A growth-promoting bacterium for promoting the growth of Caulerpa lentillifera and its application

Abstract

The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a growth-promoting bacterium for promoting the growth of Caulerpa lentillifera and its application. The present invention discloses a co-associated bacterium capable of promoting the growth of algae. The strain is Bacillus sp. Cl-9. The morphological characteristics of strain Cl-9 are: light pink, opaque, smooth surface, smooth edge, and circular. This co-associated bacterium not only has the function of dissolving inorganic phosphorus and can be prepared into a phosphorus solubilizer for increasing the phosphorus content in water bodies, but also can colonize on the algal thalli, significantly increase the biomass of algae, and promote the growth of the upright branches of algae. It can be further developed into a microbial inoculant or microbial fertilizer for promoting the growth of algae. The present invention provides a reference for the application and development of co-associated bacteria in promoting the growth of algae, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a growth-promoting bacterium for promoting the growth of long-stem grape fern algae and an application thereof. Background Art

[0002] Caulerpalentillifera is an edible, economically large seaweed with high nutritional value. The algae consists of three parts: upright branches, stolons, and rhizomes. The upright branches are the main part sold as a commodity. At present, long-stem grape fern algae is mainly produced using a controllable land-based factory-based artificial cultivation model. However, this model has two bottleneck problems that restrict the high-quality development of the long-stem grape fern cultivation industry. One is the contamination of harmful attached microorganisms, which leads to slow growth of the algae. The other is that after a large number of upright branches are harvested, the stolons of the algae recover slowly, and the number of new upright branches is small, resulting in unstable commercial output rate of upright branches, thus affecting the seaweed cultivation cycle and economic benefits.

[0003] During their natural growth, eukaryotic organisms harbor a rich diversity of microorganisms, forming symbiotic functional associations. These associations can generate mutualistic or antagonistic interactions through factors such as extracellular metabolites. This suggests that beneficial microorganisms can be selected from eukaryotic organisms to manipulate the microbiome, thereby improving host growth and health. Clearly, identifying suitable strains is a key issue in the application of microbial manipulation techniques. While microbial manipulation techniques have been used to improve the health and growth performance of several crops, they have not yet been explored in large seaweeds. Recent studies have found that beneficial seaweed microorganisms can promote growth and / or enhance disease resistance. Therefore, seaweed microbiome manipulation techniques hold promise as a potential and effective approach to promoting seaweed aquaculture production. The controlled, land-based, factory-based cultivation of long-stemmed grape fern provides a practical opportunity for applying beneficial microbial manipulation techniques to improve seaweed growth and health.

[0004] In summary, it can be seen that screening out beneficial microorganisms that can promote the growth of long-stem grape ferns from the tissues of healthy long-stem grape ferns has great potential application value. Summary of the Invention

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a Bacillus sp. Cl-9, which has the ability to dissolve inorganic phosphorus and promote algae growth, and has great potential application value.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] The first aspect of the present invention provides the use of Bacillus Cl-9 in promoting algae growth. The Bacillus Cl-9 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 11, 2022, with the deposit number: CGMCC No. 26129.

[0008] Preferably, the algae include Pteris longifolia.

[0009] The present invention screens beneficial microorganisms from the tissues of healthy long-stem grape ferns. A beneficial microorganism, namely strain Cl-9, which can be used to promote the growth and development of the long-stem grape ferns, especially the growth and development of the upright branches of the long-stem grape ferns, is obtained from the screening. The strain is classified and named Bacillus sp., and provides technical support for land-based factory cultivation of long-stem grape ferns, shortening the cultivation cycle and increasing the commercial output rate of upright branches, thereby improving the economic benefits of seaweed cultivation. It also provides technical support for the further commercial development of seaweed growth-promoting microbial live bacteria agents.

[0010] Preferably, promoting the growth of algae includes promoting the accumulation of algae biomass and / or promoting the differentiation of upright branches of algae runners.

[0011] Preferably, the salinity of the algae growth environment is less than 35.

[0012] Preferably, the algae are grown in an environment free of antibiotics.

