Preparation method and application of actinomyces agardhii active substance

Abstract

The present application relates to the technical field of eutrophication water treatment, and discloses a preparation method and application of active substances of algicidal actinomycetes, which comprises the following steps: (1) inoculating actinomycetes into a shaking flask containing a culture medium, wherein the culture medium comprises carbohydrates, salts, glutamic acid and cyanobacteria, and then placing the shaking flask in a culture box for oscillation culture to obtain a first-stage seed liquid; (2) inoculating the first-stage seed liquid into a first-stage seed tank according to a volume fraction of 10-20% to obtain a second-stage seed liquid; (3) inoculating the second-stage seed liquid into a fermentation tank according to a volume fraction of 10-20% to obtain an actinomycete fermentation liquid; and (4) centrifuging the actinomycete fermentation liquid to obtain a supernatant and mixing the supernatant with a stabilizer. Glutamic acid can promote the actinomycetes to secrete more active substances during the fermentation process, and cyanobacteria can enable the actinomycetes to maintain the ability to remove algae during the fermentation process to secrete corresponding active substances. The stabilizer can inhibit the growth of miscellaneous bacteria to maintain the stability of algae removal.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental protection, in particular to the field of eutrophication water treatment, and more particularly to a preparation method and application of actinomycete active substance. BACKGROUND

[0002] Algal blooms refer to a natural ecological phenomenon of algal mass propagation in freshwater bodies, which is a characteristic of water eutrophication. It is mainly caused by the entry of a large amount of waste water containing nitrogen and phosphorus from life and industrial and agricultural production into water bodies, and a large number of blue-green algae (also known as cyanobacteria, including microcystis, fishy algae, tremella, pearl algae, blue ball algae, and hair algae), green algae, and diatoms propagate to make the water body appear blue or green.

[0003] The current domestic and foreign methods for removing algae include physical methods, chemical methods, and biological methods. The physical methods mainly include mechanical dredging, filtration, clay removal, and air floatation, which are expensive, high in cost, and difficult to apply to large areas of blue-green algae control, and are limited in practical application. The chemical method mainly uses chemical agents such as copper sulfate, hydrogen peroxide, surfactants, and sodium hypochlorite. The chemical method is easy to cause secondary pollution and has potential harm to water organisms. The biological method uses natural enemies of algae such as bacteria, viruses, planktonic animals, aquatic plants, large algae, and some substances produced by them to control the increase of the number of algae, so as to achieve the purpose of inhibition. The biological method has the advantages of simple operation, low cost, and no secondary pollution, and has become a research hotspot at home and abroad. Therefore, it is of great significance to find a high-efficiency biological algae-removing agent.

[0004] In the biological method, the use of algicidal bacteria to remove harmful algae in water bodies is a direction that is currently studied more. It is found that more algicidal bacteria remove algae by secreting extracellular active substances. At present, the research on algicidal bacteria is more in the laboratory stage, and the production culture is relatively less. SUMMARY

[0005] In view of the above problems, the present application aims to provide a preparation method and application of algicidal actinomycete active substance, which is simple, can effectively promote the secretion of algicidal actinomycete active substance, and has obvious and stable algae-removing effect.

[0006] To achieve the above-mentioned purpose, the present application provides a preparation method of algicidal actinomycete active substance, which comprises the following steps:

[0007] (1) inoculating actinomycetes into a flask containing a culture medium, wherein the culture medium comprises carbohydrates, salts, glutamic acid, and blue-green algae, and then placing the flask in a culture box for shaking culture to obtain a first seed liquid;

[0008] (2) inoculating the primary seed liquid into a primary seed tank for cultivation to obtain a secondary seed liquid, with the inoculation amount being 10-20% by volume fraction;

[0009] (3) inoculating the secondary seed liquid into a fermentation tank for cultivation to obtain an actinomycete fermentation liquid;

[0010] (4) centrifuging the actinomycete fermentation liquid to obtain a supernatant and mixing the supernatant with a stabilizer.

