Organic waste degradation complex microbial inoculant and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and provides an organic waste degradation complex microbial inoculant and application thereof. The composting bacillus DF-3 and the bacillus subtilis YB-5 disclosed by the invention can generate a large amount of cellulase activity, and the yields of beta-glucosidase, exoglucanase and endoglucanase are 50.65 U / mL, 45.73 U / mL and 69.08 U / mL respectively, and the yields of beta-glucosidase, exoglucanase and endoglucanase are 69.08 U / mL, 48.74 U / mL and 94.1 U / mL respectively. The compost bacillus DF-3 and the bacillus subtilis YB-5 have a high corn straw degradation rate, a microbial agent compounded by the compost bacillus DF-3 and the bacillus subtilis YB-5 shows a high corn straw degradation effect, the degradation rate can reach 42.64% after the corn straw is fermented for 14 days, the straw returning degradation speed can be greatly increased, meanwhile, the compost bacillus DF-3 and the bacillus subtilis YB-5 can be used for pretreatment of compost straw auxiliary materials, and the application range of the compost bacillus DF-3 and the bacillus subtilis YB-5 is widened. The resource utilization rate is improved. The compounded microbial inoculum can accelerate in-situ decomposition of straw returned to the field, can promote temperature rise and decomposition of a compost body in the composting (retting) process, and improves the quality of the composting (retting) fertilizer.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically, it relates to a compound microbial agent for the degradation of organic waste and its application. Background Technology

[0002] Currently, my country's annual output of organic solid waste such as livestock and poultry manure and crop straw reaches as high as 4 billion tons (wet basis). Composting is the main method for treating livestock and poultry manure and straw. Under normal circumstances, straw can be directly or separately composted and then returned to the field. Statistics show that fertilization, mainly through returning straw to the field, accounts for 54.70% of the resource utilization of collectable straw, making it the most important method of straw resource utilization. Livestock and poultry manure has characteristics such as high moisture content and dense structure, making it difficult to heat up quickly during composting. Straw is usually added as an auxiliary material to adjust the compost conditions. However, straw itself is mainly composed of lignocellulose, with a dense and complex structure, resulting in high degradation resistance and slow decomposition. This problem is significant in the processes of directly returning straw to the field, composting it alone, or composting it with livestock and poultry manure, manifesting as slow temperature rise in the compost pile, low straw degradation efficiency, insignificant degradation in the short term, long fermentation cycle, and low maturity. Therefore, accelerating the degradation of straw is key to straw return to the field, composting of livestock and poultry manure, soil fertilization, and resource utilization.

[0003] Whether straw is returned to the field or used as a composting agent, the degradation of straw mainly depends on the quantity and degradation capacity of the microbial community. Typically, the number of microorganisms capable of degrading straw in farmland soil and livestock manure is small, and their degradation capacity is weak. Utilizing microorganisms to produce cellulase to degrade straw is not only more efficient but also has advantages such as being environmentally friendly. For example, CN118995469B discloses a facultative anaerobic bacterial community that promotes both direct straw return to the field and rice growth, achieving a straw degradation rate of approximately 40% after 40 days. CN118389297B discloses a compound microbial agent that promotes the fermentation and decomposition of corn straw, significantly increasing the degradation rate of lignocellulose in the straw; after 28 days of treatment, the lignocellulose degradation rate reached 31.04%-39.38%. CN119144516A discloses a microbial agent primarily composed of Bacillus subtilis; after 30 days of treatment, the corn straw decomposition rate reached 41.7%-50%, but after 15 days, the straw degradation rate was only about 20%, indicating a slow degradation rate that falls far short of the requirements for straw resource utilization. Therefore, finding a rapid and efficient straw-degrading microorganism and compounding a straw-degrading agent is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a compound microbial agent for the degradation of organic waste and its application.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides a composite microbial agent for the rapid degradation of organic waste, the composite microbial agent being composed of Bacillus stercoris DF-3 bacterial solution and Bacillus subtilis YB-5 bacterial solution.

[0006] Furthermore, the compound microbial agent contains 10% Bacillus composting DF-3 bacteria. 8 -10 9 CFU / mL, Bacillus subtilis YB-5 bacterial count was 10. 8 -10 9 The CFU / mL ratio of the two bacteria is preferably 1:1.

