Compound microbial agent and application thereof

Abstract

The invention discloses a compound microbial agent and application thereof, and belongs to the technical field of microbial agents. The method specifically comprises the following steps: adding clostridium butyricum fermentation centrifugate into a fermentation tank, adding a nitrogen source, a carbon source and inorganic salt into the fermentation tank, then adding a bacillus subtilis solution and a bacillus licheniformis solution, stirring at a certain temperature and a certain rotating speed, fermenting for a period of time, then adding a lactic acid bacteria solution, and fermenting for a period of time at a certain rotating speed; stirring and fermenting at a certain rotating speed at a certain temperature, detecting the pH value after fermenting for a period of time, and when the pH value is reduced to 4.3-4.5, obtaining the compound microbial agent. The prepared compound microbial agent can improve the saline-alkali soil, effectively improve the pH of the saline-alkali soil, consume soil salinity and improve the crop germination rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial agents, and in particular relates to a composite microbial agent and application thereof. Background Art

[0002] China's saline-alkali land covers approximately 170 million hectares, accounting for over one-sixth of its total land area. Saline-alkali land refers to soil containing excessive amounts of salt and alkaline substances, making it unsuitable for plant growth. Its formation is primarily due to a variety of environmental factors, including drought, water shortages, and poor drainage. Furthermore, excessive use of chemical fertilizers and pesticides is also a significant contributor to its development. The formation of saline-alkali land has severely impacted agricultural production and ecological improvements. Currently, the main methods for improving saline-alkali land are physical, hydraulic, and chemical methods. Physical methods require land leveling, deep plowing, and sun-drying, which require significant manpower and resources. Hydraulic methods require large amounts of water flushing, which is inconsistent with national water conservation standards. Chemical methods, which apply large amounts of amendments such as gypsum and black tar to reduce or directly eliminate soil alkalinity, can easily lead to new problems such as soil pollution. While these three methods can improve saline-alkali land, they lack long-term and effective results. In recent years, with the continuous development of microbial technology, its application in saline-alkali land remediation has gradually demonstrated its unique advantages.

[0003] Microbial agents are an important means of treating saline-alkali land. These agents usually contain a variety of beneficial microorganisms that can work synergistically to improve the soil environment. By applying the corresponding composite microbial agents, not only can the number of microorganisms in the soil be increased, but the decomposition of soil organic matter and the formation of aggregate structures can also be promoted, thereby breaking up soil compaction, regulating and improving the soil's water retention, fertilizer retention, and air permeability, making it more suitable for crop growth. At the same time, inorganic salts are nutrients required for microbial growth and can effectively consume the salt rich in saline-alkali land. Some microorganisms have acid-producing properties to regulate soil pH. However, the current preparation cost of microbial fertilizers is high, the microorganisms have a short shelf life, the strains are of poor quality, and the stability is poor, which has not played a corresponding role in improving saline-alkali land.

[0004] Clostridium butyricum boasts a long logarithmic phase, early acid production, high acid production, high conidia conversion rate, high enzyme activity, strong antibacterial properties, acid and alkali resistance, bile salt tolerance, high temperature resistance, and high stability. As the "King of Probiotics," it combines the advantages of other probiotics and is the preferred choice for solving saline-alkali soil problems. Currently, most C. butyricum fermentations are produced using liquid submerged fermentation. Typical C. butyricum preparations undergo liquid fermentation and are then centrifuged, freeze-dried, or spray-dried to produce bacterial powder. However, the centrifugation used in the preparation process does not remove all bacteria and their metabolites, and some bacteria remain in the centrifuge. Acetic acid, butyric acid, amylase, vitamin K, and B vitamins produced during fermentation, as well as unused culture medium components, also remain in the centrifuge. Direct discharge of the centrifuge not only pollutes water bodies but also results in significant waste. Therefore, effective utilization of the C. butyricum fermentation centrifuge is crucial. Summary of the Invention

[0005] The use of Clostridium butyricum fermentation centrifuge to prepare bacterial fertilizer can improve the quality of bacterial fertilizer and reduce costs. In view of this, the present invention aims to provide an application of a composite microbial agent in bacterial fertilizer, which utilizes Clostridium butyricum fermentation centrifuge with Bacillus subtilis, Bacillus licheniformis, and lactic acid bacteria for mixed fermentation. By selecting the Bacillus subtilis, Bacillus licheniformis, and lactic acid bacteria varieties and supplementing them with nutrients, the live bacteria of Clostridium butyricum, Bacillus subtilis, Bacillus licheniformis, and lactic acid bacteria are of high quality and strong stability, and the cost is low and the preparation method is simple, effectively improving saline-alkali land.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a composite microbial agent, the specific method being:

