A compound microbial agent for reducing greenhouse gas emissions during membrane composting and its application

Abstract

This invention belongs to the field of nitrogen retention and synergistic emission reduction technology in composting. Specifically, it discloses a compound microbial agent for reducing greenhouse gas emissions during membrane composting and its application. This compound microbial agent consists of fermentation broth containing *Leifsonia shinshuensis* N37, *Oceanobacillus sp.* M11, and *Bacillus subtilis* M8 in a mass ratio of 1:8:1. Compared with existing technologies, this compound microbial agent can reduce NH3 emissions during membrane composting, promote nitrogen fixation, and improve the nitrogen fertilizer efficiency of the product; reduce greenhouse gas emissions during membrane composting, improving the environmental benefits of membrane composting; and promote the maturation of membrane compost products, improving their fertilizer efficiency.

Description

Technical Field

[0001] This invention relates to the field of composting nitrogen retention and synergistic emission reduction technology, and in particular to a compound microbial agent for reducing greenhouse gas emissions during membrane composting and its application. Background Technology

[0002] Composting is a method of resource utilization of waste, converting it into organic fertilizer. Traditional aerobic composting in stacks primarily results in nitrogen loss through ammonia volatilization, and also produces greenhouse gases such as CO2, CH4, and N2O, causing environmental pollution and reducing the nitrogen fertilizer efficiency of the product. Inoculating the composting process with microbial agents can reduce nitrogen loss and promote the maturation of the compost product. Nanofilm aerobic composting technology can effectively mitigate odor emissions during composting. In response to national carbon reduction policies and to reduce ammonia emissions during membrane composting, as well as improve the fertilizer efficiency of membrane compost products, it is necessary to develop corresponding microbial agents to regulate the membrane composting process, reduce greenhouse gas emissions, and increase the economic value of membrane compost products. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a composite microbial agent for reducing greenhouse gas emissions during membrane composting and its application.

[0004] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0005] One of the objectives of this invention is to provide a compound microbial agent for reducing greenhouse gas emissions during membrane composting. This compound microbial agent is composed of fermentation broth from Leifsonia shinshuensis N37, Oceanobacillus sp. M11, and Bacillus subtilis M8.

[0006] Among them, the actinomycete (Leifsonia shinshuensis) N37 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 8, 2024, with the accession number GDMCC No: 64848; the accession number of the Bacillus sp. (Oceanobacillus) M11 is GDMCC No: 64847; and the accession number of the Bacillus subtilis M8 is GDMCC No: 64849.

[0007] Preferably, the mass ratio of the fermentation broth of Leifsonia shinshuensis N37, Oceanobacillus sp. M11, and Bacillus subtilis M8 in the compound microbial agent is 1:8:1.

[0008] Furthermore, the fermentation broth of *Leifsonia shinshuensis* N37, *Oceanobacillus sp.* M11, and *Bacillus subtilis* M8 is prepared by inoculating *Leifsonia shinshuensis* N37 / *Oceanobacillus sp.* M11 / *Bacillus subtilis* M8 into LB broth medium at an inoculum of 1%, and then incubating statically for 48 hours or on a shaker until CFU > 10. 8 It was obtained.

[0009] The second objective of this invention is to provide an application of a compound microbial agent for reducing greenhouse gas emissions during membrane composting in the preparation of membrane compost, comprising the following steps:

[0010] S1. Mix mushroom residue and hen manure at a mass ratio of 2:5 evenly, then mix the compound microbial agent with the pile evenly, adjust the moisture content of the pile to 60%, and cover the surface of the pile with a polymer semi-permeable membrane; wherein, the amount of the compound microbial agent added is 2% of the wet weight of the pile.

[0011] S2. Lay ventilation ducts at the bottom of the compost pile, then cover the compost pile with the ventilation ducts. Do not turn the compost pile. Use the static composting mode for a total of 30 days to obtain the finished membrane compost.