[0007] The *Bacillus compostingus* DF-3 and *Bacillus subtilis* YB-5 of this invention are derived from compost fermentation products made from cow dung and straw, as well as humus and decaying wood, and were obtained through directional domestication and screening using CMC liquid culture medium. *Bacillus compostingus* DF-3 is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 35657, deposit date August 18, 2025. *Bacillus subtilis* YB-5 is also currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 35800, deposit date August 18, 2025.

[0008] Secondly, the present invention provides the application of the compound microbial agent in the degradation of agricultural and forestry waste.

[0009] The agricultural and forestry waste includes crop straw, preferably corn straw.

[0010] Thirdly, the present invention provides a method for returning corn stalks to the field, wherein the corn stalks are crushed, mixed with the compound microbial agent, and then covered with 10-12cm of soil for returning the stalks to the field.

[0011] Furthermore, the corn stalks and compound microbial agent are mixed at a ratio of 0.5g:1mL to 1g:0.5mL, preferably at a ratio of 1g:1mL.

[0012] Fourthly, the present invention provides the application of the compound microbial agent in the composting of crop straw and manure.

[0013] Fifthly, the present invention provides a method for composting crop straw and manure, wherein the compound microbial agent is directly inoculated into the mixture of crop straw and manure for composting fermentation; or,

[0014] First, the crop straw is pretreated with the compound microbial agent, and then the pretreated crop straw is mixed with manure for composting.

[0015] Preferably, crop straw and manure are mixed at a wet weight ratio of 1:9 to 3:7.

[0016] Furthermore, the crop straw can be selected from various agricultural and forestry straws such as corn straw, rice straw, and wheat straw.

[0017] Furthermore, the excrement comes from various livestock and poultry manure such as pig manure, cow manure, and sheep manure, and is preferably manure rich in lignocellulose.

[0018] In a sixth aspect, the present invention provides the application of the compound microbial agent in the production of β-glucosidase, exoglucanase, endoglucanase, protease and lignin-degrading enzyme.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0020] (i) The composting Bacillus DF-3 and Bacillus subtilis YB-5 of the present invention can produce a large amount of cellulase activity, and the yields of β-glucosidase, exoglucanase and endoglucanase are 50.65, 45.73 and 69.08 U / mL and 69.08, 48.74 and 94.1 U / mL, respectively.

[0021] (ii) The composting Bacillus DF-3 and Bacillus subtilis YB-5 of the present invention have a high degradation rate of corn straw. After 14 days of fermentation, the degradation rate of corn straw can reach 20.74%-26.32% and 16.11%-17.2% respectively, which is higher than the 5.57% of the control group.

[0022] (III) The compounded microbial agent of this invention exhibits a higher degradation effect on corn straw. When Bacillus composting DF-3 is compounded with Bacillus subtilis YB-5, the degradation rate of corn straw after 14 days of fermentation can reach 42.64%, which can greatly improve the degradation speed of straw returning to the field. At the same time, it can be used as a pretreatment of compost straw auxiliary material to improve its resource utilization rate.

[0023] (iv) The compound microbial agent of this invention can not only accelerate the in-situ decomposition of straw returned to the field, but also promote the temperature rise and maturity of the compost pile during the composting process. During the return to the field, the straw weight reduction rate of corn straw returned to the field for 40 days was as high as 56.3%, while the CK treatment without adding microbial agent was only 9.4%. During the composting process, the compound microbial agent can accelerate the degradation of organic matter, promote the temperature rise of the compost pile, and improve the seed germination rate of the composted products. Among them, in the pure straw composting process, compared with the treatment group without adding microbial agent, the treatment group with the microbial agent of this invention increased the pile temperature by 13℃, and the lignocellulose degradation rate and seed germination rate index increased by 30% and 32%, respectively; when the microbial agent of this invention was added during the combined natural composting of straw and pig manure or forced continuous ventilation aerobic composting, the pile temperature, the duration of high temperature, and the seed germination rate index increased by 5~8℃, 4~9 days, and 29~37%, respectively. Furthermore, the use of the microbial agent of this invention during composting or aerobic composting can significantly increase the degradation rate of lignocellulose by 8.5%-31%, thus improving the quality of compost. In particular, the effect is even more pronounced when straw is pre-treated and fermented with the microbial agent before being used as an auxiliary material for composting or aerobic composting, with seed germination rates reaching over 120%.

