Microbial brick for promoting plant growth as well as preparation method and application of microbial brick

Abstract

The invention discloses a microorganism bacterium brick for promoting plant growth and a preparation method and application thereof, the microorganism bacterium brick is composed of vinasse obtained after compound microorganism fermentation, coco coir and a binder, and the compound microorganism bacterium brick comprises 30 wt%-35 wt% of bacillus subtilis, 30 wt%-35 wt% of geotrichum candidum and 30 wt%-35 wt% of aspergillus niger. After fermentation with the compound microbial agent, the content of nutrient elements such as nitrogen, phosphorus and potassium in the vinasse is obviously increased, and the content of crude fibers is reduced. The invention further discloses a production process for preparing the microbial bacterial brick from the vinasse fermented by the compound microbial bacterial agent and application of the microbial bacterial brick to plant cultivation. The prepared microbial bacterial brick can effectively promote the growth of ryegrass. The method not only solves the problem of disposal of waste materials after wine brewing and recycles the waste materials, but also can be widely applied to the fields of agriculture, horticulture, ecological restoration and the like, and has a wide market prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation and solid waste resource utilization, and particularly relates to a microbial brick for promoting plant growth, a preparation method and an application thereof. Background Art

[0002] Distillers' grains are a major waste product in the brewing industry, with approximately three tons of them discharged for every ton of wine produced, and annual national production exceeding 80 million tons. Fresh distillers' grains have a high moisture content, making them difficult to store and dry, prone to mold and spoilage. Their utilization rate is less than 50%, and a large amount of them is discarded as waste, causing environmental pollution, wasting resources, and poor social and economic benefits. Currently, distillers' grains are primarily dried and crushed for use as a feed additive or solid-state fermentation to produce roughage. However, these products are sold at low prices and have limited economic benefits. Therefore, exploring new technologies for distillers' grain utilization is crucial to improving their efficiency and protecting the environment.

[0003] Distillers' grains are rich in crude protein, soluble sugars, and essential plant nutrients such as nitrogen, phosphorus, and potassium. Their total nutrient content is 2-3 times that of ordinary soil, making them a natural candidate for conversion into a plant cultivation medium. However, anti-nutritional factors such as tannins in fresh distillers' grains can bind to proteins to form stable complexes, reducing nutrient availability. Furthermore, the acidic environment can easily lead to mold and cause rhizosphere diseases. Therefore, using them directly as a plant cultivation medium presents technical bottlenecks.

[0004] Many microorganisms in nature have strong biocatalytic enzyme systems, which can decompose macromolecular substances such as crude fiber that are difficult for plants to digest and absorb into small molecular substances that are easily absorbed. Some microorganisms also have the effect of preventing and controlling pests and diseases. Therefore, using microorganisms to ferment distiller's grains is a green and environmentally friendly technical means that can effectively solve the current problems in the resource utilization of distiller's grains and has great potential.

[0005] Based on this, the present invention proposes a method for directed construction of functional bacterial communities to achieve synergistic transformation of lees components to prepare functional bacterial bricks. Summary of the Invention

[0006] The purpose of the present invention is to provide a microbial brick for promoting plant growth, a preparation method thereof, and application of the microbial brick in plant cultivation.

[0007] To achieve the above purpose, the following technical solution is adopted: a microbial brick for promoting plant growth, which is made by pressing wine grains fermented with a composite microbial agent, coconut bran and a binder, wherein the composite microbial agent is composed of Bacillus subtilis, Geotrichum candidum and Aspergillus niger.

[0008] It is further defined that the distribution ratio of each component in the composite microbial agent is 30wt%-35wt% of Bacillus subtilis, 30wt%-35wt% of Geotrichum candidum and 30wt%-35wt% of Aspergillus niger.

[0009] It is further defined that the weight ratio of the vinasse after fermentation with the composite microbial agent to the coconut bran is 1:1-3:1.

[0010] It is further defined that the binder is sodium alginate and calcium chloride.

[0011] It is further defined that the composite microbial agent is used to degrade cellulose, hemicellulose and crude protein.

