Bacillus velez and its use

Abstract

The present invention belongs to the field of microbial technology and discloses a strain of Bacillus Velezii and its use. Bacillus velezensis ) strain YYTF, whose deposit number is CGMCC No. 31436. The Velez Bacillus of the present invention has a wide tolerance range for salinity, effectively degrades ammonia nitrogen or nitrite in water, and can be applied to the denitrification treatment of nitrogen-containing wastewater. In addition, the Velez Bacillus of the present invention has a significant inhibitory effect on blue algae and green algae.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Velezella sp. strain and application thereof. Background Art

[0002] Bacillus velezensis is a bacterium belonging to the genus Bacillus. It is widely found in soil, plant rhizosphere and some extreme environments. It is widely used in agriculture, biological control, environmental protection and biotechnology. Bacillus velezensis can be used as a biological fertilizer to promote plant growth by secreting plant growth hormones, amino acids and other substances; control fungal plant diseases by producing antibacterial substances; decompose organic pollutants to repair the environment; improve animal digestion efficiency as a feed additive; treat polluted water and soil to improve water and soil, etc.

[0003] Cyanobacteria bloom refers to the increase of nutrients such as nitrogen and phosphorus in the water, which leads to eutrophication of the water body, causing the rapid reproduction and spread of certain cyanobacteria, and a large number of blue-green algae gather on the surface of the water body to form a blue-green algae layer visible to the naked eye, which is a pollution phenomenon that covers the water surface in an oily manner, forming a bloom. Cyanobacteria (blue-green algae) are a type of prokaryotes that carry out oxygenic photosynthesis. Their intracellular pigments mainly include chlorophyll, phycocyanin and phycoerythrin, so they absorb less blue-green light and are mainly blue-green in color; for this reason, cyanobacteria are often called blue-green algae, and blue-green algae blooms are also dominated by blue-green water color. Cyanobacteria belong to the phylum Cyanobacteria, which is divided into four orders: Chroococcales, Oscillatoriales, Nostocales and Stig onematales. Common algae species in cyanobacteria blooms include Microcystis of the Chrococoales and Anabaena of the Oscillatorales. Anabaenaflosaquae and Anabaenaspiroides are one of the main causes of cyanobacteria hydration and belong to the genus Anabaena. Microcystis aeruginosa is one of the main causes of cyanobacteria blooms and belongs to the genus Microcystis. It reproduces quickly and grows vigorously at 28-32℃, making the water gray-green and forming blooms that are visible to the naked eye. Its floating film resembles copper-green paint and has a bad smell. The microcystin toxins it produces can cause liver and gallbladder lesions.

[0004] Green algae are beneficial algae, but too much is not good. When green algae grow excessively, it may have a negative impact on water quality and even cause the water quality to deteriorate rapidly. This is because the massive reproduction of green algae will consume oxygen in the water, causing hypoxia in the water body, which in turn affects the survival of other aquatic organisms. In addition, excessive green algae will also produce toxins, causing harm to aquatic animals such as fish, and in severe cases may cause their death.

[0005] At present, the three main methods used in China to control blue algae blooms and green algae are physical, chemical and biological methods: (1) Physical method: water exchange, aeration, mechanical salvage and algae collection; (2) Chemical method: Chemical algae removal is to control the reproduction of algae in water through chemical agents, usually used in combination with flocculants; (3) Biological method: mainly planting aquatic higher plants, breeding aquatic animals, adding microbial strains, etc. Among them, physical salvage is the most widely used, but it is costly and inefficient, and the salvage speed is far less than the reproduction speed of blue algae. Once a blue algae bloom breaks out in a large area of ​​lakes and reservoirs, it is basically impossible to control it; chemical algaecides can achieve good short-term results in controlling the outbreak of blue algae, but there is a risk of secondary pollution; although the use of higher plants can effectively remove blue algae without the risk of secondary pollution, the treatment time is long and the effect is slow, and it will also cause other environmental problems due to the lack of natural enemies of alien species; and the existing microbial agents have poor ecological stability, especially after the blue algae release a large amount of algae toxins, the agents are inactivated in large quantities and cannot continue to grow, and the blue algae cannot be completely suppressed.

[0006] CN109576179A discloses a strain of Bacillus Velezii and its application to rice false smut. The colony is white or light yellow, opaque, rough and wrinkled, and its cells are straight rod-shaped. The inhibition zone for rice false smut is 33.33 mm. CN116286557A discloses a salt-tolerant Bacillus Velezii strain producing cellulase and its cultivation method. The colony is roughly milky white and circular, with irregular edges, a slightly convex center, and a rough and wrinkled surface. It can grow in LB medium with a salinity of 0-10% and survive in a water bath at 50-90°C for 10 minutes. The strain produces a high amount of cellulase and has a strong tolerance to salt, high temperature, simulated gastrointestinal fluid and bile salt, indicating that it has good application potential for cellulose degradation and can be used as a candidate strain for fermented feed with high-salt substances as raw materials, such as soy sauce residue. CN114890552A discloses the application of a multifunctional Bacillus velezensis AP3 strain in the cultivation of Penaeus vannamei in saline-alkali water, wherein the Bacillus velezensis AP3 strain isolated from the bottom mud of the shrimp cultivation pond is reported, which can significantly reduce the nitrite and nitrate content in the shrimp cultivation water and the outbreak rate of Microcystis algae blooms, and improve the weight gain rate and survival rate of Penaeus vannamei. CN112875872A discloses the application of Bacillus velezensis in improving phosphorus pollution in water bodies, which can remove phosphorus from water samples, remove phosphorus quickly, and maintain phosphorus removal for a long time.

