Microbial agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a microbial agent as well as a preparation method and application thereof. The microbial agent is composed of a microbial fermentation broth and a carrier, the microbial fermentation broth is a mixed fermentation broth of Bacillus velezensis MN14, Bacillus velezensis CC09 and Bacillus subtilis SDZHYB-1, and the carrier is muscovite powder and hexadecyl trimethyl ammonium bromide. According to the method, the muscovite powder and the hexadecyl trimethyl ammonium bromide are used as microbial carriers, so that the workload of repeatedly preparing the microbial fermentation liquid can be reduced, the microbial fermentation agent is convenient to store and transport, the colonization and reproduction of the bacillus velezensis in the waste vinasse-sludge solid substrate are ensured, and the fermentation efficiency and the yield of the fermentation product are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a microbial inoculant, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, numerous microorganisms with plant growth-promoting functions have been discovered. These microorganisms are prepared into fermentation broth through direct liquid fermentation, or mixed with agricultural by-products (such as straw) and industrial by-products (such as discarded grains and mushroom residues) for solid-state fermentation to prepare microbial fertilizers. When the microbial fermentation broth is directly applied to the field, the growth-promoting effect is unstable because it is difficult to colonize in the field soil. The microbial fertilizers prepared by solid-state fermentation have problems such as a long preparation period and difficulty in matrix degradation.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] To solve the problems in the background art, the present invention provides a microbial inoculant, a preparation method thereof, and an application thereof.

[0005] To achieve the above object, the first technical solution adopted by the present invention is: A microbial inoculant is composed of a microbial fermentation broth and a carrier. The microbial fermentation broth is a mixed fermentation broth obtained by separately fermenting Bacillus velezensis MN14, Bacillus velezensis CC09, and Bacillus subtilis SDZHYB-1, and the carrier is obtained by swelling mica powder with cetyltrimethylammonium bromide carried by supercritical CO2.

[0006] Preferably, the volume ratio of the fermentation broths of Bacillus velezensis MN14, Bacillus velezensis CC09, and Bacillus subtilis SDZHYB-1 is (1-3):1:1.

[0007] Preferably, the mass ratio of mica powder to cetyltrimethylammonium bromide is (10-15):1.

[0008] Preferably, the preparation method of the carrier is: mixing mica powder and cetyltrimethylammonium bromide and then performing a supercritical CO2 reaction, and after the reaction is completed, washing and drying the product.

[0009] Preferably, the temperature of the supercritical CO2 reaction is 35-45°C, the pressure is 20-25 Mpa, and the reaction time is 90-120 min.

[0010] Preferably, the volume-mass ratio of the microbial fermentation broth to the carrier is 1:(4-8).

[0011] The second technical solution adopted by the present invention is: Preparation method of microbial inoculum, mixing microbial fermentation broth with a carrier and then drying under vacuum.

[0012] The third technical solution adopted by the present invention is: A microbial fertilizer, comprising any one of the microbial inoculums in the first technical solution and spent grains substrate, wherein the spent grains substrate contains distillery dehydrated sludge with a wet weight of (2-8)%, and the mass ratio of the microbial inoculum to the spent grains substrate is 1:(50-150).

[0013] The fourth technical solution adopted by the present invention is: Preparation method of microbial fertilizer, mixing the microbial inoculum and the spent grains substrate and then carrying out composting fermentation, followed by drying and pulverizing after the fermentation is completed; the temperature of the composting fermentation is 50-70 °C, and the time of the composting fermentation is 40-50 days; the water content of the microbial fertilizer is not higher than 5%, and the particle size is 1-3 mm.

[0014] The preparation method of the spent grains substrate is: using spent grains with a humidity of 40-60% as the raw material, adjusting its pH to 6.0, and then adding distillery dehydrated sludge with a wet weight of (2-8)%, and mixing evenly.