[0011] Compared with the prior art, the present application uses actinomycetes for inoculation and cultivation, and the actinomycetes can produce more bioactive substances and have obvious algae inhibiting effect. The addition of glutamic acid and blue algae in the culture medium can promote the actinomycetes to secrete more active substances in the fermentation process, because part of the bioactive substances produced by the actinomycetes are amino acid substances. The addition of blue algae can make the actinomycetes maintain the ability to remove algae during the fermentation process to secrete corresponding active substances. After fermentation, the supernatant is taken and mixed with a stabilizer, which can effectively inhibit the growth of miscellaneous bacteria to maintain the stability of the algae removal performance.

[0012] In some embodiments, the culture medium comprises soluble starch 10-35 g / L, sodium acetate 5-10 g / L, sodium nitrate 0.5-2.0 g / L, potassium nitrate 0.2-1.5 g / L, K2HPO4 0.1-1 g / L, NaH2PO4 0.1-1 g / L, MgSO4·7H2O 0.2-0.8 g / L, NaCl 0.2-0.8 g / L, FeSO4·7H2O 0.005-0.05 g / L, glutamic acid 0.2-2 g / L, and blue algae 5-10 g / L.

[0013] In some embodiments, the blue algae comprises at least one of Microcystis aeruginosa, Oscillatoria and Nostoc.

[0014] In some embodiments, the actinomycetes are Streptomyces microflavus.

[0015] In some embodiments, the culture conditions in step (1) comprise shaking culture in a culture box at 25-35°C for 48-96 h, and the shaking speed is 100-200 rpm.

[0016] In some embodiments, the culture conditions in step (2) comprise temperature 25-35°C, speed 100-200 rpm, tank pressure 0.05-0.1 MPa, dissolved oxygen 20-60%, pH 6.5-8.5, and culture time 48-96 h.

[0017] In some embodiments, the conditions of the culture in step (3) include temperature 25-35℃, rotation speed 100-200rpm, tank pressure 0.05-0.1MPa, dissolved oxygen 20-60%, pH 6.5-8.5, and culture time 48-96h.

[0018] In some embodiments, the rotation speed of the centrifugation is 2000-6000rpm.

[0019] In some embodiments, the stabilizer includes sodium ethyl p-hydroxybenzoate 0.5-2.5g / L, sodium acetate 10-50g / L, and an acid, and the acid adjusts the pH to 5.0-6.5.

[0020] Another aspect of the present application provides the use of the active substance of Actinopolyspora agarolytica in algae removal in water bodies. DETAILED DESCRIPTION

[0021] When the active substance of Actinopolyspora agarolytica is used for algae removal in water bodies, different dosages can be set according to the different periods of cyanobacterial outbreak, so as to improve the algae removal effect. For example, in the early stage of cyanobacterial outbreak, the dosage of the active substance of Actinopolyspora agarolytica is 0.01‰-0.05‰ of the volume fraction of the water body; in the middle stage of cyanobacterial outbreak, the dosage of the active substance of Actinopolyspora agarolytica is 0.01‰-0.05‰ of the volume fraction of the water body; in the late stage of cyanobacterial outbreak, the dosage of the active substance of Actinopolyspora agarolytica is 0.05‰-0.05‰ of the volume fraction of the water body.

[0022] The preparation method of the active substance of Actinopolyspora agarolytica includes the following steps:

[0023] (1) inoculate Actinopolyspora into a flask containing a culture medium, the culture medium including a carbohydrate, a salt, glutamic acid and cyanobacteria, and then place the flask in a culture box for shaking culture to obtain a first-stage seed liquid;

[0024] (2) inoculate the first-stage seed liquid into a first-stage seed tank at an inoculation amount of 10-20% of the volume fraction to obtain a second-stage seed liquid;

[0025] (3) inoculate the second-stage seed liquid into a fermentation tank at an inoculation amount of 10-20% of the volume fraction to obtain an Actinopolyspora fermentation liquid;

[0026] (4) centrifuge the Actinopolyspora fermentation liquid to obtain a supernatant and mix the supernatant with a stabilizer.

[0027] In step (1), the Actinopolyspora is Streptomyces microflavus, which has a high inhibitory effect on cyanobacteria.