[0007] Although it has been reported that Bacillus Velez can inhibit blue algae blooms, there are no reports that Bacillus Velez can be used to inhibit green algae. Summary of the invention

[0008] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a strain of Bacillus Velezii and its use, in order to solve the problems existing in the prior art.

[0009] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0010] One of the purposes of the present invention is to protect a Bacillus velezensis strain YYTF, whose deposit number is CGMCC No.31436.

[0011] In certain embodiments, the 16S rRNA sequence of the Bacillus Velezii strain YYTF comprises the sequence shown in SEQ ID No.1.

[0012] In certain embodiments, the Bacillus Velez strain YYTF tolerates salt at a concentration of 0 to 100 g / L.

[0013] The second object of the present invention is to protect a fermentation culture obtained by culturing the Bacillus Velez strain YYTF as described above.

[0014] In certain embodiments, the culture temperature is 20-37°C.

[0015] In certain embodiments, during culturing, the culture medium used comprises tryptone and yeast powder.

[0016] The third object of the present invention is to protect a microbial agent comprising the Bacillus Velez strain YYTF as described above or the fermentation culture as described above.

[0017] In certain embodiments, the microbial agent is a lyophilized powder.

[0018] In certain embodiments, the effective viable count of Bacillus Velez strain YYTF in the microbial agent is at least 8×10 9 CFU / g.

[0019] The fourth object of the present invention is to protect the use of the Bacillus Velez strain YYTF as described above, or the fermentation culture as described above, or the microbial agent as described above in at least one of the following: A1) denitrification of nitrogen-containing wastewater or preparation of water quality improver; A2) inhibition of algae growth or preparation of products that inhibit algae growth; A3) degradation of cellulose or preparation of products that degrade cellulose; A4) preparation of cellulase.

[0020] In certain embodiments, the nitrogen-containing wastewater contains nitrogen, which refers to one or more of ammonia nitrogen, nitrate or nitrite.

[0021] A fifth object of the present invention is to protect a method for removing blue algae or green algae in water, comprising: applying the Bacillus Velez strain YYTF as described above or the fermentation culture as described above or the microbial agent as described above to the water.

[0022] The sixth object of the present invention is to protect a method for denitrification of nitrogen-containing wastewater, comprising: applying the Bacillus Velez strain YYTF as described above or the fermentation culture as described above or the microbial agent as described above to water.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The Bacillus Velez YYTF of the present application can grow well in a culture medium with a salinity of 100 g / L, and can effectively inhibit the growth of Microcystis aeruginosa and green algae, providing a new microbial treatment method for the prevention and control of cyanobacterial blooms and green algae. In addition, the Bacillus Velez YYTF of the present application can effectively degrade the ammonia nitrogen and nitrite content in nitrogen-containing wastewater, and has good application value and application prospects in wastewater treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is an optical microscope picture of Microcystis aeruginosa cultured in BG11 liquid culture medium in Example 1 of the present invention.

[0026] Figure 2 This is the growth curve of Microcystis aeruginosa.

[0027] Figure 3 Shown are actual photos of a river sewage sample and a yellowed water sample after one week of cultivation of Microcystis aeruginosa in Example 1 of the present invention.

[0028] Figure 4 Shown is a morphological observation diagram of the Bacillus Velez strain YYTF in Example 1 of the present invention.

[0029] Figure 5 It shows a real picture of the fermentation broth of Bacillus velez strain YYTF with an inoculation amount of 2 mL / 150 mL in Example 2 of the present invention, which was cultured in a liquid of Microcystis aeruginosa on the same day.

[0030] Figure 6 It shows a real shot of the fermentation broth of Bacillus velez strain YYTF with an inoculation amount of 2 mL / 150 mL in Example 2 of the present invention being cultured in a liquid of Microcystis aeruginosa for 3 days.

[0031] Figure 7 It shows a real shot of the fermentation broth of Bacillus velez strain YYTF with an inoculation amount of 2 mL / 150 mL in Example 2 of the present invention being cultured in a liquid of Microcystis aeruginosa for 7 days.

[0032] Figure 8 It shows a real shot of the fermentation broth of Bacillus Velez strain YYTF with an inoculation amount of 2 mL / 150 mL in Example 2 of the present invention being cultured in a liquid of Microcystis aeruginosa for 30 days.

[0033] Fig. 9 It shows the growth curve of Microcystis aeruginosa after adding the fermentation liquid of strain YYTF in Example 2 of the present invention.