[0024] (v) In addition to having a good ability to produce cellulase, the composting Bacillus DF-3 and Bacillus subtilis YB-5 of the present invention can also produce a certain amount of protease and a small amount of lignin-degrading enzymes, such as laccase, manganese oxidase and peroxidase, which synergistically promote the resource utilization of straw.

[0025] (vi) Compared to other reports on straw degradation, such as the SX-7-2 *Phanerochaete chrysosporium* strain in CN119307384A which showed a 31.2% degradation rate in rice straw after 15 days of treatment, this is slightly higher than the degradation rate of corn straw after 14 days of treatment with single strains of *Bacillus compostii* DF-3 and *Bacillus subtilis* YB-5, but far lower than the 42.64% degradation rate of corn straw achieved by the compound microbial agent of this invention. Furthermore, the straw degradation rate of the microbial agent composed of single strains of *Bacillus compostii* DF-3 and *Bacillus subtilis* YB-5 is significantly higher than that of commercially available straw degradation microbial agents. In composting applications, for example, the microbial agent composed of Bacillus NJAU-190 and Xanthomonas pseudoxanthomonas NJAU-273 in CN119162059B only maintains a high temperature duration of 6 days during composting, which is shorter than that of the microbial agent of this invention. Specifically, the microbial agents composed of Bacillus composting DF-3 and Bacillus subtilis YB-5, as well as their combination, all show good degradation effects and potential for corn straw, and the combination agents are also effective in promoting the temperature rise and decomposition of the compost pile. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of strain antagonism in a preferred embodiment of the present invention, where a, b, c, and d represent different strains.

[0027] Figure 2 The Congo red cellulose plates of Bacillus compostingus DF-3 and Bacillus subtilis YB-5, which were initially screened in a preferred embodiment of the present invention.

[0028] Figure 3 The strain morphology of Bacillus compostii DF-3 and Bacillus subtilis YB-5 is shown in the preferred embodiment of the present invention.

[0029] Figure 4 The results of the antagonistic experiment between Bacillus compostii DF-3 and Bacillus subtilis YB-5 in a preferred embodiment of the present invention are shown.

[0030] Figure 5 The cellulase activity of Bacillus compostii DF-3 and Bacillus subtilis YB-5 in a preferred embodiment of the present invention is shown.

[0031] Figure 6 The weight reduction rate of corn stalks in a preferred embodiment of the present invention.

[0032] Figure 7 This is a preferred embodiment of the present invention showing the degradation of corn stalks treated as a control (CK) after 15 days.

[0033] Figure 8 This is an example of the degradation of corn straw treated with Bacillus composting DF-3 in Experiment 1 of the preferred embodiment of the present invention after 15 days.

[0034] Figure 9 This is Experiment Example 2, a preferred embodiment of the present invention, showing the degradation of corn stalks after 15 days of treatment with Bacillus subtilis YB-5.

[0035] Figure 10 This is an example of the degradation of corn straw treated with Bacillus compostii DF-3 and Bacillus subtilis YB-5 in Experiment 3 of the preferred embodiment of the present invention after 15 days.

[0036] Figure 11 The degradation of corn stalks after 15 days of treatment with a commercially available straw degradation microbial agent 1 is shown in Comparative Example 1, a preferred embodiment of the present invention.

[0037] Figure 12 The degradation of corn stalks after 15 days of treatment with a commercially available straw degradation microbial agent 2, which is a preferred embodiment of the present invention, is shown in Comparative Example 2.

[0038] Figure 13 The weight reduction rate of corn stalks during the returning process in a preferred embodiment of the present invention.

[0039] Figure 14 The changes in temperature, lignocellulose degradation, and seed germination rate during the natural composting of corn stalks in a preferred embodiment of the present invention are shown.

[0040] Figure 15 The changes in temperature, lignocellulose degradation, and seed germination rate during the combined natural composting of corn stalks and pig manure after microbial agent pretreatment are shown in a preferred embodiment of the present invention.

[0041] Figure 16 The changes in temperature, lignocellulose degradation, and seed germination rate during the combined natural composting of corn stalks and pig manure in a preferred embodiment of the present invention are shown.