[0015] Preferably, during the composting process of the microbial fertilizer, the pile is turned every 4 days in the first two weeks, and once a week from the 3rd week to the 20th week; 30 days before composting, an appropriate amount of tap water is added to ensure that the moisture content is within a suitable range (40-60%); temperature: during summer composting, it can reach above 60 °C the next day and remain for about 40 days.

[0016] The fifth technical solution adopted by the present invention is: Application of microbial fertilizer in promoting the growth of crops.

[0017] Preferably, the crops include at least one of Solanaceae tobacco, tomato, sorghum, and corn. Compared with the prior art, the present invention has the following beneficial effects: The present invention uses mica powder and cetyltrimethylammonium bromide as the microbial carrier, which can reduce the workload of repeatedly preparing microbial fermentation broth, facilitate the storage and transportation of microbial fermentants, and enhance the stability of the inoculated microorganisms during the fermentation process, as well as their persistence, survival, and colonization ability in the soil and plant roots.

[0018] The present invention applies a microbial inoculum composed of specific components to the fermentation of spent grains to prepare a microbial fertilizer, which has a growth-promoting function for a variety of crops, can effectively promote the growth of the above-ground parts and underground roots of crops, and can improve the physical and chemical properties and microbial flora of the crop root soil.

[0019] The microbial fertilizer of the present invention adds a small amount of sludge during solid-state fermentation, which has a good effect on maintaining moisture during the fermentation process and also helps to improve the stability of microorganisms during fermentation and application.

[0020] The present invention obtains muscovite powder and cetyltrimethylammonium bromide through a supercritical CO2 reaction, which significantly increases the specific surface area and the openness of the interlayer structure of the muscovite powder, helps to improve the adsorption performance and biological activity of the microbial carrier, and can achieve better effects with fewer types and amounts of loaded microorganisms. Description of the Drawings

[0021] 图1 Growth conditions of tobacco under different treatments; 图2 Column chart of GO enrichment of up-regulated genes in tobacco leaves; 图3 Column chart of GO enrichment of down-regulated genes in tobacco leaves. Detailed Embodiments

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] The sources or purchase information of some reagents used in the following examples are as follows: Bacillus velezensis MN14 (product number HZB131640), Bacillus velezensis CC09 (product number HZB224578)), Bacillus subtilis SDZHYB-1 (product number HZB119293), all purchased from Huizao Biology.

[0024] Example 1 Preparation of Microbial Inoculum 1) Preparation of microbial fermentation broth: Bacillus velezensis MN14, Bacillus velezensis CC09, and Bacillus subtilis SDZHYB-1 were respectively fermented and cultured in LB liquid medium for 24 - 36 h (the rotation speed of the constant-temperature shaker was set at 150 rpm, and the temperature was set at 28 °C). The cell concentration in the fermentation broth was adjusted by dilution with an ultraviolet spectrophotometer to 3*10 8 CFU, and then the fermentation broths were mixed at a volume ratio of 2:1:1 to obtain a composite microbial fermentation broth.

[0025] 2) Preparation of microbial carrier: Mica powder and cetyltrimethylammonium bromide were added to the closed reaction equipment of the CO2 supercritical fluid system according to a weight ratio of 12:1. After discharging the air, CO2 was introduced, and then the system conditions were set as: temperature 40°C, pressure 22 Mpa, and swelling treatment was carried out for 100 minutes. After depressurization, ethanol washing and drying were carried out to obtain the carrier.

[0026] 3) Preparation of microbial inoculum: The composite microbial fermentation broth and the carrier were mixed according to a mass ratio of 1:6, and then vacuum dried to obtain the microbial inoculum.

[0027] Example 2 Preparation of microbial inoculum 1) Preparation of microbial fermentation broth: Bacillus velezensis MN14, Bacillus velezensis CC09, and Bacillus subtilis SDZHYB-1 were respectively fermented and cultured in LB liquid medium for 24 - 36 h (the rotation speed of the constant temperature shaker was set at 150 rpm, and the temperature was set at 28°C). With the help of an ultraviolet spectrophotometer, the cell concentration in the fermentation broth was diluted and adjusted to 3*10 8 CFU, and then the fermentation broths were mixed according to a volume ratio of 3:1:1 to obtain the composite microbial fermentation broth.