[0034] Fig.10 Shown is an optical microscope image of green algae after being cultured in BG11 medium in Example 2 of the present invention.

[0035] Fig.11 It shows the growth of strain YYTF in Example 3 of the present invention in Congo red cellulose medium.

[0036] Fig.12 Shown are actual photos before and after freeze-dried powder containing Bacillus Velez strain YYTF was added to river water with vigorous blue algae growth in Example 4 of the present invention.

[0037] Fig.13 Shown are actual photos of the koi pond with an outbreak of green algae in Example 4 of the present invention before and after the freeze-dried powder containing the Velezac strain YYTF was applied. DETAILED DESCRIPTION

[0038] The present invention separates and selects a strain YYTF with high algae inhibition activity from river sewage, provides bacterial resources for controlling the outbreak of cyanobacteria blooms and green algae and provides a theoretical basis for exploring the mechanism of algae inhibition, and contributes to the application of biological methods to control harmful cyanobacteria blooms, green algae, denitrification, and improve water quality.

[0039] One of the purposes of the present invention is to protect a Bacillus velezensis strain YYTF, whose deposit number is CGMCC No.31436.

[0040] Bacillus velezensis strain YYTF was deposited in the General Microbiology Center of the China National Depository and Clearing Corporation on July 24, 2024. The address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31436. It was isolated from river sewage.

[0041] In certain embodiments, the 16S rRNA sequence of the Bacillus Velezii strain YYTF comprises the sequence shown in SEQ ID No.1.

[0042] The morphological characteristics of the Bacillus Velez strain YYTF of the present application are: the colony is milky yellow, the surface is wrinkled, dry, collapsed in the middle, and convex at the edge. The homology of its 16S rDNA with the model strain Bacillus Velez in GenBank reaches 99.73%.

[0043] In certain embodiments, the tolerance concentration of the Bacillus Velez strain YYTF is 0-100 g / L of salt. The salt is sodium chloride, and the Bacillus Velez strain YYTF of the present application can grow in LB medium with a salinity of 0-100 g / L.

[0044] The second object of the present invention is to protect a fermentation culture obtained by culturing the Bacillus Velez strain YYTF as described above.

[0045] In certain embodiments, the culture temperature is 20-37°C, 20-28°C, 25-32°C, 30-37°C, or 25°C, 28°C, or 30°C.

[0046] In certain embodiments, during culturing, the culture medium used comprises tryptone and yeast powder.

[0047] In some specific embodiments, the culture medium further comprises one or both of sodium chloride and agar.

[0048] In some specific embodiments, the culture medium comprises 5-15 g / L tryptone, 1-10 g / L yeast powder, and 5-15 g / L sodium chloride. In a specific embodiment, it is 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 1 L of water. The pH value of the culture medium is 7.0-7.2.

[0049] The third object of the present invention is to protect a microbial agent comprising the Bacillus Velez strain YYTF as described above or the fermentation culture as described above.

[0050] In certain embodiments, the dosage form of the microbial agent is selected from freeze-dried powder, capsule, tablet or granule. In a specific embodiment, it is freeze-dried powder.

[0051] In certain embodiments, the effective viable count of Bacillus Velez strain YYTF in the microbial agent is at least 8×10 9 CFU / g. CFU / g is interpreted as the common meaning in the art, indicating the number of microbial colonies contained in gram of test sample, and CFU means colony forming unit.

[0052] In certain embodiments, the microbial agent further comprises an auxiliary material.

[0053] In certain specific embodiments, the auxiliary material is selected from one or more of skimmed milk powder or hydrolyzed starch. In a specific example, it is skimmed milk powder.

[0054] The present application also provides a method for preparing the microbial agent as described above, comprising the following steps:

[0055] 1) Inoculating the Bacillus Velez strain YYTF into a culture medium and culturing the medium to obtain a fermentation liquid;

[0056] 2) The fermentation liquid is mixed with auxiliary materials and then freeze-dried to obtain the microbial agent.

[0057] In certain embodiments, the Bacillus Velez strain YYTF is activated by inoculating the strain from the strain collection center onto an LB plate and placing it in a constant temperature incubator at 20-37°C for activation. Preferably, the temperature is 30°C.

[0058] In certain embodiments, the mass volume ratio of the fermentation broth and the auxiliary material can be (1-10) g:10 mL, (1-5) g:10 mL, (4-8) g:10 mL, (6-10) g:10 mL, or 1 g:10 mL.

[0059] In certain embodiments, the culture medium comprises the following raw materials in parts by weight: 5-15 g / L tryptone, 1-10 g / L yeast powder, and 5-15 g / L sodium chloride. In a specific example, it is 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 1 L of water.

[0060] In certain embodiments, the culture temperature is 8-37°C, or 20-28°C, or 25-32°C, or 30-37°C, or 25°C, 28°C, or 30°C.

[0061] In certain embodiments, the culture time is 1 to 10 days, or 1 to 5 days, or 4 to 8 days, or 7 to 10 days, or 3 days.