[0042] Figure 17 The changes in temperature, lignocellulose degradation, and seed germination rate during the combined aerobic composting of corn stalks and pig manure in a preferred embodiment of the present invention are shown. Detailed Implementation

[0043] This invention provides a microbial agent composed of Bacillus compostii DF-3 and Bacillus subtilis YB-5 and its application in straw degradation.

[0044] The composting Bacillus of the present invention belongs to a subspecies of Bacillus subtilis, and is classified as Bacillus stercoris DF-3, with accession number CGMCC No. 35657 and accession date of August 18, 2025.

[0045] The Bacillus subtilis YB-5 of this invention has the accession number CGMCC No. 35800 and the accession date is August 18, 2025.

[0046] Bacillus compostii DF-3 and Bacillus subtilis YB-5 can produce cellulase efficiently. After being cultured at 35℃ and 160r / min for 48h, the yields of β-glucosidase, exoglucanase, and endoglucanase were 50.65, 45.73, and 69.08 U / mL and 69.08, 48.74, and 94.1 U / mL, respectively.

[0047] Both Bacillus compostingus DF-3 and Bacillus subtilis YB-5 of the present invention have good straw degradation performance. When the OD600 of 150 mL of Bacillus compostingus DF-3 and Bacillus subtilis YB-5 bacterial cultures is increased to 3, 5 g of corn straw (2-8 cm) is added to each culture and cultured at 35℃ and 160 r / min for 14 days, the weight reduction rate of corn straw can reach 26.32% and 17.2%, respectively.

[0048] Bacillus compostingus DF-3 and Bacillus subtilis YB-5 were cultured for 5-7 days respectively. 150 mL of bacterial solution was prepared with an OD600=3 ratio (the ratio of the two bacteria was 1:1). 5 g of corn stalks with a length of 2-8 cm was added. After culturing at 35℃ and 160 r / min for 14 days, the weight loss rate of the corn stalks could reach as high as 42.64%.

[0049] The microbial agent of this invention, through straw return to the field experiment and four types of natural composting and aerobic composting experiments, showed that it was effective in accelerating in-situ decomposition of straw and promoting the warming and maturation of the compost pile. When applied to straw returned to the field, the microbial agent showed that after 40 days, the straw weight reduction rate of corn straw reached as high as 56.3%, far exceeding the 9.4% of the control treatment. The microbial agent of this invention, through application to four types of composting experiments, showed that it was effective in promoting the warming and decomposition of the compost pile. These four composting experiments were: 2% compound microbial solution (based on straw weight) + pure corn straw composting alone; 2% compound microbial solution + corn straw-pig manure combined composting (straw and pig manure mixed at a wet weight ratio of 1:9); corn straw-pig manure combined composting after 2% microbial solution pretreatment; and 2% compound microbial solution + corn straw-pig manure combined aerobic composting process (straw and pig manure mixed at a wet weight ratio of 1:9). The use of microbial agents in these four composting processes can significantly promote the temperature rise of the compost pile, increase the duration of the high-temperature period, the degradation rate of lignocellulose, and the seed germination rate. In particular, when straw pretreated with the microbial agent of this invention is used as an auxiliary material in the co-composting and aerobic composting of pig manure, the temperature rises rapidly, reaching a high temperature of 58-61℃ by the end of the first day of composting, with a high-temperature period lasting up to 15 days. The lignocellulose degradation rate can reach up to 43.5%, and the seed germination rate index reaches over 120%.

[0050] In addition to their excellent cellulase-producing ability, the Bacillus compostii and Bacillus subtilis of the present invention can also produce a certain amount of protease and a small amount of lignin-degrading enzymes, such as laccase, manganese oxidase and peroxidase, to synergistically promote the resource utilization of high lignocellulose wastes such as straw.

[0051] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0052] The culture medium used in the following examples:

[0053] Screening medium (CMC liquid medium): 2.5g K2HPO4, 2.5g Na2HPO4, 20.0g sodium carboxymethyl cellulose, 2.0g peptone, 0.5g yeast extract powder, add deionized water to a final volume of 1 L, and sterilize at 121℃ for 30 min.

