Method and device for testing concentration and particle size of microbial aerosol for low-pressure sprinkling irrigation of biogas slurry

Abstract

The invention provides a biogas slurry low-pressure sprinkling irrigation microbial aerosol concentration and particle size testing method and device. The method comprises the following steps: testing the concentration of microorganisms in diluted biogas slurry by adopting a plate coating method; three kinds of low-pressure spray heads are selected, and the mounting height of the spray heads is set to be 1.2 m; the method comprises the following steps: by taking a nozzle as a starting point, arranging microbial aerosol measuring points at intervals of 8m along the radial direction, and selecting the average breathing height 1.64 m of a human body as the collection height at each measuring point; 4, counting microbial colonies on the culture dish by adopting anaerobic fermentation biogas slurry; counting microbial colonies growing on the culture plate by using a colony counting instrument, and correcting the number of the colonies by using a positivity-hole method, so as to obtain the concentration of the air microbial aerosol and the particle size distribution characteristics of the air microbial aerosol. According to the invention, deep research on the dissipation mechanism of the microbial aerosol in the biogas slurry low-pressure sprinkling irrigation process is realized, and a scientific and effective method and system support are provided for parameter optimization and health risk prevention and control of a biogas slurry low-pressure sprinkling irrigation system.

Description

Technical Field

[0001] This invention relates to the field of agricultural engineering and environmental microbiology detection technology, specifically a method and apparatus for testing the concentration and particle size of microbial aerosols in low-pressure biogas irrigation. Background Technology

[0002] Biogas projects are one of the main means of promoting the resource utilization of livestock and poultry manure in my country. Applying biogas slurry fertilizer to farmland can effectively supplement crops with a variety of nutrients, helping to reduce the use of chemical fertilizers and increase efficiency. Therefore, low-pressure sprinkler irrigation of biogas slurry for returning to the field is considered one of the important ways to utilize biogas slurry organic fertilizer on a large scale and scientifically, and it has been widely used in many integrated crop and livestock farming enterprises across the country.

[0003] During low-pressure sprinkler irrigation with biogas slurry, the release of microbial aerosols can pollute the surrounding atmosphere, leading to increased concentrations of harmful microorganisms in the air. These aerosols can also enter the human body through respiration, posing health risks such as respiratory illnesses, and may adversely affect the soil ecosystem and the growth of surrounding plants and animals. Therefore, monitoring the release of microbial aerosols during this process is crucial for implementing targeted preventative measures. The release process of microbial aerosols generated by sprinkler irrigation is highly complex, influenced by numerous factors. Previous studies on microbial aerosol release using sprinkler types, operating pressures, and irrigation water sources differ from those used in current integrated crop-livestock low-pressure sprinkler irrigation with biogas slurry, resulting in significant differences in the release characteristics and underlying mechanisms of microbial aerosols. Previous research results are not suitable for direct application in the design of low-pressure sprinkler irrigation projects with biogas slurry and for health risk control. Therefore, a method for testing the concentration and particle size of microbial aerosols and optimizing system parameters for low-pressure sprinkler irrigation with biogas slurry is urgently needed.

[0004] In the process of realizing this invention, the inventors discovered that the prior art has at least the following problems: Traditional testing methods are mostly designed for urban sewage or reclaimed water sprinkler irrigation, failing to fully consider the unique characteristics of the microbial community specific to livestock and poultry manure in biogas slurry. Existing technologies lack analysis of the coupled effects of low-pressure sprinkler irrigation system parameters, such as nozzle type, operating pressure, and nozzle diameter. These factors significantly influence the atomization effect and spray uniformity of low-pressure biogas slurry sprinkler irrigation, thereby determining the diffusion range and survival status of the unique microbial community in livestock and poultry manure. Summary of the Invention

