CN112625952B - Bacillus subtilis subspecies schoenoprasu and application thereof in inhibiting cyanobacterial bloom - Google Patents

Abstract

The invention belongs to the technical field of biology, and mainly relates to a bacillus subtilis subspecies strain with algae inhibiting activity and application thereof in inhibiting and preventing cyanobacterial bloom in a shrimp and crab culture pond. The strain is specifically Bacillus subtilis subsp. spizozenii TK812, is preserved in China general microbiological culture Collection center in 11 months and 3 days 2020, and has a preservation number of: CGMCC NO. 20996. In the invention, the bacillus subtilis subspecies is adopted for preventing and controlling the cyanobacterial bloom for the first time, and is separated from the nature, so that the method has no gene modification and no secondary pollution threat; the microbial inoculum provided by the invention does not contain preservatives and chemical additives, has no threat of chemical pollution, and has low cost and wide application prospect.

Description

Bacillus subtilis subspecies schoenoprasu and application thereof in inhibiting cyanobacterial bloom

The technical field is as follows:

the invention belongs to the technical field of biology, and mainly relates to a Bacillus subtilis subsp.

Background art:

cyanobacteria, also known as cyanobacteria, are prokaryotic microorganisms that are widely distributed in surface water and can perform photosynthesis. The cyanobacterial Bloom refers to a phenomenon that cyanobacterial algae abnormally and rapidly proliferate in a short time due to eutrophication of water, and is classified as "Harmful Algal Bloom (HAB)" because most cyanobacterial algae can generate Algal toxins Harmful to aquatic organisms and human beings. In China, the research on blue algae bloom is mainly concentrated in fresh water areas at present, from the last 80 century, with the rapid development of industry and agriculture, the blue algae bloom is generated in large scale in fresh water lakes in China, particularly in lakes in middle and downstream flow areas of Yangtze river and Yunnan pond, wherein the blue algae are mainly Microcystis aeruginosa, Anabaena flos-aquae and Oscillating algae. When the blue algae bloom occurs, the transparency of the water body is reduced, the variety of aquatic plants is reduced, and the water body becomes black due to rottenness in severe cases, which brings great harm to local tourism industry, water for people and livestock and aquaculture industry. In foreign countries, cyanobacterial blooms are classified as phytoplankton blooms. At present, how to prevent and treat the cyanobacterial bloom becomes a worldwide problem, and a great number of scientific researchers invest funds and energy in the aspect of preventing the cyanobacterial bloom every year. The basic reason for the occurrence of the blue algae bloom is eutrophication of water, under normal conditions, the eutrophication of the water is a slow natural process, but with the development of modern industry and agriculture, more and more substances such as nitrogen, phosphorus and the like are discharged into the natural world, so that the process of the eutrophication of the water is accelerated, particularly in the cultivation of modern aquatic products such as shrimps and crabs, the blue algae bloom is extremely easy to form in the cross-conversation and late summer of the shrimps and crabs every year due to the fact that a large amount of organic nutrient substances are put into the cultivation process of the aquatic products, fish fries can be regularly cleared in a shrimp and crab cultivation pond to ensure the high yield of the shrimps and crabs, and algae-eating fishes are seriously deficient, so that the blue algae bloom is extremely easy to form in the shrimp and crab cultivation pond every year, and the most direct consequence of the blue algae bloom is that the shrimps and crabs die due to oxygen deficiency, and serious economic loss is caused. In addition, the persistent influence also has a series of ecological environment problems of cyanobacteria toxin pollution of the aquaculture water body and cyanobacteria toxin pollution of aquatic products caused by the biological accumulation effect of the food chain, toxic gas pollution generated by the putrefaction of cyanobacteria, and the like. In order to reduce the loss caused by the cyanobacterial bloom, at present, copper sulfate is generally splashed to a pond for algae inhibition and algae killing in the first-line aquaculture of shrimps, crabs and the like, but excessive copper can cause the heavy metals in aquatic products to exceed the standard and also threaten the safety of drinking water, so the development and application of a safe and efficient cyanobacterial prevention and control mode is a problem to be solved urgently at present, the existing cyanobacterial bloom prevention and control mode mainly comprises biological prevention and control, physical prevention and control, chemical prevention and control and combined prevention and control of three modes, wherein the microbial prevention and control is one of the hot fields of cyanobacterial bloom prevention and control research at present.

Currently, many algal-inhibiting microorganisms have been found, but only a few have been put into practical production, such as those disclosed in publication nos: CN109609405A invention Bacillus producing algae inhibiting active substance and its use discloses a method for producing algae inhibiting substance by fermenting Bacillus (Bacillus sp) dhs 330 producing algae inhibiting active substance and its use. The publication numbers are: CN102308852A discloses a microbial algae inhibitor prepared by mixing bacillus laterosporus VKPM B-10531 and an expanded perlite particle carrier, which can inhibit the growth of microalgae in a water body and the formation of a microalgae biofilm, and finally cause the death of algae. The Bacillus subtilis subsp.