[0012] Furthermore, the reactor core is intermittently ventilated via ventilation ducts, with ventilation intervals of 10 minutes followed by 50-minute intervals, at a ventilation rate of 0.6 m / s². 3 / h.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The compound microbial agent of the present invention can reduce NH3 emissions during membrane composting, promote nitrogen fixation, and improve the nitrogen fertilizer efficiency of the product; reduce greenhouse gas emissions during membrane composting, and improve the environmental benefits of membrane composting; promote the maturation of membrane compost products, and improve the fertilizer efficiency of membrane compost products. Attached Figure Description

[0015] Figure 1 This is a temperature change graph for composting in Example 2.

[0016] Figure 2 This is a diagram showing the changes in humus in the compost from Example 2.

[0017] Figure 3 This is a graph showing the change in ammonium nitrogen in the compost of Example 2.

[0018] Figure 4 This is a graph showing the change in nitrate nitrogen in the compost of Example 2.

[0019] Figure 5 The germination index of cucumber seeds grown in compost in Example 2 is the result of different number of days.

[0020] Figure 6 This illustrates the CO2 emission pattern of composting in Example 2.

[0021] Figure 7 The CH4 emission pattern of the compost in Example 2 is shown.

[0022] Figure 8 The N2O emission pattern of composting in Example 2 is shown.

[0023] Figure 9 The emission pattern of ammonia during the composting process in Example 2 is shown.

[0024] Figure 10 The effect of microbial fertilizer on the growth promotion of peanut seedlings. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0026] Example 1: Preparation of compound microbial inoculant.

[0027] 1) Screening of actinomycete (Leifsonia shinshuensis) N37

[0028] ① Preparation of solid culture medium: Weigh 8.64g of nitrogen-free culture medium (N11764-250G model nitrogen-free culture medium purchased from Beijing Puxitang Biotechnology Co., Ltd.) (43.2g / L) into a 250mL Erlenmeyer flask, add 200mL of deionized water, stir until dissolved, sterilize in an autoclave at 121℃ for 15min, and after sterilization, pour into sterile petri dishes, adding 20mL of nitrogen-free culture medium to each dish.

[0029] ② Preparation of supernatant: Weigh 2g of soybean soil sample (field soil sample collected in 2022 at longitude 113.85, latitude 29.70) into a 50mL centrifuge tube, add 20mL of PBS, shake to mix, place on a shaker (37℃, 120r) for 30min, let stand for 10min, and take 5mL of supernatant.

[0030] ③ Streak plating: Use a sterile inoculation loop to pick up the supernatant and streak it on nitrogen-free medium using the four-zone streak method. Then incubate at 37°C for 48 hours.

[0031] ④ Purification and Propagation: Select single colonies that can grow on nitrogen-free medium, pick single bacteria with an inoculation loop, and propagate them in sterilized LB broth medium (purchased from Guangzhou Huankai Biotechnology Co., Ltd., formula: peptone 10g / L, sodium chloride 5g / L, glucose 1g / L, yeast extract powder 5g / L, final pH 7.0±0.2) for 24h. Divide the propagated bacterial solution into two portions and transfer them to sterile centrifuge tubes. Add an equal volume of 50% glycerol to one portion, and use the other portion for DNA extraction. Store the DNA at -20℃ and the bacterial solution at -80℃.

[0032] ⑤ PCR amplification: The amplification system consisted of 25 μL of culture medium, containing 12.5 μL of enzyme, 0.5 μL of upstream universal primer (27F), 0.5 μL of downstream universal primer (1492R), 1 μL of DNA, and 10.5 μL of sterile water. The PCR program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 10 s, extension at 72°C for 10 s, for 34 cycles; final extension at 72°C for 10 min; and storage at 4°C.