[0054] Fermentation enzyme production medium (used only during enzyme activity quantification in step 2): 3 g peptone, 2 g (NH4)2SO4, 0.3 g CaCYB-5(CaCO3)·2H2O, 0.3 g MgSO4, 4 g KH2PO4, 1% Tween-80, 20 g microcrystalline cellulose, add deionized water to a final volume of 1 L, adjust pH to 5.5, and sterilize at 121℃ for 30 min.

[0055] Example 1: Obtaining Bacillus compostii DF-3 and Bacillus subtilis YB-5

[0056] Step 1: Initial Screening

[0057] 10g of compost sample, mainly composed of cow dung and wheat straw fermented at high temperature, and 10g of humus and decaying wood were weighed and placed in 250mL Erlenmeyer flasks. 90mL of sterile water was added, and the samples were incubated at 30℃ and 160rpm in a constant-temperature shaking incubator for 30min. 5mL of the bacterial culture was then added to another 250mL Erlenmeyer flask, followed by 95mL of sodium carboxymethyl cellulose (CMC) liquid culture medium. The samples were incubated at 30℃ and 160rpm in a constant-temperature shaking incubator for 7 days to selectively acclimate and screen for cellulose-degrading strains. The samples were then treated using a serial dilution method, with the concentration of the sample solution progressively diluted to 10 using sterile physiological saline. -1 -10 -7 The samples were spread onto screening medium (cellulose Congo red agar) plates and incubated upside down at 30°C. Strains with strong cellulose degradation capabilities were initially screened based on colony size and hydrolysis zones. The candidate strains were then purified by streaking on cellulose Congo red agar plates 2-4 times, numbered, and temporarily stored at 4°C. Some preliminary screening results are shown below. Figure 2 As shown.

[0058] Step 2: Secondary screening

[0059] The strains obtained from the initial screening were inoculated into 50 mL of seed culture medium and cultured at 30℃ and 140 rpm for 1-2 days. The cultured seed culture was then inoculated into CMC liquid medium (i.e., fermentation enzyme production medium) at an inoculation rate of 3%-5% (OD600=1). Cellulase activities such as β-glucosidase, exoglucanase, and endoglucanase were measured (quantitative enzyme activity results are shown in the figure). Figure 5 As shown), after secondary screening, two strains with high cellulase activity, DF-3 and YB-5, were obtained (colony morphology as shown). Figure 3 (As shown).

[0060] Step 3: Molecular biological identification

[0061] The rescreened strains were selected and streaked onto LB agar plates to preliminarily distinguish between bacteria and fungi. The purified rescreened strains were then sent to a sequencing company for 16S rDNA sequencing. Subsequently, the obtained sequences were uploaded to NCBI for BLAST alignment and strain identification. The identification results showed that strain DF-3 was Bacillus compostii (SEQ ID NO:1) and strain YB-5 was Bacillus subtilis (SEQ ID NO:2).

[0062] Example 2: Compound preparation of microbial agents

[0063] The strains obtained from the secondary screening, such as Bacillus subtilis and Bacillus compostii, were subjected to antagonistic experiments. Bacillus compostii and Bacillus subtilis were respectively streaked onto CMC plates, as shown in the streaking pattern. Figure 1 As shown, the strains were inverted and incubated in a 35°C incubator for 2-5 days to observe the antagonism between the strains.

[0064] Experimental results showed that there was no significant antagonistic effect between Bacillus compostii and Bacillus subtilis. Figure 4 ).

[0065] Example 3: Application of microbial agents in straw degradation

[0066] The straw used was corn straw. Bacillus compostingus DF-3 and Bacillus subtilis YB-5 strains were streaked onto LB agar plates and incubated at 35℃ for 1-2 days. Single colonies were then inoculated into LB liquid medium and cultured at 30-35℃ and 160 rpm for 3-5 days to obtain the fermentation broth. Commercially available straw-degrading microbial agents 1 and 2 (agent 1 purchased from Shandong Junde Biotechnology Co., Ltd., mainly composed of Bacillus, Bacillus natto, Actinomycetes, Trichoderma, and yeast; agent 2 purchased from Henan Yuchuangfeng Agricultural Materials Co., Ltd., mainly composed of Bacillus, Trichoderma, yeast, and actinomycetes) were inoculated into LB liquid medium at a 3% inoculation ratio and cultured at 30-35℃ and 160 rpm for 3-5 days to obtain the fermentation broth. Accurately weigh 5 g of corn stalks dried at 65℃, with a particle size of 2-8 cm, and add 150 ml of cellulose-degrading bacterial agent (i.e., the three bacterial solutions from Experiments 1-3 and the two bacterial agents from Comparative Examples 1-2). Incubate at 30-35℃ and 160 r / min. Observe the degradation of straw at 0, 3, 7, and 14 days, and measure the straw weight loss rate on day 14. A total of 8 treatments were set up, with 3 replicates for each treatment. The composition of different bacterial strains is shown in Table 1.