[0005] This invention aims to provide a method and apparatus for testing the concentration and particle size of microbial aerosols in low-pressure biogas slurry irrigation, in order to solve the problem of missing parameter analysis in the prior art, realize the effective testing of microbial aerosols in the process of low-pressure biogas slurry irrigation, and provide a scientific basis for the optimization of parameters of low-pressure biogas slurry irrigation system and the prevention and control of health risks.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A method for testing the concentration and particle size of microbial aerosols in low-pressure sprinkler irrigation of biogas slurry, comprising: Step 1: Before testing, the concentration of microorganisms in the diluted biogas slurry was tested multiple times using the plate coating method, and the average concentration was taken as the concentration of microorganisms in the biogas slurry. Step 2: Select 3 types of low-pressure nozzles with working pressures of 69 kPa, 103 kPa and 138 kPa, and nozzle diameters of 3.97 mm, 5.95 mm and 7.93 mm, forming 27 different working condition combinations in 3×3×3, with each combination repeated 3 times; Step 3: Set the nozzle installation height to 1.2m; starting from the nozzle, set up microbial aerosol measurement points every 8m along the radial direction, and select the average human breathing height of 1.64m as the collection height for each measurement point; Step four: Anaerobic fermentation biogas slurry is diluted with water at a ratio of 1:1 and then sprayed under pressure through a low-pressure nozzle. The concentration and particle size of the released aerosols are collected using a FA-1H microbial collector. After culturing the culture dishes from the collector, the microbial colonies on the culture dishes are counted. Step 5: Each test lasts 20 minutes; use a colony counter to count the microbial colonies growing on the culture plate, and use the positive-hole method to correct the colony count, thereby obtaining the concentration and particle size distribution characteristics of airborne microbial aerosols.

[0007] 1. Optionally, the formula for determining the microbial concentration in the biogas slurry in step one is: ; in: (Microbial concentration per unit volume, unit: CFU / mL) The number of colonies counted on the petri dish, in units of cells. The volume of liquid used during coating, in mL. This refers to the dilution factor; Each diluted sample was plated, and the number of colonies on each petri dish was recorded. The microbial concentration of each sample was calculated. The average of these values ​​was taken as the concentration of microorganisms in the biogas slurry.

[0008] Optionally, step five involves correcting the colony count using the positive-hole method, including: ; In the formula, The corrected colony counts for each layer of agar culture plate. N The number of sampling wells for each layer of agar culture plate. The actual colony counts for each layer of agar culture plates were measured. The concentrations of microbial aerosols at each level in the air were determined based on the corrected colony counts of each layer of agar plates, the sampling airflow rate, and the sampling time. ; In the formula, The concentration of microbial aerosols at each level, CFU / m³ 3 , To sample air flow rate in L / min, t The sampling time is in minutes.

[0009] 2. Optionally, the particle size is obtained using a laser particle size analyzer or a microbial collector, and the log-normal distribution characteristic of the particle size distribution in step five is obtained using the following formula: ; in: Particle size frequency, The logarithmic mean of the particle size. The standard deviation of particle size is the logarithm of particle size. Particle size distribution is usually calculated using particle size analysis. Specifically, an aerosol particle size analyzer can be used to obtain the aerosol concentration at different particle sizes, and then a particle size distribution map can be drawn.

[0010] 3. Optionally, the airborne microbial aerosol concentration distribution characteristics in step five are obtained using the following formula: ; in: Distance to nozzle Aerosol concentration at the location, CFU / m³ This refers to the aerosol concentration near the nozzle, in CFU / m³. Let m be the distance from the nozzle to the nearest measuring point. It is the concentration decay index, which is usually obtained based on experience or experimental data.

[0011] Optionally, step four specifically includes: 1) Pretreatment of biogas slurry: Mix biogas slurry and water at a volume ratio of 1:1 and stir evenly to obtain homogeneous biogas slurry; perform serial dilution of the homogeneous biogas slurry, take each serial dilution and drop it onto a petri dish and spread it evenly, incubate at 37℃ for 24 hours, and count the number of colonies to determine the concentration of microorganisms in the biogas slurry. 2) Sampling preparation: Place LB agar medium in each impact chamber of the six-stage sieve impact sampling unit, connect the six-stage sieve impact sampling unit with the FA-1H microbial collector, turn on the FA-1H microbial collector and adjust it to the preset flow rate; 3) Sprinkler irrigation and sampling: The homogenized biogas slurry is pressurized by a pressurizing device and then atomized and sprayed out through a low-pressure nozzle for continuous irrigation for 20 minutes. During the irrigation process, the escaping microbial aerosols are collected by a FA-1H type microbial collector. 4) Cultivation and Analysis: After the sprinkler irrigation is completed, the culture dishes in the six-stage sieve impact collection unit are removed under aseptic conditions and incubated at a constant temperature of 37℃ for 24 hours. The number of microbial colonies on each stage of the culture dish is counted. Combined with the corresponding microbial particle size range of each stage of the six-stage sieve impact collection unit, the concentration and particle size distribution of microbial aerosols are analyzed.