[0033] ⑥ Identification: The PCR product was sent to Guangzhou Sangon Biotech Co., Ltd. for sequencing. The sequence is as follows:

[0034]

[0035] 2) Screening of Oceanobacillus sp. M11

[0036] ①1① Weigh 4.2g of LB agar medium (21g / L) into a 250mL Erlenmeyer flask. The LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd. The formula of LB agar medium is: peptone 10g / L, sodium chloride 5g / L, glucose 1g / L, yeast extract powder 5g / L, agar 15g / L, final pH 7.0±0.2. Add 200mL of deionized water and stir until dissolved. Add 0.02g of aniline blue solid and shake until the aniline blue solid dissolves. Sterilize in an autoclave at 121℃ for 15min. After sterilization, pour into sterile petri dishes, adding 20mL of medium to each dish to obtain aniline blue medium.

[0037] ② Weigh 4.2g of LB agar medium (21g / L) into a 250mL Erlenmeyer flask. The LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd. The formula of LB agar medium is: peptone 10g / L, sodium chloride 5g / L, glucose 1g / L, yeast extract powder 5g / L, agar 15g / L, final pH 7.0±0.2. Add 200mL of deionized water and stir until dissolved. Add 20ul of guaiacol and shake well. Sterilize in an autoclave at 121℃ for 15min. After sterilization, pour into sterile petri dishes, adding 20mL of medium to each dish to obtain guaiacol medium.

[0038] ③ Preparation of supernatant: Weigh 2g of samples from the early, middle and late stages of composting (membrane composting test samples collected in 2022 from Runtian Composting Plant in Xinxing County, Yunfu City, Guangdong Province) into a 50mL centrifuge tube, add 20mL of PBS, shake to mix, place on a shaker (37℃, 120r) for 30min, let stand for 10min, and take 5mL of supernatant.

[0039] ④ Dilute the supernatant to 10. -5 10 -6 10 -7 To dilute the liquid, take 100 μL of the diluted liquid and place it in aniline blue medium and guaiacol medium. Spread it evenly with a spreader and then incubate it upright in a 37°C incubator for 2 hours, followed by inverted incubation for 48 hours.

[0040] ⑤ Select single colonies that produce a clear zone around the colony on aniline blue medium and colonies that produce a red color zone on guaiacol medium. Pick single colonies using an inoculation loop and propagate them in LB broth for 24 hours. Divide the propagated bacterial solution into two portions and transfer them to sterile centrifuge tubes. Add an equal volume of 50% glycerol to one portion and use the other portion for DNA extraction. Store the DNA at -20℃ and the bacterial solution at -80℃.

[0041] ⑥ PCR amplification: The amplification system consisted of 25 μL of culture medium, containing 12.5 μL of enzyme, 0.5 μL of upstream universal primer (27F), 0.5 μL of downstream universal primer (1492R), 1 μL of DNA, and 10.5 μL of sterile water. The PCR program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 10 s, extension at 72°C for 10 s, for 34 cycles; final extension at 72°C for 10 min; and storage at 4°C.

[0042] ⑦ Identification: The PCR product was sent to Guangzhou Sangon Biotech Co., Ltd. for sequencing. The sequence is as follows:

[0043]

[0044] 3) Screening of Bacillus subtilis M8

[0045] ① Weigh 4.2g of LB agar medium (21g / L) into a 250mL Erlenmeyer flask. The LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd. The formula of LB agar medium is: peptone 10g / L, sodium chloride 5g / L, glucose 1g / L, yeast extract powder 5g / L, agar 15g / L, final pH 7.0±0.2. Add 200mL of deionized water and stir until dissolved. Add 0.02g of aniline blue solid and shake until the aniline blue solid dissolves. Sterilize in an autoclave at 121℃ for 15min. After sterilization, pour into sterile petri dishes, adding 20mL of medium to each dish to obtain aniline blue medium.