[0067] Table 1 Composition of different bacterial strains

[0068] deal with Microbial composition CK No additional bacterial agent added Experimental Example 1 Bacillus subtilis in compost Experiment Example 2 Bacillus subtilis bacterial culture Experimental Example 3 A compound bacterial culture of Bacillus compostii and Bacillus subtilis Comparative Example 1 Commonly available straw degradation microbial agents 1 Comparative Example 2 Commonly available straw degradation microbial agents 2

[0069] Note: CK represents the control treatment without added inoculant, with 150 mL of LB liquid medium added. The bacterial count of Bacillus compost in Experiment 1 was approximately 10. 8 CFU / mL, the bacterial count of Bacillus subtilis in Experiment 2 was approximately 10. 8 CFU / mL, the bacterial content of Bacillus compostii and Bacillus subtilis in the compound bacterial solution of Experiment Example 3 was approximately 10 CFU / mL. 8 CFU / mL, 10 8 CFU / mL.

[0070] After fermentation, the bacterial solution is filtered with filter paper, and the filtered straw residue is repeatedly rinsed with deionized water to ensure that the bacteria are removed. The collected straw residue is then placed in a 65℃ oven for 12-24 hours, and the straw weight loss rate is calculated using the weight reduction method.

[0071] Calculation formula:

[0072] In the formula: R is the dry mass of straw residue after fermentation, in g; R0 is the initial dry mass of straw, in g.

[0073] Experimental results showed that adding microbial agents significantly promoted straw degradation, especially in Experiment 3 where the straw degradation rate reached 42.64%. Figures 6-12 (), which is much higher than other treatments.

[0074] Example 4: Application of microbial agents in the process of returning corn straw to the field

[0075] Corn stalks were crushed to 2-3 cm thickness. 5 g of corn stalks dried at 65℃ were accurately weighed and placed into nylon mesh bags with dimensions of 20 cm x 10 cm. In a field in Yutian County, Tangshan City, the nylon mesh bags containing the corn stalks were placed in 12 cm deep pits. A compound bacterial solution (the same compound bacterial solution used in Experimental Example 3 of Example 3) or sterile water was poured onto the surface of the stalks. The inoculation amount was 1 ml / g of corn stalks. Then, approximately 10 cm of soil was covered on the surface of the nylon bags. The treatment with added bacterial solution served as the experimental group, and the treatment with added sterile water served as the control group. Each treatment was replicated 12 times. The ambient temperature for straw return to the field was maintained between 20-30℃ for 40 days. During the first week, sterile water was applied to the soil surface daily, and then sterile water was applied every other day to keep the soil submerged by 2-3 cm. On the 10th, 20th, 30th, and 40th days after straw return to the field, three nylon bags were removed according to the treatment. The surface of the straw and the microorganisms were repeatedly rinsed with deionized water to ensure that the soil and microorganisms were removed. The collected straw residue was then placed in a 65℃ oven for 12-24 hours, and the straw weight loss rate was calculated using the weight reduction method.

[0076] Experimental results show that adding the microbial agent of this invention during the straw return process can significantly improve the straw decomposition effect, with a straw weight reduction rate as high as 56.3% after 40 days of return to the field. Figure 13 The result was significantly higher than that of the CK treatment, which had a result of only 9.4%.

[0077] Example 5: Application of microbial agents in the natural composting process of straw

[0078] Corn stalks were crushed to 2-3 cm, and water was added to adjust the moisture content to 60%. Based on the stalk weight, 2% of the stalk weight of bacterial solution (the same compound bacterial solution used in Experiment 3 of Example 3) was added. The stalks and bacterial solution were mixed, a certain amount of urea was added, and the mixture was thoroughly stirred. A control group without the added bacterial solution was used, and 2% of the stalk weight of sterile water was added and mixed thoroughly. After thorough mixing, the mixture was piled in a sealed container with dimensions of 52 cm (length), 39 cm (width), and 37 cm (height), with three replicates for each treatment. The entire composting period was 30 days. The pile temperature was recorded daily. The pile was turned over on days 7 and 15. Multiple sampling points were used on days 0 and 30, and at each turning, solid samples were uniformly collected for seed germination rate and lignocellulose content determination.

