CN113429227A - Application of bacillus subtilis in biological fermentation - Google Patents

Application of bacillus subtilis in biological fermentation Download PDF

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Publication number
CN113429227A
CN113429227A CN202110971100.9A CN202110971100A CN113429227A CN 113429227 A CN113429227 A CN 113429227A CN 202110971100 A CN202110971100 A CN 202110971100A CN 113429227 A CN113429227 A CN 113429227A
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China
Prior art keywords
parts
enteromorpha
bacillus subtilis
fermentation
biological
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CN202110971100.9A
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Chinese (zh)
Inventor
徐琳琳
郑秋玲
李成强
郭琳
唐美玲
王建萍
肖慧琳
徐维华
沙玉芬
刘万好
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Yantai Shangshui Pharmaceutical Technology Co ltd
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Yantai Shangshui Pharmaceutical Technology Co ltd
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Priority to CN202110971100.9A priority Critical patent/CN113429227A/en
Publication of CN113429227A publication Critical patent/CN113429227A/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
    • C05D9/00—Other inorganic fertilisers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention relates to the field of microbial fermentation engineering, and discloses an application of bacillus subtilis in biological fermentation.

Description

Application of bacillus subtilis in biological fermentation
Technical Field
The invention relates to the field of microbial fermentation engineering, in particular to application of bacillus subtilis in biological fermentation.
Background
With the gradual popularization of chemical fertilizers in agricultural applications, although beneficial effects such as yield increase and income increase are brought, a plurality of environmental problems such as soil hardening, soil acidification, soil fertility reduction and the like are brought, heavy metals and hormones contained in the chemical fertilizers also pose great threats to human health, and therefore the use of the chemical fertilizers is increasingly limited. The organic fertilizer is an environment-friendly fertilizer, can improve the crop yield and the crop quality, and can avoid the soil hardening and acidification by applying the organic fertilizer. Therefore, the demand of organic fertilizer is increasing and the production scale of organic fertilizer is also increasing. However, how to improve the fertility of the organic fertilizer, particularly, how to improve the fertility and simultaneously inhibit the occurrence of plant diseases and insect pests is a technical problem to be solved in the field.
The prior granted patent CN111732483B of the applicant of the invention discloses a fermentation method of an enteromorpha biological fertilizer; the method comprises the following steps: hydrolyzing polysaccharide, inoculating and fermenting and preparing biological fertilizer; according to the invention, the supported heteropoly acid hydrolysis auxiliary agent is adopted to dissolve enteromorpha polysaccharide on the cell surface of enteromorpha under the condition of the existence of hydrogen peroxide and inorganic salt, and the substances such as coated alga cellulose and protein are dissociated, so that the nutrient elements in the enteromorpha are effectively released, and the growth and the quality improvement of crops can be effectively promoted; the authorized invention takes the large green tide algae enteromorpha solid with wide sources and huge biological resource amount as the main raw material, adopts the fermentation method to prepare the biological fertilizer, and has the characteristics of high product conversion rate and suitability for large-scale preparation; the fermentation method has the characteristics of good biological safety, no potential harm to the environment, high content of active ingredients of the product, rapidness, high efficiency and specificity. However, the microorganisms adopted in the issued patents are bacteria powder, fungi powder and actinomycetes powder which are prepared according to the mass ratio of 0.8-1.5:0.6-1.1:1, and the disadvantages of high cost, long fermentation time and the like exist. In addition, although the enteromorpha biological fertilizer produced by fermentation has a higher promotion effect on the average length of the main roots and the average length of the tendrils of the loofah in the farmland, the enteromorpha biological fertilizer still has a space for further improvement on the yield increase of the loofah and the unit weight increase of the loofah.