[0012] 7. The method for testing the concentration and particle size of microbial aerosols in low-pressure sprinkler irrigation of biogas slurry according to claim 6, characterized in that the gradient dilution in step 1) specifically involves: mixing to prepare 10⁻¹, 10⁻², 10⁻³, and 10⁻¹ particles. 4 and 10⁻ 5 Diluent; When counting colonies in step 1), select petri dishes with colony counts between 30 and 300 for counting; In step 2), the preset flow rate of the FA-1H microbial collector is 28.3 L / min.

[0013] A device for testing the concentration and particle size of microbial aerosols in low-pressure sprinkler irrigation of biogas slurry, which implements any of the methods described in this invention, includes a fertilizer storage device (1), a pressure regulating and spraying device (2), and an FA-1H type intelligent microbial sampling device (3); the fertilizer storage device (1) is used to store biogas slurry and stir and mix it; the pressure regulating and spraying device (2) is used to apply a specific pressure to the biogas slurry and atomize and spray it out; and the FA-1H type intelligent microbial sampling device (3) is used to collect microbial aerosols that escape during sprinkler irrigation.

[0014] Optionally, the fertilizer storage device (1) includes a fertilizer storage tank (12), a mixer (11), and a ball valve (13); the mixer (11) is installed on the top of the fertilizer storage tank (12), and its mixing component extends into the inside of the fertilizer storage tank (12); the ball valve (13) is installed at the liquid outlet of the fertilizer storage tank (12), and is connected to the pressure regulating and spraying device (2) through a water pipe. The connection parts of the water pipe to the ball valve (13) and the pressure regulating and spraying device (2) are all threaded and wrapped with sealing raw material tape.

[0015] Optionally, the FA-1H type intelligent microbial sampling device (3) includes an FA-1H type microbial collector (31), a rubber hose (32), a six-stage sieve impact collection unit (33), and a tripod support frame (34); the six-stage sieve impact collection unit (33) is detachably installed on the top of the tripod support frame (34); one end of the rubber hose (32) is sealed to the air outlet of the six-stage sieve impact collection unit (33), and the other end is sealed to the air inlet of the FA-1H type microbial collector (31), and all connection parts are sealed joints.

[0016] The beneficial effects of this invention are: This invention integrates multiple disciplines such as agricultural water conservancy (low-pressure sprinkler irrigation) and environmental microbiology (aerosol collection), and for the first time establishes a whole-chain test method for the dispersion of biogas slurry aerosols during low-pressure biogas slurry sprinkler irrigation, clarifying the concentration and particle size distribution of microbial aerosols during the process of biogas slurry low-pressure sprinkler irrigation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram of the testing device for the concentration and particle size of biogas slurry low-pressure sprinkler irrigation microbial aerosols according to the present invention. Figure 2 For the present invention Figure 2 Enlarged view of point A in the middle; Figure 3 For the present invention Figure 2 Enlarged view of point B in the middle; Figure 4 For the present invention Figure 2 Enlarged view of point C in the middle; Figure 5 The results of data accuracy measurement of the method of the present invention using the support vector machine algorithm in Example 1 are the training set results; Figure 6 The results of the data accuracy measurement of the method of the present invention using the support vector machine algorithm in Example 1 are the test set results; Figure 7 Images of microbial colonies in various petri dishes under a typical operating condition (R3000-10psi-#30) in this invention; In the diagram: 1-Fertilizer storage device, 11-Mixer, 12-Fertilizer storage tank, 13-Ball valve; 2-Pressure regulation and spraying device, 21-220V self-priming centrifugal pump, 22-Pressure gauge, 23-Pressure regulator and nozzle; 3-FA-1H type intelligent microbial sampling device, 31-FA-1H type microbial collector, 32-rubber hose, 33-six-stage sieve impact sampling unit, 34-tripod support frame. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0020] The present invention provides a method for testing the concentration and particle size of microbial aerosols used in low-pressure sprinkler irrigation of biogas slurry, comprising five steps: Step 1: Before testing, the concentration of microorganisms in the diluted biogas slurry is tested multiple times using the plate coating method, and the average concentration is taken as the concentration of microorganisms in the biogas slurry. Step 1: Determination of biogas slurry microbial concentration; The microbial concentration in the diluted biogas slurry is tested using the plate coating method, and the average concentration is calculated. The colony counting method (plate coating method) is used to determine the concentration of microorganisms per unit volume. The formula is: ; in: Microbial concentration per unit volume (unit: CFU / mL). The number of colonies counted on the petri dish. This refers to the volume of liquid used during coating (unit: mL). This represents the dilution factor. Calculation process: Each diluted sample is plated, and the number of colonies on each petri dish is recorded. The microbial concentration of each sample was calculated. The average of these values ​​was taken as the concentration of microorganisms in the biogas slurry.

