A composite bacterial agent for promoting lignocellulose degradation and humus generation in mulberry twig and cow dung composting process and its application

Abstract

The present invention belongs to the field of microbial technology and specifically relates to a composite microbial agent for promoting lignocellulose degradation and humus production during mulberry twig and cow dung composting, and its application. The composite microbial agent comprises a mixture of strains B1, F1, and F2, wherein the ratio of effective viable bacteria among strains B1, F1, and F2 is 1:1:1. The deposit number of strain B1 is GDMCC No. 65049; the deposit number of strain F1 is GDMCC No. 65050; and the deposit number of strain F2 is GDMCC No. 65051. The Bacillus cereus B1, Phanerochaete chrysosporium F1, and Sphaerotheca fusca F2 in the composite microbial agent are all effective fertilizer compost-promoting microorganisms. These three microorganisms can synergize with each other during aerobic composting, fully achieving the conversion and degradation of raw materials and producing high-quality organic fertilizer. Furthermore, the composite microbial agent can promote lignocellulose degradation and humus production during composting.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a composite bacterial agent that promotes the degradation of lignocellulose and the generation of humus in the process of composting mulberry branches and cow dung, and application thereof. Background Art

[0002] According to reports, the Guangxi Zhuang Autonomous Region slaughtered 1.4282 million cattle in 2022, a 6.3% increase over the previous year. Sericulture in the region produces approximately 3 million tons of dry mulberry branches and over 4 million tons of silkworm feces annually. Efficiently degrading and transforming mulberry straw is a pressing issue facing modern agriculture and a focus of considerable research. In the past, most cut mulberry straw was burned as firewood or simply discarded in the fields, resulting in significant resource waste and environmental pollution. Only a small portion of mulberry straw was repurposed for use in the production of health foods, pharmaceuticals, and edible fungi (utilization rate approximately 5%). Therefore, to repurpose mulberry straw waste, composting can be used to mix mulberry branches with cattle manure in a specific ratio. Microorganisms then degrade the waste and transform it into useful, mature organic products. However, traditional composting processes have long fermentation cycles, slow degradation of lignocellulose, and low humus content. They also produce greenhouse gases such as CO2, CH4, and N2O, leading to carbon and nitrogen losses and reduced compost quality. Therefore, in-depth research on the technology to improve the efficiency of mulberry branch and cow dung composting is of great significance to solving the technical bottleneck of mulberry branch and cow dung fertilizer utilization.

[0003] Humus, a byproduct of composting, is a key indicator for evaluating compost stability and quality. Furthermore, humus improves soil quality, absorbs heavy metals, and increases crop yields. Converting more organic matter into humus is crucial for improving compost quality and environmental benefits. High-temperature composting is an effective way to achieve harmless utilization of cow manure and mulberry branches. However, cow manure and mulberry branches contain large amounts of complex and difficult-to-degrade lignocellulose, resulting in low composting efficiency, long composting cycles, and incomplete degradation. This severely limits the resource utilization of mulberry and silkworm waste. Therefore, promoting the degradation of lignocellulose for humus synthesis is of great significance.

[0004] Bioaugmentation has attracted widespread attention for its potential to improve humus synthesis. Inoculating with microorganisms with specific functions can promote humus synthesis. Multiple studies have shown that adding lignocellulose-degrading microorganisms to compost can increase lignocellulose degradation and humus content. Bacteria are more inclined to degrade cellulose, while fungi tend to decompose hemicellulose and lignin. Different strains have varying degradation effects on different components, and studies have also shown that different strains can synergize to degrade the same substance. However, humus formation is regulated by a complex microbial network. Interactions between these microorganisms may influence bacterial communities, leading to bacterial community succession. During the composting process, microorganisms decompose organic matter and convert it into humus through community succession. Changes in physical and chemical parameters such as temperature, pH, and EC can alter the microbial community structure, thereby affecting compost quality. Changes in environmental factors can influence microbial community succession. When the microbial community changes, its metabolic function also changes, affecting cellulose degradation and humus synthesis.