[0028] 2) Preparation of microbial carrier: Mica powder and cetyltrimethylammonium bromide were added to the closed reaction equipment of the CO2 supercritical fluid system according to a weight ratio of 10:1. After discharging the air, CO2 was introduced, and then the system conditions were set as: temperature 35°C, pressure 20 Mpa, and swelling treatment was carried out for 120 minutes. After depressurization, ethanol washing and drying were carried out to obtain the carrier.

[0029] 3) Preparation of microbial inoculum: The composite microbial fermentation broth and the carrier were mixed according to a mass ratio of 1:8, and then vacuum dried to obtain the microbial inoculum.

[0030] Example 3 Preparation of microbial inoculum 1) Preparation of microbial fermentation broth: Bacillus velezensis MN14, Bacillus velezensis CC09, and Bacillus subtilis SDZHYB-1 were respectively fermented and cultured in LB liquid medium for 24 - 36 h (the rotation speed of the constant temperature shaker was set at 150 rpm, and the temperature was set at 28°C). With the help of an ultraviolet spectrophotometer, the cell concentration in the fermentation broth was diluted and adjusted to 3*10 8 CFU, and then the fermentation broths were mixed according to a volume ratio of 1:1:1 to obtain the composite microbial fermentation broth.

[0031] 2) Preparation of microbial carrier: Mica powder and cetyltrimethylammonium bromide are added to a closed reaction device of a CO2 supercritical fluid system according to a weight ratio of 15:1. After discharging the air, CO2 is introduced, and then the system conditions are set as follows: temperature 45°C, pressure 25 Mpa, and swelling treatment is carried out for 80 minutes. After depressurization, ethanol washing and drying are performed to obtain the carrier.

[0032] 3) Preparation of microbial inoculant: The composite microbial fermentation broth and the carrier are mixed according to a mass ratio of 1:4, and then vacuum dried to obtain the microbial inoculant.

[0033] Comparative Example 1 Compared with Example 1, the difference is only that the supercritical CO2 method is not used in the preparation of the microbial carrier, but direct mixing is carried out; the remaining steps and parameters are the same.

[0034] Comparative Example 2 Compared with Example 1, the difference is only that only mica powder is used as the microbial carrier; the remaining steps and parameters are the same.

[0035] Comparative Example 3 Compared with Example 1, the difference is only that no microbial carrier is used, and the composite microbial fermentation broth is directly mixed and fermented with the distiller's grains substrate; the remaining steps and parameters are the same.

[0036] Test Example 1 Preparation of microbial fertilizer In this example, the microbial fermenters obtained in the above Examples 1 to 3 and Comparative Examples 1 to 3 are applied to the fermentation of the distiller's grains substrate to investigate their fermentation effects.

[0037] The compost fermentation and detection specifically include the following processes: 1) Compost raw materials: Fresh distiller's grains from a Luzhou-flavor liquor factory (the main raw material is sorghum) are taken, and it is measured whether the humidity of the distiller's grains is within the range of 50-60%. The humidity can be adjusted by means such as sun drying or adding water; then 0.7% (wet weight) of quicklime is used to adjust the pH of the distiller's grains to 6.0, and 5% (wet weight) of the dehydrated sludge from the liquor factory is added for mixing to obtain the distiller's grains substrate.

