Algae-controlling bioactive preparation as well as preparation method and application thereof

Abstract

The invention discloses an algae-controlling bioactive preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: inoculating bacillus velezensis HY16 in a bioactive substance fermentation culture medium and culturing; the culture temperature is 35-40 DEG C, and the culture time is 32-48 hours. The algae-controlling bioactive preparation provided by the invention has good capability of inhibiting growth of microcystis aeruginosa, and the algae-lysing rate of the algae-controlling bioactive preparation on microcystis aeruginosa in an aquarium system can reach 97% or above. The algae-controlling bioactive preparation prepared from the bacillus velezensis HY16 fermentation active liquid is stable in property, safe, environment-friendly and simple to apply, can be used for effectively and biologically controlling and repairing an aquarium water body with microcystis aeruginosa, is remarkable in effect, and has good market application prospect and value.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an algae-controlling biologically active preparation and a preparation method and application thereof. Background Art

[0002] With the intensification of climate change and eutrophication, cyanobacterial blooms have become one of the major challenges facing the global water environment. In recent years, many freshwater lakes and reservoirs in my country have experienced cyanobacterial blooms to varying degrees, especially in the middle and lower reaches of the Yangtze River. The main cyanobacteria in bloom include Microcystis, Aphanizomenon, Dolichospermum, Oscillatoria and Cylindrospermopsis raciborskii.

[0003] In water bodies rich in nutrients, when the temperature is suitable and the light is sufficient, cyanobacteria can reproduce rapidly and form sufficient biomass. Under the combined effects of biological factors (such as predation by zooplankton and the effects of microcystins) and non-biological factors (such as temperature, nutrients and metal ions, etc.), cyanobacteria gather and float up in large quantities to form water blooms. This water bloom phenomenon has a significant impact on the ecological environment: on the one hand, the dominant growth of cyanobacteria reduces the algae diversity in the water body; on the other hand, water blooms lead to a decrease in dissolved oxygen concentration and water transparency, seriously damaging the living environment of bottom organisms and causing the death of a large number of fish and shrimp. In addition, the algal toxins produced by some toxic cyanobacteria cause damage to the liver, digestive tract and nervous system of birds and mammals, and accumulate through the food chain, indirectly threatening human health.

[0004] At present, the technologies for preventing and controlling blue algae blooms are mainly divided into three categories: physical, chemical and biological. At present, the common physical algae control methods mainly include shading, fishing, ultrasound, flotation, filtration and sedimentation. Common chemical algae removal methods mainly include chemical oxidation algae removal, coagulation algae removal, electrochemical and photocatalytic methods. Both physical and chemical methods have many disadvantages in application, and are restricted by many factors such as construction methods, costs, effects and secondary pollution. At present, the biological algae control method in the microbial method is the most studied and popular one. Microbial algae control is an algae control technology with good effect, low cost and eco-friendly environment. The advantages of this technology are in line with the current environmental protection concept and have huge market application prospects and value. Therefore, it is of great significance to screen and develop efficient algae control microbial agents. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a bioactive algae control agent and a preparation method and application thereof, wherein the bioactive algae control agent has a significant algae control effect, and has a mature process that can realize industrial-scale production, as well as an application scheme for effectively controlling algae in water bodies.

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

[0007] The invention provides a method for preparing an algae-controlling bioactive agent. The method comprises the following steps: inoculating and culturing Bacillus Velez HY16 in a bioactive substance fermentation medium; the culturing temperature is 35-40° C., and the culturing time is 32-48 hours.

[0008] Preferably, the classification name of the Bacillus velezensis HY16 is Bacillus velezensis, and its deposit number is CGMCC No.31645.

[0009] Preferably, the preparation method further comprises obtaining a high-yield algae-controlling active liquid by fermentation, and then obtaining the HY16 algae-controlling biologically active preparation powder by freeze-drying.

[0010] Preferably, the formula of the bioactive substance fermentation medium is: 20-25 g / L glycerol, 45-50 g / L soybean meal powder, 4-5 g / L magnesium sulfate, and the balance is water, and the pH of the medium is 7.0-7.5.

[0011] Preferably, the fermentation inoculation amount of the Bacillus Velezii HY16 is 4-5%.

[0012] The present invention also provides an algae-controlling biologically active preparation prepared by the preparation method described above.

[0013] Preferably, the algae-controlling biologically active agent is capable of inhibiting and killing Microcystis aeruginosa.

[0014] The present invention also provides an application of the above-mentioned algae-controlling biologically active preparation in controlling and killing Microcystis aeruginosa.