Currently, Bacillus subtilis contains four subspecies, namely Bacillus subtilis subsp.subtilis, Bacillus subtilis subsp.inaquosum, Bacillus subtilis subsp.spizonii and Bacillus subtilis subsp.steroris, and scientific research in recent years finds that all the four subspecies can be used as an independent strain, but four strains cannot be distinguished in a conventional manner due to the lack of clearly distinguishable phenotypes, and three people, such as a Christopher a.nlap.michael j. bowman. daniel r.zeigler, compare four strains from a genome to a metabolic composition, and find that all the four strains have differences in the metabolic composition, and the main differences are as follows: bacillus Subtilis subsp. Subtilis produces only 3, 3' -neothalosadiamine; bacillus subtilis subsp. inaquosum produces Bacillus mycin F, fengycin and an unknown PKS/NRPS structural cluster; bacillus subtilis subsp.spizofacienii produces subtilisin, bacteriocin and 3, 3' -neothapsidiamine; stercoris can produce both plumocin and unknown PKS/NRPS structural clusters. In addition, in the comparison of genome differences, three found that four species of Bacillus subtilis are unilineage branches, i.e., they share a common ancestor, but are currently far from evolutionary relationship with each other.

The Bacillus subtilis subsp. spizonii is named for commemorating american bacillicians j.spizonii, has a single or chain rod-shaped cell with the size of 0.5 x 2-3 μm, flagella, gram-positive staining, metaplasia elliptica, facultative anaerobic, can tolerate 7% concentration sodium chloride, produce amylase and protease, can reduce nitrate, has genes for encoding Bacillus subtilis, bacteriocin, lipopeptide, mycotin, surfactin and 3, 3' -neotrehaladimine, is one of strains with extremely strong development potential, and is used for the first time for preventing and treating cyanobacterial bloom.

The invention content is as follows:

one of the purposes of the present invention is to provide a Bacillus subtilis subsp.spizozenii TK812 strain with good algae inhibition, wherein the strain is stored in the common microorganism center of the china committee for culture and management of microorganisms at 11-3/2020 with the storage address: the microbial research institute of the national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation numbers are as follows: CGMCC NO. 20996.

The second purpose of the invention is to provide application of a Bacillus subtilis subsp.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the invention provides a microecological preparation for preventing cyanobacterial bloom in a fish and shrimp culture pond prepared by using bacillus subtilis subspecies TK812, and the specific method comprises the following steps:

(1) strain activation and first-order seed liquid preparation

Inoculating a strain TK812 glycerol pipe into a seed liquid culture medium according to the inoculation amount of 1-5%, wherein the primary seed liquid culture condition is 30-40 ℃, and the primary seed liquid culture condition is 200-250rpm for shake cultivation for 24-48 h;

further, the preparation method of the strain TK812 glycerol pipe comprises the following steps: diluting the enriched liquid of nutrient broth culture medium of strain TK812 with sterile normal saline to strain concentration of 10⁷Inoculating the CFU/ml into a 500ml baffle bottle containing 100ml of glycerol tube culture medium according to the proportion of 1-5%, culturing for 48-60h under the conditions of 35-40 ℃ and 200-plus 250rpm shaking table, after spores are generated, killing live bacteria in a fermentation liquor baffle bottle in water bath at the temperature of 80-100 ℃ for 20-50min, retaining the spores, and finally, after the culture solution is cooled to room temperature, adding 10-50% of sterile glycerol in the volume of the bacteria solution to prepare the glycerol tube for industrial production;

preferably, the glycerol tube culture medium consists of: 1-2% of glucose, 2-3% of corn flour and Dada corn1-2% of bean flour, 0.5-1% of yeast extract, 1-2% of tryptone and mineral salt solution (0.5-1% of NaCl, K)₂HPO₄ 0.5-1％，MgSO₄0.01-0.05％，MnSO₄ 0.01-0.05％，(NH₄)₂SO₄0.1-0.5%, the rest is water) 1-2%, the rest is water, pH is 7.4, sterilizing at 115 deg.C for 15 min;

preferably, the glycerol pipe for industrial production is connected with an amount of 1%;

preferably, in the preparation of the glycerin pipe, the optimal water bath temperature of the fermentation liquid is 82 ℃, the treatment time is 30min, and the addition proportion of glycerin is 32%;