[0005] By screening existing microorganisms, the inventors have constructed a composite bacterial agent with efficient fermentation function. By adding the composite bacterial agent, the composting process is optimized, thereby accelerating the composting process and increasing the humus content of the compost, which can provide scientific and technological support for promoting the resource utilization of agricultural solid waste and the production of new bio-organic fertilizers.

[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0007] The purpose of the present invention is to provide a composite bacterial agent and its application for promoting the degradation of lignocellulose and the generation of humus during the composting process of mulberry branches and cow dung, so as to solve the problems of long fermentation cycle, slow degradation of lignocellulose and low humus content in traditional composting in the background technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A composite bacterial agent, the composite bacterial agent is composed of a mixture of strain B1, strain F1 and strain F2, wherein the ratio of the effective viable counts of strain B1, strain F1 and strain F2 is 1:1:1; wherein the taxonomic name of strain B1 is Bacillus cereus, and it was deposited in Guangdong Provincial Microbial Culture Collection on August 23, 2024, with the deposit number: GDMCC No: 65049;

[0010] The strain F1 is taxonomically named Phanerochaete chrysosporium and was deposited in Guangdong Provincial Microbiological Culture Collection on August 23, 2024, with the deposit number: GDMCC No: 65050.

[0011] The taxonomic name of the strain F2 is Bjerkandera adusta, and it was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2024, with the deposit number: GDMCC No: 65051.

[0012] More specifically, the effective viable count of the strains B1, F1 and F2 was 1.0×10 8 spore / mL.

[0013] The present invention also provides the use of the composite bacterial agent in promoting the degradation of lignocellulose and the generation of humus during the composting process of mulberry branches and cow dung.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The Bacillus cereus B1, Phanerochaete chrysosporium F1 and Pseudomonas aeruginosa F2 in the microbial composite agent of the present invention are all effective microorganisms for promoting fertilizer composting. The three microorganisms can cooperate with each other in the aerobic composting process to fully realize the conversion and degradation of raw materials and produce high-quality organic fertilizer.

[0016] (2) The microbial composite agent of the present invention can achieve rapid compost temperature rise, with the high temperature period lasting more than 15 days, and can effectively kill pathogens and weed seeds. The total carbon content reaches 35.51%, and the total nitrogen content can reach 2.75%, meeting the national industry standard for organic fertilizers. It can also improve the seed germination index, lignin degradation rate, cellulose degradation rate, hemicellulose degradation rate, and humus content. It can be seen that the microbial composite agent of the present invention can promote the degradation of lignocellulose and the generation of humus during the composting process.

[0017] Preservation Information

[0018] The taxonomic name of strain B1 is Bacillus cereus, and it was deposited in Guangdong Microbial Culture Collection on August 23, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, deposit number: GDMCC No: 65049;

[0019] The strain F1 is taxonomically named Phanerochaete chrysosporium and was deposited in Guangdong Microbial Culture Collection on August 23, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, with the deposit number: GDMCC No: 65050.

[0020] The taxonomic name of strain F2 is Bjerkandera adusta, and it was deposited in the Guangdong Provincial Microbial Culture Collection on August 23, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, deposit number: GDMCC No: 65051. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For strain screening and plate confrontation;

[0022] Figure 2 The phylogenetic tree of the strain and its growth in culture medium;

[0023] Figure 3 The degradation of mulberry straw by the composite microbial agent;

[0024] Figure 4 This is a temperature change diagram during aerobic composting of cow dung and mulberry branches;

[0025] Figure 5 This is the pH change diagram during aerobic composting of cow dung and mulberry branches;

[0026] Figure 6 This is the total carbon change diagram during aerobic composting of cow dung and mulberry branches;

[0027] Figure 7 This is the change of total nitrogen during aerobic composting of cow dung and mulberry branches;

[0028] Figure 8 This is a graph showing the carbon-nitrogen ratio changes during aerobic composting of cow dung and mulberry branches;

[0029] Figure 9 This is a graph showing the changes in seed germination index during aerobic composting of cow dung and mulberry branches;

[0030] Figure 10 This is a diagram showing the changes in lignocellulose components during aerobic composting of cow dung and mulberry branches;

[0031] Figure 11 This is a diagram showing the changes in humus components during aerobic composting of cow dung and mulberry branches.