[0062] The fourth object of the present invention is to protect the use of the Bacillus Velez strain YYTF as described above, or the fermentation culture as described above, or the microbial agent as described above in at least one of the following: A1) denitrification of nitrogen-containing wastewater or preparation of water quality improver; A2) inhibition of algae growth or preparation of products that inhibit algae growth; A3) degradation of cellulose or preparation of products that degrade cellulose; A4) preparation of cellulase.

[0063] In certain embodiments, the algae are selected from one or both of cyanobacteria and green algae.

[0064] In some specific embodiments, the cyanobacteria is selected from one or more of Microcystis viridis, Microcystis aeruginosa, Cyanothece sp. ATCC 51142, Microcysti swesenbergii and Oscillatoria. Preferably, it is Microcystis aeruginosa. Cyanobacteria are prokaryotic plants.

[0065] In some specific embodiments, the green algae is selected from Selenastrum bibraianum. Green algae are eukaryotic plants.

[0066] In certain embodiments, the cellulose-degrading product is a probiotic or a cellulose-degrading agent, the probiotic is a probiotic that produces extracellular digestive enzymes, and the extracellular digestive enzymes are cellulases.

[0067] In certain embodiments, the product that inhibits the growth of algae is an algaecide or an algaecide.

[0068] In certain embodiments, the water quality improver can reduce one or more of ammonia nitrogen, nitrate or nitrite in water, which can regulate water quality and keep the water clear.

[0069] The Bacillus Velezii strain YYTF of the present application can produce a transparent zone in a Congo red medium solid plate, and the ratio of the transparent zone diameter D to the colony diameter d is 3.38, indicating that it has the ability to produce cellulase extracellularly. It can provide a new strain or new method for the industrial production of cellulase, and provide technical support for agricultural waste resource utilization, bioenergy preparation, and industrial cellulase source development.

[0070] In certain embodiments, the nitrogen-containing wastewater contains nitrogen, which refers to one or more of ammonia nitrogen, nitrate or nitrite. Preferably, it is ammonia nitrogen or nitrite.

[0071] A fifth object of the present invention is to protect a method for removing blue algae or green algae in water, comprising: applying the Bacillus Velez strain YYTF as described above or the fermentation culture as described above or the microbial agent as described above to the water.

[0072] In certain embodiments, based on the mass of the water body, the amount of the microbial preparation added is 1g / ton to 200g / ton of water, or 1g / ton to 70g / ton of water, or 40g / ton to 120g / ton of water, or 100g / ton to 200g / ton of water, preferably 10g / ton to 100g / ton of water, or 100g / ton of water or 10g / ton of water.

[0073] The inhibition rate of the Velez Bacillus strain YYTF or fermentation culture or microbial agent on cyanobacteria is at least 45%, and the strain YYTF is used to 680 The inhibition rate of liquid Microcystis aeruginosa with a value of 0.789 is 45.9-90.5%; using a microbial preparation containing strain YYTF, the inhibition rate of Microcystis aeruginosa in river polluted water is at least 75%.

[0074] The inhibition rate of the Velez Bacillus strain YYTF or fermentation culture or microbial agent on green algae is at least 70%, and the strain YYTF is used to 680 The inhibition rate of the green algae liquid with a value of 0.756 was 72.2% to 95.8%.

[0075] The sixth object of the present invention is to protect a method for denitrification of nitrogen-containing wastewater, comprising: applying the Bacillus Velez strain YYTF as described above or the fermentation culture as described above or the microbial agent as described above to water.

[0076] In certain embodiments, based on the mass of the water body, the amount of the microbial preparation added is 1g / ton to 200g / ton of water, or 1g / ton to 70g / ton of water, or 40g / ton to 120g / ton of water, or 100g / ton to 200g / ton of water, preferably 10g / ton to 100g / ton of water, or 100g / ton of water or 10g / ton of water.

[0077] The Bacillus Velez subtilis strain YYTF of the present application was inoculated into a nitrification medium with an ammonia nitrogen concentration of 52 mg / L. After 48 hours of treatment with the strain YYTF, the ammonia nitrogen concentration was reduced by 98.46%; the Bacillus Velez subtilis strain YYTF of the present application was inoculated into a denitrification medium with a nitrite concentration of 20 mg / L, and the nitrite concentration was reduced by 99.9%; the microbial agent of the present application was applied to the water body, and the ammonia nitrogen in the water body was reduced by at least 75% after 30 days, and could reach 96.5% after 30 days.

[0078] The Bacillus Velez, fermentation culture or microbial agent of the present application has the function of inhibiting the growth of blue algae and green algae, and has the function of degrading ammonia nitrogen and nitrite, and also has the performance of producing cellulase. The above effects enable the microbial agent of the present application to significantly improve the aquaculture water body, and provide a new strain or new method for the industrial production of cellulase.

[0079] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0080] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0081] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0082] In the examples of the present application, Microcystis aerugincsa was purchased from the freshwater algae species bank of the Chinese Academy of Sciences, and Selenastrum bibraianum among the green algae was provided by the laboratory of Ningbo University.