(2) fermenting with second-stage seed liquid and 50L fermentation tank

Inoculating the first-stage seed liquid after 24-48h culture into a 5L conical flask containing 1L of seed liquid culture medium according to 1-10% of inoculation amount, putting the conical flask into a shaking table at 30-40 ℃ for culture, rotating at the speed of 200 plus 250rpm for 24-36h culture, inoculating the conical flask into a 50L fermentation tank containing 30L of fermentation medium according to 1-5% of inoculation amount after the culture is finished, rotating at the speed of 100 plus 150rpm and at the temperature of 30-40 ℃, ventilating, wherein the ventilation amount is 2-3:1 (V/V.min), fermenting for 36-60h, and flexibly supplementing acid, alkali and defoaming agent according to requirements so that the pH value of the fermentation liquid in the tank is stabilized between 7.0 and 7.4;

preferably, the seed liquid culture medium comprises: 2-3% of glucose, 2-3% of corn flour, 1-2% of bran, 1-2% of soybean flour, 0.1-0.5% of yeast extract and mineral salt solution (K)₂HPO₄0.5-1％，MgSO₄ 0.01-0.05％， MnSO₄0.01-0.05％，(NH₄)₂SO₄0.1-0.5%, and the balance of water) 1-2%, and the balance of water, wherein the pH is 7.0, and the mixture is sterilized at 115 ℃ for 15 min.

Preferably, the fermentation medium consists of: 2-5% of glucose, 1-3% of corn flour, 1-2% of bran, 1-2% of soybean meal, 1-2% of yeast extract and mineral salt solution (K)₂HPO₄ 0.5-1％，MgSO₄ 0.02-0.03％， MnSO₄ 0.02-0.05％，(NH₄)₂SO₄0.2-0.5%, and the balance water) 0.5-1%, and the balance water, with a pH of 7.0, sterilizing at 115 deg.C for 25 min;

preferably, the culture temperature of the primary and secondary seed liquids and the fermentation tank is 37 ℃;

preferably, the culture time of the primary and secondary seed liquid is 36h, and the fermentation time of the fermentation tank is 48 h;

preferably, the rotating speed of the primary and secondary seed liquid shaking tables is 220rpm, the rotating speed of the fermentation tank is 150rpm, and the ventilation volume is 3:1 (V/V.min);

preferably, the inoculation amount of the first-stage seed liquid is 2%, the inoculation amount of the second-stage seed liquid is 5%, and the inoculation amount of the fermentation tank is 1%;

(3) preparation of high-efficiency algae inhibiting active bacteria powder and preparation of algae inhibiting active bacteria emulsion

Preparing high-efficiency algae inhibiting active bacteria powder: centrifuging the zymophyte liquid at 7000rpm of 5000-⁹-10¹⁰CFU/g, obtaining the bacillus subtilis subspecies Sphaete high-efficiency algae-inhibiting active bacterial powder;

preferably, the supernatant of the fermentation liquor is taken and sprayed onto the clay for several times, the clay is stirred in the spraying process, and the clay is placed in an environment with the temperature of 30-40 ℃ for air drying after being sprayed each time until the moisture content is 1-10%;

more preferably, the centrifugal speed is 6000rpm, the sprayed clay is dried at 35 ℃ until the moisture content is 5%, and the viable count is not less than 10 after crushing⁹CFU/g；

Preparing the algae inhibiting active emulsion: centrifuging the fermentation bacteria liquid at 7000rpm of 5000-;

preferably, the adding amount of the Tween 80 is 3 percent, and the adding amount of the glycerol is 14 percent;

(4) preparation of algae inhibiting active granule

Mixing the fermentation liquor and clay according to the mass ratio of 1: 1.5-2, then pressing into granules through a granulator, wherein the algae inhibiting granules have larger specific surface area and can improve the adsorption effect;

preferably, the mass ratio of the fermentation liquor to the clay in the preparation process of the algae inhibiting active particles is 1: 1.5, the diameter of the pressed granule is 4 mm.

Further, the application method of the preparation containing the bacillus subtilis subspecies TK812 in the control of blue algae is as follows:

(1) when macroscopic algae bloom particles appear in the water body, adding the algae inhibiting active bacteria powder or the algae inhibiting active emulsion, wherein the using amount is that every 1kg of the active bacteria powder is used in 260 cubic meters of water body, or every 1kg of the algae inhibiting active emulsion is splashed with 200 cubic meters of water body and 300 cubic meters of water body;

(2) within 24 hours after the blue algae outbreak, the algae inhibiting active particles are applied, the dosage is that 300 plus 400 cubic meters of water is splashed for every 1kg, and the equal amount is splashed for the second time after 48 hours;

the blue algae outbreak is mainly characterized in that the turbidity of the water body is rapidly increased within 24 hours and is more than 60 NTU. The invention has the beneficial effects that:

1) the bacillus subtilis subspecies used by the invention is separated from the nature, has no gene modification and no secondary pollution threat.

2) In the invention, the bacillus subtilis subspecies stressful is adopted for the first time to prevent and control the cyanobacterial bloom.

3) The microbial inoculum provided by the invention does not contain preservatives and chemical additives, has no threat of chemical pollution, and has low cost and wide application prospect.

Description of the drawings:

in order to better show the bacillus subtilis subspecies TK812 used in the invention and the inhibition effect of the bacillus subtilis subspecies TK812 on blue-green algae, the strain property, the growth property and the algae inhibition property of the strain TK812 are shown in the following figures.

FIG. 1 is a photograph showing the colony morphology of Bacillus subtilis subspecies Ski 812.