[0032] Description of main reference numerals:

[0033] Figure 1 In the figure, (a) shows the fading of different strains in aniline blue medium; (b) shows the fading of different strains in Congo red medium; (c) shows the antagonism between strains, from left to right: antagonism between bacteria and bacteria, fungi and fungi, and bacteria and fungi;

[0034] exist Figure 4-11 In the table, Control is the control group, and SynCom is the inoculated group;

[0035] exist Figure 5-Figure 11 In the composting process, IPP is the initial stage, MEP is the temperature rise stage, THP is the high temperature stage, and MAP is the mature stage. DETAILED DESCRIPTION

[0036] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.

[0037] 1. Screening of strains

[0038] Weigh 5g of silkworm excrement and mulberry branch compost sample and dissolve it in 95mL of sterile water. Oscillate at 30℃ and 180rpm for 30min. Pipette 5mL of bacterial suspension and place it in a conical flask containing 95mL of enrichment medium. Oscillate at 30℃ and 180rpm for 72h. Perform 10-fold gradient dilution. Pipette a small amount of bacterial suspension and evenly spread it on the primary screening medium (aniline blue medium and Congo red medium). Place it at 28℃ and 37℃ and culture it at constant temperature for 24h-48h. Observe the growth of the colony to determine whether there is a hydrolysis zone. Select the strains in the degradation zone and pick the bacteria separately (bacteria are streaked and fungi are gradient diluted and spread) to purify the strains and store them at 4℃ for later use.

[0039] Bacterial cakes with a diameter of 1 cm for each strain were inoculated onto CMC-Na plate culture medium and cultured for 24-72 h. A blank control (CK) without adding any strain was used. The cells were stained with 0.1% Congo red aqueous solution for 30 min, and the staining solution was discarded.

[0040] Dp=(D / d) 2 , D is the diameter of the hydrolysis zone, d is the diameter of the colony, and the average value is obtained by measuring four times from different directions. According to the size of the hydrolysis zone, two bacterial strains and two fungal strains were selected respectively. The results are shown in Table 1 and Figure 1 .

[0041] Table 1 Dp values of cellulose-degrading bacteria

[0042]

[0043] Note: The data in the column represent the mean ± standard error

[0044] 2. Strain Antagonism

[0045] The different strains isolated were inoculated on carboxymethyl cellulose solid culture medium plates, and the bacteria were crossed in pairs. The antagonism between bacteria and fungi was achieved by first applying bacteria and then inoculating fungi, and punching holes between fungi. Three parallel tests were set up. The strains were cultured in a constant temperature incubator at the corresponding temperature, and the growth of the strains was observed to see whether there were antagonistic or spore-inhibiting phenomena. Strains with antagonistic effects could not be combined. Strains without antagonistic effects on each other were selected for the construction of composite bacterial agents. The results are shown in Table 2 and Figure 1 shown.

[0046] Table 2 Direct antagonistic relationship between strains

[0047]

[0048] Note: + indicates antagonism, - indicates no antagonism

[0049] As shown in Table 2, there was no antagonistic effect between bacteria B1 and the two fungal strains F1 and F2.

[0050] 3. Strain Identification

[0051] Genomic DNA of the strains was extracted according to the kit protocol. PCR amplification was performed using universal primers for fungi and bacteria, using the genomic DNA of the strains as a template. The amplified results were sequenced. The nucleotide sequences of each strain were analyzed for homology in NCBI, and a phylogenetic tree was constructed using MEGA11.0 software.

[0052] According to the morphological identification of the strains and combined with the phylogenetic tree, the results are as follows Figure 2 shown.