[0046] ② Weigh 4.2g of LB agar medium (21g / L) into a 250mL Erlenmeyer flask. The LB agar medium was purchased from Guangzhou Huankai Biotechnology Co., Ltd. The formula of LB agar medium is: peptone 10g / L, sodium chloride 5g / L, glucose 1g / L, yeast extract powder 5g / L, agar 15g / L, final pH 7.0±0.2. Add 200mL of deionized water and stir until dissolved. Add 20ul of guaiacol and shake well. Sterilize in an autoclave at 121℃ for 15min. After sterilization, pour into sterile petri dishes, adding 20mL of medium to each dish to obtain guaiacol medium.

[0047] ③ Preparation of supernatant: Weigh 2g of samples from the early, middle and late stages of composting (membrane composting test samples collected in 2022 from Runtian Composting Plant in Xinxing County, Yunfu City, Guangdong Province) into a 50mL centrifuge tube, add 20mL of PBS, shake to mix, place on a shaker (37℃, 120r) for 30min, let stand for 10min, and take 5mL of supernatant.

[0048] ④ Dilute the supernatant to 10. -5 10 -6 10 -7 To dilute the liquid, take 100 μL of the diluted liquid and place it in aniline blue medium and guaiacol medium. Spread it evenly with a spreader and then incubate it upright in a 37°C incubator for 2 hours, followed by inverted incubation for 48 hours.

[0049] ⑤ Select single colonies that produce a clear zone around the colony on aniline blue medium and colonies that produce a red color zone on guaiacol medium. Pick single colonies using an inoculation loop and propagate them in LB broth for 24 hours. Divide the propagated bacterial solution into two portions and transfer them to sterile centrifuge tubes. Add an equal volume of 50% glycerol to one portion and use the other portion for DNA extraction. Store the DNA at -20℃ and the bacterial solution at -80℃.

[0050] ⑥ PCR amplification: The amplification system consisted of 25 μL of culture medium, containing 12.5 μL of enzyme, 0.5 μL of upstream universal primer (27F), 0.5 μL of downstream universal primer (1492R), 1 μL of DNA, and 10.5 μL of sterile water. The PCR program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 10 s, extension at 72°C for 10 s, for 34 cycles; final extension at 72°C for 10 min; and storage at 4°C.

[0051] ① Identification: The PCR product was sent to Guangzhou Sangon Biotech Co., Ltd. for sequencing. The sequence is as follows:

[0052]

[0053] Leifsonia shinshuensis N37, Oceanobacillus sp. M11, and Bacillus subtilis M8 were inoculated into LB broth medium at a 1% inoculum and incubated statically for 48 hours or on a shaker until CFU > 10⁻⁶. 8 Fermentation broths of three strains were prepared. The fermentation broths of Leifsonia shinshuensis N37, Oceanobacillus sp. M11, and Bacillus subtilis M8 were mixed in a mass ratio of 1:8:1 to obtain a compound microbial agent.

[0054] Example 2: Verification of the effect of compound microbial agents on reducing greenhouse gas emissions during membrane composting

[0055] Mushroom residue and hen manure were mixed evenly at a mass ratio of 2:5. Then, a compound microbial agent was mixed evenly with the pile, and the moisture content of the pile was adjusted to 60%. The amount of compound microbial agent added was 2% of the wet weight of the pile. A polymer semi-permeable membrane was then placed on the surface of the pile, designated as group PB4. A control group (CK) was also set up: mushroom residue and hen manure were mixed evenly at a mass ratio of 2:5, the moisture content of the pile was adjusted to 60%, and a polymer semi-permeable membrane was placed on the surface of the pile. Ventilation ducts were laid at the bottom of the pile, and the pile was then covered over the ventilation ducts. The pile was ventilated intermittently through the ventilation ducts, with a ventilation rate of 0.6 m / s². 3 / h; without turning the compost pile, the static composting mode is used for a total of 30 days to obtain the membrane compost product.