[0021] Step two: Select three commonly used low-pressure nozzles for circular sprinkler irrigation machines: the D3000 nozzle from Nelson (USA), the R3000 nozzle from Nelson (USA), and the KPT nozzle from Komet (Austria). Pair these with the working pressures of the three commonly used circular sprinkler irrigation machines (69 kPa, 103 kPa, and 138 kPa) and the nozzle diameters of the three commonly used circular sprinkler irrigation machines (3.97 mm, 5.95 mm, and 7.93 mm) to form 27 different working condition combinations (3×3×3). Each combination is repeated three times.

[0022] Step 3: Based on Step 2, the nozzle installation height is set to 1.2m, the same as the nozzle height of most circular sprinkler irrigation machines. Starting from the nozzle, microbial aerosol measurement points are set up radially every 8m. The average human breathing height of 1.64m is selected for each measurement point (the average height of men and women aged 18-44 in my country is 169.7cm and 158.0cm, respectively. This invention selects the average height of the two sexes as the average human breathing height) as the collection height to analyze the inhalation of microbial aerosols by people of average height.

[0023] Step four, the specific steps are as follows: 1) Anaerobic fermentation biogas slurry is used, which is diluted with clean water at a ratio of 1:1 and then transported under pressure through PVC pipes.

[0024] 2) The biogas slurry is sprayed out through low-pressure nozzles, and the concentration and particle size of the aerosols released during the low-pressure sprinkler irrigation process are collected by the FA-1H type microbial collector.

[0025] 3) After culturing the culture dishes from the collector, count the microbial colonies on the culture dishes.

[0026] Step 5: Building upon Step 4, each test lasts 20 minutes. After collection, the culture plate is removed and covered under aseptic conditions, then incubated upside down in a constant temperature incubator for 24 hours. The colony count is then performed using a colony counter. Because microbial aerosol particles overlap when settling within the agar plate, the positive-hole method is used to correct the colony count, thereby determining the concentration and particle size distribution characteristics of airborne microbial aerosols.

[0027] In this invention, step five uses the positive-hole method to correct the colony count, including: ; In the formula, The corrected colony counts for each layer of agar culture plate. N The number of sampling wells for each layer of agar culture plate. The actual colony counts for each layer of agar culture plates were measured. The concentrations of microbial aerosols at each level in the air were determined based on the corrected colony counts of each layer of agar plates, the sampling airflow rate, and the sampling time. ; In the formula, The concentration of microbial aerosols at each level, CFU / m³ 3 , To sample air flow rate in L / min, t The sampling time is in minutes.

[0028] 5. In this invention, the particle size is obtained by a laser particle size analyzer or a microbial collector, and the log-normal distribution of the particle size distribution characteristic in step five is obtained by the following formula: ; in: Particle size frequency, The logarithmic mean of the particle size. The standard deviation of particle size is the logarithm of particle size. Particle size distribution is usually calculated using particle size analysis. Specifically, an aerosol particle size analyzer can be used to obtain the aerosol concentration at different particle sizes, and then a particle size distribution map can be drawn.

[0029] 6. In this invention, the concentration distribution characteristics of airborne microbial aerosols in step five are obtained by the following formula: ; in: Distance to nozzle Aerosol concentration at the location, CFU / m³ This refers to the aerosol concentration near the nozzle, in CFU / m³. Let m be the distance from the nozzle to the nearest measuring point. It is the concentration decay index, which is usually obtained based on experience or experimental data.

[0030] In this invention, step four specifically includes: 1) Pretreatment of biogas slurry: Mix biogas slurry and water at a volume ratio of 1:1 and stir evenly to obtain homogeneous biogas slurry; perform serial dilution of the homogeneous biogas slurry, take each serial dilution and drop it onto a petri dish and spread it evenly, incubate at 37℃ for 24 hours, and count the number of colonies to determine the concentration of microorganisms in the biogas slurry. 2) Sampling preparation: Place LB agar medium in each impact chamber of the six-stage sieve impact sampling unit, connect the six-stage sieve impact sampling unit with the FA-1H microbial collector, turn on the FA-1H microbial collector and adjust it to the preset flow rate; 3) Sprinkler irrigation and sampling: The homogenized biogas slurry is pressurized by a pressurizing device and then atomized and sprayed out through a low-pressure nozzle for continuous irrigation for 20 minutes. During the irrigation process, the escaping microbial aerosols are collected by a FA-1H type microbial collector. 4) Cultivation and Analysis: After the sprinkler irrigation is completed, the culture dishes in the six-stage sieve impact collection unit are removed under aseptic conditions and incubated at a constant temperature of 37℃ for 24 hours. The number of microbial colonies on each stage of the culture dish is counted. Combined with the corresponding microbial particle size range of each stage of the six-stage sieve impact collection unit, the concentration and particle size distribution of microbial aerosols are analyzed.