[0038] 2) Preparation of microbial bacterial fertilizer: The microbial inoculants prepared in each example and comparative example were respectively mixed with the discarded grains substrate at a mass ratio of 1:100, and then compost fermentation was carried out. The uniformly mixed materials were placed in a trapezoidal bin (2.4×1.7×1.6m). In the first two weeks, the pile was turned every 2 - 3 days. From the 3rd week to the 7th week, the pile was turned once every 5 days. The temperature of compost fermentation was 50 - 70°C, and the time of compost fermentation was 40 - 50 days. The fermented mature discarded grains were dried and crushed to obtain dry distiller's grains fermentation materials. The water content of the obtained microbial bacterial fertilizer after drying was not higher than 5%, and the particle size of the dry distiller's grains fermentation materials after crushing was 1 - 3mm. Temperature and humidity monitoring: 30 days before composting, an appropriate amount of tap water was added to ensure that the moisture content was within a suitable range (40 - 60%). Temperature: During summer composting, the temperature could reach above 60°C the next day and remain for about 40 days.

[0039] 3) Detection method Determination of organic matter content: After composting was completed, mixed samples were collected from multiple points. The K2Cr2O7 volumetric method was used to determine the organic matter content (dry basis, %) in each mixed sample.

[0040] Preparation of leaching solution: Fresh compost samples were leached with deionized water at a solid-liquid ratio (mass / volume) of 1:10 for 3 hours, and then the compost leaching solution was obtained by filtration.

[0041] Determination of absorbance: Using an ultraviolet spectrophotometer, the absorbance values (denoted as E4 and E6) of the leaching solution were measured at wavelengths of 465nm and 665nm respectively.

[0042] Calculation of humification parameters: Calculate the E4 / E6 value, which is used as one of the indicators to evaluate the degree of compost maturity. The smaller the E4 / E6 value, the higher the degree of humification of the compost.

[0043] The test results are shown in Table 1: Table 1 Detection of the maturity degree of bacterial fertilizer 。

[0044] Application Example 1 Apply the microbial bacterial fertilizer prepared in Example 1 to tobacco planting: When applying the microbial bacterial fertilizer to tobacco planting, when the tobacco seedlings were in the state of "three leaves and one heart", the first fertilization was carried out. The microbial bacterial fertilizer was covered on the surface of the tobacco soil, and 500g of fertilizer was applied to each tobacco plant, with a covering thickness of 1 - 2cm. And 30 days after the first fertilization, the second fertilization was carried out, and the fertilizer was covered on the upper layer of the substrate of the first fertilization. The amounts of the two fertilizations were kept the same. 2g / plant of urea was used as the chemical fertilizer control, and clear water was used as the experimental blank control.

[0045] The relevant agronomic traits were measured 60 days after the first fertilization of tomato plants, including plant height, stem diameter, number of compound leaves, leaf area, SPAD, and nitrogen content. Measurement of plant height: The distance from about 1 cm near the base of the main stem to the growth point of the tomato apical bud was measured with a ruler from the surface soil to the highest stem growth point, accurate to 0.1 cm; Measurement of stem diameter: The diameter 1 cm above the cotyledons was measured with a digital vernier caliper, and the average value was taken as the stem diameter by the cross method, accurate to 0.01 cm; Measurement of leaf area: Measured from the second leaf above the cotyledons. The leaf length (L) was calculated from the leaf base (where it connects to the main stem) to the leaf tip, and the leaf width (D) was measured with a ruler at the widest part of the leaf. The leaf area was calculated using the formula S = L * D * 0.5468; Measurement of leaf SPAD and nitrogen content: Referring to the national standard "YCT 142-2010", a chlorophyll meter was used to measure one true leaf from the upper, middle, and lower parts of the plant respectively, and the average value was taken. Avoid the leaf veins during the measurement process; Dry and fresh weight: The fresh weight was measured after rinsing the plants with clear water and drying them. The samples were put into a ventilated drying oven, blanched at 105 °C for 30 min, and then dried to a constant weight at 75 °C and weighed.