[0079] The composted product was air-dried and pulverized to determine the lignocellulose content. Lignin, cellulose, and hemicellulose contents were determined using a cellulose analyzer (ANKOM2000, ANKOM Corporation, USA) via the Pantheon acid-base washing method. The composted product was mixed with deionized water at a ratio of 1 g:10 mL (mass / volume) for water extraction. After shaking for 30 min, the extract was filtered. 5 mL of the extract (with 5 mL of deionized water as a blank control) was poured into a petri dish lined with filter paper to determine the seed germination index (GI). Ten radish seeds were evenly placed in each petri dish and cultured at 25 ± 1 ℃ for 48 h. The number of germinated seeds and root length were then measured. The GI value was calculated using the following formula:

[0080] GI 100%

[0081] Experimental results show that adding the microbial agent of this invention during the natural composting process of straw can significantly promote the temperature rise of the compost pile and improve the composting effect. Specifically, the maximum temperature of the compost pile during the process can reach 52℃, the maximum lignocellulose degradation rate can reach 41%, and the seed germination rate index can reach 95%, which are respectively 13℃, 30%, and 32% higher than the treatment group without the microbial agent. Figure 14 ).

[0082] Example 6: Application of microbial agents in straw pretreatment during straw-pig manure co-composting

[0083] Corn stalks were crushed to 2-3 cm, and water was added to adjust the moisture content of the stalks to 60% before use. Based on the stalk weight, 2% bacterial solution (the same compound bacterial solution as in Experiment 3 of Example 3) was added, and the mixture was placed in an open box measuring 52.0 cm × 38.5 cm × 37.0 cm for pretreatment at room temperature in summer for 5 days. After pretreatment, the pretreated corn stalks and fresh pig manure were mixed at a wet weight ratio of 1:9. Using untreated corn stalks as a control, corn stalks and fresh pig manure were mixed at a wet weight ratio of 1:9, water was added to adjust the moisture content of the fermentation materials to 60%, and the mixture was thoroughly mixed. The mixture was then placed in a closed box with dimensions of 52 cm (length), 39 cm (width), and 37 cm (height), with three replicates for each treatment. The entire composting cycle was 30 days. The temperature of the compost pile was recorded daily. The pile was turned over on the 5th, 10th and 20th days. On the 0th day, the 30th day and each time the pile was turned, a multi-point sampling method was used to uniformly collect solid samples for the determination of seed germination rate and lignocellulose content.

[0084] Experimental results show that in straw-pig manure co-composting, pretreatment of straw with the microbial agent of this invention significantly promotes the temperature rise of the compost pile, prolongs the duration of the high-temperature period, and improves the quality of composting. Specifically, the pile temperature reached as high as 58℃ at the end of the first day of composting, the high-temperature period lasted for 15 days, and the seed germination rate at the end of composting reached 120%, far exceeding the 50℃, 6-day, and 73% of the experimental group without pretreatment with the microbial agent of this invention. Figure 15 ).

[0085] Example 7: Application of microbial agents in the composting process of straw and pig manure

[0086] Corn stalks crushed to 2-3 cm and fresh pig manure were mixed at a wet weight ratio of 1:9, and water was added to adjust the moisture content of the fermentation materials to 60%. Based on the weight of the fermentation materials, 2% of the bacterial solution (the same compound bacterial solution as in Experiment 3 of Example 3) was added, with a control group not receiving the mixed bacterial solution. 2% of the straw weight of sterile water was also added and mixed thoroughly. After thorough mixing, the mixture was placed in an open container with dimensions of 52 cm (length), 39 cm (width), and 37 cm (height), with three replicates for each treatment. The entire composting period was 30 days. The pile temperature was recorded daily. The pile was turned over on days 5, 10, and 20. On day 0, day 30, and each time the pile was turned, multiple sampling points were used to uniformly collect solid samples for seed germination rate and lignocellulose content determination.