[0031] In this invention, the gradient dilution in step 1) specifically involves mixing to prepare 10⁻¹, 10⁻², 10⁻³, and 10⁻¹ solutions. 4 and 10⁻ 5 Diluent; When counting colonies in step 1), select a petri dish plate with a colony count between 30 and 300 for counting; The preset flow rate of the FA-1H microbial collector in step 2) is 28.3 L / min.

[0032] The present invention also provides a device for testing the concentration and particle size of microbial aerosols in low-pressure sprinkler irrigation of biogas slurry, comprising the following devices: a fertilizer storage device for storing biogas slurry and uniformly stirring it; a pressure regulating and spraying device for spraying the biogas slurry after applying a specific pressure; and a FA-1H type intelligent microbial sampling device for collecting microbial aerosols released during low-pressure sprinkler irrigation.

[0033] include: Step 1) Mix biogas slurry and clean water in a 1:1 ratio and stir until homogeneous biogas slurry is obtained.

[0034] Step 2): The biogas slurry is serially diluted and spread onto petri dishes. After incubation at 37°C for 24 hours, the colony count is recorded. The microbial concentration of the biogas slurry is obtained.

[0035] Step 3): Place LB agar medium in the six-stage sieve impact collection unit and turn on the collector.

[0036] Step 4) After pressurizing the homogenized biogas slurry, it is atomized and sprayed out through the nozzle for continuous irrigation for 20 minutes.

[0037] Step 5) After the irrigation is completed, remove the petri dish and incubate at 37°C for 24 hours. Count the number of colonies at each level and analyze the microbial aerosol dispersion pattern.

[0038] Combination Figure 1-4 The biogas slurry low-pressure sprinkler irrigation microbial aerosol concentration and particle size testing device provided by the present invention includes a fertilizer storage device 1, a pressure regulating and spraying device 2, and a FA-1H type intelligent microbial sampling device 3.

[0039] The fertilizer storage device 1 consists of a mixer 11, a fertilizer storage tank 12, and a ball valve 13. The mixer 11 is installed on the top of the fertilizer storage tank 12, and the mixing component of the mixer 11 extends into the fertilizer storage tank 12. When it is necessary to mix the fertilizer in the fertilizer storage tank 12, the mixer 11 is started, and the mixing component rotates, which can make the fertilizer and water fully mixed to form a uniform fertilizer solution.

[0040] Ball valve 13 is installed at the outlet of the fertilizer storage tank 12. Ball valve 13 is threaded to the water pipe, and sealing tape is wrapped around the threaded connection. The sealing tape effectively fills the thread gaps, enhancing the seal and preventing fertilizer solution leakage from the threaded connection. Ball valve 13 is also threaded to the inlet of the 220V self-priming centrifugal pump 21 via the water pipe, and sealing tape is also wrapped around the threaded connection. This design organically integrates the fertilizer storage device 1 with the pressure regulating and spraying device 2 into a single unit, ensuring tight connection and operational stability throughout the entire process of biogas slurry storage, mixing, pressurization, and irrigation.

[0041] The pressure regulating and spraying device 2 consists of a 220V self-priming centrifugal pump 21, a pressure gauge 22, a pressure regulator, and a nozzle 23. The outlet of the 220V self-priming centrifugal pump 21 is connected to the inlet of the pressure gauge 22 through a pipeline. The pipeline and the outlet of the 220V self-priming centrifugal pump 21 are connected by a thread. The threaded connection is wrapped with sealing tape to enhance the sealing of the connection and prevent biogas slurry from leaking from the threaded connection gap.

[0042] The outlet of pressure gauge 22 is connected to the inlet of pressure regulator and nozzle 23 via a pipeline. The outlet of the pipeline and pressure gauge 22 is made of PVC adhesive. The inlet of the pipeline and pressure regulator and nozzle 23 is connected by a threaded connection. The threaded connection is wrapped with sealing PTFE tape to further ensure the sealing of the entire pipeline system.

