsi is used as an index to analyze the influence of each interstitial bacteria on the growth of Stephanodinium williamsi. Finally, one strain with obvious promoting effect on the growth of Stephanodinium williamsi is screened.

[0016] Example 2: Identification of the screened symbiotic strain The identification of the screened symbiotic strain of the present application mainly involves the following steps: The genomic DNA of the strain is extracted by using TIANamp Bacteria DNA Kit kit, and after PCR amplification by using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), it is sent to Qingdao Ruibo Xingke Biotechnology Co., Ltd. for sequencing identification.

[0017] Flagellimonas The sequencing sequence result of the sp. Bacteroidetes is SEQ ID NO:1.

[0018] The 16S rDNA sequence (SEQ ID NO: 1) measured was uploaded to NCBI for BLAST alignment analysis, and the alignment result showed that the screened algicola strain of the application is a Xanthomonas (Xanthomonas sp.) strain, named Xanthomonas (Xanthomonas sp.) FLG-A strain. The preservation number is CGMCC No. 34666, the preservation date is May 23, 2025, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Flagellimonas Flagellimonas The biological characteristics of Xanthomonas (Xanthomonas sp.) FLG-A strain: the colony is round, orange, the surface is moist and smooth, and it is a gram-negative bacterium.

[0019] The biological characteristics of Xanthomonas (Xanthomonas sp.) FLG-A strain: the colony is round, orange, the surface is moist and smooth, and it is a gram-negative bacterium. Flagellimonas Figure 1 .

[0020] Example 3: Application of the strain in promoting the reproduction of W. viridis First, prepare the algal-bacterial expansion liquid: f / 2 liquid medium (provided by Shanghai Guangyu Company), which is composed of 100 mg / L KNO3, 10 mg / L KH2PO4, 3 mg / L C6H5O7Fe, 0.25 mg / L MnCl2 4H2O, 20 mg / L NaEDTA, 5 mg / L CH4N2O, and 20 mg / L NaSiO3.

[0021] In a 100 L conical flask, add 50 mL of the above sterilized algal-bacterial expansion liquid, inoculate W. viridis with a final concentration of 5×10 4 cell / mL, and activate the FLG-A strain with a final concentration of OD 600 =0.1, mix uniformly, and cultivate at 20℃, light intensity of 150 μmol·m -2 ·s -1 , light cycle of 12L:12D for 15 days. Each culture solution is set in triplicate, and the culture density is counted every 3 days using a hemocytometer to evaluate the influence of the growth density of the strain.

[0022] In order to compare the effect of the above expansion method, algal-bacterial expansion liquid, algal-bacterial expansion liquid added with non-algicola isolated Xanthomonas sp. purchased from Marine Microbial and Culture Collection Center (http: / / mccc.org.cn / ); and algal-bacterial expansion liquid added with algicola bacteria FLG-A isolated in the application with growth-promoting effect are used for cultivation, each culture group is set in triplicate, and the inoculation density is 5×10 4 ​​cell / mL, and the culture density was counted every 3 days using a hemocytometer Figure 2 The culture results show that the growth density of the algae cells of W. cordiformis is obviously higher than that of the rest of the groups when the interalgal bacteria screened in the application are added, indicating that the strains screened in the application have a good effect of promoting the growth of W. cordiformis.

Claims

1. A flagellate monocytogenes, characterized in that The deposit number of the flagellate monocytogenes is CGMCC No.34666.

2. The flagellate monocytogenes according to claim 1, wherein The sequence of 16S rDNA of the flagellate monocytogenes is SEQ ID NO:

1.

3. Use of the flagellate monocytogenes according to claim 1 in promoting the growth of Thalassiosira wiseri.

4. Use of the flagellate monocytogenes according to claim 1 in preparing a product for propagation of Thalassiosira wiseri.

5. The use according to claim 4, characterized in that The product is a microbial bacterial liquid.

6. A method for propagating Thalassiosira wiseri, characterized in that: The method comprises adding the flagellate monocytogenes according to claim 1 to a culture medium for culturing Thalassiosira wiseri.

7. The method according to claim 6, wherein Described culture medium, its composition is as follows: 100mg / L KNO3, 10mg / L KH2PO4, 3mg / L C6H5O7Fe, 0.25 mg / L MnCl24H2O, 20 mg / LNaEDTA, 5 mg / L CH4N2O, 20 mg / L NaSiO3.

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