[0087] Experimental results show that the use of the microbial agent of this invention in straw-pig manure co-composting can significantly promote the temperature rise of the compost pile, prolong the duration of the high-temperature period, and improve the quality of composting. Specifically, the pile temperature reached as high as 55℃ at the end of the first day of composting, the high-temperature period lasted for 15 days, and the seed germination rate at the end of composting reached 109%, far exceeding the 51℃, 11 days, and 90% of the experimental group without the added mixed microbial solution. Figure 16 ).

[0088] Example 8: Application of microbial agents in the aerobic composting process of straw and pig manure

[0089] Corn stalks crushed to 2-3 cm and fresh pig manure were mixed at a wet weight ratio of 1:9, and water was added to adjust the moisture content of the fermentation materials to 60%. Based on the weight of the fermentation materials, 2% of bacterial solution (the same compound bacterial solution as in Experiment 3 of Example 3) was added, with a control group not containing the mixed bacterial solution. 2% of the straw weight of sterile water was also added and mixed thoroughly. The mixture was then added to a 60 L stainless steel insulated, forced-ventilation, sealed composting fermentation tank for forced continuous aerobic composting at a ventilation rate of 0.24 L·kg·DM. -1 ·min -1 A layer of insulation cotton is placed between the double-layer stainless steel plates on the walls of the fermentation tank to prevent heat loss. The fermentation cycle is 25 days. The pile is turned manually every 5 days. The temperature of the pile is recorded daily. On day 0, day 25, and each time the pile is turned, a multi-point sampling method is used to uniformly collect solid samples for the determination of seed germination rate and lignocellulose content.

[0090] Experimental results show that the use of the microbial agent of this invention in aerobic composting of straw and pig manure can significantly shorten the composting cycle and improve the composting effect. Specifically, the pile temperature reached as high as 61℃ at the end of the first day of composting, with a high-temperature period lasting up to 15 days. The seed germination rate at the end of composting reached 124%, far exceeding the 51℃, 11 days, and 90% of the experimental group without added mixed microbial solution. Figure 17 ).

[0091] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Organic waste degrading complex microbial inoculum, characterized in that, The compound microbial agent is composed of Bacillus stercoris DF-3 bacterial solution and Bacillus subtilis YB-5 bacterial solution; The preservation number of Bacillus composting DF-3 is CGMCC No. 35657, and the preservation number of Bacillus subtilis YB-5 is CGMCC No. 35800.

2. The complex bacterial agent according to claim 1, characterized by, The compound microbial agent contains 10% Bacillus composting DF-3 bacteria. 8 -10 9 CFU / mL, Bacillus subtilis YB-5 bacterial count was 10. 8 -10 9 The CFU / mL ratio of the two bacteria is preferably 1:

1.

3. The application of the compound microbial agent according to claim 1 or 2 in the degradation of agricultural and forestry waste; The agricultural and forestry waste includes crop straw, preferably corn straw.

4. A method of corn stalk field returning characterized by, The corn stalks are crushed, mixed with the compound microbial agent described in claim 1 or 2, and then covered with 10-12 cm of soil for straw return to the field.

5. The method of claim 4, wherein, Corn stalks and compound microbial agents are mixed at a ratio of 0.5g:1mL to 1g:0.5mL, preferably at a ratio of 1g:1mL.

6. The application of the compound microbial agent according to claim 1 or 2 in the composting or fermentation of crop straw and manure.

7. A method for composting or vermicomposting crop residues mixed with manure, characterized in that, The compound microbial agent described in claim 1 or 2 is directly inoculated into a mixture of crop straw and manure for composting or fermentation; or, First, pretreat crop straw with the compound microbial agent described in claim 1 or 2, and then mix the pretreated crop straw with manure for composting or fermentation.

8. The method of claim 7, wherein, Crop straw and manure are mixed at a wet weight ratio of 1:9 to 3:

7.

9. The method according to claim 7 or 8, characterized in that, The crop straw is selected from corn straw, rice straw, and wheat straw; The feces are derived from pig manure, cow manure, or sheep manure, and are preferably feces rich in lignocellulose.

10. The application of the compound microbial agent according to claim 1 or 2 in the production of β-glucosidase, exoglucanase, endoglucanase, protease and lignin-degrading enzyme.

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