FIG. 2 shows a photomicrograph and a scanning electron micrograph of a Bacillus subtilis subspecies TK 812.

FIG. 3 shows the growth curve of Bacillus subtilis subspecies TK812 in nutrient broth.

FIG. 4 is a phylogenetic tree of Bacillus subtilis subspecies Sk 812.

FIG. 5 is a photograph of the anabaena culture medium with the addition of Bacillus subtilis subspecies TK812 and a blank control group (the left side is an experimental group, the right side is a blank control, and the culture is carried out for 4 d).

FIG. 6 is a graph showing the inhibition curve of Bacillus subtilis subspecies Sk 812 against anabaena.

FIG. 7 is a graph of the inhibition of Microcystis aeruginosa by strain TK 812.

The specific implementation mode is as follows:

the objects, technical solutions and advantages of the present invention are described in detail below with reference to specific embodiments, and unless otherwise noted, all technical means used in the present invention are the methods currently disclosed in the field, and the present invention is specifically described below with reference to the accompanying drawings, tables and examples. It should be noted that the following specific examples are merely illustrative of the present invention, but the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various modifications or variations can be made in the specific parameters such as raw materials and their contents in the following examples based on the present invention.

In the following examples, the reference "%" refers to mass percent of material, concentration percent of solution refers to grams of solute contained in each 100mL of solution, and the percentage between liquids refers to the volume ratio of the solution at 25 ℃, if not otherwise specified. Embodiments are described in terms of mass to volume ratios "W/V" or "m/V", where not specifically noted, units are g/mL; the mass ratio is "m/m".

In the following examples, BG11 Medium (Medium for Blue Green Algae, BG11) and Nutrient Broth Medium (Nutrient Broth, NB) was used for the Bacillus subtilis subspecies TK 812.

BG11 culture medium mainly comprises (g/L): NaNO₃ 1.5，K₂HPO₄ 0.04，MgSO₄.7H₂O 0.075，CaCl₂.7H₂O 0.036，Na₂CO₃0.02, citric acid 0.006, ferric ammonium citrate 0.006, trace element solution A₅1ml, 1L of distilled water.

Microelement solution A₅(g/L)：H₃BO₄ 2.86，MnCl₂.4H₂O 1.81，ZnSO₄ 0.222， Na₂MoO₄0.39，CuSO₄.5H₂O 0.079，Co(NO₃)₂.6H₂O0.494, distilled water 1L.

Nutrient Broth (NB): tryptone 1%, beef extract 0.5%, NaCl 0.5%, distilled water 100ml, pH 7.6.

Example 1 isolation and characterization of Bacillus subtilis subspecies Sk 812 with algal inhibiting Activity

The Bacillus subtilis subsp. spizozenii TK812 with algae inhibiting activity in the invention is separated from wild earthworm excrement collected from the campus of Tianjin scientific university.

The specific method comprises the following steps:

1. sample processing

Taking a captured wild earthworm, washing the surface of the wild earthworm with sterile normal saline, placing the wild earthworm in a test tube, taking out the earthworm after the earthworm produces excrement, adding a proper amount of sterile normal saline into the test tube, shaking uniformly, diluting and coating the earthworm on a nutrient broth culture medium, placing a coated flat plate in an incubator at 30 ℃ for culturing for 48 hours, and picking a single colony for shake flask culture. The culture conditions are 220rpm, 37 ℃, and the algae inhibiting active microorganism is screened after being cultured for 24 hours.

2. Screening of microorganisms having algal inhibiting activity

Mixing a culture solution of Microcystis aeruginosa (Microcystis aeruginosa) FACHB-978 in logarithmic phase with a BG11 culture medium containing 1.5% agar, pouring a blue algae plate, culturing for 3 days under an illumination condition (3000lx and 28 ℃), punching with a puncher with the diameter of 1cm, adding 100 mu L of enriched bacterial liquid into each hole, airing the bacterial liquid under an aseptic condition, then placing the bacterial liquid into an illumination incubator for culturing, observing whether an algae inhibition ring exists after culturing for 3-7 days, and selecting a bacterial colony with an obvious algae inhibition ring for microorganism species identification.

3. Identifying strains of microorganisms with algae inhibiting activity:

taking 1ml of the microorganism enrichment solution, extracting the genome of the bacteria by a phenol-copy method, amplifying by using a universal 16S rDNA primer (the sequence of the universal primer is 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), obtaining a specific gene sequence after sequencing an amplification product, then uploading gene data to compare with an NCBI database, and showing that the similarity is most similar to a subspecies Stahnsonii of bacillus subtilis and reaches 99 percent according to a comparison result, wherein the name is as follows: bacillus subtilis subsp. spizofacieni) TK812, the 16S rDNA sequence of the bacterial part being:

CTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTT AGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGA TAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGT TCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGC ATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATGCGTAGCCGA CCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCT ACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGA GCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTA GGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAAC CAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG GCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTCCTT AAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACT GGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGA AATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTC TGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATA CCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCC GCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGG TCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTG GAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGAC ATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACA GGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTC TAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAAT CATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACA AAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAG TTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC CCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTT AGGAGCCAGCCGCCGAAGTGAC

4. colony and microscopic morphological characteristics

As shown in figures 1 and 2, the bacterial colony on the nutrient broth solid medium is milky white, yellowish, flat and flaky, has a special odor of bacillus, is rod-shaped gram-positive bacteria observed under a microscope, and is mostly in a serial connection state with two bacteria, and spores are oval and end-growing or end-growing.