[0028] As an example, the culture medium comprises soluble starch 10-35 g / L, sodium acetate 5-10 g / L, sodium nitrate 0.5-2.0 g / L, potassium nitrate 0.2-1.5 g / L, K2HPO4 0.1-1 g / L, NaH2PO4 0.1-1 g / L, MgSO4·7H2O 0.2-0.8 g / L, NaCl 0.2-0.8 g / L, FeSO4·7H2O 0.005-0.05 g / L, glutamic acid 0.2-2 g / L and cyanobacteria 5-10 g / L. The content of soluble starch can be specifically but not limited to 10 g / L, 13 g / L, 15 g / L, 17 g / L, 20 g / L, 23 g / L, 25 g / L, 27 g / L, 30 g / L, 33 g / L, 35 g / L. The content of sodium acetate can be specifically but not limited to 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L. The content of sodium nitrate can be specifically but not limited to 0.5 g / L, 0.7 g / L, 0.9 g / L, 1.1 g / L, 1.3 g / L, 1.5 g / L, 1.7 g / L, 1.9 g / L, 2.0 g / L. The content of potassium nitrate can be specifically but not limited to 0.2 g / L, 0.3 g / L, 0.5 g / L, 0.7 g / L, 0.9 g / L, 1.1 g / L, 1.3 g / L, 1.5 g / L. The content of K2HPO4 can be specifically but not limited to 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L. The content of NaH2PO4 can be specifically but not limited to 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L. The content of MgSO4·7H2O can be specifically but not limited to 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L. The content of NaCl can be specifically but not limited to 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L. The content of FeSO4·7H2O can be specifically but not limited to 0.005 g / L, 0.01 g / L, 0.015 g / L, 0.02 g / L, 0.025 g / L, 0.03 g / L, 0.035 g / L, 0.04 g / L, 0.045 g / L, 0.05 g / L. The content of glutamic acid can be specifically but not limited to 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, 2 g / L.The content of cyanobacteria can be, but is not limited to, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L.

[0029] As an example, the cyanobacteria include at least one of Microcystis aeruginosa, Oscillatoria and Nostoc.

[0030] The culture conditions in step (1) include shaking culture in an incubator at 25-35 °C for 48-96 h, and the shaking speed is 100-200 rpm. As an example, the culture temperature can be, but is not limited to, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C. The culture time can be, but is not limited to, 48 h, 50 h, 53 h, 57 h, 60 h, 64 h, 68 h, 72 h, 75 h, 78 h, 80 h, 82 h, 84 h, 86 h, 88 h, 90 h, 92 h, 94 h, 96 h. The speed can be, but is not limited to, 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm.

[0031] The culture conditions in step (2) include temperature 25-35 °C, speed 100-200 rpm, tank pressure 0.05-0.1 MPa, dissolved oxygen 20-60%, pH 6.5-8.5, and culture time 48-96 h. As an example, the culture temperature can be, but is not limited to, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C. The speed can be, but is not limited to, 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm. The tank pressure can be, but is not limited to, 0.05 MPa, 0.06 MPa, 0.07 MPa, 0.08 MPa, 0.09 MPa, 0.1 MPa. The dissolved oxygen can be, but is not limited to, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. The pH can be, but is not limited to, 6.5, 7.0, 7.5, 8.0, 8.5. The culture time can be, but is not limited to, 48 h, 50 h, 53 h, 57 h, 60 h, 64 h, 68 h, 72 h, 75 h, 78 h, 80 h, 82 h, 84 h, 86 h, 88 h, 90 h, 92 h, 94 h, 96 h.

[0032] The conditions for culturing in step (3) include temperature 25-35℃, rotation speed 100-200rpm, tank pressure 0.05-0.1MPa, dissolved oxygen 20-60%, pH 6.5-8.5, and culturing for 48-96h. As examples, the culturing temperature can be, but is not limited to, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃. The rotation speed can be, but is not limited to, 100rpm, 120rpm, 140rpm, 160rpm, 180rpm, 200rpm. The tank pressure can be, but is not limited to, 0.05MPa, 0.06MPa, 0.07MPa, 0.08MPa, 0.09MPa, 0.1MPa. The dissolved oxygen can be, but is not limited to, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. The pH can be, but is not limited to, 6.5, 7.0, 7.5, 8.0, 8.5. The culturing time can be, but is not limited to, 48h, 50h, 53h, 57h, 60h, 64h, 68h, 72h, 75h, 78h, 80h, 82h, 84h, 86h, 88h, 90h, 92h, 94h, 96h.