The bacillus is used as the dominant biological population of soil and plant micro-ecological system, and has high stress resistance and antibacterial and disease-preventing effects. Many isolated strains with excellent properties have been successfully applied to the field of biological control of plant diseases. The bacillus subtilis competes with pathogenic bacteria for nutrients by colonizing to plant rhizosphere, body surface or body, and secretes antibacterial substances to inhibit the growth of the pathogenic bacteria; and simultaneously, a defense system of the plant is induced to resist the invasion of pathogenic bacteria, thereby achieving the purpose of biological control.
The prior patent application CN111733119A of the applicant of the invention discloses a high-density fermentation method of bacillus subtilis, which comprises the following specific steps: and (2) carrying out shake flask culture on the bacillus subtilis, inoculating the bacillus subtilis to a primary seed tank, carrying out fermentation culture for 18 hours under the conditions of temperature of 30-35 ℃, rotation speed of 160-200 rpm/min and pH of 6.7-7.9, inoculating the bacillus subtilis to a secondary seed tank as a secondary culture seed solution, carrying out fermentation culture for 36 hours under the conditions of temperature of 30-35 ℃, rotation speed of 160-200 rpm/min and pH of 6.7-7.9, inoculating the bacillus subtilis to a fermentation tank as a fermentation seed solution, carrying out fermentation culture for 48 hours under the conditions of temperature of 30-35 ℃, rotation speed of 160-200 rpm/min and pH of 6.7-7.9, and obtaining the bacillus subtilis high-density fermentation broth.
The inventor discovers that the fertility of the enteromorpha biological fertilizer can be further improved by taking the high-density bacillus subtilis as the fermentation microorganism of the enteromorpha in the later research process, and particularly, the method is favorable for improving the yield of the towel gourd and simultaneously improving the unit weight of the towel gourd.
Disclosure of Invention
The invention aims to solve the defects, provides the application of the bacillus subtilis in biological fermentation, and is beneficial to improving the fertility of the enteromorpha biological fertilizer by using the bacillus subtilis. The invention adopts the following technical scheme:
the invention relates to an application of bacillus subtilis in biological fermentation, wherein the bacillus subtilis takes enteromorpha as a raw material to prepare enteromorpha biological fertilizer by fermentation, and the application comprises the following steps:
step one, preprocessing the enteromorpha by the following steps: hydrolyzing polysaccharide, selecting 100-200 parts of enteromorpha, primarily cutting the enteromorpha, putting the enteromorpha into a hydrolysis kettle, adding 20-50 parts of water, 0.1-0.5 part of inorganic salt, 2-8 parts of hydrogen peroxide and 0.1-0.6 part of supported heteropolyacid hydrolysis auxiliary agent, uniformly mixing and stirring, reacting at 50-80 ℃ for 2-6 hours, and cooling to room temperature after the reaction is finished; the supported heteropolyacid hydrolysis auxiliary agent adopts 11-bromoundectyl trimethoxysilane to carry out surface treatment on the titanium silicalite molecular sieve; the preparation method of the supported heteropolyacid hydrolysis auxiliary agent comprises the following steps: according to the mass parts, 100-150 parts of titanium silicalite molecular sieve is soaked in 200-400 parts of 3.5-7% hydrochloric acid solution, the temperature is controlled to be 40-60 ℃ for treatment for 10-15h, the titanium silicalite molecular sieve is dispersed into 600-1000 parts of deionized water after washing, 15-20 parts of sodium tungstate dihydrate and 1-4 parts of 11-bromoundectyl trimethoxysilane are added, and the mixture is stirred and mixed for 20-30 min; adjusting the pH value of the solution to 6-8, continuously stirring for 60-120min, transferring the solution into a high-pressure reaction kettle, reacting for 5-10h at 120-150 ℃, cooling to room temperature after the reaction is finished, adjusting the pH value of the system to 2-3 by using 0.1-0.5mol/L phosphoric acid, stirring for 3-7h, filtering, and drying to obtain a supported heteropoly acid hydrolysis auxiliary agent;
step two, biological fermentation: inoculating and fermenting, namely adding 200 parts of 100-one of water, 2-6 parts of nitrogen, phosphorus and potassium fertilizers, 10-15 parts of bean pulp, 6-12 parts of diatomite, 0.5-3 parts of humic acid, 0.1-0.5 part of potassium fulvate and 5-18 parts of stone straw into enteromorpha cooled to room temperature, uniformly mixing, inoculating 0.8-3% of high-density bacillus subtilis, controlling the fermentation temperature to be 30-35 ℃, and fermenting for 4-6 days; the high-density Bacillus subtilis has viable count of 1.0-2.0 × 1010cfu/mL of Bacillus subtilis;
and step three, performing solid-liquid separation on the obtained fermentation product to obtain a filtrate part, namely the enteromorpha biological fertilizer.