5. Common antibiotic resistance sensitivity

Adding ampicillin, kanamycin, lincomycin, amikacin, tetracycline and chloramphenicol into a nutrient broth culture medium respectively to make the final concentration of the nutrient broth culture medium be 100mg/mL, arranging two groups of antibiotics in parallel, arranging a blank control group at the same time, inoculating the strain TK812 according to 1% inoculation amount, culturing at 37 ℃ for 48h, measuring the absorbance value of the culture solution at 600nm every 12h, and judging the antibiotic sensitivity of the strain TK812 according to the OD600 size. The test results were as follows:

TABLE 1 Strain TK812 antibiotic resistance

In the table "+" indicates that the strain TK812 has the antibiotic resistance and is insensitive to the antibiotic. "-" indicates that the strain TK812 is sensitive to this antibiotic.

As can be seen from the experiment, the strain TK812 has a certain resistance to ampicillin and no obvious resistance to other antibiotics.

6. Drawing of growth Curve for Strain TK812

Inoculating the strain enrichment culture solution into a blank nutrient broth culture medium according to the inoculation amount of 1%, setting three groups of parallel culture media, culturing at 37 ℃ and 220rpm, sampling every two hours, measuring the absorbance of the culture solution under the condition of 600nm wavelength, and finally drawing a growth curve graph by using data processing software. OD₆₀₀The values measured are as follows:

TABLE 2 Strain TK812 OD₆₀₀Measured value

As shown in figure 3, the growth rate of the strain is extremely high, the exponential growth period begins after 2 hours of inoculation, the growth rate is reduced after 8 hours, and the stationary period begins after about 20 hours.

7. Construction of an evolutionary relationship diagram of Strain TK812

In order to reflect the evolutionary position of the strain in the bacterial evolutionary relationship, 16S rDNA fragments with similar relativity and representative strains are obtained from a NCBI bacterial strain 16S rDNA database, a system evolutionary relationship diagram is constructed by using the maximum reduction method of MEGA-X software, and the construction result is shown in figure 4.

Example 2 inhibition experiment of Bacillus subtilis subspecies Ski 812 on Anabaena flos-aquae

Taking 6 bottles of healthy anabaena culture solution (specification: 100ml/250ml conical flask) in logarithmic growth phase, numbering 1-6, wherein No. 1-3 is used as a blank control group, adding a blank nutrient broth culture medium according to the inoculation amount of 5%, No. 4-6 is used as an experimental group, adding a strain TK812 enrichment culture solution (strain TK812 enrichment solution: inoculating in the nutrient broth culture medium, performing enrichment culture in a shaking table at 37 ℃ and under the condition of 220 rpm), and culturing until each ml of enrichment culture solution contains 10 TK812 viable bacteria⁸Respectively), culturing the anabaena culture solution inoculated with the TK812 enriched culture solution at 28 ℃, 100rpm for 12h, under the conditions of 12h light dark cycle and 3000lx light intensity, sampling every 24h, and determining the content of chlorophyll a. FIG. 5 is a photograph of anabaena culture medium with Bacillus subtilis subspecies TK812 added and a blank control group.

Chlorophyll a content determination mode: carrying out suction filtration on anabaena flos-aquae culture solution by using a nylon filter membrane with the aperture of 0.22 mu m, washing the anabaena flos-aquae culture solution for 2-3 times by using sterile PBS buffer solution after the suction filtration is finished, then placing the nylon filter membrane into 90% acetone solution, extracting the nylon filter membrane for 20-24h at 4 ℃ in a dark condition, finally centrifuging the nylon filter membrane for 5-10min at low temperature under the condition of 10000g of centrifugal force, measuring the absorbance of supernatant at the wavelengths of 664nm and 630nm by using an enzyme-labeling instrument, and finally according to a formula:

chlorophyll a (μ g/L) ═ 11.47 XOD_664nm-0.40×OD_630nm

Calculating to obtain the chlorophyll concentration:

TABLE 3 influence of Strain TK812 on chlorophyll content of anabaena flos-aquae culture fluid (chlorophyll content (. mu.g/L))

After the 2 nd day, 5% of blank nutrient broth culture medium or strain TK812 enriched culture solution is added, the continuously-reduced chlorophyll probably causes the inhibition effect of partial substances in tryptone on the anabaena on the third day, the chlorophyll content in the culture solution returns to the original level on the fourth day, but after the 5 th day, the blank anabaena starts to grow rapidly, the anabaena in the experimental group starts to wither, and the difference of chlorophyll content reaches a significant level. The effect of the example on the anabaena culture solution is shown in the attached figure 5, the left side is the anabaena culture solution added with the strain TK812 enriched culture solution for 4 days, and the right side is not blank control. The corresponding algae inhibition curve of this example is shown in figure 6.

