CN114477469B - Preparation method of compound microbial preparation for aquaculture - Google Patents

Preparation method of compound microbial preparation for aquaculture Download PDF

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CN114477469B
CN114477469B CN202210079455.1A CN202210079455A CN114477469B CN 114477469 B CN114477469 B CN 114477469B CN 202210079455 A CN202210079455 A CN 202210079455A CN 114477469 B CN114477469 B CN 114477469B
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preparing
culture
preparation
microbial preparation
photosynthetic bacteria
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CN114477469A (en
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潘逢文
张松柏
杨洪战
徐新煌
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Hunan Kunyuan Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/347—Use of yeasts or fungi
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/06—Controlling or monitoring parameters in water treatment pH
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/14—NH3-N
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/22—O2
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2305/00—Use of specific compounds during water treatment
    • C02F2305/06—Nutrients for stimulating the growth of microorganisms

Abstract

The invention discloses a preparation method of a composite microbial preparation for aquaculture. In addition, the purple non-sulfur photosynthetic bacteria are separated and purified after a purple non-sulfur photosynthetic bacteria enrichment culture solution is obtained by adopting a physical oxygen-removing anaerobic culture method, so that purple non-sulfur photosynthetic bacteria with better effect can be obtained, the synergistic effect with other bacteria powder is stronger after the composite microbial preparation is prepared, and a good action effect is provided for purification treatment of water and healthy growth of aquatic products.