[0083] Example 1 Screening and Isolation of Bacillus Velez

[0084] In this embodiment, screening and separation to obtain Bacillus Velezii includes the following steps:

[0085] 1.1. Sample collection

[0086] 1) Use a sterile water sample collection bag to collect 500 mL of sewage sample from the Longweishan Ancestral Hall River in Shaoxing City, Zhejiang Province, and then filter it through a 1.2 μL water system membrane (Tianjin Keyilong Experimental Equipment Co., Ltd.) to remove large particle impurities. The filtrate obtained is then filtered through a 0.45 μL sterile filter element (Tianjin Jinteng Experimental Equipment Co., Ltd.). After filtration, remove the filter membrane, cut the filter membrane into pieces and place it in LB liquid culture medium. Shake it at room temperature and shake it at 150 r / min for 2 days to obtain the filter membrane fermentation liquid.

[0087] 2) Take 100 mL of Microcystis aeruginosa and inoculate it into 400 mL of sterilized BG11 liquid culture medium, then culture it at 25°C with a light intensity of 2000 Lux 2M (2M means the distance between the algae and the light source is 2 meters), with a light-dark ratio of 1:1, and culture for 45 days. Take samples every 5 days to test the OD 680 The growth curve of Microcystis aeruginosa is calculated by the culture time and the OD value corresponding to the culture time. 680 Indicates that, specifically Figure 2 shown.

[0088] from Figure 2 It can be seen that a high concentration of Microcystis aeruginosa liquid can be obtained after culturing for 30 days, and its corresponding OD 680 The value is 0.789, reserved. 30d photos of Microcystis aeruginosa under ordinary optical microscope are as follows Figure 1 shown.

[0089] Take 150 mL of the 30-day-old cultured Microcystis aeruginosa and place it in a sterile triangular flask. Inoculate 1.5 mL of the membrane fermentation broth from step 1) and culture together. If the algae broth turns yellow after one week, Figure 3 As shown, it indicates that the fermentation broth contains algae-inhibiting bacteria.

[0090] 3) The yellowed water sample from step 2) was diluted with sterile saline in a gradient manner (10 2 , 10 3 , 10 4 ), and then spread on LB solid plate, after repeated streaking, several strains were obtained, and the obtained strains were inoculated into LB liquid culture medium for 3 days, and inoculated into 150mL of Microcystis liquid cultured in advance according to the inoculation amount of 1% (v / v), and cultured under the conditions of 25℃ light intensity of Lux 2M, and the light-dark ratio was 1:1. Finally, a strain that can make the liquid Microcystis aeruginosa yellow was screened out. The morphological characteristics of the colony on the LB plate are: the colony is milky yellow, the surface is wrinkled, dry, collapsed in the middle, and the edge is convex, such as Figure 4 shown.

[0091] The formula of LB liquid medium is: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, water 1L, pH 7.1±0.1, sterilized at 121℃ for 30min. Add 2% agar when solid.

[0092] BG11 liquid culture medium formula: sodium nitrate 1.5g / L, potassium phosphate dihydrate 0.04g / L, magnesium sulfate heptahydrate 0.075g / L, calcium chloride dihydrate 0.036g / L, citric acid 0.006g / L, ammonium ferric citrate 0.006g / L, ethylenediaminetetraacetic acid (EDTA) 0.001g / L, sodium carbonate 0.02g / L, boric acid 0.00286g / L, manganese chloride monohydrate 0.00181g / L, zinc sulfate heptahydrate 0.000222g / L, copper sulfate pentahydrate 0.000079g / L, sodium molybdate dihydrate 0.00039g / L, cobalt nitrate hexahydrate 0.000049g / L, pH 7.1±0.1, sterilization at 121℃ for 15min.

[0093] 1.2. Species determination by 16S rDNA gene sequencing

[0094] The purified strain was sent to Shanghai Saiheng Biotechnology Co., Ltd. for 16S rDNA sequencing. The obtained sequence was compared with the National Center for Bioinformation (NCBI) database of the United States. It was found that the 16S rDNA sequence was highly homologous to the model strain Bacillus velezensis in GenBank, with a homology of 99.73%.

[0095] The sequence of 16S rDNA is shown below:

[0096]

[0097] Based on the strain morphology and 16S rDNA, the strain is Bacillus velezensis. The classification name of the strain is Bacillus velezensis strain YYTF, the deposit number is CGMCC No.31436, and it was deposited in the General Microbiology Center of the Preservation Administration Committee on July 24, 2024. The address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0098] Example 2 Study on the salt tolerance and algae inhibition properties of strain YYTF

[0099] The inhibitory effect of strain YYTF on Microcystis aeruginosa and green algae and its salt tolerance were investigated.

[0100] 2.1. Investigation of the inhibitory effect of strain YYTF on Microcystis aeruginosa

[0101] 1) To eliminate the interference of LB medium on the experiment, the LB liquid medium was diluted 8 times with sterile water, i.e., 1 / 8 LB liquid medium, and then the strain YYTF obtained in Example 1 was inoculated into 1 / 8 LB liquid medium, placed in a constant temperature shaker at 28°C, 150 rpm, and cultured for 2 days to obtain YYTF fermentation broth.