Example 3 inhibition of Bacillus subtilis subspecies Sk 812 against Microcystis aeruginosa

Taking six microcystis aeruginosa bottles in logarithmic growth phase, numbering 1-6, evenly dividing into two groups, adding 5% of nutrient broth blank culture medium into the first group, adding 5% of TK812 enriched culture solution into the second group, mixing, placing in an illumination incubator at 28 deg.C for 24h and 4000lx continuously, sampling every 24h to determine absorbance at 730nm, and determining the absorbance according to OD₇₃₀Determining the growth condition, OD of Microcystis aeruginosa according to the change₇₃₀The measurement is as follows:

TABLE 4 Strain TK812 vs. Microcystis aeruginosa OD₇₃₀Influence of (2)

It can be seen that the difference from anabaena is that after the culture medium is added with a blank broth, the growth rate of the microcystis aeruginosa in the experimental group is slightly lower than that of the blank control group, the cell number of the microcystis aeruginosa starts to decrease in the 4 th to 5 th days, the difference gradually increases in the subsequent 5 th to 6 th days, the microcystis aeruginosa in the experimental group withers obviously, and the corresponding microcystis aeruginosa inhibition curve of the embodiment is shown in the attached figure 7.

Example 4 Effect of the Strain TK812 on Chlorella (commercial product) growth

The chlorella is a unicellular eukaryotic green alga, different from a cyanobacteria which can cause water bloom, is a good food source for aquatic animals, and is one of important algae which can perform photosynthesis to provide oxygen for aquatic animals except the cyanobacteria in an aquaculture pond.

The specific experimental process is that nine bottles of chlorella culture fluid (BG11 culture medium, 100ml/250ml conical flask) in logarithmic growth phase are taken, numbered 1-9, and divided into three groups on average, starting from the first day: the first group is used as a natural control group, 5% of sterile normal saline is added, 5% of sterile blank culture medium is added in the second group, 5% of strain TK812 enrichment culture solution is added in the third group, the strain TK812 enrichment culture solution is placed in a shaker at 28 ℃, 100rpm for culture, the illumination intensity is 3000lx, the illumination dark cycle is 12h, samples are taken every 24h, and the chlorella is counted by a blood counting plate.

TABLE 5 Lo Effect of Strain TK812 on Chlorella growth conditions (CFU/ml)

From the data, the addition of the culture medium can generate a certain inhibition effect on the chlorella, but the TK812 has no obvious inhibition effect on the chlorella, and the microscope examination finds that the commercialized chlorella contains trace epiphytic bacteria, and the bacteria can proliferate in large quantity after being added into the blank nutrient broth culture medium to cause the death of part of the chlorella, but the experimental group does not find the same situation as the blank control group in the microscope examination and can possibly relate to the generation of antibacterial substances by the similar strains reported in the literature.

Example 5 Industrial production preparation of Bacillus subtilis subspecies Ski 812 high efficiency algae inhibiting active bacteria powder and algae inhibiting active bacteria emulsion

(1) Strain activation and first-order seed liquid preparation

Inoculating 2% of strain TK812 glycerol into a 500ml baffle bottle with 100ml of seed liquid culture medium as a first-stage seed liquid, wherein the first-stage seed liquid culture condition is 37 ℃, and the shaking table aeration culture is carried out at 220rpm for 36 h.

The preparation method of the strain TK812 glycerol pipe comprises the following steps: diluting nutrient broth culture medium enrichment solution of strain TK812 with sterile normal saline to concentrate to 10⁷Inoculating the CFU/ml into a 500ml baffle bottle containing 100ml of glycerol tube culture medium according to the proportion of 1 percent, culturing for 48 hours under the conditions of 37 ℃ and 220rpm shaking table, after spores are produced, carrying out water bath on the baffle bottle containing fermentation liquor for 30 minutes under the condition of 82 ℃ so as to kill live bacteria and retain the spores, and finally, after the culture solution is cooled to room temperature, adding 32 percent sterile glycerol to carry out the glycerol tube production for industrial production.

The formula of the glycerol tube culture medium is as follows: 1.5% of glucose, 2% of corn flour, 1% of soybean flour, 1% of yeast extract, 1% of tryptone and 0.5% of mineral salt solution (NaCl, K)₂HPO₄ 0.5％，MgSO₄ 0.02％， MnSO₄ 0.02％，(NH₄)₂SO₄0.2%), 1% and ph 7.4, and sterilized at 115 ℃ for 15 min.