The invention also relates to the enteromorpha biological fertilizer prepared according to the application.
The invention also relates to application of the enteromorpha biological fertilizer in improving the yield of the towel gourd and/or the single weight of the towel gourd.
Has the advantages that:
according to the invention, the enteromorpha biological fertilizer is prepared by fermenting the high-density bacillus subtilis, so that the fertility of the enteromorpha biological fertilizer is improved, the produced enteromorpha biological fertilizer can effectively promote the growth of towel gourds, is beneficial to improving the towel gourd yield and the towel gourd single weight, and has important economic value.
Detailed Description
In order to further understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1:
(1) with reference to the example disclosed in CN111733119A, the viable cell count obtained after 48 hours of culture was 1.7X 1010cfu/mL, and the sporulation rate is 90 percent.
(2) Hydrolyzing polysaccharide, namely selecting 100g of enteromorpha, primarily cutting up the enteromorpha, putting the enteromorpha into a hydrolysis kettle, adding 20g of water, 0.1g of inorganic salt, 2g of hydrogen peroxide and 0.1g of supported heteropoly acid hydrolysis auxiliary agent, uniformly mixing and stirring, reacting at 50 ℃ for 2 hours, and cooling to room temperature after the reaction is finished;
(3) inoculating and fermenting, namely adding 100g of water, 2g of nitrogen-phosphorus-potassium fertilizer, 10g of soybean meal, 6g of diatomite, 0.5g of humic acid, 0.1g of potassium fulvate and 5g of stone straw into enteromorpha cooled to room temperature, uniformly mixing, inoculating 0.8% volume of high-density bacillus subtilis, controlling the fermentation temperature to be 32 ℃, and fermenting for 5 days;
(4) and (3) preparing the biological fertilizer, namely performing solid-liquid separation on the obtained fermentation product to obtain a filtrate part, namely the enteromorpha biological fertilizer.
The preparation method of the supported heteropolyacid hydrolysis auxiliary agent comprises the following steps:
soaking 100g of titanium silicalite molecular sieve in 200g of hydrochloric acid solution with the mass percent concentration of 3.5%, controlling the temperature at 40 ℃ for processing for 10h, dispersing the titanium silicalite molecular sieve in 600g of deionized water after washing, adding 15g of sodium tungstate dihydrate and 1g of 11-bromoundecyl trimethoxy silane, and stirring and mixing for 20 min; adjusting the pH value of the solution to 6, continuously stirring for 60min, transferring the solution into a high-pressure reaction kettle, reacting for 5h at 120 ℃, cooling to room temperature after the reaction is finished, adjusting the pH value of the system to 2 by using 0.1mol/L phosphoric acid, stirring for 3h, filtering, and drying to obtain the supported heteropoly acid hydrolysis auxiliary agent.
The inorganic salt is zinc sulfate.
The concentration of the hydrogen peroxide in percentage by mass is 10%.
And the solid-liquid separation adopts a plate-frame filter for filtration.
Example 2
(1) With reference to the example disclosed in CN111733119A, the viable cell count obtained after 48 hours of culture was 1.7X 1010cfu/mL, and the sporulation rate is 90 percent.