[0012] The preparation method of the plant growth-promoting microbial brick of the present invention comprises the following specific steps:

[0013] Step S1, primary culture of the strain: Bacillus subtilis, Geotrichum candidum, and Aspergillus niger were inoculated into LB medium, YPD medium, and PDB medium, respectively, and cultured in a 30° C. incubator for 2 days for Bacillus subtilis and Geotrichum candidum, and 3 days for Aspergillus niger, respectively, to obtain plate cultures of the three strains;

[0014] Step S2, secondary culture of the strain: inoculating the plate cultures of Bacillus subtilis, Geotrichum candidum, and Aspergillus niger into liquid culture medium respectively, wherein Bacillus subtilis and Geotrichum candidum are cultured in a shaker at 220 r / min and 29° C. for 48-72 h, and Aspergillus niger is cultured in a shaker at 220 r / min and 29° C. for 96-120 h, to obtain secondary cultures of the three strains, i.e., a composite microbial agent;

[0015] Step S3, vinasse pretreatment: using a concentration of 3.75 mol·L -1 The pH value of the unfermented lees is adjusted to 6.5-7.5 with sodium hydroxide solution;

[0016] Step S4, uniformly mixing the vinasse pretreated in step S3, the nutrients, and the composite microbial agent obtained in step S2, controlling the moisture content of the material to be 35 wt%-45 wt%, wherein the nutrients are glucose and ammonium chloride, aerobically fermenting the mixture at 30° C. for 5-7 days, turning the mixture twice a day, and then placing the mixture in a breathing bag with a breathing valve and anaerobically fermenting it at 30° C. for 2-4 days to obtain fermented vinasse with increased nitrogen, phosphorus, and potassium contents;

[0017] Step S5: evenly mix the fermented wine lees obtained in step S4 with coconut husks, add binders such as sodium alginate and calcium chloride, and press into shape through a mold and dry to obtain a plant growth-promoting microbial brick.

[0018] It is further defined that the fermentation formula in step S4 is 500 g of the pretreated lees in step S3, with glucose accounting for 3% by mass, ammonium chloride accounting for 3% by mass, and composite microbial agent accounting for 6% by mass.

[0019] It is further defined that in step S5, the mass ratio of fermented lees to coconut bran is 1:1-3:1, the mass proportion of sodium alginate is 5%, the mass proportion of calcium chloride is 0.5%, and the moisture content of the mixture is 40wt%-50wt%.

[0020] It is further defined that the drying conditions in step S5 are a temperature of 30-40° C., a humidity of 50%-70%, and a drying time of 24-48 hours.

[0021] The invention relates to the application of the plant growth-promoting microbial brick in plant cultivation.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] 1. Wine lees itself contains a large amount of nutrients such as protein, amino acids, vitamins, minerals and unfermented sugars. Making them into fungus bricks can not only realize the recycling of waste resources, but also provide long-term fertility for plant growth, reducing the need for frequent fertilization.

[0024] 2. The synergistic and complementary effects of multiple strains of Bacillus subtilis, Geotrichum candidum, and Aspergillus niger increased the cellulose degradation rate and crude protein content, thereby improving the nutrient balance of fermented distiller's grains.

[0025] 3. The antimicrobial peptides produced during the complex microbial fermentation process have a significant inhibitory effect on bacteria and fungi, reducing the use of chemical fertilizers and pesticides. DETAILED DESCRIPTION

[0026] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0027] Example 1

[0028] The microbial brick in this embodiment is formed by pressing lees fermented with a composite microbial agent, coconut chaff, and a binder, wherein the composite microbial agent is composed of Bacillus subtilis, Geotrichum candidum, and Aspergillus niger, and the proportion of each component in the composite microbial agent is 34wt% of Bacillus subtilis, 33wt% of Geotrichum candidum, and 33wt% of Aspergillus niger.

[0029] The specific preparation steps are as follows:

[0030] Step S1, primary culture of the strain: Bacillus subtilis, Geotrichum candidum, and Aspergillus niger were inoculated into LB medium, YPD medium, and PDB medium, respectively, and cultured in a 30° C. incubator for 2 days for Bacillus subtilis and Geotrichum candidum, and 3 days for Aspergillus niger, respectively, to obtain plate cultures of the three strains;

[0031] Step S2, secondary culture of the strain: inoculating the plate cultures of Bacillus subtilis, Geotrichum candidum, and Aspergillus niger into liquid culture medium respectively, wherein Bacillus subtilis and Geotrichum candidum are cultured in a shaker at 220 r / min and 29° C. for 60 h, and Aspergillus niger is cultured in a shaker at 220 r / min and 29° C. for 100 h, to obtain secondary cultures of the three strains, i.e., a composite microbial agent;