[0043] The FA-1H intelligent microbial sampling device 3 consists of an FA-1H microbial collector 31, a rubber hose 32, a six-stage sieve impact sampling unit 33, and a tripod support frame 34. The six-stage sieve impact sampling unit 33 is detachably installed on the top of the tripod support frame 34. The adapter interface at the top of the tripod support frame 34 is matched with the connection structure at the bottom of the six-stage sieve impact sampling unit 33 to achieve a stable connection. This ensures that the six-stage sieve impact sampling unit 33 remains horizontal and stable during the sampling process, avoiding the impact of shaking and other factors on the sampling accuracy. One end of the rubber hose 32 is sealed to the outlet of the six-stage sieve impact collection unit 33. The connection is made with a sealed joint to ensure that the airflow does not leak during transmission, so that the airflow containing microbial aerosols discharged from the six-stage sieve impact collection unit 12 can all enter the rubber hose 32. The other end of the rubber hose 32 is sealed to the inlet of the FA-1H type microbial collector 31, also through a sealed joint, so that the airflow can smoothly enter the FA-1H type microbial collector 31, and the FA-1H type microbial collector 31 collects and processes the microorganisms in the airflow.

[0044] The following detailed description of the method for testing the concentration and particle size of biogas slurry low-pressure sprinkler irrigation microbial aerosols according to the present invention is provided with reference to specific embodiments.

[0045] Example 1: Experiment on the test method of microbial aerosol concentration and particle size of biogas slurry low-pressure sprinkler irrigation in Yangling area.

[0046] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the technical conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0047] The experiment was conducted in the Modern Agricultural Demonstration Park of Yangling District, Xianyang City, Shaanxi Province (34°15′55.44″N, 108°05′43.44″E). The area is located in the western part of Xianyang City and has a temperate monsoon climate. Winters are long, dry and cold, springs are dry and windy, summers are short, hot and rainy, and autumns are short, cool, with a sharp drop in temperature and early frost. Before the experiment, the biogas slurry low-pressure sprinkler irrigation and microbial aerosol collection device were installed at the experimental site, and in-depth research experiments were conducted on the microbial aerosol dispersion mechanism during the biogas slurry low-pressure sprinkler irrigation process under different working conditions.

[0048] Step 1: Accurately measure biogas slurry and clean water into the storage tank 12 of the fertilizer storage device 1 at a volume ratio of 1:1, and add them into the storage tank 12.

[0049] Then, the mixer 11 on top of the fertilizer storage tank 12 is started, so that the mixing component of the mixer 11 rotates inside the fertilizer storage tank 12 at a preset speed to continuously mix the biogas slurry and clean water until they are evenly mixed to obtain homogeneous biogas slurry for the test, providing a uniform matrix condition for the stable release of microbial aerosols during subsequent sprinkler irrigation.

[0050] Step 2: First, take an appropriate amount of diluted biogas slurry (e.g., 10 mL), shake it thoroughly to ensure that the microorganisms are evenly distributed.

[0051] Use a 1mL sterile pipette to draw 1mL of biogas slurry and inject it into 9mL of sterile physiological saline. Mix thoroughly to prepare... Diluent.

[0052] Subsequently, replace with a new sterile pipette, accurately draw 1 mL of liquid from the above diluent, and slowly inject it into a container pre-filled with 9 mL of sterile diluent. Mix thoroughly to prepare the next concentration gradient diluent. Repeat the above steps to complete the gradient dilution operations of 1:10, 1:100, 1:1000... (the specific dilution gradient is adaptively adjusted according to the actual concentration of microorganisms in the biogas slurry to avoid inaccurate counting due to excessive colonies during subsequent colony counting).

[0053] Take 0.1 mL of each graded dilution (e.g., 1:10, 1:100, 1:1000) and drop them onto the surface of a sterile petri dish. Spread the solution evenly using a sterile spreader. The incubation conditions are as follows: invert the plate (to prevent condensation from affecting colony morphology) and incubate at 37°C for 24 hours. This temperature is the optimal growth temperature for most microorganisms in the biogas slurry.

[0054] Select plates with colony counts between 30 and 300 (this range has the smallest counting error), and use colony counting software to count the microbial colonies growing on the culture plates to obtain the microbial concentration in the biogas slurry.

[0055] In step two, the microbial concentration of the diluted biogas slurry is tested using the plate coating method, and the average concentration is calculated. The relevant theoretical calculations are as follows: Colony counting (plate method) is used to determine the concentration of microorganisms per unit volume. The formula is: ; In the formula, Microbial concentration per unit volume (unit: CFU / mL). The number of colonies counted on the petri dish. This refers to the volume of liquid used during coating (unit: mL). This represents the dilution factor.