[0013] Preferably, the application method is as follows: Cl-9 is inoculated into 2216E liquid culture medium, cultured at 25°C with constant temperature and shaking until the logarithmic growth phase, the bacterial liquid is centrifuged to collect the cells, the supernatant is removed, and the cells are washed three times with sterile seawater and resuspended, and the OD value of the bacterial suspension is adjusted. 600 After the pH value is 0.8, it is mixed with seawater at a volume ratio of 1:400 and then used for algae cultivation.

[0014] More preferably, the 2216E liquid culture medium comprises 1000 mL of distilled water, 5.0 g of peptone, 1.0 g of yeast extract powder, 0.1 g of ferric citrate, 19.45 g of NaCl, 5.98 g of MgCl2, 3.24 g of Na2SO4, 1.8 g of CaCl2, 0.55 g of KCl, 0.16 g of Na2CO3, 0.08 g of KBr, 0.034 g of SrCl2, 0.022 g of H3BO3, 0.004 g of Na2SiO3, 0.0024 g of NaF, 0.0016 g of NaNO3, and 0.008 g of NaH2PO4, with a pH of 7.6±0.2.

[0015] The second aspect of the present invention provides the use of Bacillus Cl-9 in dissolving inorganic phosphorus. The Bacillus Cl-9 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 11, 2022, with the deposit number: CGMCC No. 26129.

[0016] The present invention has found through research that the strain Cl-9 can also dissolve inorganic phosphorus and is expected to be developed into a phosphorus dissolving agent for increasing the phosphorus content in water bodies.

[0017] Preferably, the inorganic phosphorus includes but is not limited to Ca3(PO4)2.

[0018] The third aspect of the present invention provides a bacterial agent for promoting algae growth, wherein the bacterial agent contains Bacillus Cl-9 as the main active ingredient. The Bacillus Cl-9 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 11, 2022, with the deposit number: CGMCC No. 26129.

[0019] The fourth aspect of the present invention provides a phosphate dissolving agent for dissolving inorganic phosphorus, wherein the phosphate dissolving agent contains Bacillus Cl-9 as the main active ingredient. The Bacillus Cl-9 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 11, 2022, with the deposit number: CGMCC No. 26129.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention discloses a symbiotic bacterium capable of promoting the growth of algae. The strain is Bacillus sp. Cl-9, and the morphological characteristics of strain Cl-9 are: light pink, opaque, smooth surface, smooth edges, and round. The symbiotic bacteria not only have the function of dissolving inorganic phosphorus and can be prepared into a phosphate dissolving agent for increasing the phosphorus content in water bodies, but also can be colonized on algae bodies, significantly increasing the biomass of algae and promoting the growth of upright branches of algae. It can be further developed into a microbial agent or microbial fertilizer that promotes the growth of algae. The present invention provides a reference for the application and development of symbiotic bacteria in promoting the growth of algae, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the microscopic structure of strain Cl-9;

[0023] Figure 2 is the colony morphology of strain Cl-9;

[0024] Figure 3 is the phylogenetic tree of strain Cl-9;

[0025] Figure 4This is the result of the phosphorus solubility test of strain Cl-9;

[0026] Figure 5 The effect of adding strain Cl-9 on the specific growth rate of long-stem grape fern;

[0027] Figure 6 The effect of strain Cl-9 on the morphological development of the runners of Pteris longifolia;

[0028] Figure 7 This is a comparison of the growth status of the long-stem grape fern runners 15 days after inoculation with strain Cl-9;

[0029] Figure 8 This is the effect of adding strain Cl-9 on the specific growth rate of long-stem grape fern under indoor open culture conditions;

[0030] Figure 9 This is a graph showing the growth promotion effect of adding strain Cl-9 on long-stem grape fern algae under different salinities (the control is 30 salinity without bacteria). DETAILED DESCRIPTION

[0031] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0033] Example 1 Isolation and identification of long-stem grape fern algae growth-promoting bacteria Cl-9