[0056] 1) Use a TP101 metal probe thermometer to measure the temperature changes of the compost pile and the ambient temperature. Measure the core temperature of the compost pile at 9:00 AM and 5:00 PM daily, and take the average of the morning and evening temperatures as the daily temperature of the pile. Simultaneously record the morning and evening ambient temperatures, and take their average as the daily temperature. The compost temperature changes are as follows: Figure 1As shown, days 0-2 were the warming period, days 2-25 were the high-temperature period, and days 25-30 were the cooling period. The highest temperature in group PB4 occurred on day 2, reaching 63.9℃, while the highest temperature in group CK also occurred on day 2, reaching 64.4℃. However, there was no significant difference in the highest temperatures between the two groups (P < 0.05). The high-temperature period lasted 24 days for both groups, but the temperature in group PB4 was higher than that in group CK for 16 days during this period, indicating that the addition of microbial agents was more conducive to maintaining the high-temperature period. The duration of the high-temperature period (>50℃) for both groups was greater than 7 days, which meets the standards for harmless treatment of livestock and poultry (GB36195-2018). During the cooling period, the temperature in group PB4 decreased faster, indicating that the addition of microbial agents facilitated the faster maturation of the compost pile, shortened the composting time, and improved the production efficiency of membrane compost products.

[0057] 2) Humus mainly includes humic acid and fulvic acid, which are complex and stable macromolecular organic compounds formed by the decomposition and transformation of organic matter during composting. The content of humus has a positive impact on regulating soil fertility, soil microbial structure and community, and crop growth. Humus was determined using the sodium pyrophosphate-sodium hydroxide extraction-high-temperature external heating potassium dichromate oxidation capacity method. Total humus content increased with the composting process. Figure 2 As shown, the humic content decreased in the early stage of composting, increased on the 14th day, and the highest humic content appeared in the compost sample on the 21st day. The highest humic content in the PB4 group was 22.5%, and the highest humic content in the CK group was 21.1%. The results indicate that the addition of microbial agents promoted the generation of humic substances and improved the maturity of the compost pile.

[0058] 3) Ammonium nitrogen, as an important component of nitrogen in organic fertilizers, has a significant impact on soil nitrogen supply and plant growth and development after organic fertilizer is returned to the field. Increasing the ammonium nitrogen content in organic fertilizers can reduce the amount of organic fertilizer used per unit area while meeting the crop's nitrogen requirements, thereby improving the utilization efficiency of organic fertilizers and increasing production benefits. Changes in ammonium nitrogen are shown below. Figure 3 The overall trend of ammonium nitrogen during composting was upward. The ammonium nitrogen content decreased slightly from 7 to 21 days. At the end of composting, the ammonium nitrogen content in the CK group was 12.016 mg / g, and the ammonium nitrogen content in the PB4 group was 13.536 mg / g, an increase of 12.65%. This indicates that the application of microbial agents increased the ammonium nitrogen content of the membrane compost products, which is beneficial to the retention of nitrogen in the products.

[0059] For the determination method, take 0.5 g of sample, add 5 mL of deionized water, vortex for 10 min, centrifuge at 13000 r / min for 5 min, take 50 μL of supernatant, add 10 mL of 0.01 mol / L dilute sulfuric acid, add 0.5 mL of potassium sodium tartrate solution, add 0.5 mL of Nash reagent, shake well, develop color for 10 min, and measure the absorbance of the solution at 420 nm.

[0060] Preparation of the standard curve: Weigh 0.7855g of ammonium chloride and dissolve it in a 250mL volumetric flask to obtain an ammonium chloride standard working solution with a concentration of 1mg / L. Pipette 0, 0.1, 0.3, 0.5, 1, 1.5, and 2mL of the ammonium chloride standard working solution, respectively, with ammonium nitrogen contents of 0, 2, 6, 10, 20, 30, and 40ug. Add 0.01mol / L dilute sulfuric acid to a final volume of 10mL. Add 0.5mL of potassium sodium tartrate solution and 0.5mL of Nash reagent. Shake well and allow to develop color for 10min. Measure the absorbance of the solution at 420nm.