[0046] At the tobacco harvest stage, the upper leaves of tobacco were sampled and analyzed for tobacco leaf chemical components. The contents of nicotine, total sugar, reducing sugar, total nitrogen, potassium ions, and chloride ions were mainly determined. Among them, the determination of nicotine was carried out by ultraviolet spectrophotometry; the determination of total sugar was carried out by the phenol-sulfuric acid colorimetric method of GB / T 15672-2009; the determination of reducing sugar was carried out by the 3,5-dinitrosalicylic acid colorimetric method; the determination of total nitrogen was carried out by the sulfuric acid-hydrogen peroxide digestion Kjeldahl nitrogen analyzer method of NY / T 2017-2011; the determination of potassium ions was carried out by the first method of GB 5009.268-2016; the determination of chloride ions was carried out by ion chromatography.

[0047] Test results: By monitoring the growth status and agronomic traits of tobacco plants, the results showed that the bacterial agent could significantly promote the growth of tobacco plants. After 60 days of treatment, the plant height, stem diameter, number of compound leaves, leaf area, leaf SPAD value, and leaf nitrogen content of tobacco plants reached 56.62 cm, 26.15 mm, 14.22, 769.90 cm², 39.69, and 12.47 mg / g respectively, which were increased by 90.00%, 87.59%, 17.42%, 201.00%, 41.65%, and 39.49% compared with the blank control group. At the same time, the fresh weight of the stem and leaf reached 224.19 g and 493.30 g respectively, which were increased by 486.12% and 651.64% compared with the blank control group; the dry weight of the stem and leaf reached 147.38 g and 148.53 g respectively, which were increased by 553.86% and 858.88% compared with the blank control group (Table 2). The growth conditions of each group on the 15th day after the first fertilization of tomato plants are as 图1 shown.

[0048] Table 2 Determination of tobacco growth indexes under different treatments 。

[0049] Analysis of the chemical components of tobacco leaves after 60 days of treatment found that the contents of nicotine, total sugar, reducing sugar, total nitrogen and chloride ion in the tobacco leaves of Application Example 1 were 1.22 mg / g, 148.10 g / kg, 47.76 g / kg, 3.74 g / kg and 1.68 g / kg respectively, which were increased by 454.55%, 39.98%, 55.42%, 44.96% and 5.00% respectively compared with the blank control group. The potassium ion content was decreased by 6.56% compared with the control group, indicating that the bacterial agent could improve the chemical components of tobacco leaves (Table 3).

[0050] Table 3 Determination of the main chemical component contents of tobacco leaves after different treatments 。

[0051] By measuring and analyzing the chemical properties, enzyme activities and microbial community structures of tobacco planting soil, the results showed that the treatment with distiller's grains bacterial fertilizer could reduce the soil pH value and significantly increase the soil nutrient content. After 45 days of treatment, the contents of total nitrogen, hydrolyzable nitrogen, available phosphorus and organic matter in the soil reached 4.34 g / kg, 194.25 mg / kg, 65.18 mg / kg and 41.68 g / kg respectively, which were increased by 284.07%, 493.49%, 538.39% and 512.94% respectively compared with the control group. At the same time, the activities of catalase, sucrase, phosphatase and urease reached 0.14 mL / (g·min)-1, 808.37 mg / g, 75.13 μg / (g·h)-1 and 2.01 mg / (g·24 h)-1 respectively, which were increased by 40.00%, 498.57%, 113.32% and 378.57% respectively compared with the control group (Table 4).

[0052] Table 4 Physicochemical determination of tobacco soil after different treatments 。

[0053] The analysis results of the microbial community structure showed that the diversity and richness of bacteria and fungi in the soil of Application Example 1 were significantly reduced. At the bacterial phylum level, the relative abundances of Proteobacteria ( Proteobacteria ), Actinobacteria ( Actinobacteria ), Bacteroidetes ( Bacteroidota ), Patellibacteria ( Planctomycetota ), and Firmicutes ( Firmicutes ) increased, while the relative abundances of Chloroflexi ( Chloroflexota ) and Acidobacteria ( Acidobacteriota ) decreased. At the fungal phylum level, Ascomycota ( Ascomycota ), and the relative abundance of Basidiomycota ( Basidiomycota ) increased, while that of Chytridiomycota ( Chytridiomycota ) and Zygomycetes ( Zygomycotina ) decreased. At the bacterial genus level, the abundances of Bacillus and unclassified_f__ Micrococcaceae increased; at the fungal genus level, the abundances of Vanrija , Wickerhamomyces and Meyerozyma increased.