[0034] (1) Strain isolation: The long-stem grape fern algae cultured in the outdoor pool of the Donghai Island Experimental Base of Guangdong Ocean University was rinsed with sterile seawater, placed in a sterile sealed bag and transported to the laboratory at 4°C. Rinse with sterile seawater three times in a clean bench, chop the long-stem grape fern algae with sterile dissecting scissors, grind it with a sterile mortar, and dilute it with sterile seawater to make 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6For solutions of different dilution concentrations, 100 μL of each gradient dilution solution was spread on 2216E agar medium (distilled water 1000 mL, peptone 5.0 g, yeast extract powder 1.0 g, ferric citrate 0.1 g, NaCl 19.45 g, MgCl2 5.98 g, Na2SO4 3.24 g, CaCl2 1.8 g, KCl 0.55 g, Na2CO3 0.16 g, KBr 0.08 g, SrCl2 0.034 g, H3BO3 0.022 g, Na2SiO3 0.004 g, NaF 0.0024 g, NaNO3 0.0016g, NaH2PO40.008g, agar 15.0g, pH = 7.6 ± 0.2, autoclaved at 121 ° C for 15min), and cultured in a constant temperature incubator at 28 ° C until no new colonies were generated. The obtained bacteria were further purified to obtain a long-stem grape fern symbiotic bacterial strain (named Cl-9), and the strain morphology was observed ( Figure 1 ), and the purified bacteria symbiotic with long-stem grape fern algae were stored in 15% glycerol at -80°C for later use.

[0035] (2) Identification of Cl-9 bacteria symbiotic with long-stem grape fern algae

[0036] 1) Observation of colony morphology of strain Cl-9

[0037] The strain Cl-9 obtained above was streaked onto 2216E solid medium to observe the colony morphology. Figure 2 As shown, the colonies of this strain are light pink, opaque, with smooth edges, smooth surface, and round shape.

[0038] 2) 16rDNA sequencing of strain Cl-9

[0039] A bacterial genomic DNA extraction kit (purchased from Anhui Tiangen Biochemical Technology Co., Ltd.) was used to perform PCR amplification using the universal primers 27F (5′-AGAGTTTGATCCGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) of the bacterial 16S rRNA gene sequence.

[0040] The PCR amplification products were separated by 1% agarose gel electrophoresis for 15 min, and the PCR products with clear bands were sent to Shanghai Bioengineering Co., Ltd. for DNA sequencing. The strain sequences obtained by sequencing were compared and BLAST analyzed in NCBI, and the phylogenetic tree was constructed using MEGA11.0 software. Figure 3 It was found that strain Cl-9 belonged to a new Bacillus sp.

[0041] The 16S rRNA sequence of strain Cl-9 is as follows:

[0042] aatcttcaggtggggtgctatacatgcaagtcgagcggacggatgggagcttgctccctgaagtcagcggcggacgggtgagta

[0043] acacgtgggcaacctgcctgtaagactgggataacttcgggaaaccggagctaataccggataatgcacagcctctcatgaggctatgct

[0044] gaaagatggtttcggctatcacttacagatgggcccgcggcgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcgtag

[0045] ccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatg

[0046] gacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaaactctgttgttcagggaagaacaagtgccggagt

[0047] aactgccggcaccttgacggtacctgaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgt

[0048] ccggaattattgggcgtaaagcgcgcgcaggcggtctcttaagtctgatgtgaaagcccacggctcaaccgtggagggtcattggaaact

[0049] gggggacttgagtgcagaagaggaaagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaagg

[0050] cgactttctggtctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgat

[0051] gagtgctaagtgttagagggtttccgccctttagtgctgcagcaaacgcattaagcactccgcctggggagtacggccgcaaggctgaaa

[0052] ctcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatct

[0053] cctgacaaccctagagatagggcgttccccttcgggggacaggatgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttg

[0054] ggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggagg

[0055] aaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaaagggctgcaagaccgc

[0056] gaggttaagcgaatcccataaaaccattctcagttcggattgcaggctgcaactcgcctgcatgaagctggaatcgctagtaatcgcggat

[0057] cagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtggggtaa

[0058] cctttggagccagccgcctaaggacggctaaccgcc。

[0059] 3) Physiological and biochemical identification of strain Cl-9

[0060] Physiological and biochemical identifications such as Gram staining were performed on strain Cl-9, and the results are shown in Table 1.