(2) Hydrolyzing polysaccharide, namely selecting 100g of enteromorpha, primarily cutting up the enteromorpha, putting the enteromorpha into a hydrolysis kettle, adding 20g of water, 0.1g of inorganic salt, 2g of hydrogen peroxide and 0.1g of supported heteropoly acid hydrolysis auxiliary agent, uniformly mixing and stirring, reacting at 50 ℃ for 2 hours, and cooling to room temperature after the reaction is finished;
(3) inoculating and fermenting, namely adding 100g of water, 2g of nitrogen-phosphorus-potassium fertilizer, 10g of soybean meal, 6g of diatomite, 0.5g of humic acid, 0.1g of potassium fulvate and 5g of stone straw into enteromorpha cooled to room temperature, uniformly mixing, inoculating 1.2 volume percent of high-density bacillus subtilis, controlling the fermentation temperature to be 32 ℃, and fermenting for 5 days;
(4) and (3) preparing the biological fertilizer, namely performing solid-liquid separation on the obtained fermentation product to obtain a filtrate part, namely the enteromorpha biological fertilizer.
Example 3: dividing the towel gourd planting area into 12 areas with equal area, wherein each area is 100 plants, and randomly dividing the 12 areas into 3 groups; the enteromorpha biological fertilizer prepared in the examples and the control group (CN 111732483B example 1) is respectively used, the application amount is 5g of fertilizer per plant, the fertilizer is applied once every 7 days, the specific growth condition of towel gourd in a farmland is detected after 60 days, and the towel gourd is harvested within 180 days, so that the good and bad fertilizer efficiency performance of the enteromorpha biological fertilizer is represented, and the experimental results are shown in the following table.
TABLE 1 Effect of biological fertilizers on Luffa cylindrica
Group of N (number of experimental replicates) Mean length of the Main root (cm) Average melon vine length (cm) Mu yield (kg) Loofah weight (g)
Example 1 4 38.9±12.5 197.6±36.8 3219±123 410.3±108.3
Example 2 4 39.2±11.8 200.3±42.9 3259±129 416.8±113.4
Control group 4 31.8±12.8 185.6±38.4 3102±153 380.3±123.8
From the experimental results, compared with a control group, the enteromorpha biological fertilizer can effectively promote the growth of the towel gourd, is beneficial to improving the yield of the towel gourd and the single weight of the towel gourd, and has important economic value.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims (3)

1. The application of the bacillus subtilis in biological fermentation is characterized in that: the bacillus subtilis is used for preparing the enteromorpha biological fertilizer by fermenting enteromorpha as a raw material, and comprises the following steps:
step one, preprocessing the enteromorpha by the following steps: hydrolyzing polysaccharide, selecting 100-200 parts of enteromorpha, primarily cutting the enteromorpha, putting the enteromorpha into a hydrolysis kettle, adding 20-50 parts of water, 0.1-0.5 part of inorganic salt, 2-8 parts of hydrogen peroxide and 0.1-0.6 part of supported heteropolyacid hydrolysis auxiliary agent, uniformly mixing and stirring, reacting at 50-80 ℃ for 2-6 hours, and cooling to room temperature after the reaction is finished; the supported heteropolyacid hydrolysis auxiliary agent adopts 11-bromoundectyl trimethoxysilane to carry out surface treatment on the titanium silicalite molecular sieve; the preparation method of the supported heteropolyacid hydrolysis auxiliary agent comprises the following steps: according to the mass parts, 100-150 parts of titanium silicalite molecular sieve is soaked in 200-400 parts of 3.5-7% hydrochloric acid solution, the temperature is controlled to be 40-60 ℃ for treatment for 10-15h, the titanium silicalite molecular