[0032] Step S3, vinasse pretreatment: using a concentration of 3.75 mol·L -1 The pH value of the unfermented lees is adjusted to 6.5-7.5 with sodium hydroxide solution;

[0033] Step S4, uniformly mixing the pretreated vinasse obtained in step S3, the nutrients, and the composite microbial agent obtained in step S2, and controlling the moisture content of the material to be 35wt%-45wt%, wherein the nutrients are glucose and ammonium chloride, and the fermentation formula is 500g of pretreated vinasse, 3% by weight of glucose, 3% by weight of ammonium chloride, and 6% by weight of the composite microbial agent, aerobically fermenting at 30°C for 6 days, turning the material twice a day, and then placing the material in a breathing bag with a breathing valve and anaerobically fermenting at 30°C for 3 days to obtain fermented vinasse with increased nitrogen, phosphorus, and potassium contents;

[0034] Step S5, mixing the fermented lees obtained in step S4 with coconut chaff and adding binders of sodium alginate and calcium chloride, wherein the mass ratio of fermented lees to coconut chaff is 2:1, the mass proportion of sodium alginate is 5%, the mass proportion of calcium chloride is 0.5%, and the moisture content of the mixture is 40wt%-50wt%. The mixture is pressed into shape by a mold and dried to obtain a microbial brick that promotes plant growth, wherein the drying conditions are a temperature of 35°C, a humidity of 60%, and a drying time of 32h.

[0035] Comparative Example 1

[0036] Preparation of Bacillus subtilis:

[0037] Primary culture: Bacillus subtilis was inoculated into LB medium and cultured in a 30°C incubator for 2 days to obtain a plate culture of Bacillus subtilis.

[0038] Secondary culture: inoculate the plate culture of Bacillus subtilis into LB liquid culture medium, and culture in a shaker at 29° C. and 220 rpm for 60 h to obtain bacterial strains for use.

[0039] Preparation of fermented lees:

[0040] Pretreatment of lees: using a concentration of 3.75 mol·L -1 The pH value of the unfermented grains is adjusted to 6.5-7.5 with sodium hydroxide solution.

[0041] Fermentation substrate: 500 g of pretreated vinasse, 3% by weight of glucose, 3% by weight of ammonium chloride, 6% by weight of Bacillus subtilis, and a moisture content of 35 wt% to 45 wt% of the mixture.

[0042] The pretreated vinasse, nutrients (glucose and ammonium chloride) and bacterial agent were evenly mixed and the moisture content of the material was controlled at 35wt%-45wt%. The material was aerobically fermented at 30°C for 6 days, with the material turned over twice a day. The material was then placed in a breathing bag with a breathing valve and anaerobically fermented at 30°C for 3 days to obtain a single-strain fermented vinasse.

[0043] Preparation of mushroom bricks:

[0044] The mass ratio of single-strain fermentation wine grains to coconut bran is 2:1, the mass proportion of sodium alginate is 5%, the mass proportion of calcium chloride is 0.5%, and the moisture content of the mixture is 40wt%-50wt%.

[0045] The single-strain fermented wine grains and coconut husks are mixed in proportion and sodium alginate and calcium chloride as binders are added. The mixture is pressed into shape by a mold and dried to obtain a microbial brick. The drying conditions are a temperature of 35° C., a humidity of 60%, and a drying time of 32 hours.

[0046] Comparative Example 2

[0047] The method is basically the same as Comparative Example 1, except that the strain is Geotrichum candidum and the culture medium is YPD.

[0048] Comparative Example 3

[0049] The method is basically the same as Comparative Example 1, except that the strain is Aspergillus niger, the culture medium is PDB, the primary culture time is 3 days, and the secondary culture time is 100 hours.

[0050] Determination of total nitrogen, total phosphorus, potassium and crude fiber content in distiller's grains before and after fermentation

[0051] Total nitrogen is tested using the Kjeldahl method; total phosphorus is tested using the method described in the national standard GB / T6437-2018; potassium is tested using the method described in GB / T13885-2017; crude fiber is tested using the filter bag method described in GB / T6434-20226.

