Method for reducing greenhouse gas emission in compost maturing process by using nematodes
By adding cultured nematodes to the compost to regulate microbial changes, the problem of amplification of traditional compost greenhouse gas emissions is solved, and lower greenhouse gas emissions and more efficient compost calcification is achieved.
Patent Information
- Application Number
- CN202510262769.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-06-13
AI Technical Summary
Traditional aerobic compost has large greenhouse gas emissions and long composting time. Although existing bacterial agents promote compost calcification efficiency, they greatly increase greenhouse gas emissions to pollute the environment.
By culturing and expanding the market-purchased nematodes, adding them to the composting, using nematodes as bacteria agents, adjusting microbial changes in the composting and reducing greenhouse gas emissions.
The greenhouse gas emission rates and cumulative emissions of the composting process are significantly reduced, and air pollution is reduced, providing a diversified method of compost corruption.
Smart Images
Figure CN120136585A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial research, and more particularly to a method for reducing greenhouse gas emissions during the composting process by using nematodes. Background Art
[0002] Composting is a biochemical process in which microorganisms act on organic matter for mineralization and harmless treatment, accelerating the decomposition of organic matter in livestock and poultry manure, and ultimately forming stable compounds containing N, P, K and humus. Traditional aerobic composting has been greatly restricted by problems such as large greenhouse gas emissions and long composting time. At present, most at home and abroad are committed to using microbial agents to solve composting problems. Although the composting efficiency is promoted, the greenhouse gas emissions are greatly increased, polluting the environment. Therefore, it is hoped to optimize the composting process, solve the problem of greenhouse gas emissions, and transform livestock and poultry manure into harmless and resourceful products.
[0003] Soil nematodes are the most widely distributed and abundant invertebrate metazoans in terrestrial ecosystems, and are an important part of soil animals. They are active at different positions in the food web, occupying multiple trophic levels of the food web, interacting with soil microorganisms and plants, and making important contributions to maintaining the stability of the soil biological community. Therefore, whether nematodes can reduce greenhouse gas emissions during the composting process, especially considering their functions and importance in the microbial food web, has become an urgent challenge in the fields of composting technology and biology. By comprehensively considering the current situation of composting and the functional roles of bacterivorous nematodes, how to use such bacterivorous nematodes to reduce greenhouse gas emissions during composting is an urgent problem to be solved. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for reducing greenhouse gas emissions during the composting process by using nematodes. By culturing and propagating the nematodes purchased from the market, it can be popularized and applied to composting to reduce the content of greenhouse gas emissions during its composting process.
[0005] In order to achieve the above purpose, the present invention adopts the following technical measures: A method for reducing greenhouse gas emissions during the composting process by using nematodes, including the following steps:
[0006] A. Take Escherichia coli strains and nematodes. Use Escherichia coli OP50 as the food for nematodes. Inoculate Escherichia coli OP50 into LB liquid medium, and then culture the LB liquid medium containing Escherichia coli OP50 in a shaker until the OD value is close to 2.5. Then, take 200 microliters of the LB liquid medium (containing cultured Escherichia coli OP50) and evenly coat it on the NGM nematode growth solid medium. Then, pick the nematodes onto the NGM nematode growth solid medium coated with Escherichia coli OP50. Finally, culture the NGM nematode growth solid medium inoculated with Escherichia coli and nematodes in an incubator.
[0007] B. Use an inverted microscope to count the number of nematodes growing in the NGM nematode growth solid medium. Add 2 ml of nematode washing solution in a laminar flow hood to obtain the washed nematodes.
[0008] C. Repeat step B 3 - 4 times. Put the washed nematodes into a 10 - ml centrifuge tube to obtain a nematode solution with a certain concentration of nematodes.
[0009] D. Disinfect the nematodes with alcohol. First, use alcohol with a volume fraction of 75 ± 5%, and the dosage is 1 ml of alcohol corresponding to 500 - 550 nematodes. Mix evenly and let stand for 4.5 - 5 hours. During the standing period, place it in an incubator at 28°C. After disinfection, centrifuge to remove the supernatant.
[0010] E. Crush the organic materials, humidify them, adjust the water content to 65%, pile them into a compost pile with a length × width × height of 1.5 m × 1.5 m × 1 m. Aspirate the nematode solution with a pipette and evenly drip it into each position of the compost when the compost is first piled up. The nematode addition amount is 10 - 100 nematodes / 100 g of compost; when the temperature completely drops to room temperature and the germination index is greater than 95%, it indicates that the organic fertilizer is completely decomposed.