[0102] 2) Take the liquid of Microcystis aeruginosa cultured for 30 days in step 1.1 of Example 1, and its OD 680 The value is 0.789, and it is packaged into sterile 250mL Erlenmeyer flasks, with each portion containing 150mL.

[0103] 3) According to the volume ratio of YYTF fermentation liquid to Microcystis aeruginosa liquid of 2mL / 150mL, the YYTF fermentation liquid of step 1) was inoculated into the conical flask of step 2), and three sets of parallel experiments were set up.

[0104] At the same time, the same volume of sterile water inoculated with Microcystis aeruginosa liquid was set as a blank control group.

[0105] 4) Then, each group of flasks was cultured at 25℃ and 2000Lux 2M for 3d, 7d, and 15d, and photographed. Figures 5 to 8 At the same time, the OD values ​​of the cells were measured on the same day, 3 days, 7 days, and 15 days of culture. 680 Value, get the growth curve, see Fig. 9 .

[0106] Figure 5 , 6 , 7, and 8 are actual photos of inoculating 2 mL YYTF fermentation liquid into 150 mL Microcystis aeruginosa liquid on the first day, 3 days, 7 days, and 15 days of culture, respectively.

[0107] from Figure 5-8 It can be seen that the color of the blank control group became greener as the culture time went by; after inoculation with strain YYTF, the color of the Microcystis aeruginosa liquid became lighter as the culture time went by, from dark green on the day of inoculation to light yellow on the 7th day of culture, and to clear and transparent on the 15th day of culture.

[0108] OD was measured on the day of inoculation. 680 The value was 0.786, and the OD was measured on day 3 of culture. 680 The value was 0.425, and the OD was measured after 7 days of cultivation. 680 The value was 0.286, and the OD was measured after culturing for 15 days. 680 The value is 0.075, such as Fig. 9 shown.

[0109] In summary, the Velez subtilis strain YYTF of the present application can inhibit Microcystis aeruginosa. Compared with the inoculation, the inhibition rate of Microcystis aeruginosa after inoculation with strain YYTF is 45.9% to 90.5%, and the inhibition rate is (OD after inoculation). 680 -OD on the day of inoculation 680 ) / OD on the day of inoculation 680 .

[0110] 2.2. Investigation of the inhibitory effect of strain YYTF on green algae

[0111] 1) To eliminate the interference of LB culture medium on the experiment, the LB liquid culture medium was diluted 8 times with sterile water, i.e., 1 / 8 LB liquid culture medium, and then the strain YYTF obtained in Example 1 was inoculated into the 1 / 8 LB liquid culture medium, placed in a constant temperature shaker at 28°C, 150 rpm, and cultured for 3 days to obtain YYTF fermentation broth.

[0112] 2) Inoculate 100 mL of the crescent algae seed into 400 mL of sterilized BG11 liquid culture medium, and then culture it at 25°C with a light intensity of 2000 Lux 2M, a light-dark ratio of 1:1, and culture for 7 days to obtain the cultured crescent algae liquid, and its OD 680 The value is 0.756, and it is divided into sterile 250mL Erlenmeyer bottles, each containing 150mL. Fig.10 shown.

[0113] 3) According to the volume ratio of YYTF fermentation liquid to Lunar Algae liquid of 2mL / 150mL, the YYTF fermentation liquid was inoculated into the conical flask containing Lunar Algae liquid in step 2), and three groups of parallel experiments were set up, namely, Replicate 1#, Replicate 2#, and Replicate 3#.

[0114] At the same time, the liquid of Lunar Algae inoculated with sterile water was set as a blank control group (CK group).

[0115] 4) Then, each group of triangular flasks was cultured at 25°C with a light intensity of 2000 Lux 2M for 1, 2, and 3 days, and the OD 680 The values ​​are shown in Table 1.

[0116] Table 1

[0117]

[0118] From Table 1, we can see that the strain has a strong inhibitory effect on the green algae. Compared with the blank control group, the inhibition rate of the green algae after inoculation with strain YYTF was 72.2% to 95.8%, and the inhibition rate was (OD 680 -Blank control group OD 680 ) / blank control group OD 680 .

[0119] 2.3. Investigation of salt tolerance of strain YYTF

[0120] The YYTF fermentation broth in step 2.1 was inoculated into LB liquid culture medium with different concentrations of sodium chloride (based on the total volume of LB liquid culture medium, the concentrations of sodium chloride were 10g / L, 20g / L, 40g / L, 60g / L, 80g / L and 100g / L, respectively). The culture was carried out at room temperature and shaken at 150r / min. After 3 days of culture, the absorbance of each fermentation broth at a wavelength of 600nm was measured with an ultraviolet spectrophotometer. Three parallel experiments were set up for each experimental group. The results are shown in Table 2. The inoculation amount of YYTF fermentation broth in each experimental group was: 2mL / 150mL, that is, 150mL of liquid culture medium containing sodium chloride LB was inoculated with 2mL of YYTF fermentation broth.

[0121] At the same time, a blank LB medium with the same salinity but not inoculated with any strain was used as a blank control group.