[0061] Sodium hydroxide reagent: Weigh 12g sodium hydroxide (NaOH) and dissolve it in 60mL of water, then cool. Weigh 1.7g mercuric chloride (HgCl2) and dissolve it in 30mL of water. Weigh 3.5g potassium iodide (KI) and dissolve it in 10mL of water. While stirring, slowly add the above mercuric chloride solution to the potassium iodide solution until the red precipitate no longer dissolves. While stirring, slowly add the sodium hydroxide solution cooled to room temperature to the above mixture of mercuric chloride and potassium iodide, then add the remaining mercuric chloride solution. Mix well and let stand in the dark for 24 hours. Pour off the supernatant and store it in a brown bottle.

[0062] Potassium sodium tartrate solution: Weigh 50g of potassium sodium tartrate (KNaC4H6O6·4H2O) and dissolve it in 100mL of water. Heat to boiling to remove ammonia, cool, and then dilute to 100mL.

[0063] 4) Nitrate nitrogen, as one of the nitrogen elements that plants can directly absorb and utilize, is mainly affected by nitrifying and denitrifying bacteria during composting. Therefore, increasing the nitrate nitrogen content in compost products is worthwhile research in order to improve the nitrogen fertilizer efficiency of the products. Figure 4 As shown, in the early stage of composting, the nitrate nitrogen content increased. On day 3, the nitrate nitrogen content in the CK group was 3.92 mg / g, and that in the PB4 group was 3.2 mg / g. This was mainly due to the large-scale proliferation of microorganisms inside the compost pile during the early stage of composting, leading to the formation of anaerobic zones and the generation of nitrate nitrogen through anaerobic nitrification. During the high-temperature period, the nitrate nitrogen content decreased. On day 21, the nitrate nitrogen content in both the CK and PB4 groups was 3.57 mg / g. At the end of composting, the nitrate nitrogen content in the PB4 group was 4.12 mg / g, and that in the CK group was 3.84 mg / g. The application of microbial agents increased the nitrate nitrogen content by 7.29%, which is beneficial for improving the nitrogen fertilizer efficiency of the product.

[0064] 5) Germination index is an indicator for evaluating the toxicity of compost products. High-quality organic fertilizer can promote seed germination and root development, so the germination index can indirectly reflect the maturity of compost products. Take 2g of compost samples from days 0, 1, 3, 7, 14, 21, and 28, add 20mL of deionized water, vortex for 30min, let stand for 30min, place a filter paper at the bottom of a petri dish, and add 10 cucumber seeds (Yuexiu No. 3). Each group has three replicates. Add 10mL of supernatant to the petri dish. Add 10mL of deionized water to the blank control group. Incubate in the dark at 25℃ for 48h, then record the seed germination rate and root length. The germination index calculation formula is as follows:

[0065] GI(%)=(A1*A2) / (B1*B2);

[0066] In the formula: A1 is the germination rate of the experimental group; A2 is the average root length of the experimental group; B1 is the germination rate of the control group; B2 is the average root length of the control group.

[0067] Germination index at different times as follows Figure 5 As shown, in the early stages of composting (0, 1, 4, 7 days), the germination index of the product was less than 80%, indicating that the product had a strong inhibitory effect on seed germination. The germination index was above 80% on day 10, indicating that the inhibitory effect on seed germination was relieved. The highest germination index was observed on day 21 of composting, with the CK group at 114.62% and the PB4 group at 138.34%. The germination index of the microbial agent-added groups was 23.72% higher than that of the CK group on day 21. Although the germination index decreased slightly on day 28, PB4 still had a higher germination index (126.48%) than CK's 114.62%, an increase of 11.86%. This indicates that the addition of microbial agents promoted the maturation of the compost, reduced the toxicity of the product, and promoted seed germination.