[0056] Calculation process: Each diluted sample was plated, and the number of colonies on each petri dish was recorded. The microbial concentration of each sample was calculated. The average of these values ​​was taken as the concentration of microorganisms in the biogas slurry. Step 3: Take sterile LB agar medium and place it steadily into each impact chamber of the six-stage sieve impact collection unit (corresponding to the aforementioned device 33), ensuring that the distance between the medium and the impact plate in each impact chamber meets the collection requirements.

[0057] After placing the culture medium, turn on the FA-1H microbial collector that is matched with the six-stage sieve impact collection unit, and adjust the collector to the preset flow rate (usually 28.3 L / min) to put the collector into standby mode and wait to capture the microbial aerosols that escape during the irrigation process.

[0058] Step 4: Open the ball valve 13 at the outlet of the fertilizer storage tank 12. Under the combined action of its own gravity and the suction of the subsequent water pump, the homogenized biogas slurry in the fertilizer storage tank 12 flows along the connecting water pipe to the pressure regulating and spraying device 2. Start the 220V self-priming centrifugal pump 23 in the device 2. The 220V self-priming centrifugal pump 23 pressurizes the biogas slurry so that the biogas slurry pressure reaches the preset irrigation pressure.

[0059] The pressurized biogas slurry is transported along the water pipe to the pressure regulator and nozzle 21, and sprayed out in atomized form through the nozzle 21 to start a continuous 20-minute sprinkler irrigation operation. During this period, the 220V self-priming centrifugal pump 23 is kept in stable operation to ensure uniform sprinkler irrigation pressure and flow.

[0060] Step 5: After the sprinkler irrigation operation has lasted for 20 minutes, first turn off the 220V self-priming centrifugal pump 23 to stop the biogas slurry sprinkler irrigation; then, using aseptic operation, quickly remove the petri dishes containing LB agar medium from each impact chamber of the six-stage sieve impact collection unit to avoid contamination of the culture medium surface by external microorganisms.

[0061] The removed petri dishes were placed in a constant temperature incubator, and the temperature was set to 37℃ for 24 hours of constant temperature incubation. After the incubation period, the petri dishes were removed, and the number of microbial colonies growing on the surface of each level of petri dish was recorded one by one using a point-and-count camera app. Based on the microbial particle size range corresponding to different levels of impact chambers, combined with the colony count results, the data on the number of microbial aerosols of different particle size ranges during biogas slurry sprinkler irrigation were obtained, and the dispersion pattern of microbial aerosols was analyzed.

[0062] This invention also provides a distribution map of bacterial colonies collected from various measurement points in petri dishes at different levels under one operating condition (10psi-#30) of the R3000 sprinkler head for low-pressure irrigation of biogas slurry, as shown in the embodiments. Figure 7 As shown.

[0063] Because microbial aerosol particles overlap when settling in agar plates, the positive-hole method is used to correct for colony counts. ; In the formula, The corrected colony counts for each layer of agar culture plate. N The number of sampling wells for each layer of agar culture plate. The actual colony counts for each layer of agar culture plates were measured.

[0064] The concentrations of microbial aerosols at each level in the air were determined based on the corrected colony counts of each layer of agar plates, the sampling airflow rate, and the sampling time. ; In the formula, The concentration of microbial aerosols at each level (CFU / m3) is given. The sampling airflow rate (L / min) is used. t Sampling time (min).

[0065] The formula for calculating the proportion of microbial aerosol particles at each level is as follows: ; In the formula, P The percentage of microbial aerosol particles at each level, n The total number of colonies in a 6-layer agar plate.

[0066] Based on the number of microbial aerosol particles at levels 1 to 6, the cumulative percentage of particle counts at each level is calculated. This percentage is then accumulated sequentially from level 6 to level 1 to derive the regression equation between the effective intercepted particle size at each level and the cumulative percentage. The particle size corresponding to a cumulative percentage of 50% is the median particle size of the microbial aerosol particles at that sampling point.

[0067] The concentration and particle size distribution of microbial aerosols under various operating conditions of the R3000 nozzle are shown in Tables 1 and 2 below: Table 1. Microbial aerosol concentrations under various operating conditions using the R3000 nozzle. (The aerosol concentrations in the table are the sum of aerosol concentrations for each particle size from level 1 to level 6.)

[0068] Table 2. Microbial aerosol particle size distribution under various operating conditions of R3000 nozzles.