[0053] After identification, the taxonomic name of strain B1 is Bacillus cereus, the taxonomic name of strain F1 is Phanerochaete chrysosporium, and the taxonomic name of strain F2 is Bjerkandera adusta. The sequences of strain B1, strain F1 and strain F2 are shown in Table SEQ ID No. 1-3.

[0054] 4. Construction of composite bacterial agents

[0055] The single-factor method was used to compare the efficiency of single strains B1, F1, F2 and composite bacterial agents in degrading lignocellulose under the same conditions, so as to optimize and construct the composite bacterial agent.

[0056] The selected bacteria and fungi were inoculated into Erlenmeyer flasks containing LB and PDA liquid culture media, respectively, and cultured under the following conditions: initial pH range of 5.0-6.0, shaker speed of 180 rpm / min, culture temperature of 37°C or 28°C, and cultured for 48h or 72h before use as strains.

[0057] The strain (effective viable count 1×10 8 spore / mL) were mixed according to Table 3 to construct a composite bacterial agent, and filter paper strip disintegration test and mulberry straw degradation test were carried out.

[0058] Filter paper disintegration test: Single and combined strains were inoculated into 50 mL of filter paper disintegration medium in an Erlenmeyer flask. A control group was set up without the addition of bacterial solution. The culture was carried out at 30°C and 130 rpm. Filter paper disintegration was regularly observed and recorded. The results are shown in Table 3.

[0059] Table 3 Degradation effect of lignocellulose-degrading bacteria on filter paper strips (3d)

[0060]

[0061] Note: “0” means almost no change in the filter paper; “+” means the filter paper edge swells; “++” means the filter paper as a whole swells; “+++” means the filter paper swells in large pieces; “++++” means the filter paper is in a paste-like state with small pieces; “+++++” means the filter paper is in a paste-like state and dispersed.

[0062] As shown in Table 3, strains B1, F1, F2 and their composite bacterial agents can all degrade filter paper strips, among which composite bacterial agents M1, M2, M4 and M5 are better than other groups; among them, after degradation of filter paper strips by M4 and M5, the filter paper becomes paste-like and dispersed, which is better than M1 and M2.

[0063] Mulberry straw degradation experiment: The strain combinations M1, M2, M4 and M5 in Table 3 were inoculated into mulberry liquid enzyme production culture medium (10 g / L crushed mulberry straw through a 20-mesh sieve, 1 g / L (NH4)2·SO4, 1 g / L KH2PO4, 0.1 g / L yeast extract, 0.5 g / LMgSO4·7H2O, 0.1 g / L CaCl2) at 6%, and the group without bacterial liquid was set as the control group. The culture was carried out at 30°C and 130 rpm / min in the dark for 7 days. After the culture was completed, the residual mycelium and calcium carbonate were removed with a mixture of hydrochloric acid and nitric acid, and the culture was repeatedly rinsed with sterile water. The culture was centrifuged at 5000 rpm for 10 minutes, and the precipitate was dried in a drying oven to constant weight. The weight loss rate of the mulberry straw was measured. Results Figure 3 .

[0064] Depend on Figure 3It can be seen that the strain combinations of M1, M2, M4 and M5 can all degrade mulberry straw, among which M4 and M5 have more significant degradation rates on mulberry straw, with mulberry straw weight loss rates of 33.60% and 32.33%, respectively. M4 was subsequently used as a microbial composite agent.

[0065] 5. Aerobic composting test of cow dung and mulberry branches

[0066] 5.1 Strain culture

[0067] Inoculate Bacillus cereus B1 into a 1L Erlenmeyer flask containing 500mL of beef extract peptone medium. Culture conditions include an initial pH range of 5.0-6.0, a shaker speed of 180 rpm / min, and a temperature of 37°C. After 48 days of incubation, the medium is ready for use. To prepare 1L of beef extract peptone medium, weigh 5g of beef extract, 10g of peptone, and 5g of NaCl, dissolve them, and then dilute to 1L with distilled water. Sterilize at 115°C for 20 minutes.