[0033] In step (4), the rotation speed for centrifugation is 2000-6000rpm, and as examples, the rotation speed for centrifugation can be, but is not limited to, 2000rpm, 2500rpm, 3000rpm, 3500rpm, 4000rpm, 4500rpm, 5000rpm, 5500rpm, 6000rpm.

[0034] The stabilizer includes sodium ethyl p-hydroxybenzoate 0.5-2.5g / L, sodium acetate 10-50g / L, and an acid. As examples, the content of sodium ethyl p-hydroxybenzoate can be, but is not limited to, 0.5g / L, 0.8g / L, 1.0g / L, 1.2g / L, 1.5g / L, 1.8g / L, 2.0g / L, 2.3g / L, 2.5g / L. The content of sodium acetate can be, but is not limited to, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L. The acid is at least one of hydrochloric acid, sulfuric acid, and acetic acid, and the pH is adjusted to 5.0-6.5 using the acid.

[0035] For better illustrating the purposes, technical solutions and beneficial effects of the present application, the present application will be further described below in combination with specific examples. It should be noted that the following described method is a further explanation of the present application and should not be regarded as a limitation of the present application. The raw materials involved in the examples and comparative examples of the present application can be all commercially available, except for special instructions, wherein the Microcystis aeruginosa and the Nostoc can be purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences. The Streptomyces microflavus can be purchased from the China General Microbiological Culture Collection Center.

[0036] Example 1

[0037] The present example is a preparation method of the active substance of the algicidal actinomycete, comprising the following steps:

[0038] (1) inoculate the Streptomyces microflavus into a shake flask containing a culture medium, the culture medium comprises soluble starch 10 g / L, sodium acetate 6 g / L, sodium nitrate 1.0 g / L, potassium nitrate 0.2 g / L, K2HPO4 0.5 g / L, NaH2PO4 0.1 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, glutamic acid 0.5 g / L and Microcystis aeruginosa 10 g / L, and then place it in a 30℃ incubator for shaking culture at a speed of 150 rpm for 48 h to obtain a primary seed liquid;

[0039] (2) inoculate the primary seed liquid into a primary seed tank for culture at a inoculation amount of 10% by volume to obtain a secondary seed liquid, the culture conditions of the primary seed tank are temperature 30℃, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture for 48 h;

[0040] (3) inoculate the secondary seed liquid into a fermentation tank for culture at a inoculation amount of 10% by volume to obtain an actinomycete fermentation liquid, the culture conditions of the fermentation tank are temperature 30℃, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture for 48 h;

[0041] (4) centrifuge the actinomycete fermentation liquid at a speed of 4000 rpm to obtain a supernatant and then mix a stabilizer, the stabilizer comprises p-hydroxybenzoic acid ethyl ester sodium 0.5 g / L, sodium acetate 10 g / L and hydrochloric acid, the concentration of the hydrochloric acid is 5% and the pH of the mixed solution is adjusted to 5.5.

[0042] Example 2

[0043] The present example is a preparation method of the active substance of the algicidal actinomycete, comprising the following steps:

[0044] (1) Streptomyces microflavus was inoculated into a flask containing a culture medium including soluble starch 30 g / L, sodium acetate 9 g / L, sodium nitrate 1.0 g / L, potassium nitrate 1.0 g / L, K2HPO4 0.8 g / L, NaH2PO4 0.5 g / L, MgSO4·7H2O 0.6 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.03 g / L, glutamic acid 1.0 g / L and Microcystis aeruginosa 5 g / L, and then placed in a 30℃ incubator for oscillation culture at a rotation speed of 200 rpm for 48 h to obtain a primary seed liquid;

[0045] (2) The primary seed liquid was inoculated into a primary seed tank in a volume fraction of 10% for culture to obtain a secondary seed liquid, and the culture conditions of the primary seed tank were temperature 25℃, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture time 80 h;

[0046] (3) The secondary seed liquid was inoculated into a fermentation tank in a volume fraction of 15% for culture to obtain an actinomycete fermentation liquid, and the culture conditions of the fermentation tank were temperature 35℃, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 50%, pH 7.0, and culture time 48 h;

[0047] (4) The actinomycete fermentation liquid was centrifuged at a rotation speed of 5500 rpm to obtain a supernatant, and a stabilizer was added for mixing, and the stabilizer included sodium ethyl p-hydroxybenzoate 2.0 g / L, sodium acetate 5 g / L and hydrochloric acid, the concentration of the hydrochloric acid was 5%, and the pH of the mixed solution was adjusted to 5.5.