[0061] Combined with colony morphology observation, physiological and biochemical reaction results, and 16S rRNA sequence analysis, strain Cl-9 was identified as Bacillus sp. Finally, on November 11, 2022, the strain was sent to the General Microbiology Center of the China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) for preservation, with the deposit number: CGMCC No. 26129, and the strain was classified as Bacillus sp.

[0062] Table 1 Physiological and biochemical identification of strain Cl-9

[0063]

[0064] Example 2 Phosphate Solubilization Ability Test of Bacillus Cl-9

[0065] (1) Qualitative analysis of phosphate solubility of strain Cl-9

[0066] After activation, the strain was inoculated on inorganic phosphorus (seawater) solid medium (glucose 10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, MgSO4 0.3 g, MnSO4 0.03 g, K2SO4 0.3 g, FeSO4 0.03 g, Ca3(PO4)2 5.0 g, agar 15.0 g, 1000 mL of aged seawater, pH (7.0-7.5), autoclaved at 115 °C for 30 min) and organic phosphorus (seawater) solid medium (glucose 10 g, (NH4)2SO4 0.5 g, yeast extract 0.5 g, NaCl 0.3 g, MgSO4 0.3 g, MnSO4 0.03 g, KCl 0.3g, FeSO40.03g, lecithin0.2g, CaCO31.0g, agar15.0g, Chenhai water1000mL, pH(7.0-7.5), autoclave at 121℃ for 15min), culture in a 28℃ constant temperature incubator, observe the plate culture medium after 5 days to see if there is a phosphate dissolution zone.

[0067] As shown in Table 2, a phosphate-dissolving zone appeared in the inorganic phosphate plate culture medium, indicating that the strain Cl-9 has a phosphate-dissolving effect.

[0068] Table 2 Phosphate solubilization ability of strain Cl-9

[0069]

[0070] (2) Quantitative analysis of phosphate solubility of strain Cl-9

[0071] The molybdenum antimony colorimetric method was used to test whether Cl-9 could dissolve inorganic phosphorus. The isolated Cl-9 was activated and inoculated into an inorganic phosphorus (seawater) liquid culture medium (glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4 0.3g, MnSO4 0.03g, K2SO4 0.3g, FeSO4 0.03g, Ca3(PO4)2 5.0g, 1000mL of Chenhai water, pH (7.0-7.5), autoclaved at 115°C for 30min). The culture was shaken at 28°C and 150r / min for 3 days. The resulting culture was centrifuged at 4°C and 6000r / min for 5 minutes. The supernatant was filtered through a 0.22μm filter membrane and the total phosphorus content was determined. Uninoculated inorganic phosphorus culture medium was used as a control.

[0072] like Figure 4 As shown in Figure 2, strain Cl-9 was found to be able to dissolve inorganic phosphorus, with a conversion rate of 15.37 mg·L -1 .

[0073] Example 3 Experiment on the Growth Promotion of Long-stem Grape Fern by Bacillus Cl-9