Description

Preparation method of compound microbial preparation for aquaculture
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to a preparation method of a compound microbial preparation for aquaculture.
Background
The compound microbial preparation, also called 'compound microbial ecological preparation', is a microbial preparation for enhancing the growth, the immunity and the like of aquaculture organisms, and two or more microbes are cultured together according to a proper proportion, so that the advantages of the groups are fully exerted, and the best application effect is obtained. The microorganism used usually has multiple effective microorganism groups such as photosynthetic bacteria group, actinomycetes group, yeast group and lactic acid bacteria group. The compound microbial preparation can decompose organic matters in the culture water body, so that ammonia nitrogen, nitroso nitrogen and the like are converted into harmless substances, and the compound microbial preparation plays an important role in purifying the water body and establishing a good ecological balance environment; the flora can generate a plurality of vitamins, amino acids and physiological active substances in the metabolism process, thereby promoting the nutrient absorption and growth of animals and plants, improving the immunity and disease resistance and enhancing the culture effect.
When various strains are cultured, glucose, methanol or sucrose is usually adopted as a carbon source for culturing the strains, so that the cost is overhigh, and the photosynthetic bacteria commonly adopted by the conventional compound microbial preparation are common photosynthetic bacteria, so that the effect is deficient and limited when the photosynthetic bacteria and other microorganisms are used for treating water quality in a synergistic manner.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the preparation method of the compound microbial preparation for aquaculture, which avoids the increase of the preparation cost caused by the overhigh cost of the adopted carbon source when each bacterial strain is cultured, and improves the synergy of the microbes in the water quality treatment.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a compound microbial preparation for aquaculture comprises the following steps of preparing raw materials of the compound microbial preparation, wherein the raw materials comprise purple non-sulfur photosynthetic bacteria, saccharomyces cerevisiae, bacillus subtilis, bdellovibrio bacteriovorus, compound EM bacteria and a carbon source:
step one, preparing a carbon source culture medium: adding an alkali solution into crop plant fibers for soaking pretreatment, adding cellulose decomposing bacteria into the pretreated cellulose to obtain a carbon source mixed solution, and preparing the carbon source mixed solution into a culture medium;
step two, screening and purifying the purple non-sulfur photosynthetic bacteria: obtaining an enrichment culture solution of the purple non-sulfur photosynthetic bacteria by adopting a physical oxygen-removing anaerobic culture method, and then separating and purifying the purple non-sulfur photosynthetic bacteria;
step three, liquid fermentation of strains: activating and culturing each strain under a proper culture condition, and then respectively inoculating the strain into the culture medium prepared in the step one to perform amplification culture to a logarithmic phase;
step four, mixing the strain liquid: modulation of OD 600 =1.5, then mixing the culture solutions of the respective strains for standby;
step five, vacuum freeze drying of strains: centrifuging the mixed bacteria liquid obtained in the fourth step, removing supernatant, adding a freeze-drying protective agent, pre-freezing for 1-5 hours at-76 ℃, and then transferring the frozen sample into a vacuum freeze-drying machine for freeze-drying to obtain bacteria powder;
step six, packaging the fungus powder: wrapping the fungus powder with water soluble material, packaging, and storing in a cool, dry, ventilated and clean warehouse.
In a preferred technical scheme of the invention, in the first step, the plant fiber is one or more of reed fiber, straw fiber and corncob fiber.
As a preferable technical scheme of the invention, in the step one, the alkali solution is a NaOH solution with the mass fraction of 1-5%.
In the second step, a single colony in the test tube is purified by a double-layer plate method, and the purified strain is preserved.
As a preferred embodiment of the present invention, in step four, if OD 600 >1.5 dilution to 0D with culture broth 600 =1.5; if OD 600 <1.5, removing part of the supernatant after centrifugation, and adjusting to 0D 600 ＝1.5。
As a preferred technical scheme of the invention, in the fourth step, each strain liquid is mixed according to 20-30 parts of purple non-sulfur photosynthetic bacteria, 5-10 parts of saccharomyces cerevisiae, 1-5 parts of bacillus subtilis, 1-5 parts of bdellovibrio and 5-10 parts of composite EM bacteria for preparation and mixing.
In the fifth step, the temperature of the centrifugal equipment is regulated to 1-5 ℃, the rotating speed is 3000-6000r/min, and the centrifugal time is 10-15min.
As a preferred technical scheme of the invention, in the sixth step, the temperature of the storage environment of the bacterial powder is above 35 ℃, and the bacterial powder is packaged in vacuum or by filling nitrogen.
Compared with the prior art, the invention can achieve the following beneficial effects:
in addition, after the purple non-sulfur photosynthetic bacteria enrichment culture solution is obtained by adopting a physical oxygen-removing anaerobic culture method, the purple non-sulfur photosynthetic bacteria are separated and purified, so that purple non-sulfur photosynthetic bacteria with better effect can be obtained, after the composite microbial preparation is prepared, the synergistic effect of the purple non-sulfur photosynthetic bacteria and other bacteria powder is stronger, and a good effect is provided for purification treatment of water bodies and healthy growth of aquatic products.
Detailed Description
Technical means for implementing the present invention; authoring features; the purpose served by the disclosure is to provide a thorough understanding of the invention, and is to be construed as being a limitation on the scope of the invention as defined by the appended claims. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, materials used in the following examples; reagents and the like are commercially available unless otherwise specified.
Example 1
Step one, preparing a carbon source culture medium: taking a certain amount of corncob fiber, placing the corncob fiber into a container, adding a NaOH solution with the mass fraction of 2% for soaking pretreatment, placing the pretreated cellulose into another container, adding cellulose decomposing bacteria simultaneously to obtain a carbon source mixed solution, and preparing the carbon source mixed solution into a culture medium;
step two, screening and purifying purple non-sulfur photosynthetic bacteria: after a purple non-sulfur photosynthetic bacteria enrichment culture solution is obtained by adopting a physical oxygen-removing anaerobic culture method, the purple non-sulfur photosynthetic bacteria are separated and purified, and the method comprises the following steps:
s1, taking 1mL of purple non-sulfur photosynthetic bacteria enrichment culture solution for gradient dilution, taking a plurality of purple non-sulfur photosynthetic bacteria enrichment culture solutions with different dilution degrees, respectively inoculating the culture solutions on a semi-solid separation culture medium, fully and uniformly mixing, sealing with sterilized liquid paraffin, plugging a rubber plug, sealing with a sealing film, and culturing under the conditions of 2000-3000 lux (Lx) illumination intensity and 30 ℃ for 7-10 days until deep red single colonies appear in a test tube;
s2, selecting a single bacterial colony in a test tube, purifying by adopting a double-layer plate method, firstly scribing on a separation culture medium plate, then pouring a layer of molten culture medium on the plate to form an anaerobic environment, sealing by using a sealing film, culturing for 7-10 days under the conditions of 2000-3000 lux (Lx) illumination intensity and 30 ℃, repeatedly selecting the single bacterial colony, performing microscopic examination, and storing the purified bacterial strain.
Step three, liquid fermentation of strains: and (3) performing activated culture on each strain under a proper culture condition, and then respectively inoculating the strains into the culture medium prepared in the step one to perform amplification culture to a logarithmic phase.
Step four, mixing strain liquid: adjusting OD 600 =1.5, if OD 600 >1.5 dilution to 0D with culture broth 600 =1.5; if OD 600 <1.5, removing part of the supernatant after centrifugation, and adjusting to 0D 600 =1.5; then mixing culture solutions (25 parts of purple non-sulfur photosynthetic bacteria, 8 parts of saccharomyces cerevisiae, 3 parts of bacillus subtilis, 2 parts of bdellovibrio and 5 parts of composite EM bacteria) of the strains purified and cultured in the third step for later use;
step five, vacuum freeze drying of the strains: centrifuging the mixed bacteria liquid obtained in the fourth step by using a centrifugal device (the centrifugation temperature is 5 ℃, the rotation speed is 5000r/min, and the centrifugation time is 15 min), removing the supernatant, adding trehalose serving as a freeze-drying protective agent, pre-freezing for 2h at-76 ℃, transferring the frozen sample into a vacuum freeze-drying machine, and freeze-drying to obtain bacteria powder;
step six, packaging the fungus powder: wrapping the fungus powder with water-soluble material, packaging, and storing in a cool, dry, ventilated and clean warehouse.
And (3) testing the purification effect:
the method adopts water for a certain prawn farm in Liuyang city of Hunan province as an experimental water source, wherein: the dissolved oxygen is 3.82mg/L, the ammonia nitrogen is 0.25mg/L, and the pH value is 7.05. In the experiment, 4 water buckets are used, the volume is 15L, the bucket No. 1 is a blank group, the bucket No. 2 is a saccharomycete group, the bucket No. 3 is a lactobacillus group, the bucket No. 4 is a composite microbial preparation group, the strain input amount of each bucket is 15mL/L, the temperature is controlled to be about 25 ℃, the dissolved oxygen, ammonia nitrogen and pH value of each bucket are tested when the buckets are cultured for 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days and 16 days respectively, samples are taken from the surface layer, the middle layer and the bottom layer of each bucket during sampling, and the average value is taken as the measurement result, and the result is shown in the following table 1:
TABLE 1
Figure BDA0003485323410000061
Figure BDA0003485323410000071
As can be seen from the above table 1, the composite microbial preparation prepared by the invention provides good effects for water purification treatment and healthy growth of aquatic products.
The foregoing shows and describes the general principles of the present invention; the main features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (6)