[0008] Take 30-70L, the viable bacteria count is 1.0×10 6 ~5.0×10 8 CFU / mL of Clostridium butyricum fermentation centrifuge was added to the fermenter. After adding nitrogen source, carbon source and inorganic salt to the fermenter, 1-4 L of the fermenter with a viable bacterial count of 1.0×10 9 ~1.0×10 10 CFU / mL of Bacillus subtilis liquid, 1-4L, viable bacteria count 1.0×10 9 ~1.0×10 10 CFU / mL of Bacillus licheniformis solution was stirred at 100-300 rpm at 35-40 °C for 15-20 h, and then 1-4 L of 2.0×10 9 ~3.0×10 9CFU / mL of lactic acid bacteria liquid is stirred and fermented at 35-40°C at a speed of 100-300 rpm, and the pH is detected after 20-24 hours of fermentation. When the pH value drops to 4.3-4.5, the composite microbial agent is obtained.

[0009] Furthermore, the Clostridium butyricum fermentation centrifuge is obtained by fermenting Clostridium butyricum at 30-40° C., a rotation speed of 100-150 r / min, and anaerobic conditions for 5-15 hours, and then centrifuging at a rotation speed of 6000-7000 r / min.

[0010] Furthermore, the number of viable Clostridium butyricum in the Clostridium butyricum fermentation centrifuge was 3.6×10 7 CFU / mL, the number of viable Bacillus subtilis in the Bacillus subtilis liquid is 1.0×10 10 CFU / mL, the number of viable Bacillus licheniformis in the Bacillus licheniformis bacterial solution is 1.0×10 10 CFU / mL.

[0011] Furthermore, the lactic acid bacteria liquid consists of Lactobacillus acidophilus, Enterococcus faecalis and Lactobacillus casei.

[0012] Furthermore, the viable count of Lactobacillus acidophilus is 1.0×10 9 ~1.0×10 10 CFU / mL, the viable count of Enterococcus faecalis was 7.0×10 8 ~7.0×10 9 CFU / mL, the viable count of Lactobacillus casei is 1.0×10 9 ~1.0×10 10 CFU / mL.

[0013] Furthermore, the nitrogen source is added in an amount of 0.5-2.0% by mass of the Clostridium butyricum fermentation centrifuge, the carbon source is added in an amount of 3.0-7.0% by mass of the Clostridium butyricum fermentation centrifuge, and the inorganic salt is added in an amount of 0.44-1.32% by mass of the Clostridium butyricum fermentation centrifuge.

[0014] Furthermore, the nitrogen source is one or two of corn steep liquor, peptone, yeast extract, beef powder, beef extract, and peptide powder.

[0015] Furthermore, the carbohydrate carbon source is a mixture of glucose and molasses; the amount of glucose added is 2.0% to 3.5% of the mass of the Clostridium butyricum fermentation centrifuge; and the amount of molasses added is 1.0% to 3.5% of the mass of the Clostridium butyricum fermentation centrifuge.

[0016] Furthermore, the inorganic salts are MgSO4, MnSO4, K2HPO4 and sodium acetate, wherein the mass fractions of MgSO4, MnSO4, K2HPO4 and sodium acetate in the Clostridium butyricum fermentation centrifuge are: 0.025% to 0.075% MgSO4, 0.015% to 0.045% MnSO4, 0.1% to 0.3% K2HPO4, and 0.3% to 0.9% sodium acetate.

[0017] Furthermore, the content of Bacillus subtilis in the composite microbial agent is greater than or equal to 1.0×10 8 CFU / mL, Bacillus licheniformis content is greater than or equal to 1.0×10 8 CFU / mL, lactic acid bacteria content is greater than or equal to 2.5×10 8 CFU / mL, Clostridium butyricum content is greater than or equal to 1.0×10 6 CFU / mL.

[0018] In a second aspect, the present invention provides a microbial agent, which is prepared by the preparation method.

[0019] In a third aspect, the present invention further provides a microbial fertilizer comprising the microbial agent.

[0020] Furthermore, the bacterial fertilizer is in liquid form.

[0021] In a fourth aspect, the present invention also provides the use of the microbial agent or the microbial fertilizer in improving saline-alkali soil.