sieve is dispersed into 600-1000 parts of deionized water after washing, 15-20 parts of sodium tungstate dihydrate and 1-4 parts of 11-bromoundectyl trimethoxysilane are added, and the mixture is stirred and mixed for 20-30 min; adjusting the pH value of the solution to 6-8, continuously stirring for 60-120min, transferring the solution into a high-pressure reaction kettle, reacting for 5-10h at 120-150 ℃, cooling to room temperature after the reaction is finished, adjusting the pH value of the system to 2-3 by using 0.1-0.5mol/L phosphoric acid, stirring for 3-7h, filtering, and drying to obtain a supported heteropoly acid hydrolysis auxiliary agent;
step two, biological fermentation: inoculating and fermenting, namely adding 200 parts of 100-one of water, 2-6 parts of nitrogen, phosphorus and potassium fertilizers, 10-15 parts of bean pulp, 6-12 parts of diatomite, 0.5-3 parts of humic acid, 0.1-0.5 part of potassium fulvate and 5-18 parts of stone straw into enteromorpha cooled to room temperature, uniformly mixing, inoculating 0.8-3% of high-density bacillus subtilis, controlling the fermentation temperature to be 30-35 ℃, and fermenting for 4-6 days; the high-density Bacillus subtilis has viable count of 1.0-2.0 × 1010cfu/mL of Bacillus subtilis;
and step three, performing solid-liquid separation on the obtained fermentation product to obtain a filtrate part, namely the enteromorpha biological fertilizer.
2. An enteromorpha biological fertilizer prepared according to the application of claim 1.
3. The use of the enteromorpha biological fertilizer of claim 2 for improving the yield of towel gourds and/or the unit weight of towel gourds.
CN202110971100.9A 2021-08-24 2021-08-24 Application of bacillus subtilis in biological fermentation Pending CN113429227A (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103352016A (en) * 2013-06-27 2013-10-16 中国海洋大学 Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha
CN104262008A (en) * 2014-09-30 2015-01-07 青岛嘉瑞生物技术有限公司 Process for preparing liquid seaweed fertilizer through enzymolysis and fermentation
CN110615698A (en) * 2019-11-06 2019-12-27 中国农业科学院烟草研究所 Preparation method and application of biofertilizer taking enteromorpha and bacillus as raw materials
CN111732483A (en) * 2020-08-25 2020-10-02 烟台水禾土生物科技有限公司 Fermentation method of enteromorpha biological fertilizer
CN111733119A (en) * 2020-08-25 2020-10-02 烟台水禾土生物科技有限公司 High-density fermentation method of bacillus subtilis
CN112266300A (en) * 2020-12-14 2021-01-26 烟台水禾土生物科技有限公司 Method for fermenting algae microorganism using bacillus
US20210206698A1 (en) * 2020-01-03 2021-07-08 Nano And Advanced Materials Institute Limited Method for producing a microbial-enhanced organic liquid fertilizer for hydroponics cultivation

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103352016A (en) * 2013-06-27 2013-10-16 中国海洋大学 Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha
CN104262008A (en) * 2014-09-30 2015-01-07 青岛嘉瑞生物技术有限公司 Process for preparing liquid seaweed fertilizer through enzymolysis and fermentation
CN110615698A (en) * 2019-11-06 2019-12-27 中国农业科学院烟草研究所 Preparation method and application of biofertilizer taking enteromorpha and bacillus as raw materials
US20210206698A1 (en) * 2020-01-03 2021-07-08 Nano And Advanced Materials Institute Limited Method for producing a microbial-enhanced organic liquid fertilizer for hydroponics cultivation
CN111732483A (en) * 2020-08-25 2020-10-02 烟台水禾土生物科技有限公司 Fermentation method of enteromorpha biological fertilizer
CN111733119A (en) * 2020-08-25 2020-10-02 烟台水禾土生物科技有限公司 High-density fermentation method of bacillus subtilis
CN112266300A (en) * 2020-12-14 2021-01-26 烟台水禾土生物科技有限公司 Method for fermenting algae microorganism using bacillus

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