Bacillus velezensis HY16 and application thereof

Abstract

The invention discloses bacillus velezensis HY16 and application of the bacillus velezensis HY16. The strain preservation number of the bacillus velezensis HY16 is CGMCC (China General Microbiological Culture Collection Center) No.31645. The invention further discloses a preparation method of the bacillus velezensis HY16. The bacillus velezensis HY16 provided by the invention belongs to spore bacteria and has good biological safety, and compared with physical, chemical and other algae control modes, the bacillus velezensis HY16 does not cause secondary pollution, is friendly to the ecological environment and accords with the current environmental protection concept. According to the invention, the bacillus velezensis industrial production and preparation process and the algae control application scheme are simultaneously combined for the first time, the method has huge market transformation potential and application value, and river algae control experiment results show that the algae lysing rate can reach more than 96%. The microbial agent prepared by using the bacillus velezensis HY16 is stable in property, safe, environment-friendly and simple to apply, can be used for effectively performing biological control and remediation on a water body with blue-green algae, and has a wide application prospect and value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Bacillus Velez HY16 and applications thereof. Background Art

[0002] Eutrophication of water bodies leads to the widespread occurrence of Harmful Algal Blooms (HABs), of which cyanobacterial blooms are the most common type. Microcystis aeruginosa is a common cyanobacteria in freshwater. When Microcystis aeruginosa reproduces in large numbers in eutrophic water bodies, it will cause water deterioration and form the so-called "water bloom". The over-reproduction of Microcystis aeruginosa is one of the global water environmental problems and has become the main cause of eutrophication in many freshwater and coastal waters. Due to the combined factors of water pollution caused by human activities and global climate change, the frequency and duration of Harmful Cyanobacterial Blooms (HCBs) are on the rise worldwide. Harke and Ndlela et al. counted the bloom outbreak information of 257 countries and regions in 2015 and 2016 respectively, and found that 108 countries and regions in the world reported the occurrence of Microcystis blooms, and in Africa, about 39% of the countries counted had research information on cyanobacteria blooms, of which 11 countries had records of toxicity and physical and chemical parameters related to cyanobacteria blooms. In addition, research in China showed that the frequency of harmful algal blooms in coastal areas increased at a rate of 40±4% per decade from 1970 to 2015, and was significantly correlated with the average warming rate.

[0003] The occurrence of cyanobacterial blooms has caused a series of ecological, economic and health problems, including the destruction of the ecological balance of water bodies, the impact on the quality of drinking water, the production of algal toxins and economic losses. Therefore, it is of great practical significance to study safe, environmentally friendly and efficient methods to control and kill Microcystis aeruginosa. At present, the methods for controlling cyanobacterial blooms mainly include physical, chemical and biological methods. Physical methods include shading, fishing, ultrasound, flotation, filtration and sedimentation, while chemical methods include chemical oxidation, coagulation, electrochemistry and photocatalysis. However, physical and chemical methods have many limitations in terms of construction methods, costs, effects and possible secondary pollution.

[0004] Among the many methods of controlling algae, biological algae control technology has received particular attention, especially the microbial method. Microbial algae control is not only effective and low-cost, but also in line with the current environmental protection concept and has good eco-friendliness. The development and application prospects of this technology are broad, so the screening and development of efficient algae control microbial agents and solutions are of great significance. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a Bacillus Velez HY16 and its application, which has a significant algae control effect, can solve the current problems of eutrophication of water bodies and algal blooms, and has a good mature process for realizing industrial-scale production.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] The invention provides Bacillus velezensis HY16, and the strain collection number of the Bacillus velezensis HY16 is CGMCC No.31645.

[0008] Preferably, the colonies of the Bacillus Velezii HY16 are nearly circular, light yellow, with dot-like protrusions, opaque, irregular edges, and wrinkled surfaces.

[0009] Preferably, the growth carbon source of the Bacillus Velezii HY16 includes glycerol;

[0010] The concentration of the growth carbon source of the Velez Bacillus HY16 is 20-25 g / L;

[0011] The growth nitrogen source of the Bacillus Velez HY16 includes soybean meal;

[0012] The concentration of the growth nitrogen source of the Velez Bacillus HY16 is 40-45 g / L;

[0013] The inorganic salt for the growth of Bacillus Velez HY16 includes manganese sulfate;

[0014] The concentration of inorganic salts for the growth of Bacillus Velez HY16 is 1.4-1.5 g / L;

[0015] The growth temperature of the Velez Bacillus HY16 is 35-37°C;

[0016] The growth pH value of the Velez Bacillus HY16 is 6.0 to 6.5;

[0017] The inoculation amount of the Bacillus Velez HY16 is 4-5%;

[0018] The culture time of the Bacillus Velez HY16 is 32 to 40 hours.