[0011] F. Each gas sampling starts at about 12:00 on the same day. Use a 30 - mL syringe for sample collection. Collect samples once every 10 minutes within a 30 - minute time period, and a total of 4 gas samples are collected. The gas samples are placed in a 0.1 - L aluminum - plastic composite gas bag and analyzed for gas concentration simultaneously with an Agilent 7890 gas chromatograph in the laboratory.
[0012] Preferably, the static chamber method is used for gas sampling. The sampling chamber is made of PVC material and adopts a standard combination design, consisting of two parts: a chamber body and a base. The chamber body is capped and equipped with a fan, a sampling tube, and a temperature - measuring port. Before the experiment, drive the base into the sampling site for sealing, and the top of the chamber body is parallel to the compost surface.
[0013] Furthermore, the preparation method of the LB liquid medium is as follows: Add 10.0 g of peptone, 5.0 g of yeast extract powder, 1.0 g of glucose to 1 L of sterile water, and the pH is 7.0 ± 0.2.
[0014] Furthermore, the preparation method of the NGM nematode growth solid medium is as follows: Add 2.5 g of peptone, 3.0 g of sodium chloride, 0.111 g of calcium chloride, 0.12 g of magnesium sulfate, 0.005 g of cholesterol, 3.4 g of potassium dihydrogen phosphate, and 17.0 g of agar to 1 L of sterile water.
[0015] Furthermore, the culture conditions of the LB liquid medium inoculated with Escherichia coli in step A are: culture in a shaker at 25°C and 180 rmp for 48 hours.
[0016] Further, the culture conditions of the NGM nematode growth solid medium inoculated with Escherichia coli and nematodes in step A are: dark culture for 48 hours in an incubator at 28°C and a relative humidity of 50%.
[0017] Preferably, the gradient of nematode addition in step E (for three treatments) is: add 10, 50, and 100 nematodes / 100 g of compost respectively.
[0018] Thus, the key steps of the present invention are: adding nematodes to the compost and gas collection. When piling up the compost, add nematodes to the compost, and ensure that the nematode abundance is uniform at all positions of the compost during the addition process (try to keep nematodes present everywhere). And when collecting gas, it should be noted that the base of the static chamber should be parallel to the compost surface, and half of the static chamber should be embedded in the compost to ensure consistent gas collection concentration.
[0019] Compared with the prior art, the present invention has the following advantages and effects:
[0020] This method uses nematodes as inoculants, creating the first method of treating compost with nematode agents. Based on the rich microorganisms in the compost, it can provide a food source for nematodes, possibly providing a more suitable living environment, and according to the predatory characteristics of nematodes, it can regulate the microbial changes in the compost. Compared with traditional compost inoculants, the method of using nematodes instead of inoculants is more diversified, makes better use of the diversity in the compost maturity method, can reduce greenhouse gas production, and thus is expected to reduce air pollution. Description of the Drawings
[0021] Figure 1 Compost greenhouse gas emission rates (bar chart) and cumulative emissions (line chart) with and without nematode addition (CK).
[0022] Figure 2 Compost greenhouse gas emission rates (bar chart) and cumulative emissions (line chart) with inoculant addition and nematode addition. Detailed Embodiments
[0023] The following description provides specific application scenarios and requirements of the present application, aiming to enable those skilled in the art to manufacture and use the content in the present application. For those skilled in the art, various partial modifications to the disclosed embodiments are obvious, and the general principles defined here can be applied to other embodiments and applications without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure is not limited to the disclosed embodiments, but covers the broadest scope consistent with the claims.
[0024] Example 1:
[0025] A method for reducing greenhouse gas emissions during the composting process using nematodes, comprising the following steps:
[0026] A. Take Escherichia coli strains and nematodes. Use Escherichia coli OP50 as food for the nematodes. Inoculate Escherichia coli into LB liquid medium, and then culture the LB liquid medium containing Escherichia coli OP50 in a shaker at 25 °C and 180 rmp for 48 h until the OD value (abbreviation of Optical Density, used to describe the light absorption degree of the detected substance) is close to 2.5. Then, take 200 μL of the LB liquid medium (containing cultured Escherichia coli OP50) and evenly coat it on the NGM nematode growth solid medium. Then, pick the nematodes onto the NGM nematode growth solid medium coated with Escherichia coli OP50. Finally, culture the NGM nematode growth solid medium with Escherichia coli and nematodes in the dark in an incubator at 28 °C and a relative humidity of 50% for 48 hours.