[0074] (1) Effect on the specific growth rate of long-stem grape fern

[0075] The long-stemmed grape fern algae used in this example was purchased from Shenzhen Lanting Dingzhi Biotechnology Co., Ltd. in Guangdong Province. Well-grown, bright green algae were selected, and upright branches of (25±5) mm were cut off as experimental subjects. The upright branches were placed in a 480 mL tissue culture flask as a culture container, and 400 mL of filtered sterilized natural seawater was added, and 1 mL of NaNO3 nutrient solution (42.5 g·L) was added. -1 ) and 1 mL KH2PO4 nutrient solution (13.6 g·L -1 ) as nutrient salt. The isolated strain Cl-9 was activated and cultured on 2216E agar medium, and then placed in 2216E liquid medium (distilled water 1000 mL, peptone 5.0 g, yeast extract powder 1.0 g, ferric citrate 0.1 g, NaCl 19.45 g, MgCl2 5.98 g, Na2SO4 3.24 g, CaCl2 1.8 g, KCl 0.55 g, Na2CO3 0.16 g, KBr 0.08 g, SrCl2 0.034 g, H3BO3 0.022 g, Na2SiO3 0.004 g, NaF 0.0024 g, NaNO3 0.0016 g, NaH2PO4 0.008 g, pH = 7.6 ± 0.2, autoclaved at 121 ° C for 15 min) at 25 ° C, 150 r·min-1 The cells were cultured to the logarithmic growth phase and then incubated at 4°C and 6000 r·min -1 The bacterial cells were obtained by centrifugation for 5 minutes, and the obtained bacterial pellet was resuspended and washed three times with sterile seawater to adjust the bacterial solution OD 600 After the value reaches 0.8, 1 mL of the prepared bacterial solution is added to the tissue culture bottle. At the same time, sterile seawater with nutrient salts is used as a control. The culture conditions are (25±1)℃, illumination is (4000±200) lux, and the light-dark cycle is 12L:12D. The bacterial solution is added once every three days. After 15 days of culture, the algae are removed and the fresh weight of the algae in the experimental group and the control group is weighed. According to the specific growth rate formula (SGR=100×(LnW 末重量 -LnW 初始重量 ) / t 养殖天数 ) Calculate the specific growth rate of long-stemmed grape fern.

[0076] See the results Figure 5 The specific growth rate of the Cl-9 group increased by 32.15% compared with the control group, indicating that strain Cl-9 can promote the accumulation of biomass of the long-stem grape ferns.

[0077] (2) Promoting the growth of upright branches on the stolons of the long-stem grape fern

[0078] Before the experiment began, long-stemmed grape fern algae with good growth conditions were selected for stolons. The stolons of the long-stemmed grape fern algae were cut into a length of 5±0.5 cm using sterile dissecting scissors and placed in a sterile culture dish with 30 mL of sterile seawater for 48 hours under the conditions of 200 lux, 25℃, and 12L:12D to allow the wounds to heal.

[0079] Two treatments were set up (CK: sterile seawater treatment group; sterile seawater + Cl-9 bacterial suspension), and each treatment was set up with 5 replicates. Three long-stemmed grape fern runners were placed in each replicate conical flask. The volume of the conical flask was 250 mL, and the liquid volume was 200 mL. In the experimental treatment group, 10 mL of Cl-9 bacterial suspension (OD 600 The culture medium was replaced every three days and the growth index was measured after 15 days. Figure 6 、 7 shown.

[0080] After the long stem grape fern algae stolons grew for 15 days, the algae were taken out to measure the algae fresh weight, the number of new upright branches, the length of new upright branches, the number of new runners, the length of new runners, the number of new globular branches, the diameter of new globular branches, and the number of new rhizoids. Figure 6 、 7It can be seen that after the addition of strain Cl-9, the number of upright branches, upright branch length and spherical branchlet number of long-stem grape fern algae were significantly increased by 122.22%, 252.37% and 187.35% respectively compared with the control group, indicating that strain Cl-9 can promote the differentiation of upright branches of the creeping stems of long-stem grape fern algae.

[0081] (3) Effects of adding strain Cl-9 on the growth of long-stem grape fern under different salinities under indoor open culture conditions

[0082] Before the experiment began, long-stemmed grape fern algae with good growth conditions were selected and divided into four groups (salinity 25 with bacteria group A, salinity 30 with bacteria group B, salinity 35 with bacteria group C, and salinity 30 without bacteria group CK). Each group of algae weighed (50±2)g and was placed in a 25L plastic barrel. 15L of seawater was added and the salinity was adjusted to 25, 30, and 35 respectively using natural sea salt. 1mL of NaNO3 nutrient solution (42.5g·L) was added to each group. -1 ) and 1 mL KH2PO4 nutrient solution (13.6 g·L -1 ) as nutrient salts, and cultured under the conditions of (3000±200) lux, (28±1)℃, 12L:12D, with three replicates per group. Every three days, 75mL OD 600 The Cl-9 bacterial solution was cultured at a concentration of 1.0, and its fresh weight was measured after 15 days of culture to calculate its specific growth rate.

