Method for promoting the multiplication of mayflies and microorganisms used

Abstract

The application provides a method for promoting the propagation of Stephanodinium williamsii and a microorganism used in the method, wherein the microorganism is a strain of Flavobacterium sp. Flagellimonas FLG-A, and the preservation number of the strain is CGMCC No. 34666. The Flavobacterium sp. FLG-A provided by the application can effectively improve the propagation effect of the Stephanodinium williamsii, and has a good production application prospect.

Description

Technical Field

[0001] This invention belongs to the field of marine microalgae cultivation technology, specifically relating to a method for promoting the propagation of *Streptococcus viridans* and the microorganisms used therein. Background Technology

[0002] Weiss's seaweed ( Thalassiossira weissflogii *Ceratophyllum demersum* is a typical species of centroplanktonic diatom. Due to its small size, short growth cycle, high degree of cell wall silicification, and strong survival ability, it is widely distributed in various aquatic environments. As a typical microalga, *Ceratophyllum demersum* is rich in ω-3 unsaturated fatty acids (EPA and DHA) and fucoxanthin, the latter being an important carotenoid widely found in diatoms and brown algae, possessing antioxidant, anti-inflammatory, and anti-tumor biological activities. In aquaculture, *Ceratophyllum demersum* is widely used as feed, not only promoting the growth and development of aquatic animals and providing essential nutrients, but also enhancing their immunity and antioxidant capacity, thus being considered a potentially high-quality feed.

[0003] Currently, domestic and international research on the cultivation of prey microalgae such as *Streptococcus wildae* mainly focuses on the control of environmental factors such as light, salinity, and temperature, as well as the optimization of organic and inorganic nutrients in the culture medium. Despite some progress in these areas, the reproductive density of *Streptococcus wildae* remains insufficient in actual aquaculture, failing to meet the feed requirements for large-scale seedling breeding of aquatic animals.

[0004] Recent studies have revealed complex interactions between algal bacteria and microalgae, which significantly influence microalgal proliferation. For example, Haematococcus pluvialis (… Haematococcus pluvialis )Culturable bacteria of the genus *Methylobacterium* in the algal community ( Methylobacterium sp.) can significantly increase the biomass of Haematococcus pluvialis; Chlorella (Chlorella) phycolytic bacteria Rhodopseudomonas hainanensis ( Dinoroseobacter shiba It promotes the growth of Chlorella and can be used as a biological inoculant for the aquaculture industry; Sulfitebacterium ( Sulfitobacter sp. can promote the reproduction of diatoms by secreting indoleacetic acid.

[0005] Current propagation techniques for *Streptococcus wildae* do not consider the influence of inter-thallophyte bacteria. Therefore, screening for inter-thallophyte bacteria that can promote the growth of *Streptococcus wildae* is of significant research importance and promising application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a method for promoting the propagation of *Streptococcus wildae* and the microorganisms used therein, thereby overcoming the shortcomings of the prior art.

[0007] This invention first provides a flagellated monocytogenes bacterium (Flagellimonas strain FLG-A (sp.) with accession number CGMCC No.34666, deposited on May 23, 2025, deposited at the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0008] The provided flagellated monoclonal bacteria ( Flagellimonas The 16S rDNA sequence of strain FLG-A (sp.) is SEQ ID NO:1;

[0009] The present invention also provides the aforementioned flagellated monoclonal bacteria ( Flagellimonas One application of strain FLG-A (sp.) is in promoting the growth of *Streptomyces wilfordii*.

[0010] The present invention also provides the aforementioned flagellated monoclonal bacteria ( Flagellimonas Another use of sp.) FLG-A strain is in the preparation of products for the propagation of Leymus chinensis;

[0011] One of the products is a microbial liquid.

[0012] In another aspect, the present invention provides a method for propagating *Streptococcus westermani*, wherein the method involves adding the aforementioned flagellated bacteria to the culture medium for culturing *Streptococcus westermani*. Flagellimonas (sp.) FLG-A strain;

[0013] The culture medium, as a specific example described herein, has the following composition:

[0014] 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.

[0015] The flagellated monoclonal bacteria provided by this invention ( Flagellimonas The FLG-A strain (sp.) can effectively improve the propagation effect of *Streptomyces wilfordii* and has a promising prospect for production application. Attached Figure Description

[0016] Figure 1 The flagellated monoclonal bacteria screened in this invention ( Flagellimonas Photograph of FLG-A strain colony (sp.);

[0017] Figure 2 : Data chart of the propagation effect of *Streptomyces wilfordii*. Detailed Implementation

[0018] The application will be described in detail below with reference to the drawings.