[0048] Example 3

[0049] The present embodiment is a preparation method of an active substance of Microcystis aeruginosa, which comprises the following steps:

[0050] (1) Streptomyces microflavus was inoculated into a flask containing a culture medium including soluble starch 20 g / L, sodium acetate 6 g / L, sodium nitrate 2.0 g / L, potassium nitrate 0.2 g / L, K2HPO4 0.5 g / L, NaH2PO4 0.1 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.8 g / L, FeSO4·7H2O 0.01 g / L, glutamic acid 1.0 g / L and Nostoc 10 g / L, and then placed in a 35℃ incubator for oscillation culture at a rotation speed of 200 rpm for 85 h to obtain a primary seed liquid;

[0051] (2) The primary seed liquid was inoculated into a primary seed tank in a volume fraction of 15% for culture to obtain a secondary seed liquid, and the culture conditions of the primary seed tank were temperature 25℃, rotation speed 200 rpm, tank pressure 0.1 MPa, dissolved oxygen 50%, pH 8.0, and culture time 48 h;

[0052] (3) the secondary seed liquid is inoculated into the fermentation tank at a volume fraction of 10% to obtain an actinomycete fermentation liquid, and the culture conditions of the fermentation tank are temperature 30°C, rotation speed 200 rpm, tank pressure 0.1 MPa, dissolved oxygen 45%, pH 8.0, and culture time 60 h;

[0053] (4) the actinomycete fermentation liquid is centrifuged at a rotation speed of 5500 rpm to obtain a supernatant, and a stabilizer is added for mixing, the stabilizer including sodium ethyl p-hydroxybenzoate 0.5 g / L, sodium acetate 10 g / L, and acetic acid, the concentration of the acetic acid being 20%, and the pH of the mixed solution being adjusted to 5.1.

[0054] Comparative Example 1

[0055] The present example is a preparation method of an active substance of an algicidal actinomycete, comprising the steps of:

[0056] (1) the Streptomyces microflavus is inoculated into a flask containing a culture medium, the culture medium including soluble starch 10 g / L, sodium acetate 6 g / L, sodium nitrate 1.0 g / L, potassium nitrate 0.2 g / L, K2HPO4 0.5 g / L, NaH2PO4 0.1 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, and Microcystis aeruginosa 10 g / L, and then the flask is placed in a 30°C incubator and shaken at a rotation speed of 150 rpm for 48 h to obtain a primary seed liquid;

[0057] (2) the primary seed liquid is inoculated into a primary seed tank at a volume fraction of 10% to obtain a secondary seed liquid, and the culture conditions of the primary seed tank are temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture time 48 h;

[0058] (3) the secondary seed liquid is inoculated into the fermentation tank at a volume fraction of 10% to obtain an actinomycete fermentation liquid, and the culture conditions of the fermentation tank are temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture time 48 h;

[0059] (4) the actinomycete fermentation liquid is centrifuged at a rotation speed of 4000 rpm to obtain a supernatant, and a stabilizer is added for mixing, the stabilizer including sodium ethyl p-hydroxybenzoate 0.5 g / L, sodium acetate 10 g / L, and hydrochloric acid, the concentration of the hydrochloric acid being 5%, and the pH of the mixed solution being adjusted to 5.5.