[0083] See the results Figure 8 and Figure 9 Under the salinity of 25, the growth effect of the long-stem grape fern algae with the addition of Cl-9 was the best; there was no significant difference in the specific growth rate of the long-stem grape fern algae between the salinity 30 bacteria group and the salinity 25 bacteria group, but the specific growth rate of the salinity 30 bacteria group increased by about 11.77 times compared with the salinity 30 no bacteria group.

[0084] Example 4 Antibiotic Sensitivity Analysis of Bacillus Cl-9

[0085] After activation, strain Cl-9 was cultured in 2216E liquid medium overnight. The bacterial concentration was adjusted to 0.5 McFarland units using physiological saline and then evenly spread on 2216E agar medium. Sterile tweezers were used to stick paper strips containing antibiotics (purchased from Hangzhou Microbiological Reagent Co., Ltd.) on the flat culture medium. Six paper strips (diameter 6.0 mm × 0.7 mm) were stuck on each culture dish. After incubation at 28°C for 3 days, the inhibition zone was observed to determine the sensitivity of strain Cl-9 to 20 antibiotics.

[0086] As shown in Table 3, strain Cl-9 is sensitive to multiple antibiotics and is suitable for use in an antibiotic-free environment.

[0087] Table 3 Antibiotic sensitivity test of Bacillus Cl-9

[0088]

[0089]

[0090] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. Application of Bacillus sp. Cl-9 in promoting the growth of algae, characterized in that, the Bacillus sp. Cl-9 was deposited on November 11, 2022 at the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No. 26129; the algae is Caulerpa lentillifera.

2. The application according to claim 1, characterized in that, promoting the growth of algae includes promoting the accumulation of algal biomass and / or promoting the differentiation of upright branches of algal stolons.

3. The application according to claim 1, characterized in that, the salinity of the algal growth environment is less than 35.

4. The application according to claim 1, characterized in that, the growth environment of the algae does not contain antibiotics.

5. The application according to claim 1, characterized in that, The specific application method is as follows: Cl-9 was inoculated into 2216E liquid culture medium, cultured at 25°C with constant temperature shaking until the logarithmic growth phase, the bacterial liquid was centrifuged to collect the cells, the supernatant was removed and the cells were washed three times with sterile seawater and resuspended, and the OD of the bacterial suspension was adjusted. 600 After the pH value is 0.8, it is mixed with seawater at a volume ratio of 1:400 and then used for algae cultivation.

6. The application according to claim 5, characterized in that, The 2216E liquid medium includes 1000 mL of distilled water, 5.0 g of peptone, 1.0 g of yeast extract powder, 0.1 g of ferric citrate, 19.45 g of NaCl, 5.98 g of MgCl 2 , 3.24 g of Na 2 SO 4 , 1.8 g of CaCl 2 , 0.55 g of KCl, 0.16 g of Na 2 CO 3 , 0.08 g of KBr, 0.034 g of SrCl 2 , 0.022 g of H 3 BO 3 , 0.004 g of Na 2 SiO 3 , 0.0024 g of NaF, 0.0016 g of NaNO 3 , 0.008 g of NaH 2 PO 4 , and the pH is 7.6 ± 0.

2.

7. Application of Bacillus sp. Cl-9 in dissolving inorganic phosphorus, characterized in that, the Bacillus sp. Cl-9 was deposited on November 11, 2022 at the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No. 26129.

8. A bacterial agent for promoting the growth of algae, characterized in that, the bacterial agent takes Bacillus sp. Cl-9 as the main active ingredient, the Bacillus sp. Cl-9 was deposited on November 11, 2022 at the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No. 26129; the algae is Caulerpa lentillifera.

9. A phosphorus solubilizing agent for dissolving inorganic phosphorus, characterized in that, the phosphorus solubilizing agent takes Bacillus sp. Cl-9 as the main active ingredient, the Bacillus sp. Cl-9 was deposited on November 11, 2022 at the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No. 26129.

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