[0027] B. Use an inverted microscope to count the number of nematodes growing on the NGM nematode growth solid medium. Add 2 mL of nematode washing solution in a laminar flow hood to obtain washed nematodes.
[0028] C. Repeat step B four times. Put the washed nematodes into a 10 mL centrifuge tube to obtain a nematode solution with a certain concentration of nematodes.
[0029] D. Disinfect the nematodes with alcohol. First, use alcohol with a volume fraction of 75 ± 5%, and the dosage is 1 mL of alcohol corresponding to 500 - 550 nematodes. Mix evenly and let stand for 5 hours. During the standing period, place it in a constant temperature incubator at 28 °C. After disinfection, centrifuge to remove the supernatant.
[0030] E. Crush the compost raw materials (cow dung and straw, with a carbon-nitrogen ratio of 25:1), humidify them, adjust the water content to 65%, and pile them into a compost pile with a length × width × height = 1.5 m × 1.5 m × 1 m. Use a pipette to suck out the nematode solution, and according to the nematode addition gradient, add 0 (CK: no nematodes added), 10, 50, and 100 nematodes / g of compost, and evenly drip them into each position of the compost when the compost is initially piled up; when the temperature completely drops to room temperature and the germination index is greater than 95%, it indicates fully decomposed organic fertilizer.
[0031] F. Each gas sampling started at around 12:00 on the same day. A 30 mL syringe was used for sample collection. Samples were collected once every 10 minutes within a 30-minute period, and a total of 4 gas samples were collected. The gas samples were placed in a 0.1 L aluminum-plastic composite gas bag, and the gas concentrations were analyzed simultaneously using an Agilent 7890 gas chromatograph in the laboratory. The static chamber method was used for gas collection. The sampling chamber was made of PVC material and designed in a standard combination, consisting of a chamber body and a base. The chamber body was capped and equipped with a fan, a sampling tube, and a temperature measurement port. Before the experiment, the base was driven into the sampling site and sealed, and the top of the chamber body was parallel to the compost surface.
[0032] The preparation method of LB liquid medium is as follows: Add 10.0 g of peptone, 5.0 g of yeast extract powder, 1.0 g of glucose to 1 L of sterile water, and the pH is 7.0 ± 0.2.
[0033] The preparation method of NGM nematode growth solid medium is as follows: Add 2.5 g of peptone, 3.0 g of sodium chloride, 0.111 g of calcium chloride, 0.12 g of magnesium sulfate, 0.005 cholesterol, 3.4 g of potassium dihydrogen phosphate, and 17.0 g of agar to 1 L of sterile water.
[0034] The Escherichia coli OP50 was purchased from Hangzhou Hongsai Biotechnology Co., Ltd. on September 14, 2023, with the product number 1083.
[0035] The nematodes were: Caenorhabditis elegans wild type N2, purchased from Hangzhou Hongsai Biotechnology Co., Ltd. on September 17, 2023, with the product number x2034.
[0036] As Figure 1 shown, compared with adding nematodes to compost and not adding nematodes, adding nematodes significantly reduced the emission rates of greenhouse gas CO 2 during the high emission period (the 4th - 8th day of the composting process), N 2 O during the high emission period (the 8th day of the composting process), and CH 4 during the high emission period (the 4th day of the composting process). And adding nematodes can greatly reduce the cumulative emissions of greenhouse gases (CO 2 , N 2 O, and CH 4 ) during the composting process.
[0037] Example 2:
[0038] A comparative experiment on the method of adding compost starter inoculant and bacterivorous nematodes was carried out. The steps are as follows:
[0039] A. Take Escherichia coli strains and nematodes. Use Escherichia coli OP50 as the food for nematodes. Inoculate Escherichia coli into LB liquid medium, and then culture the LB liquid medium containing Escherichia coli OP50 in a shaker at 25°C and 180 rmp for 48 h until the OD value (abbreviation of Optical Density, used to describe the light absorption degree of the detected substance) is close to 2.5. Then, take 200 μL of the LB liquid medium (containing cultured Escherichia coli OP50) and evenly spread it on the NGM solid medium for nematode growth. Then, pick the nematodes onto the NGM solid medium for nematode growth coated with Escherichia coli OP50. Finally, culture the NGM solid medium with Escherichia coli and nematodes in the dark in an incubator at 28°C and a relative humidity of 50% for 48 hours.