The seed liquid culture medium comprises: glucose 3%, corn flour 2%, bran 1%, soybean flour 2%, yeast extract 0.5%, mineral salt solution (K)₂HPO₄ 0.5％，MgSO₄ 0.02％，MnSO₄ 0.02％，(NH₄)₂SO₄0.2%) 1%, ph 7.4, sterilized at 115 ℃ for 15 min.

(2) Fermenting the second-stage seed liquid in a 50L fermentation tank.

Inoculating the first-stage seed liquid after 36h culture into a 5L conical flask containing 1L of seed liquid culture medium according to the inoculation amount of 5%, placing the conical flask into a shaking table at 37 ℃ for culture, rotating at 220rpm for 36h, inoculating the conical flask into a 50L fermentation tank containing 30L of fermentation culture medium according to the inoculation amount of 1% after 36h culture, rotating at 150rpm and 37 ℃, ventilating, wherein the ventilation amount is 3:1 (V/V.min), fermenting for 48h, and flexibly supplementing acid, alkali and defoaming agent according to requirements to stabilize the pH value of fermentation liquid in the tank to be between 7.0 and 7.4.

The fermentation medium formula of the fermentation tank is as follows: 5% of glucose, 3% of corn flour, 2% of bran, 2% of soybean meal, 1% of yeast extract and mineral salt solution (K)₂HPO₄ 0.5％，MgSO₄ 0.02％，MnSO₄ 0.02％， (NH₄)₂SO₄0.2%) 1%, and sterilizing at 115 deg.C for 25 min.

(3) Preparation of high-efficiency algae inhibiting active bacteria powder and preparation of algae inhibiting active bacteria emulsion

Preparing high-efficiency algae inhibiting active bacteria powder: centrifuging the zymophyte liquid with a GQ80-LD high-speed tubular separator at 6000rpm for 15min, spraying the supernatant of the zymophyte liquid onto dry clay with the same quality for 3 times, stirring the clay in the spraying process, drying the clay in a 35 ℃ environment after each spraying, or spraying multiple batches of zymophyte liquid onto the same batch of clay, finally drying the clay of the zymophyte liquid at 40 ℃ until the water content is not higher than 5%, and crushing to obtain the zymophyte liquid with the viable count of 10⁹CFU/g is the high-efficiency algae-inhibiting active bacterial powder of the subspecies Ski 812 of the bacillus subtilis.

Preparing the algae inhibiting active emulsion: adding 3% of Tween 80 and 14% of glycerol into the precipitate of the tubular centrifuge according to the mass ratio, and uniformly mixing to obtain the algae inhibiting active bacteria emulsion.

Preparing algae inhibiting active particles: directly mixing the fermentation liquor and clay according to the mass ratio of 1: 1.5, mixing, and pressing the mixed particles into particles with the diameter of 4mm by a granulator, namely the algae inhibiting active particles.

Example 6 experiment of inhibiting natural algal bloom blue algae enriched liquid by high efficiency algae inhibiting active bacteria powder, algae inhibiting active emulsion and algae inhibiting active granule

The natural algal bloom and blue algae water sample is taken from a Changzhou shrimp and crab culture pond with the outbreak of blue algae bloom in Changzhou, the sampled sample is transported to a laboratory under the condition of normal temperature and visible light, 50ml of the sample is added into a 250ml triangular flask containing 50ml of a sterile lake BG11 culture medium (prepared according to a BG11 culture medium formula, lake water is used as a solvent, the mixture is shaken up and deposited during use and then subpackaged), the culture is carried out for one week under the condition of natural illumination, then the culture is expanded to 2L, and the main algal bloom algae is Oscillating algae or Anabaena algae through microscopic examination.

The specific implementation method comprises the following steps: shaking up the enriched algal bloom culture solution, subpackaging 15 bottles of algal bloom enriched solution into 15 conical flasks with the same 250mL specification according to the subpackaging amount of 100mL per bottle, finally averagely dividing the 15 bottles of algal bloom enriched solution into 5 groups, numbering the first group, the second group, the third group, the fourth group and the fifth group, wherein the first group is added with 0.5% (W/V) of the sterile clay used in the example 5, the second group and the third group are respectively added with 0.5% (W/V) of the high-efficiency algae inhibiting active bacteria powder prepared in the example 5, the fourth group is added with the algae inhibiting active emulsion and 0.25% (W/V) of algae inhibiting active particles, the fifth group is used as a blank control, no matter is added, samples are taken respectively on the 1 st day, the 3 rd day and the 7 th day, the chlorophyll content in the enriched solution is measured, and the measurement result is as follows:

TABLE 6 inhibition of algal bloom enrichment culture medium (chlorophyll content (μ g/L))

From the above results, it can be seen that, compared with the blank blue-green algae enriched liquid without any substance, the clay and the high-efficiency algae inhibiting active bacteria powder, the algae inhibiting active emulsion and the algae inhibiting active particles provided in example 5 all have the algae-blooms and blue-green algae inhibiting effect, but the high-efficiency algae inhibiting active bacteria powder, the algae inhibiting active emulsion and the algae inhibiting active particles provided in example 5 all have the algae inhibiting effect higher than that of the pure clay.