[0068] Phanerochaete chrysosporium F1 and F2 were inoculated into a 1L Erlenmeyer flask containing 500mL of PDA medium. The culture conditions were: an initial pH range of 5.0-6.0, a shaker speed of 180 rpm / min, and a temperature of 28°C. After 72 days of incubation, the culture was used as a starting material. For example, to prepare 1L of PDA medium, weigh 200g of peeled potatoes, cut them into small pieces, and boil them in 1L of boiling deionized water for 30 minutes. After passing through four layers of gauze, 20g of glucose was added to the filtrate, bringing the volume to 1L, and sterilizing it at 115°C for 20 minutes.

[0069] 5.2 Preparation of composite bacterial agent

[0070] The fermentation broth of Bacillus cereus B1, Phanerochaete chrysosporium F1 and F2 was adjusted to 1×10 8 spore / mL. The adjusted bacterial solution was evenly mixed according to the effective colony count ratio of Bacillus cereus B1, Phanerochaete chrysosporium F1 and F2 in a ratio of 1:1:1, and then the bacterial strain adsorbent rice bran was mixed and adsorbed in a blender at a volume-to-mass ratio of 1:10. The mixture was dried at room temperature until the moisture content was less than 20%, thereby obtaining a microbial composite agent. The agent was stored in a cool and dry place for future use.

[0071] 5.3 Aerobic composting test

[0072] Fresh cow dung and mulberry branches were collected from farmers in Dacai Township, Huanjiang Maonan Autonomous County, Hechi City, Guangxi Zhuang Autonomous Region. Their basic physical and chemical properties are shown in Table 4.

[0073] Table 4 Basic physical and chemical properties of compost materials

[0074]

[0075] Air-dry fresh cow dung and mulberry branches. Crush the dried mulberry branches and pass them through a 10-mesh sieve. Thoroughly mix the cow dung and mulberry branches in a ratio of 8:2 by dry weight, adjust the moisture content to around 60%, and build a pile measuring 1.5m x 1.5m x 1.2m in length x width x height.

[0076] An inoculation group and a control group were set up to compare their composting effects. The inoculation group was inoculated with the prepared microbial composite agent, while the control group was supplemented with an equal amount of adsorbent rice bran. At the initial stage of composting, the total fresh weight of each pile was approximately 1,000 kg, and the ratio of inoculum to pile mass was 6%. The piles were turned on the 1st, 3rd, 5th, 10th, and 15th day after creation. From 15 to 60 days, the piles were turned and aerated every 7 days to ensure uniformity. After 50 days of composting, the compost was piled in a ventilated and cool place for 40 days to enter the post-ripening stage and wait until the compost was fully decomposed. During the composting process, temperature measurements were taken at 3:00 p.m. every day, and changes in temperature and morphology of the piles were recorded over 60 days.

[0077] After the cow dung and mulberry branches were piled, samples were collected on the 1st, 3rd, 5th, 10th, 15th, 22nd, 29th, 36th, 45th, 52nd, and 60th day after creation. The collection method was as follows: the pile was divided equally into three sections: front, middle, and back. A cross-section was selected from each section. The cross-section sampling method was used: the pile was divided into multiple sections, and equal samples were collected at multiple heights within each section (upper layer: 0.05m-0.1m; middle layer: 0.5m-0.6m; lower layer: 0.8m-1.2m). The samples were then mixed and sampled three times using a quartering method to ensure representativeness. A portion was stored in a refrigerator at 4°C, while a portion was air-dried for determination of physical and chemical parameters and fertilizer maturity analysis.

[0078] Determination of carbon-nitrogen ratio during composting: A carbon-nitrogen element analyzer was used to determine the total carbon, total nitrogen and carbon-nitrogen ratio in the air-dried samples.