[0052] Table 1 shows the conversion of total nitrogen in distiller's grains by different bacterial strains. Analysis revealed that, under conditions where the initial total nitrogen content was 5.59 ± 0.06%, the total nitrogen content of the distiller's grains (Example 1) fermented with the composite microbial inoculant was significantly increased, with an increase of approximately 10%-19%. However, the total nitrogen content of the distiller's grains fermented with a single bacterial strain remained unchanged compared to the pre-fermentation level.

[0053] Table 1 Detection of total nitrogen content in lees before and after fermentation

[0054]

[0055]

[0056] Table 2 shows the total phosphorus content of vinasse after fermentation with different strains. Comparative analysis revealed that, under conditions where the initial total phosphorus content was 0.60 ± 0.02%, the total phosphorus content of vinasse (Example 1) after fermentation with the composite microbial inoculant was significantly increased, with an increase of approximately 7%-17%. However, the total phosphorus content of vinasse (Comparative Examples 1-3) after fermentation with a single strain was not significantly different from that of the vinasse before fermentation.

[0057] Table 2 Detection of total phosphorus content in lees before and after fermentation

[0058] Group Total phosphorus content (%) Example 1 0.67±0.03 Comparative Example 1 0.59±0.02 Comparative Example 2 0.61±0.01 Comparative Example 3 0.60±0.01 unfermented lees 0.60±0.02

[0059] Table 3 shows the potassium content of vinasse after fermentation with different strains. Comparative analysis revealed that, under the condition of an initial potassium content of 9600 ± 50 mg per kilogram of vinasse, the potassium content of the vinasse after fermentation with the composite microbial inoculant (Example 1) was significantly increased to 11000 mg ± 70 mg per kilogram of vinasse, an increase of approximately 14%-15%. However, the potassium content of the vinasse after fermentation with a single strain (Comparative Examples 1-3) did not change significantly compared to the vinasse before fermentation.

[0060] Table 3 Detection of potassium content in lees before and after fermentation

[0061] Group Potassium content (mg / kg) Example 1 11000±70 Comparative Example 1 9700±50 Comparative Example 2 9635±50 Comparative Example 3 9650±30 unfermented lees 9600±50

[0062] Table 4 shows the crude fiber content of vinasse after fermentation with different strains. Comparative analysis revealed that, under conditions where the initial crude fiber content was 13.31 ± 0.02%, the crude fiber content of the vinasse (Example 1) after fermentation with the composite microbial inoculant was significantly reduced to 10.32 ± 0.05%, a decrease of approximately 22%. However, the crude fiber content of the vinasse (Comparative Examples 1-3) after fermentation with a single strain showed no significant change compared to the vinasse before fermentation.

[0063] Table 4 Detection of crude fiber content in lees before and after fermentation

[0064]

[0065]

[0066] 2. Effect of fermented distiller's grains on ryegrass growth

[0067] The experiment was divided into 4 groups, namely Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3, with 3 replicates in each group. The experiment adopted broadcast sowing with a sowing rate of 175g / m 2 The sowing depth was about 3 cm. The growth performance of ryegrass was tested during the growth period. Ten ryegrass plants were selected for each repetition and the number of leaves of each ryegrass plant was counted. The representative 10 cm 2 The number of branches larger than 20 cm in the sample segment. Use a tape measure to measure plant height, which is the height from the ground to the top of the ear.

[0068] Measure the length and width of the leaf and calculate the leaf area:

[0069] Leaf area = leaf length × leaf width × 0.7

[0070] Table 5 shows the effects of fermented distiller's grains on ryegrass growth. Comparative analysis revealed that the bacterial bricks made from fermented distiller's grains with the composite microbial strain significantly promoted ryegrass growth, with leaf count, plant height, branch number, and leaf area all exceeding those of the unfermented distiller's grains and other control groups.

[0071] Table 5 Effect of fermented distiller's grains on ryegrass growth

[0072] Group Number of leaves / (pieces / plant) Plant height / cm <![CDATA[Number of branches / (branches / dm 2 )]]> <![CDATA[Leaf area / cm 2 > Example 1 5.32±0.32 31.08±2.48 12.10±0.75 8.70±0.51 Comparative Example 1 3.94±0.23 26.78±1.72 7.54±0.57 7.50±0.43 Comparative Example 2 4.28±0.31 25.89±1.67 7.61±0.54 7.25±0.39 Comparative Example 3 4.09±0.32 27.03±1.54 7.82±0.56 7.57±0.41 unfermented lees 3.58±0.27 25.32±1.56 6.98±0.52 7.09±0.36

[0073] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation utilizing the contents of the present invention specification, or any direct or indirect application in other related technical fields, shall similarly be included in the patent protection scope of the present invention.