[0068] 6) To mitigate global warming and respond to national carbon reduction policies, reducing greenhouse gas emissions during composting is urgently needed. Greenhouse gases mainly include CO2, CH4, and N2O, with CO2 being the primary contributor to the greenhouse effect and also the main greenhouse gas produced during composting. In this study, compost exhaust gases were collected daily at 5 PM using the static chamber method at 0 and 60 minutes. Aluminum foil bags were used to collect the gases, and the contents of CO2, CH4, and N2O in the exhaust gases were measured using an Agilent 7890B analyzer. The CO2 emission pattern is as follows: Figure 6 As shown, the CH4 emission pattern is as follows: Figure 7 As shown, the N2O emission pattern is as follows: Figure 8 As shown.

[0069] CO2 emissions are mainly concentrated in the early stages of composting, with the CK group reaching its maximum CO2 emission of 8.45 × 10⁻⁶ on day 3. 4 The CO2 emissions of group PB4 reached a maximum of 8.35 × 10 mg / kg on the second day. 4 mg / kg. Considering the entire composting process, the cumulative CO2 emissions of group CK were 126.11 × 10 mg / kg. 4 The cumulative CO2 emissions of group PB4 were 114.2 × 10 mg / kg. 4 mg / kg, the addition of microbial agent reduces CO2 emissions by 9.44%.

[0070] CH4 emissions are mainly concentrated during the high-temperature period of composting, with an emission peak at the end of composting. Figure 7 The results showed that CH4 emissions in group PB4 were significantly lower than those in group CK. The highest daily emission in group CK was 386.54 mg / kg on day 28, while the highest daily emission in group PB4 was 200.97 mg / kg on day 28. The lowest values ​​occurred on day 0, with group CK at 10.08 mg / kg and group PB4 at 11.28 mg / kg. In terms of cumulative emissions, group CK had 4877.37 mg / kg, while group PB4 had 2805.52 mg / kg, indicating that the addition of the microbial agent reduced CH4 emissions by 42.48%.

[0071] N2O emissions such as Figure 8 As shown, the emissions are mainly concentrated in the early and late stages of composting. The highest daily emission of the CK group was 196.15 mg / kg, and the highest daily emission of the PB4 group was 249.85 mg / kg. Considering the total emissions throughout the composting process, the cumulative emissions of the CK group were 877.47 mg / kg, and the cumulative emissions of the PB4 group were 847.98 mg / kg. In summary, the emission reduction rate of N2O by the addition of microbial agents was 3.36%.

[0072] PB4 microbial agent has a significant effect on greenhouse gas emission reduction, with the highest reduction rate of 42.48% for CH4. However, CO2 emissions are four orders of magnitude higher than those for CH4 and N2O. GHG (greenhouse gas) is significantly affected by CO2 emissions. Therefore, this microbial agent has a positive effect on greenhouse gas emission reduction in the membrane composting process and can reduce the environmental pollution caused by composting.

[0073] 7) The emission pattern of ammonia during composting is as follows: Figure 9 As shown, 0.5 mol / L dilute sulfuric acid was used as the absorbent for ammonia. Ammonia in the tail gas was absorbed using a static chamber method from 8 PM to 9 AM. The detection method was the same as for ammonium nitrogen. Ammonia emissions were mainly concentrated in the early stages of composting. As composting progressed, ammonia emissions gradually decreased, with the highest emissions occurring on day 1. The highest ammonia emission in the control group was 53.97 mg / m³. 2The highest ammonia emission in group PB4 was 53.38 mg / m³. 2 The cumulative NH3 emissions for each group were CK (235.88 ± 10.71 mg / m³). 2 ), PB4 (204.46±4.71mg / m 2 The cumulative NH3 emissions in group PB4 were significantly lower than those in group CK during the first 7 days (P < 0.05). At the end of composting, the cumulative NH3 emissions were 2882.79 ± 41.06 mg for CK and 2538.37 ± 26.38 mg for PB4. Compared with group CK, group PB4 significantly reduced the cumulative ammonia emissions (P < 0.05), with a reduction rate of 11.97%.