[0019] Preferably, the Bacillus Velezii HY16 enters the logarithmic growth phase after 6 hours of culture, enters the late logarithmic growth phase at 18 to 20 hours, and is in the stable growth phase at 20 to 26 hours.

[0020] Preferably, the Bacillus Velezii HY16 can effectively inhibit and kill Microcystis aeruginosa.

[0021] The present invention also provides a use of the above-mentioned Bacillus Velezii HY16 in controlling and killing Microcystis aeruginosa.

[0022] Preferably, the method for controlling and killing Microcystis aeruginosa by Bacillus Velez HY16 comprises the following steps:

[0023] S1. First, a culture medium for activating algae control microbial agent powder is prepared, wherein the culture medium components are 20-25 g / L glycerol, 45-50 g / L soybean meal powder, and 4-5 g / L magnesium sulfate;

[0024] S2, then inoculating the algae-controlling microbial agent powder into the above culture medium at an inoculation rate of 1-3%, and performing fermentation activation to obtain an algae-controlling activation liquid;

[0025] S3. The obtained algae control activation liquid is used for controlling and killing Microcystis aeruginosa in water bodies at a dosage of 5‰.

[0026] Preferably, the algae-lysing rate of the Bacillus Velez HY16 bacterial powder activation liquid on Microcystis aeruginosa can reach more than 96%.

[0027] Preferably, in step S2, the fermentation activation conditions are: tank pressure 0.2-0.3 MPa, temperature 35-37° C., dissolved oxygen ≥ 20%, stirring speed 200-240 rpm, and fermentation time 32-40 h.

[0028] The present invention further provides a microbial agent comprising the Bacillus Velez HY16 described above, wherein the microbial agent is used for controlling and killing Microcystis aeruginosa.

[0029] Preferably, the microbial agent further comprises a carrier, and the carrier is any one of Jiayi powder, diatomaceous earth and calcium carbonate.

[0030] Preferably, the preparation method of the microbial agent comprises the following steps: firstly, obtaining the semi-finished product powder of HY16 by spray drying, and then diluting it by the carrier to obtain the algae control microbial agent powder, wherein the content of Bacillus Velez HY16 in the powder is 2.0×10 10 ~3.0×10 10 CFU / g.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The Bacillus Velez HY16 provided by the present invention belongs to the genus Bacillus and has good biosafety. Compared with physical and chemical algae control methods, it does not cause secondary pollution, is friendly to the ecological environment, and is consistent with the current environmental protection concept. The present invention combines the industrial production and preparation process of Bacillus Velez with its algae control application scheme for the first time, and has huge market transformation potential and application value. The results of the river algae control experiment show that its algae dissolving rate can reach more than 96%. After production fermentation and post-treatment spray drying, Bacillus Velez HY16 is prepared into an algae control microbial agent together with a carrier, and then the activation liquid is used for biological algae control after activation. It can significantly inhibit and kill Microcystis aeruginosa in the water body, so it has broad market application prospects and value in the treatment and restoration of water bodies dominated by Microcystis aeruginosa outbreaks. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a characteristic diagram of the colony morphology of Bacillus Velez HY16 of the present invention;

[0034] Figure 2 is a phylogenetic tree of Bacillus Velez HY16 of the present invention;

[0035] Figure 3 is a growth curve diagram of Bacillus Velez HY16 of the present invention;

[0036] Figure 4 This is a diagram showing the evaluation results of the application scheme of the Bacillus Velez HY16 for controlling algae powder in the present invention;

[0037] Figure 5 This is a diagram showing the results of an on-site pilot demonstration and evaluation of the application of the Bacillus Velez HY16 bacteria to control algae. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0039] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0040] The formula of the culture medium required in the following examples is as follows:

[0041] 1. BG11 medium for the cultivation of Microcystis aeruginosa:

[0042]

[0043]

[0044] 2. Nutrient broth (NB) medium: 3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 1000 mL water, pH 7.2±0.2.