[0122] Table 2

[0123]

[0124] As shown in Table 2, the YYTF strain can still grow well in a culture medium with a salinity of up to 100 g / L.

[0125] Example 3 Study on the performance of strain YYTF in degrading ammonia nitrogen and nitrite

[0126] In this Example 3, strain YYTF was used to conduct nitrification performance test and denitrification performance test, which specifically included the following:

[0127] 3.1 Nitrification performance test

[0128] The strain YYTF obtained in Example 1 was inoculated into 100 mL of LB liquid medium and fermented at 30°C for 3 days to obtain a fermentation broth, which was inoculated into a nitrification medium at an inoculum of 10 v / v%, placed in a constant temperature shaker at 30°C, with a rotation speed of 150 r / min, and cultured for 48 hours. A total of 10 parallel samples were made.

[0129] The nitrification culture medium is: ammonium chloride 0.2g / L, potassium dihydrogen phosphate 0.2g / L, glucose 2g / L, magnesium sulfate 0.1g / L, ferrous sulfate 0.02g / L, pH 7.0-7.4, sterilization at 121℃ for 30min.

[0130] The culture was fermented at 26°C for 48 h, and the ammonia nitrogen content in the culture medium was detected before inoculation and after 48 h of fermentation.

[0131] Ammonia nitrogen content was detected by Nessler's reagent photometry. The specific operation is: take 5 mL of fermentation product, add it to a 50 mL colorimetric tube, dilute to the mark, add 1.0 mL of potassium tartrate solution, and measure the absorbance at 420 nm. For details, please refer to the method of Nessler's reagent photometry (A) in the "Water and Wastewater Monitoring and Analysis Methods (Fourth Edition)".

[0132] The detection principle is: the alkaline solution of mercuric iodide and potassium iodide of Nessler's reagent reacts with ammonia to form a light reddish-brown colloidal compound, which has strong absorption in a wider wavelength range.

[0133] The results showed that before inoculation, the concentration of ammonia nitrogen in the nitrification medium was 52 mg / L; after 48 hours of treatment with strain YYTF, the concentration of ammonia nitrogen in the nitrification medium was only 0.8 mg / L, a decrease of 98.46%.

[0134] 3.2 Denitrification performance test

[0135] The strain YYTF obtained in Example 1 was inoculated into 100 mL of LB liquid medium and fermented at 30°C for 3 days to obtain a fermentation broth, which was inoculated into a denitrification medium at an inoculum of 10 v / v%, placed in a constant temperature shaker at 30°C, with a rotation speed of 150 r / min, and cultured for 48 hours. A total of 10 parallel samples were made.

[0136] The denitrification culture medium includes: sodium nitrite 0.2 / L, potassium dihydrogen phosphate 0.2g / L, dipotassium hydrogen phosphate 0.1g / L, glucose 1.5g / L, magnesium chloride 0.1g / L, ferrous sulfate 0.02g / L, pH 7.0-7.4, sterilization at 121°C for 30min.

[0137] The culture was fermented at 28°C for 48 h, and the nitrite content in the denitrification medium was detected before inoculation and after 48 h of fermentation.

[0138] The nitrite content was detected by N-(1-naphthyl)-ethylenediamine photometric method. The specific operation is: take 5 mL of fermentation product, add it to a 50 mL colorimetric tube, dilute to the mark, add 1.0 mL of color developer, and measure the absorbance at 540 nm. For details, please refer to the method of N-(1-naphthyl)-ethylenediamine photometric method (A) in "Water and Wastewater Monitoring and Analysis Methods (Fourth Edition)".

[0139] The detection principle is: in a phosphoric acid medium, when the pH value is 1.8±0.3, nitrite reacts with p-aminobenzenesulfonamide to generate a diazonium salt, which is then coupled with N-(1-naphthyl)-ethylenediamine to generate a red dye.

[0140] The results showed that before inoculation, the concentration of nitrite in the denitrification medium was 20 mg / L; after 48 hours of treatment with strain YYTF, the concentration of nitrite in the aerobic denitrification medium was only 0.02 mg / L, a decrease of 99.9%.

[0141] Example 4 Study on the cellulose degradation performance of strain YYTF

[0142] The strain YYTF of Example 1 was inoculated into a cellulose Congo red medium solid plate, placed in a 30°C constant temperature incubator, and cultured for 3 days. Fig.11 .

[0143] Congo red is a commonly used stain, especially in the screening of cellulolytic bacteria, as it can form a red complex with cellulose, thus helping to identify microorganisms that produce cellulase.

[0144] Congo red staining is used to identify the decomposition of cellulose or cellulose-degrading bacteria. When cellulose is degraded by cellulase, the Congo red-cellulose complex cannot be formed, so a transparent circle centered on the cellulose-degrading bacteria will appear in the culture medium. By observing the formation of the transparent circle, cellulose-degrading bacteria can be screened out.