[0022] In a fifth aspect, the present invention also provides the use of the microbial agent or the microbial fertilizer in improving the germination rate of crops.

[0023] The composite microbial agent of the present invention is mainly obtained by fermenting Clostridium butyricum fermentation centrifuge with Bacillus subtilis strain, Bacillus licheniformis strain, lactic acid bacteria strain and carbohydrate carbon source. The growth of Clostridium butyricum and lactic acid bacteria reduces the pH value of the centrifuge, making the centrifuge convenient for storage, while also enriching the types of probiotics and metabolites. The obtained agricultural microbial fertilizer contains Clostridium butyricum, Bacillus subtilis, Bacillus licheniformis and lactic acid bacteria and their metabolites at the same time. Its application effect is the result of the combined action of Clostridium butyricum, Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria and their metabolites. By inoculating Clostridium butyricum centrifuge with Bacillus subtilis species, Bacillus licheniformis species and lactic acid bacteria species, the effect of fermenting Bacillus subtilis, Bacillus licheniformis and lactic acid bacteria using Clostridium butyricum centrifuge is achieved. While harmlessly treating the Clostridium butyricum fermentation centrifuge, a Clostridium butyricum microbial fertilizer containing a large amount of live Bacillus subtilis, Bacillus licheniformis and lactic acid bacteria was produced, which can be used to improve saline-alkali land. This effectively solves the problem of the Clostridium butyricum centrifuge being difficult to treat as sewage, and solves the technical problem of recycling residual bacteria and nutrient-rich substances in the Clostridium butyricum centrifuge.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention uses Clostridium butyricum fermentation centrifuge liquid and lactic acid bacteria to ferment together, which not only effectively utilizes the Clostridium butyricum fermentation centrifuge liquid but also allows the fermentation liquid to be stored for a long time due to the pH adjustment effect of lactic acid bacteria.

[0026] (2) The composite microbial agent obtained by the present invention has a lot of nutrients, which can not only ensure the activity of beneficial bacteria, but also increase soil fertility, and enhance the colonization ability of Clostridium butyricum, Bacillus subtilis, Bacillus licheniformis and lactic acid bacteria on plant roots.

[0027] (3) The composite microbial agent of the present invention contains a large number of highly active Clostridium butyricum, Bacillus subtilis, Bacillus licheniformis and lactic acid bacteria. The growth and reproduction of the four effectively consumes the salt in the saline-alkali land. Clostridium butyricum and lactic acid bacteria have a synergistic effect when used as fertilizers. Organic acids such as butyric acid, acetic acid and lactic acid effectively improve the pH environment of the saline-alkali land. Bacillus subtilis and Bacillus licheniformis can use the humus in the soil to reproduce in large quantities and decompose the organic matter in the soil. They can not only increase the effective nutrients in the soil and improve the soil structure, but also prevent the soil from becoming compacted. During the reproduction process, they can produce active substances such as subtilisin, polymyxin, nystatin and gramicidin. These active substances can cause the mycelium of pathogens to break, disintegrate or digest the cytoplasm, thereby killing the pathogens and effectively preventing the harm of pathogens to crops. They can also produce a variety of hormone substances and various vitamins, which can not only increase the nutrient content of the soil, but also promote crop growth, increase yield and improve the quality of agricultural products. DETAILED DESCRIPTION

[0028] To better illustrate the present invention, the following embodiments are listed. Obviously, the embodiments described are only part of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making any creative efforts are also within the scope of protection of the present invention.

[0029] The technical solution of the present invention is further described below through examples.

[0030] Example 1

[0031] (1) Prepare raw materials

[0032] Clostridium butyricum fermentation centrifuge: Clostridium butyricum with the deposit number CICC 10390 was fermented at 37°C, 120 r / min, and anaerobic conditions for 10 h. The centrifuge was centrifuged at 6600 r / min using a butterfly centrifuge to obtain the Clostridium butyricum fermentation centrifuge. The content of Clostridium butyricum in the centrifuge was 3.6×10 7 CFU / mL;

[0033] Bacillus subtilis solution: Bacillus subtilis with the deposit number of CGMCC No.19440 has a content of 1.0×10 10 CFU / mL;

[0034] The Bacillus subtilis strain solution was obtained by culturing in LB medium; the LB medium culture conditions were as follows: beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, pH 7.0-7.2, and steam sterilized at 121°C for 30 min. After the culture solution was cooled, the Bacillus subtilis strain was inoculated and cultured at 37°C, 180 r / min, for 6-10 hours. The bacterial count was determined to be 1.0×10 10 CFU / mL is sufficient.