[0045] 3. Nutrient broth (NB) solid culture medium: 3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 15 g agar powder, 1000 mL water, pH 7.2±0.2.

[0046] 4. Industrial fermentation medium: glycerol 20-25 g / L, soybean meal 40-45 g / L, disodium hydrogen phosphate 3 g / L, manganese sulfate 1.4-1.5 g / L, sodium chloride 4-6 g / L, calcium chloride 1.3-1.8 g / L, the balance is water, the pH of the culture medium is 6.5-7.0.

[0047] 5. Culture medium for activating algae-control microbial agents: 20-25 g / L glycerol, 45-50 g / L soybean meal, 4-5 g / L magnesium sulfate, and the remainder is water. The pH of the culture medium is 7.0-7.5.

[0048] Before use, the above culture media must be sterilized at 115℃ for 20 minutes and used after cooling to an appropriate temperature.

[0049] Example 1 Isolation, Identification and Preservation of Bacillus Velez HY16

[0050] 1. Screening and isolation of Bacillus Velez HY16

[0051] Water samples were collected from multiple rivers and landscape ponds with algal blooms, and each water sample was placed in a nutrient broth (NB) culture medium to prepare a bacterial solution. Each prepared bacterial solution was co-cultured with Microcystis aeruginosa in a light incubator at 25°C for 5 days. The group that produced yellowing indicated that the corresponding water sample contained algae-lytic bacteria. Subsequently, each yellowed sample was spread on a nutrient broth (NB) solid plate, and single colonies of different morphologies were picked for zoning, streaking and purification. A total of 43 strains were obtained, and their anti-algae ability was initially and rescreened using the double-layer plate method and liquid infection method. It was found that HY16 had a higher anti-algae ability. The streaking separation and purification were repeated 3 times, and finally a single colony was obtained, which was named HY16. Figure 1 As shown, the colonies of the strain HY16 on the nutrient broth (NB) solid culture medium plate are round, light yellow, dot-like, opaque, with irregular edges and wrinkles on the surface.

[0052] 2. Identification of Bacillus Velez HY16

[0053] The DNA of the strain HY16 was used as a template, and 16S rRNA universal primers were used for amplification. The amplified fragment was sequenced, and the obtained 16S rRNA sequence was shown in SEQ ID No. 1. The 16S rDNA sequencing result of the strain HY16 was compared with the sequence in GenBank and homology analysis was performed. The phylogenetic tree was constructed as shown in Figure 2 As shown, the results showed that strain HY16 had the highest homology with Bacillus velezensis, so the strain HY16 was determined to be Bacillus velezensis.

[0054] Wherein, SEQ ID No.1 sequence is as follows:

[0055]

[0056] 3. Deposit of Bacillus Velez HY16

[0057] The screened Bacillus velezensis HY16 was deposited, and the depository unit of the Bacillus velezensis HY16 is: General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC); address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: August 9, 2024; the deposit number of Bacillus velezensis is: CGMCC No.31645.

[0058] Example 2 Growth determination and physiological and biochemical characteristics of Bacillus Velez HY16

[0059] 1. Growth assay of Bacillus velez HY16

[0060] In a sterile environment, the culture solution of strain HY16 was inoculated with 1% inoculation volume into the NB medium sterilized by high temperature and high pressure, and then placed in a constant temperature shaking incubator for culture. Samples were taken every 2 hours, and the absorbance of the culture solution was measured at 600nm. The growth curve was drawn with time as the horizontal axis and optical density (OD600) as the vertical axis. Figure 3 As shown, strain HY16 is in the stagnant phase from 0 to 6 hours and grows slowly; it enters the stable phase after about 20 hours, with an OD600 value as high as 2.85, and then enters the decay phase, thus completing the entire growth cycle.

[0061] 2. Physiological and biochemical characteristics of Bacillus Velez HY16

[0062] According to the requirements, the Velez Bacillus HY16 strain was further cultured in NB medium, and then tested according to the microbial physiological and biochemical detection method. The results are shown in Table 1. The glycolysis experiment of strain HY16 showed that the bacteria was fermentative and could utilize lactose, glucose, sucrose and mannitol; in addition, the gelatin liquefaction experiment showed that the strain could also produce gelatinase and reduce nitrate; the strain VP experiment was positive. The strain methyl red was negative, indicating that the bacteria could not produce a large amount of acidic substances; the strain did not produce harmful gas hydrogen sulfide. In addition, the strain was negative for hydrogen peroxide, peptone water, rhamnose, ammonium glucose and Simon's citrate.