Claims

1. A microbial brick for promoting plant growth, characterized by: The microbial brick is formed by pressing wine dregs fermented with a composite microbial agent, coconut bran and a binder, wherein the composite microbial agent consists of Bacillus subtilis, Geotrichum candidum and Aspergillus niger.

2. The plant growth promoting microbial brick according to claim 1, characterized in that: The composition ratio of each component in the composite microbial agent is 30 wt%-35 wt% of Bacillus subtilis, 30 wt%-35 wt% of Geotrichum candidum and 30 wt%-35 wt% of Aspergillus niger.

3. The plant growth promoting microbial brick according to claim 1, characterized in that: The weight ratio of the vinasse after fermentation with the composite microbial agent to the coconut bran is 1:1-3:

1.

4. The plant growth promoting microbial brick according to claim 1, characterized in that: The binder is sodium alginate and calcium chloride.

5. The microbial brick for promoting plant growth according to claim 1, characterized in that: The composite microbial agent is used for degrading cellulose, hemicellulose and crude protein.

6. A method for preparing a plant growth-promoting microbial brick according to any one of claims 1 to 5, characterized in that The specific preparation steps are: Step S1, primary culture of the strain: Bacillus subtilis, Geotrichum candidum, and Aspergillus niger were inoculated into LB medium, YPD medium, and PDB medium, respectively, and cultured in a 30° C. incubator for 2 days for Bacillus subtilis and Geotrichum candidum, and 3 days for Aspergillus niger, respectively, to obtain plate cultures of the three strains; Step S2, secondary culture of the strain: inoculating the plate cultures of Bacillus subtilis, Geotrichum candidum, and Aspergillus niger into liquid culture medium respectively, wherein Bacillus subtilis and Geotrichum candidum are cultured in a shaker at 220 r / min and 29° C. for 48-72 h, and Aspergillus niger is cultured in a shaker at 220 r / min and 29° C. for 96-120 h, to obtain secondary cultures of the three strains, i.e., a composite microbial agent; Step S3, vinasse pretreatment: using a concentration of 3.75 mol·L -1 The pH value of the unfermented lees is adjusted to 6.5-7.5 with sodium hydroxide solution; Step S4, uniformly mixing the vinasse pretreated in step S3, the nutrients, and the composite microbial agent obtained in step S2, controlling the moisture content of the material to be 35 wt%-45 wt%, wherein the nutrients are glucose and ammonium chloride, aerobically fermenting the mixture at 30° C. for 5-7 days, turning the mixture twice a day, and then placing the mixture in a breathing bag with a breathing valve and anaerobically fermenting the mixture at 30° C. for 2-4 days to obtain fermented vinasse with increased nitrogen, phosphorus, and potassium contents; Step S5: evenly mix the fermented wine lees obtained in step S4 with coconut husks, add binders such as sodium alginate and calcium chloride, and press into shape through a mold and dry to obtain a plant growth-promoting microbial brick.

7. The method for preparing a plant growth-promoting microbial brick according to claim 6, characterized in that: The fermentation formula in step S4 is 500 g of the pretreated lees in step S3, 3% by weight of glucose, 3% by weight of ammonium chloride, and 6% by weight of the composite microbial agent.

8. The method for preparing a plant growth-promoting microbial brick according to claim 6, characterized in that: In step S5, the mass ratio of fermented vinasse to coconut bran is 1:1-3:1, the mass proportion of sodium alginate is 5%, the mass proportion of calcium chloride is 0.5%, and the moisture content of the mixture is 40wt%-50wt%.

9. The method for preparing a plant growth-promoting microbial brick according to claim 6, wherein: The drying conditions in step S5 are a temperature of 30-40° C., a humidity of 50%-70%, and a drying time of 24-48 hours.

10. Use of the plant growth promoting microbial brick according to any one of claims 1 to 5 in plant cultivation.

Images