[0015] Preferably, the method for controlling and killing Microcystis aeruginosa is: directly adding the algae-controlling biologically active agent to the water body where Microcystis aeruginosa breaks out at a dosage of 3-5% of the amount of the applied water body.

[0016] Preferably, the algae-controlling biologically active agent has an algae-dissolving rate of more than 97% for Microcystis aeruginosa.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The algae-controlling biologically active preparation provided by the present invention, as a biocontrol functional microorganism, has good biosafety. Compared with physical and chemical methods, this biologically active preparation will not cause secondary pollution when controlling algae, is more friendly to the ecological environment, and is consistent with the current environmental protection concept. The present invention combines the production process of the high-yield algae-controlling biologically active preparation of Bacillus Velez with its algae-controlling application scheme for the first time, showing huge market transformation potential and application value. The results of the aquarium algae control experiment show that the algae-dissolving rate of the preparation can be as high as 97% or more. Therefore, the algae-controlling biologically active preparation prepared by producing fermentation and post-processing freeze-dried Bacillus Velez HY16 can significantly inhibit and kill Microcystis aeruginosa in water bodies. This biological algae-controlling preparation shows broad market application prospects and value in the treatment and restoration of water bodies dominated by Microcystis aeruginosa outbreaks. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a characteristic diagram of the colony morphology of Bacillus Velez HY16 of the present invention;

[0020] Figure 2 is a phylogenetic tree of Bacillus Velez HY16 of the present invention;

[0021] Figure 3 is a growth curve diagram of Bacillus Velez HY16 of the present invention;

[0022] Figure 4 This is a diagram showing the evaluation results of the algae control application scheme of the algae control biological active preparation prepared by Bacillus Velez HY16 of the present invention;

[0023] Figure 5 This is a diagram showing the results of the demonstration and evaluation of the algae control application of the algae control biological active agent prepared by Bacillus Velez HY16 of the present invention in aquarium. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0025] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0026] The formula of the culture medium required in the following examples is as follows:

[0027] 1. BG11 medium for the cultivation of Microcystis aeruginosa:

[0028]

[0029] 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.

[0030] 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.

[0031] 4. High-yield algae-controlling bioactive substance fermentation medium: glycerol 20-25 g / L, soybean meal 45-50 g / L, magnesium sulfate 4-5 g / L, the remainder is water, and the pH of the culture medium is 7.0-7.5.

[0032] Before use, the above culture media must be sterilized at 115℃ for 20 minutes and used after cooling to an appropriate temperature.

[0033] Example 1 Isolation, Identification and Preservation of Bacillus Velez HY16

[0034] 1. Screening and isolation of Bacillus Velez HY16

[0035] 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.

[0036] 2. Identification of Bacillus Velez HY16

[0037] 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.

[0038] Wherein, SEQ ID No.1 sequence is as follows:

[0039]

[0040] 3. Deposit of Bacillus Velez HY16

[0041] 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.

[0042] Example 2 Growth determination and physiological and biochemical characteristics of Bacillus Velez HY16

[0043] 1. Growth assay of Bacillus velez HY16

[0044] 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.

[0045] 2. Physiological and biochemical characteristics of Bacillus Velez HY16

[0046] 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.

[0047] Table 1 Physiological and biochemical characteristics of Bacillus velez HY16

[0048]

[0049]

[0050] Note: <+> indicates positive, <-> indicates negative.

[0051] Example 3 Preparation of algae-controlling biologically active preparation

[0052] (1) The Bacillus Velezii 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;

[0053] (2) The fermentation liquid is inoculated into a pilot fermentation tank, cultured at 37°C, freeze-dried after fermentation, and packaged to obtain an algae-controlling biologically active preparation for controlling and killing Microcystis aeruginosa; the formula of the fermentation medium is: 22g / L glycerol, 48g / L soybean meal, 4.5g / L magnesium sulfate, the balance is water, the pH of the medium is 7.0, and the fermentation conditions are: tank pressure 0.2-0.3MPa, temperature 35-40°C, dissolved oxygen ≥20%, stirring speed 220-240rpm, fermentation time 32-48h. The fermentation medium selects the above corresponding appropriate carbon source, nitrogen source and inorganic salt, and the dosage is the above corresponding appropriate concentration.

[0054] Example 4 Study on the production process of algae control bioactive agent based on Bacillus Velez HY16

[0055] In order to test the industrial production performance of Bacillus Velezii HY16, the industrial fermentation production was verified under the conditions of a 50L fermenter system. The test was mainly based on the optimal fermentation parameters and culture medium data obtained from the shake flask experiment. After the fermentation was completed, the algae control application effect evaluation experiment of the high-yield algae control bioactive fermentation liquid of strain HY16 was carried out. The results showed that the algae lysis rate of the algae control bioactive fermentation liquid was 94.56%. In addition, the algae control bioactive fermentation liquid was freeze-dried to prepare an algae control bioactive powder preparation, and the algae control experiment was evaluated and analyzed again. The results showed that the algae lysis rate was 91.26%, and the algae control effect was significant.