[0035] Bacillus licheniformis solution: the content of Bacillus licheniformis with the deposit number ACCC 06149 is 1.0×10 10 CFU / mL;

[0036] The Bacillus licheniformis strain solution was obtained by culturing in a commonly used LB culture medium; the LB culture medium culture conditions were 3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, and a pH of 7.0-7.2. The culture medium was steam sterilized at 121°C for 30 minutes. After the culture medium was cooled, the Bacillus licheniformis strain was inoculated and cultured at 37°C, 180 r / min, for 6-10 hours. The bacterial count was determined to be 1.0×10 10 CFU / mL is sufficient.

[0037] Lactic acid bacteria solution: the content of Lactobacillus acidophilus with the deposit number ACCC 05489 is 1.0×10 9 CFU / mL, and the content of Enterococcus faecalis with the deposit number CICC 23658 was 7.0×10 8 CFU / mL, and the content of Lactobacillus casei with the preservation number CICC 20995 was 1.0×10 9 CFU / mL;

[0038] The lactic acid bacteria strain solution was cultured in MRS broth medium containing 10 g / L peptone, 4 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium citrate, 0.2 g / L MgSO4, and 0.2 g / L MnSO4, with a final pH of 6.5. The solid medium was supplemented with 15 g / L agar. The culture was statically incubated at 37°C for 24 hours. Lactobacillus acidophilus, Enterococcus faecalis, and Lactobacillus casei were all cultured under these conditions.

[0039] Carbon sources: glucose and molasses;

[0040] Nitrogen source: corn steep liquor;

[0041] Inorganic salts: MgSO4, MnSO4, K2HPO4 and sodium acetate.

[0042] (2) Preparation of composite microbial agents

[0043] The above-mentioned 50L Clostridium butyricum fermentation centrifuge was added to a fermentation tank, and 1.5kg of glucose, 1kg of molasses, 0.5kg of corn steep liquor, 12.5g of MgSO4, 7.5g of MnSO4, 50g of K2HPO4, and 300g of sodium acetate were added to the fermentation tank. Then, 2.5L of Bacillus subtilis strain solution and 2.5L of Bacillus licheniformis strain solution were added in sequence, and fermented at 37°C with stirring at a speed of 100rpm. After fermentation for 15h, 2.5L of lactic acid bacteria strain solution was added, and fermented at 37°C with stirring at a speed of 100rpm. The pH was detected during fermentation for 20-24h. When the pH value dropped to 4.3-4.5, the composite microbial agent was obtained.

[0044] The content of Bacillus subtilis in the composite microbial agent was detected to be 2.0×10 8 CFU / mL, the content of Bacillus licheniformis was 2.0×10 8 CFU / mL, lactic acid bacteria content is 2.5×10 8 CFU / mL, Clostridium butyricum content was 4.0×10 6 CFU / mL.

[0045] Experimental Example 1

[0046] Select the same saline-alkali land and divide it into two plots, each with a length of 4m 2 The plots were marked as A and B. 1 L / plot of composite microbial agent was used in plot A, and no fertilizer was applied to plot B. 2 g soil samples were taken from 5 randomly selected points after 3, 7, and 10 days, respectively, to measure soil pH, number of viable bacteria, and total soil salt content (see Table 1).

[0047] Table 1 Microbial agents for improving saline-alkali land

[0048]

[0049] As shown above, the bacterial fertilizer prepared by the composite microbial agent of the present invention can effectively improve the pH of saline-alkali land and consume soil salt, wherein the survival rate of lactic acid bacteria drops significantly in harsh environments, and the compound microbial agent is mainly used to regulate the pH of saline-alkali land. Clostridium butyricum in the agent.

[0050] Experimental Example 2

[0051] Select the same piece of light saline-alkali land and randomly select 3 plots to plant wheat, with each plot 3m 2 They were marked as A, B, and C. Plot A was not fertilized, and plot B was fertilized with a composite microbial agent (total bacterial count 6.54×10 8 CFU / mL) 1L / plot, and compound microbial agent was applied to plot C (total bacterial count 6.54×10 8 CFU / mL) 2L / block, and the soil pH value and wheat germination rate were calculated after 10 days, as shown in Table 2.

[0052] Table 2 Statistics of pH value and wheat germination rate in saline-alkali soil

[0053]

[0054] As shown in the above table, the bacterial fertilizer obtained in Example 2 of the present invention can effectively reduce the pH in saline-alkali soil, and the effect is better as the dosage increases, while also improving the germination rate of crops.