[0040] B. Use an inverted microscope to count the number of nematodes growing on the NGM solid medium for nematode growth. Add 2 mL of nematode washing solution in a laminar flow hood to obtain washed nematodes.
[0041] C. Repeat step B 4 times. Put the washed nematodes into a 10 mL centrifuge tube to obtain a nematode solution with a certain concentration of nematodes.
[0042] D. Disinfect the nematodes with alcohol. First, use alcohol with a volume fraction of 75 ± 5%, and the dosage is 1 mL of alcohol corresponding to 500 - 550 nematodes. Mix evenly and let it stand for 5 hours, during which it is placed in a constant temperature incubator at 28°C. After disinfection, centrifuge to remove the supernatant.
[0043] E. Crush the compost raw materials (cow dung and straw, with a carbon-nitrogen ratio of 25:1), humidify them, adjust the water content to 65%, and pile them into a pile with a length × width × height of 1.5 m × 1.5 m × 1 m. Aspirate the nematode solution with a pipette. According to the added nematode gradient, add 10, 50, and 100 nematodes / 100 g of compost respectively, and evenly drip the nematodes into each position of the compost when the compost is initially piled up. When the temperature completely drops to room temperature and the germination index is greater than 95%, it indicates that the organic fertilizer is completely decomposed.
[0044] F. Each gas sampling starts at around 12:00 on the same day. The sample collection uses a 30 mL syringe, and samples are collected once every 10 min within a 30 min time period, for a total of 4 gas samples. The gas samples are placed in a 0.1 L aluminum-plastic composite gas bag, and the gas concentration is analyzed simultaneously using an Agilent 7890 gas chromatograph in the laboratory. The static chamber method is used for gas collection. The sampling chamber is made of PVC material and is designed in a standard combination, consisting of a chamber body and a base. The chamber body is capped and equipped with a fan, a sampling tube, and a temperature measurement port. Before the experiment, drive the base into the sampling site for sealing, and the top of the chamber body is parallel to the compost surface.
[0045] The control experiment is as follows:
[0046] The composting raw materials are cow dung and straw. The carbon-nitrogen ratio of cow dung and straw is 25:1. The composting raw materials are crushed, humidified, and the water content is adjusted to 65%. A compost pile with a length×width×height = 1.5m×1.5m×1m is formed. The microbial mixed inoculant is evenly added to each position of the compost at the initial composting start-up. 1g of the mixed inoculant is added per 1kg of compost. The gas sampling and detection methods are the same as those in step F.
[0047] In this example, the source of the nematodes is the same as that in Example 1; the culture medium is the same as that in Example 1; the composition of the organic materials is the same as that in Example 1.
[0048] The microbial mixed inoculant includes the following strains mixed in the following proportions: Bacillus subtilis, viable count ≥ 5×107 CFU / g; Bacillus amyloliquefaciens, viable count ≥ 5×107 CFU / g; Saccharomyces cerevisiae, viable count ≥ 1×108 CFU / g; Trichoderma harzianum, viable count ≥ 1×108 CFU / g, and a microbial mixed preparation is made according to the ratio of 1:1:1:1. Purchased from: Pengyao Lianye Biotechnology Co., Ltd., Jiangyin City, Jiangsu Province. Purchase date: September 1st, 2023.
[0049] As Figure 2 shown, compared with adding nematodes to the compost and adding the microbial inoculant, adding nematodes significantly reduced the emission rates of greenhouse gases CO 2 during the high emission period (the 4th - 8th day of the composting process), N 2 O during the high emission period (the 8th day of the composting process), and CH 4 during the high emission period (the 4th day of the composting process). And compared with adding the microbial inoculant, adding nematodes can greatly reduce the cumulative emissions of greenhouse gases (CO 2 , N 2 O, and CH 4 ) during the composting process.