[0079] Determination of fertilizer germination index during composting: Add 5g of fresh compost sample to 50mL of deionized water, shake on a shaker for 24h, and filter. Add 5mL of filtrate to a culture dish lined with filter paper, add 20 plump cucumber seeds to the culture dish, spread them evenly, and culture in a dark incubator at 25℃ for 3d. Set up a control (add 5mL of sterile water to the control group), and replicate each sample three times. Count the number of germinated seeds and measure the length of the seed roots to analyze the degree of maturity of the compost material and the quality of the organic fertilizer. Calculation formula:

[0080] GI (%) = (seed germination rate of compost extract × seed root length) / (seed germination rate of distilled water × seed root length) × 100.

[0081] Determination of humic matter content during composting: Compost samples were extracted with a mixture of 0.1 M Na₄P₂O₇·10H₂O and NaOH at a ratio of 1:10 (w:v). The samples were shaken at 25°C for 24 hours and centrifuged at 11,000 rpm for 5 minutes to obtain the supernatant. The solution was then filtered through a 0.45 μm pore membrane to obtain humic matter. The pH of the humic matter solution was adjusted to 1.5 with 6 M HCl and allowed to stand at 4°C for 12 hours. The acidic solution was centrifuged at 11,000 rpm for 15 minutes. The supernatant was FA, and the precipitate was HA. The HA was washed twice with 0.1 M HCl and deionized water, alternating between the two. Finally, the precipitate was dissolved with 0.05 M NaHCO₃. Humic matter, HA, and FA concentrations were determined using a Shimadzu TOC-Vcph analyzer. All procedures were repeated in triplicate, and the average value for each condition was calculated.

[0082] Determination of lignocellulose content during composting: The contents of lignin, cellulose, and hemicellulose were determined using the Paradigm washing method using an FT12 automatic fiber analyzer.

[0083] 5.2 Results and Analysis

[0084] 5.2.1 Temperature changes during aerobic composting of cow dung and mulberry branches

[0085] See the results Figure 4 .

[0086] Depend on Figure 4 As can be seen, the compost in the inoculated group heated up faster and to a higher temperature than the compost in the control group, indicating that the microbial composite agent of the present invention can effectively accelerate the start of composting. At the same time, the temperature in the compost in the inoculated group dropped to room temperature more quickly than in the control group, indicating that the composting process stopped at room temperature, indicating that the inoculated group improved composting efficiency by accelerating the high temperature period and shortening the composting cycle.

[0087] 5.2.2 pH changes during aerobic composting of cow dung and mulberry branches

[0088] See the results Figure 5 .

[0089] Depend on Figure 5 It can be seen that the pH values of the control group and the inoculated group showed a similar pattern, increasing rapidly during the warming period, decreasing during the high temperature period, and slightly recovering during the mature period, reaching 8.60 and 8.67 respectively. The control group fluctuated more significantly than the inoculated group, which indicates that the inoculated group can effectively improve the synthesis of organic acids during the composting process.

[0090] 5.2.3 Changes in total carbon during aerobic composting of cow dung and mulberry branches

[0091] See the results Figure 6 .

[0092] Depend on Figure 6 The total carbon content of both the control and inoculated groups showed a downward trend throughout the composting process, and the total carbon degradation rate of the inoculated group was higher than that of the control group, indicating that the mineralization effect of organic matter was greater in the inoculated group than in the control group. The decrease in total carbon content in the inoculated group may be the result of enhanced interactions between microorganisms after inoculation with the synthetic bacterial consortium.

[0093] 5.2.4 Changes in total nitrogen during aerobic composting of cow dung and mulberry branches

[0094] See the results Figure 7 .

[0095] Depend on Figure 7 It can be seen that the total nitrogen content of the inoculated group and the treated group showed an overall upward trend during the entire composting process, and the total nitrogen content of the inoculated group increased significantly during the warming period, indicating that the inoculation of synthetic bacteria can reduce a certain amount of nitrogen loss.

[0096] 5.2.5 Changes in the carbon-nitrogen ratio during aerobic composting of cow dung and mulberry branches

[0097] See the results Figure 8 .