[0063] Table 1 Physiological and biochemical characteristics of Bacillus velez HY16

[0064]

[0065]

[0066] Note: <+> indicates positive, <-> indicates negative.

[0067] Example 3 Preparation of Bacillus Velez HY16 bacterial agent

[0068] (1) The Bacillus Velez HY16 isolated in Example 1 was inoculated into a 250 ml flask containing 100 ml of nutrient broth (NB) culture solution using an inoculation loop and cultured in a constant temperature incubator at 37° C. and 210 rpm for 6 h;

[0069] (2) The fermented bacterial liquid was inoculated into a pilot fermentation tank and cultured at 37°C. After the fermentation was completed, it was spray dried, packaged, and mixed with Jiayi powder or diatomaceous earth or calcium carbonate to prepare the Bacillus Velez HY16 bacterial agent for controlling and killing Microcystis aeruginosa, with a viable bacterial count of 2.0×10 10 ~3.0×10 10 CFU / g. The pilot medium formula is: glycerol 20-25g / L, soybean meal 40-45g / L, disodium hydrogen phosphate 3g / L, manganese sulfate 1.4-1.5g / L, sodium chloride 4-6g / L, calcium chloride 1.3-1.8g / L, the balance is water, and the pH of the medium is 6.5-7.0.

[0070] Example 4 Pilot production process study of Bacillus Velez HY16

[0071] In order to test the industrial production performance of Bacillus Velez HY16, the industrial fermentation production was verified under the conditions of a 15T fermenter system. The test was mainly based on the optimal fermentation parameters and culture medium data obtained from the small-scale test process. During the process, the spore rate of the strain in the fermentation broth was monitored, which was used as the basis for stopping the tank. When the fermentation reached 28h, the spore rate in the fermentation broth had reached more than 95% by microscopic examination. At this time, the fermentation was stopped, and the bacterial count of the fermentation broth was counted and analyzed. The obtained biological bacterial count of the fermentation broth was 1.22×10 10 CFU / mL, the spray drying results showed that the obtained bacterial powder was 53.8 billion / g, and the fermentation broth biomass yield was 88.2%. Based on the above pilot fermentation and post-treatment spray drying results, it is shown that strain HY16 has the potential for industrial-scale production.

[0072] Example 5 Study on the application scheme of Bacillus Velez HY16 powder for algae control

[0073] The obtained finished powder of Bacillus Velez HY16 was used as the activation seed. The bacterial content of the finished powder was 2.0×10 10 ~3.0×10 10CFU / g, firstly, a finished bacterial powder activation culture medium for Bacillus Velez HY16 is prepared, and the components of the culture medium are 20-25g / L glycerol, 45-50g / L soybean meal powder and 4-5g / L magnesium sulfate; then the finished bacterial powder of the algae control microbial agent Bacillus Velez HY16 is inoculated into the above culture medium at an inoculation rate of 1%, and activated according to the fermentation preparation conditions of the above Bacillus Velez HY16 fermentation liquid, and the fermentation conditions are: tank pressure 0.2-0.3MPa, temperature 35-37°C, dissolved oxygen ≥20%, stirring speed 200-240rpm, fermentation time 28-32h, and finally the algae control activation liquid is obtained.

[0074] The above activation liquid was added in different amounts to carry out algae control experiment analysis. The results are as follows Figure 4 As shown. When the addition amount is 0.5-5‰, the chlorophyll a concentration decreases significantly with the increase in the addition amount, and the algae lysis rate also increases significantly. When the addition amount continues to increase, the decrease in chlorophyll a and the increase in algae lysis rate are no longer obvious, and the changes are not significant. Therefore, considering the application cost and algae control effect, when algae control is applied based on this method, an activation liquid addition amount of 5‰ can be selected to control and repair the water body with microcystis outbreaks.