[0145] from Fig.11 As shown, strain YYTF can produce a large transparent circle on the Congo red medium solid plate, the diameter of the transparent circle D is 30.8mm, the colony diameter d is 9.10mm, and the ratio of the transparent circle diameter D to the colony diameter d is 3.38; the D / d ratio can reflect the activity of cellulase, and the larger the ratio, the stronger the cellulose degradation ability. Therefore, it shows that strain YYTF has the function of producing cellulase outside the cell.

[0146] Congo red medium: sodium nitrate 1.0g / L, potassium dihydrogen phosphate 0.9g / L, dipotassium hydrogen phosphate 1.2g / L, magnesium sulfate 0.5g / L, potassium chloride 0.5g / L, yeast extract powder 0.5g / L, acid hydrolyzed casein 0.5g / L, Congo red 0.2g / L, cellulose powder 5g / L, agar 15, pH 7.0±0.2.

[0147] Example 5 Microbial preparation and its application in inhibiting the growth of blue algae

[0148] The fermentation broth of strain YYTF was prepared into freeze-dried powder for river treatment.

[0149] 5.1 Preparation of lyophilized powder

[0150] The strain YYTF obtained in Example 1 was inoculated into 100 mL of LB liquid culture medium and fermented at 30° C. for 3 days to obtain a fermentation broth.

[0151] Skim milk powder was added to the fermentation broth; the skim milk powder and the fermentation broth were mixed in a mass-to-volume ratio of 1 g:10 mL, and then freeze-dried powder was prepared by freeze-drying. The live bacterial content of strain YYTF in the freeze-dried powder was 8×10 9 cfu / g.

[0152] 5.2. Inhibition of blue algae

[0153] The freeze-dried powder obtained in step 5.1 was applied to the river water body where cyanobacteria grew vigorously at an amount of 100 g / ton of water. The application was performed every three days. The water quality was observed before and 30 days after the freeze-dried powder was applied. The results were as follows: Fig.12 The OD of the river water was tested before and 30 days after application. 680 The detection of ammonia nitrogen content is the same as step 3.1 in Example 3.

[0154] from Fig.12 It can be seen that before the application, the river water was green and turbid; 30 days after the application, the river water became clear.

[0155] Before adding freeze-dried powder, the OD of river water 680 The value was 0.432, and the ammonia nitrogen content was 4.26 mg / L; the OD of the water body after 30 days of application 680 The value was 0.108, and the ammonia nitrogen content was 0.15 mg / L; after application, the ammonia nitrogen content was reduced by 96.5%, and the inhibition rate of cyanobacteria was 75%.

[0156] 5.3 Inhibition of green algae

[0157] Water samples were taken from an outdoor koi pond with an algae outbreak. A 10 μL sample was directly observed under a microscope and found to contain a lot of algae. The OD of the 5 mL sample was680 The value is 0.65, so it is judged to be a green algae outbreak. The freeze-dried powder obtained in step 5.1 was added to the koi pond at an amount of 10g / ton of water, and the water quality was observed before applying the freeze-dried powder and after 24 and 48 hours of treatment. The results are shown in Fig.13 shown.

[0158] from Fig.13 It can be seen that before application, the pond water was turbid; 24 hours after application of the freeze-dried powder, the water in the koi pond became clear; 48 hours after application of the freeze-dried powder, the water in the koi pond was clear.

[0159] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A strain of Bacillus velez Bacillus velezensis ) strain YYTF, whose deposit number is CGMCC No.31436; the 16S rDNA sequence of the Velez subtilis strain YYTF comprises the sequence shown in SEQ ID No.

1.

2. A fermentation culture, characterized in that The method is obtained by culturing the Bacillus Velez strain YYTF as claimed in claim 1.

3. The fermentation culture according to claim 2, characterized in that The culture temperature is 20-37°C; And / or, during the culturing, the culture medium used comprises tryptone and yeast powder.

4. A microbial agent, characterized in that: It comprises the Bacillus Velez strain YYTF as claimed in claim 1 or the fermentation culture as claimed in claim 2.

5. The microbial agent according to claim 4, characterized in that: The microbial agent is freeze-dried powder; and / or, the effective viable count of Bacillus Velez strain YYTF in the microbial agent is at least 8×10 9 CFU / g.

6. Use of the Bacillus Velez strain YYTF as claimed in claim 1 or the fermentation culture as claimed in claim 2 or the microbial agent as claimed in claim 4 in at least one of the following: A1) denitrification of nitrogen-containing wastewater or preparation of water quality improver; A2) inhibition of algae growth or preparation of products that inhibit algae growth; A3) degradation of cellulose or preparation of products that degrade cellulose; A4) preparation of cellulase; the nitrogen-containing wastewater contains one or more of ammonia nitrogen, nitrate or nitrite; The algae are selected from one or both of blue algae and green algae.

7. A method for removing blue algae from water, characterized in that: include: The Bacillus Velez strain YYTF according to claim 1 or the fermentation culture according to claim 2 or the microbial agent according to claim 4 is applied to water.

8. A method for denitrification of nitrogen-containing wastewater, characterized in that: include: The Bacillus Velez strain YYTF according to claim 1 or the fermentation culture according to claim 2 or the microbial agent according to claim 4 is applied to water.

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