[0055] Example 3

[0056] Select the same saline-alkali land and divide it into two plots, each with a length of 4m 2 The plots A and B were marked. The plot A was treated with composite microbial agents (total bacterial count 6.54×10 8 CFU / mL) 1L / plot, and plot B used commercially available composite microbial agents (Haowangnong soil improver purchased from Taobao with a total bacterial count of 5×10 8 CFU / mL) 1L / block, and 2g soil samples were taken from 5 randomly selected points after 3 days, 7 days, and 10 days, and the soil pH and total soil salinity were measured (see Table 3).

[0057] Table 3 Comparative statistics of composite microbial agents and commercially available microbial agents

[0058]

[0059] As shown in the table above, the bacterial fertilizer obtained in Example 3 of the present invention can effectively reduce the pH and salinity in saline-alkali soil, and its effect is better than that of commercially available composite microbial inoculants. The total viable bacterial count of the composite microbial inoculant of the present invention can gradually increase from a decrease during the process of improving saline-alkali soil, and can sustainably improve saline-alkali soil. Currently available commercial composite microbial inoculants continuously decrease in saline-alkali soil improvement and cannot achieve a sustained effect.

[0060] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a composite microbial agent, characterized in that: The specific method is: Take 30-70L, the viable bacteria count is 1.0×10 6 ~5.0×10 8 CFU / mL of Clostridium butyricum fermentation centrifuge was added to the fermenter. After adding nitrogen source, carbon source and inorganic salt to the fermenter, 1-4 L of the fermenter with a viable bacterial count of 1.0×10 9 ~1.0×10 10 CFU / mL of Bacillus subtilis liquid, 1-4L, viable bacteria count 1.0×10 9 ~1.0×10 10 CFU / mL of Bacillus licheniformis was stirred at 100-300 rpm at 35-40 °C for 15-20 h, and then 1-4 L of 2.0×10 9 ~3.0×10 9 CFU / mL of lactic acid bacteria liquid is stirred and fermented at 35-40°C at a speed of 100-300 rpm, and the pH is detected after 20-24 hours of fermentation. When the pH value drops to 4.3-4.5, the composite microbial agent is obtained.

2. The preparation method according to claim 1, characterized in that The Clostridium butyricum fermentation centrifuge is obtained by fermenting Clostridium butyricum at 30-40° C., a rotation speed of 100-150 r / min and anaerobic conditions for 5-15 hours, and then centrifuging at a rotation speed of 6000-7000 r / min.

3. The preparation method according to claim 2, characterized in that The number of viable Clostridium butyricum in the Clostridium butyricum fermentation centrifuge was 3.6×10 7 CFU / mL, the number of viable Bacillus subtilis in the Bacillus subtilis liquid is 1.0×10 10 CFU / mL, the number of viable Bacillus licheniformis in the Bacillus licheniformis bacterial solution is 1.0×10 10 CFU / mL.

4. The preparation method according to claim 1, characterized in that The lactic acid bacteria liquid consists of Lactobacillus acidophilus, Enterococcus faecalis and Lactobacillus casei.

5. The preparation method according to claim 4, characterized in that The viable bacteria count of Lactobacillus acidophilus is 1.0×10 9 ~1.0×10 10 CFU / mL, the viable count of Enterococcus faecalis was 7.0×10 8 ~7.0×10 9 CFU / mL, the viable count of Lactobacillus casei is 1.0×10 9 ~1.0×10 10 CFU / mL.

6. The preparation method according to claim 1, characterized in that The nitrogen source is added in an amount of 0.5-2.0% of the mass of the Clostridium butyricum fermentation centrifuge, the carbon source is added in an amount of 3.0-7.0% of the mass of the Clostridium butyricum fermentation centrifuge, and the inorganic salt is added in an amount of 0.44-1.32% of the mass of the Clostridium butyricum fermentation centrifuge.

7. A microbial agent, characterized in that: The microbial agent is prepared by the preparation method according to any one of claims 1 to 6.

8. A microbial fertilizer, characterized in that: The microbial fertilizer comprises the microbial agent according to claim 7.

9. Use of the microbial agent according to claim 7 or the microbial fertilizer according to claim 8 in improving saline-alkali soil.

10. Use of the microbial agent according to claim 7 or the microbial fertilizer according to claim 8 in improving the germination rate of crops.

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