[0019] Example 1: Screening of symbiotic strains

[0020] In January 2024, symbiotic strains were isolated and screened from the culture solution of Aureococcus anophageicus. The strain isolation method is as follows: gradient dilution was performed on the culture solution of Aureococcus anophageicus, and after dilution, it was coated on 2216E solid culture medium. The culture conditions were 28℃, dark, and inverted cultivation for about 1 week, and different types, morphologies and sizes of single colony colonies were obtained.

[0021] The single colony colonies with high degree of separation, clear boundary, and various morphologies were picked and transferred to new 2216E solid culture medium, and the purified strains were obtained by multiple bacterial three-zone streaking method. The purified strains were inoculated into 2216E liquid culture medium and placed in a shaking incubator for culture. The culture conditions were 28℃, dark, and the rotation speed was 200r·min -1 , and after 12h, logarithmic phase bacterial solution was obtained, and glycerol was added and stored in a-80℃ refrigerator.

[0022] Aureococcus anophageicus and the interstitial bacteria isolated were respectively cultured to the exponential growth phase. Different interstitial bacteria were added to the culture solution of Aureococcus anophageicus at a ratio of 1:200 of the cell density of the algae and bacteria, and three parallel groups were set for each group. Sterilized seawater medium was used as a control group. The cell concentration of Aureococcus anophageicus was used as an index to analyze the influence of each interstitial bacteria on the growth of Aureococcus anophageicus. Finally, a strain with obvious promoting effect on the growth of Aureococcus anophageicus was screened.

[0023] Example 2: Identification of the screened symbiotic strain

[0024] The identification of the symbiotic strain screened by the application mainly involves the following steps:

[0025] The genomic DNA of the strain was extracted by using a TIANamp Bacteria DNA Kit kit, and universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification, and then sent to Qingdao Ruibo Xingke Biotechnology Co., Ltd. for sequencing identification.

[0026] Flagellimonas The sequencing sequence result of the sp. Bacteroides is SEQ ID NO: 1:

[0027]

[0028] 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 phytostimulatory bacterial strain of the application was a Xanthomonas sp. (Xanthomonas sp.) FLG-A strain, with a preservation number of CGMCC No. 34666, a preservation date of May 23, 2025, a preservation unit of China General Microbiological Culture Collection Center, and a preservation address of No. 3, Yikhina West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Flagellimonas Flagellimonas The biological characteristics of the Xanthomonas sp. (Xanthomonas sp.) FLG-A strain are as follows: the colony is round, orange, the surface is moist and smooth, and it is a gram-negative bacterium.

[0029] The biological characteristics of the Xanthomonas sp. (Xanthomonas sp.) FLG-A strain are as follows: the colony is round, orange, the surface is moist and smooth, and it is a gram-negative bacterium. Flagellimonas Figure 1

[0030] Example 3: Application of the strain in promoting the reproduction of W. viridis

[0031] First, an algal-bacterial propagation solution was prepared: f / 2 liquid medium (provided by Shanghai Guangyu Company), with the following composition: 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.

[0032] In a 100 L conical flask, 50 mL of the above sterilized algal-bacterial propagation solution was added, and W. viridis with a final concentration of 5x10 4 cell / mL and activated FLG-A strain with a final concentration of OD 600 =0.1 were inoculated, and the mixture was uniformly mixed and cultured at 20°C, a light intensity of 150 μmol·m -2 ·s -1 -1, and a light cycle of 12L:12D for 15 days. Each culture solution was set in triplicate, and the culture density was counted every 3 days using a hemocytometer to evaluate the influence of the growth density of the strain.

[0033] To compare the effects of the above propagation methods, algal-bacterial propagation solution, algal-bacterial propagation solution with non-phyllosphere isolated Xanthomonas sp. purchased from the Marine Microbial and Culture Collection Center (http: / / mccc.org.cn / ), and algal-bacterial propagation solution with the phyllosphere bacteria FLG-A isolated in the application and having a phytostimulatory effect were used for culture, and each culture group was set in triplicate with an inoculation density of 5x10 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 strain of Pseudomonas sp. FLG-A, characterized in that, Flagellimonas The preservation number of the said flagellimonas is CGMCC No. 34666. ​ 2. The P. flagellatum of claim 1, wherein, The sequence of 16S rDNA of the said flagellimonas is SEQ ID NO:

1.

3. The flagellimonas of claim 1 is used for promoting the growth of A. cartanica.

4. The flagellimonas of claim 1 is used for preparing a product for the propagation of A. cartanica.

5. The use according to claim 4, wherein the compound is ###0002### The product is a microbial bacteria solution.

6. A method of Schizochytrium sp. strain 6698 propagation, characterized by, The method is adding the flagellimonas of claim 1 into the culture medium for culturing A. cartanica.

7. The method of claim 6, wherein, The culture medium 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, 20 mg / L NaSiO3.

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