The above embodiments only represent some embodiments of the present invention, and the embodiments of the present invention are disclosed as the first best embodiments, but the present invention is only limited to this, and any person skilled in the art can make corresponding additions or simplifications without departing from the basic scope of the present invention, and the scope of the present invention is defined by the claims.

SEQUENCE LISTING

<110> Tianjin science and technology university

<120> a bacillus subtilis subspecies schoenleinii and application thereof in inhibiting blue algae bloom

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1434

<212> DNA

<213> Bacillus subtilis subsp. spizozinii) TK812

<400> 1

ctatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg 60

gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta 120

ataccggatg cttgtttgaa ccgcatggtt caaacataaa aggtggcttc ggctaccact 180

tacagatgga cccgcggcgc attagctagt tggtgaggta atggctcacc aaggcaacga 240

tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc 300

tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg 360

cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtaccgt 420

tcgaataggg cggtaccttg acggtaccta accagaaagc cacggctaac tacgtgccag 480

cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg 540

caggcggttc cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa 600

actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt 660

agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg 720

agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg 780

agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc 840

gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc 900

ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct 960

ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt 1020

gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc 1080

ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag 1140

gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg 1200

gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt 1260

tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag 1320

catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt 1380

tgtaacaccc gaagtcggtg aggtaacctt ttaggagcca gccgccgaag tgac 1434

Claims (6)

1. A bacillus subtilis subspecies Ski is characterized in that the bacillus subtilis subspecies Ski is specifically (Bacillus subtilis subsp. spizizenii) TK812, accession number: CGMCC number 20996.

2. A bacterial preparation comprising the Bacillus subtilis subspecies TK812 of claim 1,

the microbial inoculum is active bacteria powder, and is prepared by mixing, air-drying and crushing a supernatant obtained by centrifuging TK812 fermentation liquor and clay, wherein the viable count of the crushed TK812 bacteria powder reaches 10⁹-10¹⁰CFU/g; or

The microbial inoculum is active emulsion, and is emulsion obtained by adding 1-5% of tween 80 and 10-20% of glycerol into the precipitate obtained after centrifuging TK812 fermentation liquor, and uniformly mixing; or

The microbial inoculum is active particles, and is prepared by mixing TK812 fermentation liquor and clay according to the mass ratio of 1: 1.5-2, and pressing into granules by a granulator.

3. The microbial inoculum of claim 2, wherein the fermentation broth is prepared by the following method:

(1) inoculating a strain TK812 glycerol tube into a seed liquid culture medium according to the inoculation amount of 1-5%, wherein the primary seed liquid culture condition is 30-40 ℃, and the shake cultivation is carried out for 24-48h at 200-250 rpm;

(2) inoculating the first-level seed liquid into a seed liquid culture medium according to the inoculation amount of 1-10%, culturing at 30-40 ℃ and 250rpm for 24-36h, inoculating the first-level seed liquid into a fermentation culture medium according to the inoculation amount of 1-5% after the culture is finished, wherein the rotation speed is 100rpm, the temperature is 30-40 ℃, the ventilation amount is 2-3:1, the fermentation time is 36-60h, and the pH value is stabilized between 7.0-7.4.

4. The microbial preparation according to claim 3,

the seed liquid culture medium comprises the following components: 2-3% of glucose, 2-3% of corn flour, 1-2% of bran, 1-2% of soybean flour, 0.1-0.5% of yeast extract, 1-2% of mineral salt solution and the balance of water, wherein the pH is =7.0, and the mixture is sterilized at 115 ℃ for 15 min;

the mineral salt solution consists of: k₂HPO₄0.5-1%，MgSO₄ 0.01-0.05%，MnSO₄ 0.01-0.05%，(NH₄)₂SO₄0.1-0.5 percent of water for the rest;

the fermentation medium comprises the following components: 2-5% of glucose, 1-3% of corn flour, 1-2% of bran, 1-2% of soybean meal, 1-2% of yeast extract, 0.5-1% of mineral salt solution and the balance of water, wherein the pH is =7.0, and the mixture is sterilized at 115 ℃ for 25 min;

the mineral salt solution consists of: k₂HPO₄ 0.5-1%，MgSO₄ 0.02-0.03%，MnSO₄ 0.02-0.05%，(NH₄)₂SO₄0.2-0.5% and the balance of water.

5. The use of the bacillus subtilis subspecies TK812 of claim 1 or the bacterial agent of claim 2 for inhibiting cyanobacterial bloom.

6. The use as claimed in claim 5, wherein the active bacteria powder or the active emulsion is added after macroscopic algal bloom particles appear in the water body, and the use amount is that every 1kg of the active bacteria powder is used in 260 cubic meters of the water body, or every 1kg of the algae inhibiting active emulsion is splashed in 200 cubic meters of the water body and 300 cubic meters of the water body.

Images