[0060] Comparative Example 2

[0061] The present example is a preparation method of an active substance of an algicidal actinomycete, comprising the steps of:

[0062] (1) Streptomyces microflavus was inoculated into a flask containing a culture medium including soluble starch 10 g / L, sodium acetate 6 g / L, sodium nitrate 1.0 g / L, potassium nitrate 0.2 g / L, K2HPO4 0.5 g / L, NaH2PO4 0.1 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L and glutamic acid 0.5 g / L, and then placed in a 30°C incubator for shaking culture at a rotation speed of 150 rpm for 48 h to obtain a first-stage seed solution;

[0063] (2) The first-stage seed solution was inoculated into a first-stage seed tank for culture at a volume fraction of 10% to obtain a second-stage seed solution, and the culture conditions of the first-stage seed tank were temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0 and culture time 48 h;

[0064] (3) The second-stage seed solution was inoculated into a fermentation tank for culture at a volume fraction of 10% to obtain an actinomycete fermentation liquor, and the culture conditions of the fermentation tank were temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0 and culture time 48 h;

[0065] (4) The actinomycete fermentation liquor was centrifuged at a rotation speed of 4000 rpm to obtain a supernatant, and a stabilizer was added for mixing, and the stabilizer included p-hydroxybenzoic acid ethyl ester sodium 0.5 g / L, sodium acetate 10 g / L and hydrochloric acid, the concentration of the hydrochloric acid was 5%, and the pH of the mixed solution was adjusted to 5.5.

[0066] Comparative Example 3

[0067] The present example is a preparation method of an active substance of algicidal actinomycete, which comprises the following steps:

[0068] (1) Streptomyces microflavus was inoculated into a flask containing a culture medium including soluble starch 10 g / L, sodium acetate 6 g / L, sodium nitrate 1.0 g / L, potassium nitrate 0.2 g / L, K2HPO4 0.5 g / L, NaH2PO4 0.1 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.5 g / L and FeSO4·7H2O 0.01 g / L, and then placed in a 30°C incubator for shaking culture at a rotation speed of 150 rpm for 48 h to obtain a first-stage seed solution;

[0069] (2) The first-stage seed solution was inoculated into a first-stage seed tank for culture at a volume fraction of 10% to obtain a second-stage seed solution, and the culture conditions of the first-stage seed tank were temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0 and culture time 48 h;

[0070] (3) the secondary seed liquid is inoculated into the fermenter at a volume fraction of 10% to obtain an actinomycete fermentation liquid, and the culture conditions of the fermenter are temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture time 48 h;

[0071] (4) the actinomycete fermentation liquid is centrifuged at a rotation speed of 4000 rpm to obtain a supernatant, and a stabilizer is added for mixing, wherein the stabilizer comprises sodium ethyl p-hydroxybenzoate 0.5 g / L, sodium acetate 10 g / L, and hydrochloric acid, the concentration of the hydrochloric acid is 5%, and the pH of the mixed solution is adjusted to 5.5.

[0072] Comparative Example 4

[0073] The present embodiment is a preparation method of an active substance of an algicidal actinomycete, comprising the following steps:

[0074] (1) the Streptomyces microflavus is inoculated into a flask containing a culture medium, wherein the culture medium comprises soluble starch 10 g / L, sodium acetate 6 g / L, sodium nitrate 1.0 g / L, potassium nitrate 0.2 g / L, K2HPO4 0.5 g / L, NaH2PO4 0.1 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, glutamic acid 0.5 g / L, and Microcystis aeruginosa 10 g / L, and then the flask is placed in a 30°C incubator for oscillation culture at a rotation speed of 150 rpm for 48 h to obtain a primary seed liquid;

[0075] (2) the primary seed liquid is inoculated into a primary seed tank at a volume fraction of 10% to obtain a secondary seed liquid, and the culture conditions of the primary seed tank are temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture time 48 h;

[0076] (3) the secondary seed liquid is inoculated into the fermenter at a volume fraction of 10% to obtain an actinomycete fermentation liquid, and the culture conditions of the fermenter are temperature 30°C, rotation speed 150 rpm, tank pressure 0.08 MPa, dissolved oxygen 30%, pH 7.0, and culture time 48 h;

[0077] (4) the actinomycete fermentation liquid is centrifuged at a rotation speed of 4000 rpm to obtain a supernatant.