[0050] By Figure 1 and Figure 2By comparison, it can be seen that the cumulative emissions of greenhouse gases after adding the microbial agent are higher than those without adding the microbial agent. The reason is that the microorganisms in the microbial agent have a high metabolic intensity and a fast reproduction rate. They can accelerate the decomposition of organic matter in the compost, causing the carbon source to be rapidly converted into greenhouse gases such as carbon dioxide and released. At the same time, the microbial agent promotes the temperature rise of the compost and prolongs the high-temperature period, further accelerating the decomposition of organic matter and resulting in an increase in greenhouse gas emissions. The cumulative emissions of greenhouse gases from adding nematodes are lower than those from adding the microbial agent. This may be because nematodes inhibit the excessive reproduction and activities of some microorganisms by feeding on microorganisms such as bacteria, thereby reducing the generation of greenhouse gases during the decomposition of organic matter. In addition, when nematodes move in the compost pile, they can form tiny channels, increasing the porosity of the compost pile materials and improving the aeration of the compost pile. Good aeration helps increase the oxygen content in the compost pile, promotes the activities of aerobic microorganisms, reduces methane production under anaerobic conditions, and makes the decomposition of organic matter more inclined to produce carbon dioxide rather than greenhouse gases with stronger greenhouse effects such as methane. This ecological regulation effect reduces the total emissions of greenhouse gases.
[0051] As described above, it is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope of the claims.
Claims
1. A method for reducing greenhouse gas emissions during composting using nematodes, characterized in that: The steps include: A. After inoculating E. coli into LB liquid medium for cultivation, take the LB liquid medium and evenly spread it on the NGM nematode growth solid medium, pick the nematodes into the NGM nematode growth solid medium, and finally culture the NGM nematode growth solid medium with E. coli and nematodes in the dark in an incubator; B. Use an inverted microscope to count the number of nematodes growing in the NGM nematode growth solid culture medium, add nematode cleaning solution in the clean bench to clean the nematodes; C. Repeat step B several times to obtain nematode solution; D. Disinfect the nematodes with alcohol and remove the supernatant after disinfection. E. Add the nematode solution evenly into the compost; F. Use a static chamber to measure gas emissions during composting.
2. The method according to claim 1, characterized in that In step A, the LB liquid culture medium is prepared by adding 10.0 g of peptone, 5.0 g of yeast extract powder, and 1.0 g of glucose to 1 L of sterile water, with a pH of 7.0±0.
2.
3. The method according to claim 1, characterized in that In step A, the NGM nematode growth solid culture medium is prepared by adding 2.5 g of peptone, 3.0 g of sodium chloride, 0.111 g of calcium chloride, 0.12 g of magnesium sulfate, 0.005 g of cholesterol, 3.4 g of potassium dihydrogen phosphate, and 17.0 g of agar to 1 L of sterile water.
4. The method according to claim 1, characterized in that: In step A, the culture conditions of the LB liquid culture medium are: culture in a shaker at 25° C. and 180 rpm for 48 hours; The culture conditions of NGM nematode growth solid medium are: culture in the dark in an incubator at 28°C and 50% relative humidity for 48 hours.
5. The method according to claim 1, characterized in that In step C, step B is repeated 3-4 times.
6. The method according to claim 1, characterized in that In step D, the method of alcohol disinfection is: first use alcohol with a volume fraction of 75±5%, with 1 ml of alcohol corresponding to 500-550 nematodes, mix well and let stand for 4.5-5 hours, during which time it is placed in a constant temperature box at 28°C; After disinfection, the supernatant was removed by centrifugation.
7. The method according to claim 1, characterized in that In the step E, the nematode liquid is uniformly added to the compost, comprising: crushing the organic material, humidifying, adjusting the water content to 65%, piling it into a pile with a length×width×height=1.5m×1.5m×1m, sucking out the nematode liquid with a pipette, and evenly dripping it into various positions of the compost when the compost is first piled up, the amount of nematodes added is 10-100 nematodes / 100g of compost; when the temperature is completely reduced to room temperature, the germination index is greater than 95%, indicating that the organic fertilizer is completely decomposed.
8. The method according to claim 7, characterized in that The concentrations of the added nematodes are: 10 nematodes / 100 g of compost, 50 nematodes / 100 g of compost, and 100 nematodes / 100 g of compost.
9. The method according to claim 1, characterized in that: In the step F, the gas emission during the composting process is measured by using a static box, including: each gas sampling starts at about 12:00 on the same day, the sample is collected using a 30mL syringe, a sample is collected every 10 minutes within a 30-minute period, and a total of 4 gas samples are collected. The gas samples are placed in a 0.1L aluminum-plastic composite air bag, and the gas concentration is simultaneously analyzed in the laboratory using an Agilent 7890 gas chromatograph.
10. The method according to claim 9, characterized in that The gas was collected using the static box method. The sampling box was made of PVC material and consisted of a box body and a base. The box was sealed with a fan, sampling tube and temperature measuring port installed inside. Before the experiment, the base was driven into the sampling site and sealed. The top of the box was parallel to the compost surface.