[0098] Depend on Figure 8 It can be seen that the carbon-nitrogen ratio of the control group and the inoculated group showed an overall downward trend during the composting process, and decreased rapidly during the warming period and the decomposition period. At the end of fermentation, the carbon-nitrogen ratios of the control group and the inoculated group were 13.38 and 12.91, respectively. This is because the mineralization of total carbon is stronger than that of total nitrogen.

[0099] 5.2.6 Changes in Seed Germination Index During Aerobic Composting of Cow Dung and Mulberry Branches

[0100] See the results Figure 9 .

[0101] Depend on Figure 9 The results show that the seed germination index of both the control and inoculated groups showed an upward trend during the composting process, but the seed germination index of the inoculated group was 51.32% higher than that of the control group during the mature stage. This indicates that the inoculated group has high biological activity throughout the composting process, effectively degrading organic acids, reducing compost toxicity, and increasing the germination index.

[0102] 5.2.7 Changes in lignocellulose components during aerobic composting of cow dung and mulberry branches

[0103] See the results Figure 10 .

[0104] Depend on Figure 10As shown, the lignocellulose content of both the control and inoculated groups decreased during composting, with significant differences between the two groups during the high-temperature period. The lignocellulose degradation rates of the inoculated groups were significantly higher than those of the control groups in the later stages of composting. In contrast, the inoculated groups increased lignin degradation by 19.21%, cellulose degradation by 7.89%, and hemicellulose degradation by 11.96%. These results suggest that inoculation can accelerate compost stabilization and maturation through lignocellulose conversion.

[0105] 5.2.8 Changes in Humus Components During Aerobic Composting of Cow Manure and Mulberry Branches

[0106] See the results Figure 11 .

[0107] Depend on Figure 11 It can be seen that the humus content of the control group and the inoculated group showed a trend of first decreasing and then increasing during the composting process. This is because the organic matter that can be directly utilized in the early stage of composting is rapidly decomposed. When the available organic matter is insufficient, humus will be decomposed and utilized by microorganisms. The humus content of the inoculated group increased to 117.05 mg / g in the later stage of composting, which is significantly higher than the control group (94.30 mg / g), and increased by 24.13% over the control group. During the composting process, the humic acid content of the control group and the inoculated group both showed an upward trend, while the fulvic acid content showed a trend of first decreasing and then increasing. In addition, the humic acid content of the inoculated group increased by 6.23% over the control group. These results show that the inoculated group can accelerate the stability and maturity of compost by promoting the conversion of fulvic acid to humic acid.

[0108] In summary, Bacillus cereus B1, Phanerochaete chrysosporium F1 and Bacillus fuscae F2 in the microbial composite agent of the invention are all effective microorganisms for promoting fertilizer composting. The three microorganisms can cooperate with each other in the aerobic composting process to fully realize the transformation and degradation of raw materials and produce high-quality organic fertilizer.

[0109] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An application of a composite bacterial agent in promoting the degradation of lignocellulose and the generation of humus during the composting process of mulberry twigs and cow dung, characterized in that: The composite bacterial agent is prepared by mixing strain B1, strain F1 and strain F2, wherein the ratio of the effective viable bacteria counts of strain B1, strain F1 and strain F2 is 1:1:1; Among them, the taxonomic name of the strain B1 is Bacillus cereus ( Bacillus cereus ), deposited in Guangdong Provincial Microbiological Culture Collection on August 23, 2024, with the deposit number: GDMCC No: 65049; The strain F1 is taxonomically named Phanerochaete chrysosporium ( Phanerochaete chrysosporium ), deposited in Guangdong Provincial Microbiological Culture Collection on August 23, 2024, with the deposit number: GDMCC No: 65050; The taxonomic name of the strain F2 is Bjerkandera adusta ), deposited in Guangdong Provincial Microbiological Culture Collection on August 23, 2024, with the deposit number: GDMCC No: 65051.

2. The use of the composite bacterial agent according to claim 1 in promoting the degradation of lignocellulose and the generation of humus during the composting of mulberry twigs and cow dung, characterized in that: The effective viable bacteria count in strains B1, F1 and F2 was 1.0×10 8 spore / mL.

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