[0074] 8) Figure 10 The effect of microbial fertilizer on the growth promotion of peanut seedlings. Figure 10 The results showed that the application of microbial agents had a significant growth-promoting effect on peanuts. On days 7, 14, 21, 28, 35, and 70, the growth-promoting effect of the PB4 group compared to the CK group was 9.77%, 8.55%, 2.11%, 2.41%, 4.63%, and 7.30%, respectively. Considering the total weight of above-ground plants, total weight of underground roots, and fruit weight, the growth-promoting effect of the PB4 group was significant, indicating that the application of microbial agents improved the fertilizer efficiency of organic fertilizer, promoting peanut plant growth and yield.

[0075] Table 1. Benefits of microbial inoculant application on plant growth promotion.

[0076]

[0077] In summary, the composite microbial agent of the present invention can reduce ammonia emissions during membrane composting, reduce the environmental impact of compost odor; reduce greenhouse gas emissions during composting, contributing to carbon reduction during composting; promote compost maturation, increase lignin degradation rate, increase humus content, improve the maturity of compost products, increase ammonium nitrogen content, promote nitrogen fixation, and increase fertilizer efficiency.

[0078] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A compound microbial inoculant for reducing greenhouse gas emissions during membrane composting, characterized in that, This compound microbial agent is composed of actinomycetes (… Leifsonia shinshuensis N37, Bacillus ( Oceanobacillus sp M11, Bacillus subtilis ( Bacillus subtilis Composition of the fermentation broth for M8; Among them, the actinomycetes ( Leifsonia shinshuensis N37 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 8, 2024, with accession number GDMCC No: 64848; the Bacillus ( Oceanobacillus sp. The accession number for M11 is GDMCC No: 64847; the Bacillus subtilis ( Bacillus subtilis The accession number for M8 is GDMCCNo: 64849.

2. The compound microbial agent for reducing greenhouse gas emissions during membrane composting according to claim 1, characterized in that, The compound microbial agent contains actinomycetes ( Leifsonia shinshuensis N37, Bacillus ( Oceanobacillus sp M11, Bacillus subtilis ( Bacillus subtilis The mass ratio of the fermentation broth of M8 is 1:8:

1.

3. The compound microbial agent for reducing greenhouse gas emissions during membrane composting according to claim 2, characterized in that, The actinomycetes ( Leifsonia shinshuensis N37, Bacillus ( Oceanobacillus sp M11, Bacillus subtilis ( Bacillus subtilis The fermentation broth of M8 is made from actinomycetes ( Leifsonia shinshuensis N37 / Bacillus ( Oceanobacillus sp M11 / Bacillus subtilis ( Bacillus subtilis M8 was inoculated into LB broth at a 1% inoculum and incubated statically for 48 h or on a shaker until CFU > 10. 8 Prepared at / mL.

4. The application of a composite microbial agent for reducing greenhouse gas emissions during membrane composting as described in any one of claims 1-3 in the preparation of membrane compost, characterized in that, Includes the following steps: S1. Mix mushroom residue and hen manure at a mass ratio of 2:5 until homogeneous. Then mix the compound microbial agent with the pile material until homogeneous. Adjust the moisture content of the pile material to 60%, and cover the surface of the pile material with a polymer semi-permeable membrane. The amount of the compound microbial agent added is 2% of the wet weight of the pile material. S2. Lay ventilation ducts at the bottom of the compost pile, then cover the compost pile with the ventilation ducts. Do not turn the compost pile. Use the static composting mode for a total of 30 days to obtain the finished membrane compost.

5. The application of the composite microbial agent for reducing greenhouse gas emissions during membrane composting according to claim 4 in the preparation of membrane compost, characterized in that, The reactor core is ventilated intermittently via ventilation ducts, with 10-minute ventilation intervals followed by 50-minute intervals, at a ventilation rate of 0.6 m / s². 3 / h.

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