[0078] The algicidal actinomycete active substance prepared in Examples 1-3 and Comparative Examples 1-4 was added to water bodies at different periods of the outbreak of blue algae, with an early addition amount of 0.003% by volume fraction of the water body, a middle addition amount of 0.03% by volume fraction of the water body, and a late addition amount of 0.02% by volume fraction of the water body. After 3 days of reaction, the blue algae removal rate was as shown in Table 1. The contamination and algae removal rate of the algicidal actinomycete active substance prepared in Examples 1-3 and Comparative Examples 1-4 at different storage times were tested, and the results are shown in Table 2.

[0079] Table 1 Blue algae removal rate in water bodies at different periods of the outbreak of blue algae

[0080]

[0081] Table 2 Contamination and algae removal rate at different storage times

[0082]

[0083] From the results in Table 1, it can be seen that the algicidal actinomycete active substance prepared by the preparation method of the algicidal actinomycete active substance of the present application has a high algae removal rate at the early, middle, and late periods of the outbreak of blue algae. The algae removal rates of Comparative Examples 1-3, which lack glutamic acid and / or blue algae, are greatly affected.

[0084] From the results in Table 2, it can be seen that the algicidal actinomycete active substance prepared by the preparation method of the algicidal actinomycete active substance of the present application, after the addition of a stabilizer, can effectively inhibit the growth of miscellaneous bacteria, with a contamination rate of less than 1% after 2 years (24M) of storage. It also has good algae removal stability, with an algae removal rate of more than 95% after 2 years of storage.

[0085] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, it is not limited to the embodiments listed in the examples. Those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for the preparation of an actives substance of Actinoplanes alginaticus, characterized in that, The method comprises the following steps: (1) inoculating actinomycetes into a flask containing a culture medium, the culture medium comprising carbohydrates, salts, glutamic acid and cyanobacteria, and placing the flask into a culture box for shaking culture to obtain a first seed liquid, the actinomycetes being Streptomyces microflavus, the cyanobacteria being at least one of Microcystis aeruginosa and Nostoc, and the culture medium comprising soluble starch 10-35 g / L, sodium acetate 5-10 g / L, sodium nitrate 0.5-2.0 g / L, potassium nitrate 0.2-1.5 g / L, K2HPO4 0.1-1 g / L, NaH2PO4 0.1-1 g / L, MgSO4·7H2O 0.2-0.8 g / L, NaCl 0.2-0.8 g / L, FeSO4·7H2O 0.005-0.05 g / L, glutamic acid 0.2-2 g / L and cyanobacteria 5-10 g / L; (2) inoculating the first seed liquid into a first seed tank for culture to obtain a second seed liquid, the inoculation amount being 10-20% by volume fraction; (3) inoculating the second seed liquid into a fermentation tank for culture to obtain an actinomycete fermentation liquid, the inoculation amount being 10-20% by volume fraction; (4) centrifuging the actinomycete fermentation liquid to obtain a supernatant, and mixing the supernatant with a stabilizer, the stabilizer comprising sodium ethyl p-hydroxybenzoate 0.5-2.5 g / L, sodium acetate 10-50 g / L and an acid, and the acid being used to adjust the pH to 5.0-6.

5.

2. The method of preparing an actinomycete active substance according to claim 1, characterized in that, The culture conditions in step (1) include shaking culture at 25-35°C for 48-96 h in a culture box, and the shaking speed is 100-200 rpm.

3. The method for preparing the active substance of alginolytic actinomycetes according to claim 1, characterized in that, The culture conditions in step (2) include a temperature of 25-35°C, a speed of 100-200 rpm, a tank pressure of 0.05-0.1 MPa, dissolved oxygen of 20-60%, a pH of 6.5-8.5 and a culture time of 48-96 h.

4. The method for preparing the active substance of alginolytic actinomycetes according to claim 1, characterized in that, The culture conditions in step (3) include a temperature of 25-35°C, a speed of 100-200 rpm, a tank pressure of 0.05-0.1 MPa, dissolved oxygen of 20-60%, a pH of 6.5-8.5 and a culture time of 48-96 h.

5. The method for preparing the active substance of alginolytic actinomycetes according to claim 1, characterized in that, The centrifugation speed is 2000-6000 rpm.

6. The use of the algicidal actinomycete active substance prepared by the method according to any one of claims 1-5 in removing algae in a water body.

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