[0056] Based on the results obtained from the above production fermentation and post-processing freeze-drying, it is shown that the algae-control bioactive preparation based on Bacillus Velezii HY16 has the potential for industrial-scale production and market application value.

[0057] Example 5 Study on the application of algae control bioactive preparation based on Bacillus Velez HY16

[0058] In order to determine the appropriate dosage of the algae control bioactive agent, the algae control bioactive agent was subjected to algae control experimental analysis at different addition amounts. The results are as follows: Figure 4As shown in the figure, starting from 1%, the algae-dissolving rate increases with the increase of the dosage. When the dosage is 3% or above, the algae-dissolving rate almost tends to be stable and no longer increases significantly with the increase of dosage. Considering the application cost and algae-control effect, the algae-control bioactive agent with a 3% addition ratio can be used as the dosage for the treatment and restoration of water bodies with Microcystis aeruginosa outbreaks.

[0059] Example 6 Demonstration study on the application of algae control bioactive agent prepared based on Bacillus Velez HY16 in aquarium algae control

[0060] This experiment is mainly to test and evaluate the algae control effect of the algae control biological active agent prepared based on Bacillus Velez HY16 in the aquarium system. The experimental water volume of a single aquarium is 40L. The experiment sets up 3 parallel control groups and 3 parallel experimental groups. The experimental period is 5 days. The dosage of the algae control biological active agent in the experimental group is 3%. The experimental results are as follows Figure 5 As shown. During the algae control experiment, the concentration of chlorophyll a in the control group without the addition of algae control biological active agents continued to increase over time, while the concentration of chlorophyll a in the experimental group with the addition of algae control biological active agents gradually decreased over time, indicating that the algae control active substances can effectively inhibit the growth and activity of Microcystis aeruginosa. In addition, the algae lysis rate of the experimental group also continued to increase with the extension of time, and finally reached 97.89% at the end of the experiment, indicating that the algae control biological active agent has a significant algae control effect and has the potential and application value for biological algae control in water bodies with outbreaks of Microcystis aeruginosa.

[0061] The algae-controlling biological active agent prepared based on Bacillus Velez HY16 of the present invention can be used for the repair and management of algae-burst water bodies and the prevention of algae-burst in water bodies, and has been verified to have the potential for industrial-scale production, mature preparation technology and simple and convenient application. At the same time, as a microbial agent, the application is safer and more environmentally friendly. Therefore, it has important application and promotion value and significance in the management, repair and prevention and control of algae-burst water bodies.

[0062] 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 modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. A method for preparing an algae-controlling biologically active agent, characterized in that: The preparation method comprises the following steps: inoculating Bacillus Velez HY16 in a bioactive substance fermentation medium and culturing the culture medium; the culture temperature is 35-40° C. and the culture time is 32-48 hours.

2. The preparation method according to claim 1, characterized in that The classification name of the Bacillus velezensis HY16 is Bacillus velezensis, and its deposit number is CGMCC No.31645.

3. The preparation method according to claim 1, characterized in that: The preparation method also includes obtaining a high-yield algae-controlling active liquid by fermentation, and then obtaining HY16 algae-controlling biological active preparation powder by freeze-drying.

4. The preparation method according to claim 1, characterized in that: The formula of the bioactive substance fermentation medium is: 20-25 g / L glycerol, 45-50 g / L soybean meal powder, 4-5 g / L magnesium sulfate, and the balance is water. The pH of the medium is 7.0-7.

5.

5. The preparation method according to claim 1, characterized in that: The fermentation inoculation amount of the Bacillus Velez HY16 is 4-5%.

6. A bioactive algae-controlling agent prepared by the preparation method according to any one of claims 1 to 5.

7. The algae-controlling biologically active agent according to claim 1, characterized in that: The algae-controlling biologically active agent can inhibit and kill Microcystis aeruginosa.

8. Use of the algae-controlling biologically active agent as claimed in claim 6 in controlling and killing Microcystis aeruginosa.

9. The use according to claim 8, characterized in that The method for controlling and killing Microcystis aeruginosa is as follows: the algae-controlling biological active agent is directly added to the water body where Microcystis aeruginosa breaks out at a dosage of 3-5% of the water volume of the applied water body.

10. The use according to claim 8, characterized in that The algae-controlling biologically active agent has an algae-dissolving rate of more than 97% for Microcystis aeruginosa.

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