[0075] Example 6 Pilot demonstration study on on-site algae control in rivers using Bacillus Velez HY16 bacterial powder

[0076] This experiment is mainly to test and evaluate the effect of Bacillus Velez HY16 powder on algae control in river channels. The experimental water area is about 1000m 3 In the same implementation process, the fermentation activation liquid of strain HY16 powder was used to carry out a pilot application evaluation experiment on the river channel where blue algae broke out. The entire experimental period was 10 days, the dosage of the bacterial agent activation liquid was 5‰, and the specific activation operation method was the same as the steps in Example 5. The experimental results are as follows: Figure 5 As shown. During the algae control experiment, as the action time prolonged, the concentration of chlorophyll a in the water body began to gradually decrease, and the algae dissolving rate also began to gradually increase. When the algae control experiment was on the 6th day, the algae dissolving rate reached 64.78%, and when it continued to act for 8 days, the algae dissolving rate reached 86.89%. By the 10th day when the algae control experiment ended, the algae dissolving rate was 96.35%. It was clearly visible that the water body changed from blue-green to yellow before and after the field experiment, indicating that the biological strain HY16 has the potential and application value for on-site biological algae control in waters with blue algae outbreaks.

[0077] The Bacillus Velez HY16 of the present invention can be prepared into an algae-controlling microbial agent alone or together with other carriers, and is used for the repair and management of algae-burst water bodies and the prevention of algae-burst in water bodies before the outbreak. It has been verified that it has the ability to achieve industrial-scale production, and its use method is relatively simple. At the same time, as a microbial agent, the application is safer and more environmentally friendly. Therefore, it will have important value and significance in the management, repair and control of algae-burst water bodies.

[0078] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of the present invention.

Claims

1. A Bacillus Velez HY16, characterized in that The strain deposit number of the Bacillus velezensis HY16 is CGMCC No.31645.

2. The Bacillus Velez HY16 according to claim 1, characterized in that The colonies of the Bacillus Velez HY16 are nearly circular, light yellow, dot-shaped, opaque, with irregular edges and wrinkles on the surface.

3. The Bacillus Velez HY16 according to claim 1, characterized in that The growth carbon source of the Bacillus Velez HY16 includes glycerol; The concentration of the growth carbon source of the Velez Bacillus HY16 is 20-25 g / L; The growth nitrogen source of the Bacillus Velez HY16 includes soybean meal powder; The concentration of the growth nitrogen source of the Velez Bacillus HY16 is 40-45 g / L; The inorganic salt for the growth of Bacillus Velez HY16 includes manganese sulfate; The concentration of inorganic salts for the growth of Bacillus Velez HY16 is 1.4-1.5 g / L; The growth temperature of the Velez Bacillus HY16 is 35-37°C; The growth pH value of the Velez Bacillus HY16 is 6.0 to 6.5; The inoculation amount of the Bacillus Velez HY16 is 4-5%; The culture time of the Bacillus Velez HY16 is 32 to 40 hours.

4. The Bacillus Velez HY16 according to claim 1, characterized in that The Bacillus Velez HY16 can effectively inhibit and kill Microcystis aeruginosa.

5. Use of the Bacillus Velez HY16 as claimed in claim 1 in controlling and killing Microcystis aeruginosa.

6. The use according to claim 5, characterized in that The method for controlling and killing Microcystis aeruginosa by Bacillus Velez HY16 comprises the following steps: S1. First, a culture medium for activating algae control microbial agent powder is prepared, wherein the culture medium components are 20-25 g / L glycerol, 45-50 g / L soybean meal powder, and 4-5 g / L magnesium sulfate; S2, then inoculating the algae-controlling microbial agent powder into the above culture medium at an inoculation rate of 1-3%, and performing fermentation activation to obtain an algae-controlling activation liquid; S3. The obtained algae control activation liquid is used for controlling and killing Microcystis aeruginosa in water bodies at a dosage of 5‰.

7. The use according to claim 6, characterized in that In step S2, the fermentation activation conditions are: tank pressure 0.2-0.3 MPa, temperature 35-37°C, dissolved oxygen ≥ 20%, stirring speed 200-240 rpm, and fermentation time 32-40 h.

8. A microbial agent comprising the Bacillus Velezii HY16 according to claim 1, characterized in that: The microbial agent is used for controlling and killing Microcystis aeruginosa.

9. The microbial agent according to claim 8, characterized in that: The microbial agent also includes a carrier, and the carrier is any one of Jiayi powder, diatomaceous earth and calcium carbonate.

10. The microbial agent according to claim 8, characterized in that: The preparation method of the microbial agent comprises the following steps: firstly, obtaining the semi-finished product powder of HY16 by spray drying method, and then diluting it by the carrier to obtain the algae control microbial agent powder, wherein the content of Bacillus Velez HY16 in the powder is 2.0×10 10 ~3.0×10 10 CFU / g.

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