1. A preparation method of a compound microbial preparation for aquaculture is characterized by comprising the following steps: the raw materials for preparing the compound microbial preparation comprise 20-30 parts of purple non-sulfur photosynthetic bacteria, 5-10 parts of saccharomyces cerevisiae, 1-5 parts of bacillus subtilis, 1-5 parts of bdellovibrio, 5-10 parts of compound EM bacteria and a carbon source; wherein the carbon source is crop plant fiber selected from one or more of reed fiber, straw fiber and corn cob fiber; the preparation method comprises the following steps:
step one, preparing a carbon source culture medium: adding an alkali solution into crop plant fibers for soaking pretreatment, adding cellulose decomposing bacteria into pretreated cellulose to obtain a carbon source mixed solution, and preparing the carbon source mixed solution into a culture medium;
step two, screening and purifying purple non-sulfur photosynthetic bacteria: obtaining an enrichment culture solution of purple non-sulfur photosynthetic bacteria by adopting a physical oxygen-removing anaerobic culture method, and then separating and purifying the purple non-sulfur photosynthetic bacteria;
step three, liquid fermentation of strains: activating and culturing each strain under a proper culture condition, and then respectively inoculating the strain into the culture medium prepared in the step one to perform amplification culture to a logarithmic phase;
step four, mixing strain liquid: modulation of OD 600 =1.5, then mixing the culture solutions of the respective strains for standby;
step five, vacuum freeze drying of the strains: centrifuging the mixed bacterial liquid obtained in the step four, removing supernatant, adding a freeze-drying protective agent, pre-freezing for 1-5 hours at-76 ℃, and then transferring the frozen sample into a vacuum freeze-drying machine for freeze drying to prepare bacterial powder;
step six, packaging the fungus powder: wrapping the fungus powder with water-soluble material, packaging, and storing in a cool, dry, ventilated and clean warehouse.
2. The method for preparing a complex microbial preparation for aquaculture according to claim 1, wherein the method comprises the following steps: the alkali solution is NaOH solution with the mass fraction of 1-5%.
3. The method for preparing a complex microbial preparation for aquaculture according to claim 1, wherein the method comprises the following steps: in the second step, the single colony in the test tube is purified by a double-layer plate method, and the purified strain is preserved.
4. The method for preparing a complex microbial preparation for aquaculture according to claim 1, wherein the method comprises the following steps: in step four, if OD 600 >1.5 dilution to 0D with culture broth 600 =1.5; if OD 600 <1.5, removing part of the supernatant after centrifugation, and adjusting to 0D 600 =1.5。
5. The method for preparing a complex microbial preparation for aquaculture according to claim 1, wherein the method comprises the following steps: in the fifth step, the temperature of the centrifugal equipment is adjusted to be 1-5 ℃, the rotating speed is 3000-6000r/min, and the centrifugal time is 10min.
6. The method for preparing a complex microbial preparation for aquaculture according to claim 1, wherein the method comprises the following steps: and step six, the temperature of the storage environment of the bacterial powder is above 35 ℃, and the bacterial powder is packaged in vacuum or filled with nitrogen.
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Inventor after: Pan Fengwen

Inventor after: Yang Hongzhan

Inventor after: Xu Xinhuang

Inventor before: Pan Fengwen

Inventor before: Zhang Songbai

Inventor before: Yang Hongzhan

Inventor before: Xu Xinhuang