[0054] Table 5 Bacterial Alpha diversity indices of different treatment groups .

[0055] Table 6 Alpha diversity indices of fungi in different treatment groups .

[0056] Transcriptomic analysis was performed on tobacco leaves after 60 days of treatment, and the results are as 图2-3 shown. The results indicate that the treatment with the distiller's grains bacterial fertilizer of Example 1 can up-regulate genes related to biological processes such as abscisic acid response, disaccharide biosynthesis, and amino sugar metabolism in tobacco leaves, and enhance the activities of sucrose synthase, hydrolase, and the degradation ability of O-glycoside compounds. At the same time, the expression levels of genes related to plant hormones (auxin, cytokinin, gibberellin, abscisic acid, jasmonic acid) were up-regulated, promoting the growth and development of tobacco leaves. In addition, this treatment down-regulated the expression of genes related to defense responses, enabling the tobacco plants to adapt faster to the environmental stress brought by the distiller's grains-based bacterial agent, thereby optimizing their growth state.

[0057] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A microbial inoculum, characterized in that, It is composed of a microbial fermentation broth and a carrier. The microbial fermentation broth is a fermented broth obtained by mixing the fermented broths of Bacillus velezensis MN14, Bacillus velezensis CC09, and Bacillus subtilis SDZHYB-1 respectively. The carrier is obtained by swelling mica powder with cetyltrimethylammonium bromide carried by supercritical CO2 fluid.

2. The microbial inoculum according to claim 1, characterized in that, The volume ratio of the fermentation broths of Bacillus velezensis MN14, Bacillus velezensis CC09, and Bacillus subtilis SDZHYB-1 is (1-3):1:

1.

3. The microbial inoculum according to claim 1, wherein The mass ratio of mica powder to cetyltrimethylammonium bromide is (10-15):

1.

4. The microbial inoculum according to claim 1 or 3, characterized in that, The preparation method of the carrier is: mixing mica powder and cetyltrimethylammonium bromide and then carrying out a supercritical CO2 reaction. After the reaction is completed, the product is washed and dried. The temperature of the supercritical CO2 reaction is 35-45°C, the pressure is 20-25 Mpa, and the reaction time is 90-120 min.

5. The microbial inoculant according to claim 1, wherein The volume-mass ratio of the microbial fermentation broth to the carrier is 1:(4-8).

6. The preparation method of the microbial inoculum according to any one of claims 1 to 5, characterized in that, Mix the microbial fermentation broth with the carrier and then dry it under vacuum.

7. A microbial fertilizer, characterized in that, It includes the microbial inoculant as described in any one of claims 1-6 and the spent grains substrate. The spent grains substrate contains winery dehydrated sludge with a wet weight of (2-8)%. The mass ratio of the microbial inoculant to the spent grains substrate is 1:(50-150).

8. The preparation method of the microbial bacterial fertilizer according to claim 7, wherein, Mix the microbial inoculant and the spent grains substrate and then carry out composting fermentation. After the fermentation is completed, dry and crush it. The temperature of the composting fermentation is 50-70°C, and the composting fermentation time is 40-50 days. The water content of the microbial fertilizer is not higher than 5%, and the particle size is 1-3 mm.

9. The application of the microbial fertilizer as described in claim 7 in promoting the growth of crops.

10. The application according to claim 9, wherein The crops include at least one of Solanaceae tobacco, tomato, sorghum, and corn.

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