[0069] (The aerosol concentration in the table is in CFU / m3, and the nozzle model for each operating condition is #30.) As shown in Table 1, the number of microbial colonies in the petri dish is significantly positively correlated with the nozzle diameter. The nozzle diameter significantly affects the amount of biogas slurry consumed during irrigation and the concentration of microbial aerosols released during the biogas slurry irrigation process. Therefore, the number of microbial colonies in the petri dish is significantly positively correlated with the concentration of microbial aerosols released during the biogas slurry irrigation process.

[0070] In principle: The six-stage sieve impact collection unit captures microbial aerosols of different particle sizes in the air through inertial impaction and attaches them to the surface of LB agar medium. After constant temperature incubation, each viable bacterium can form a visible colony. Therefore, the number of colonies directly reflects the number of viable microorganisms captured during the collection process. Combined with the collection volume (flow rate × duration), it can be accurately converted into the actual concentration of microbial aerosols in the air, achieving the goal of "quantifying concentration by colony count".

[0071] As shown in Table 2, the microbial aerosols generated by biogas slurry sprinkler irrigation are mainly large-sized particles (>3.3 μm). This distribution characteristic is closely related to the atomization mechanism of biogas slurry sprinkler irrigation and the characteristics of the microorganisms themselves.

[0072] Impact of atomization process: After being pressurized by a 220V self-priming centrifugal pump, the biogas slurry is sprayed and broken into droplets through the nozzle. Some droplets further break down and disperse to form microbial aerosols with even smaller particle sizes (aerosol particle sizes are much smaller than visible droplets, usually in the micrometer range, close to the particle size range of fog, and can be captured by a dedicated aerosol sampler). During this process, microorganisms diffuse into the air with the aerosols: large-particle-size microbial aerosols have a relatively higher water content and are not easily evaporated during diffusion, making it easier to maintain the humidity environment required for microbial survival, thus resulting in a higher viable bacteria retention rate; while small-particle-size microbial aerosols, although having a wider diffusion range, have extremely small particle sizes and a significantly increased total surface area per unit mass (i.e., specific surface area), making them prone to rapid evaporation and water loss after atomization and during diffusion, leading to microbial inactivation due to insufficient water. Therefore, the proportion of viable bacteria in small-particle-size microbial aerosols is significantly lower than that in large-particle-size microbial aerosols.

[0073] Using MATLAB software and different algorithms, such as BP neural network, support vector machine, and random forest, we trained the model on the existing data, using 80% of the data as a training set and the remaining 20% ​​as prediction values. The accuracy of the prediction model was then assessed.

[0074] The Support Vector Machine (SVM) algorithm was ultimately selected. Ten sets of feature values ​​were used: 1) particle size of the microbial aerosol; 2) wind speed; 3) distance from the measurement point to the nozzle; 4) pressure of the biogas slurry leaving the nozzle; 5) diameter of the irrigation jet (nozzle diameter); 6) type of different nozzles; 7) ambient temperature during measurement; 8) droplet diameter of the biogas slurry during irrigation; 9) velocity of the droplets generated during irrigation; and 10) kinetic energy of the droplets during irrigation. The output value was the number of microbial colonies. Outliers were filtered and removed. The final results are shown in [link to results]. Figure 5 and 6 It is evident that the method provided by this invention has high precision.

[0075] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced under equivalent conditions and in a wide range of scope without departing from its spirit and scope, and without requiring unnecessary experimentation. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A method for testing the microbial aerosol concentration and particle size of biogas slurry low-pressure sprinkling irrigation, characterized in that, The application relates to a method for testing the concentration and particle size distribution of microbial aerosol in the air. Step one: before testing, the concentration of microorganisms in the diluted biogas slurry is tested multiple times by adopting the plate coating method, and the average concentration is taken as the concentration of microorganisms in the biogas slurry; Step two: three low-pressure nozzles are selected, the working pressure is 69kPa, 103kPa and 138kPa, the nozzle diameter is 3.97mm, 5.95mm and 7.93mm, 3x3x3, that is, 27 different working condition combinations are formed, and each combination is repeated three times; Step three: the installation height of the nozzle is set to 1.2m; the nozzle is taken as the starting point, and the microbial aerosol measuring points are arranged every 8m along the radial direction, and the average human breathing height of 1.64m is taken as the collection height; Step four: the anaerobic fermentation biogas slurry is diluted by 1:1 clean water, is sprayed out by the low-pressure nozzle through pressurized conveying, the concentration and particle size of the diffused aerosol are collected by the FA-1H microbial collector, and the culture dishes in the collector are taken out to culture, and the microbial colonies on the culture dishes are counted; Step five: the testing time is 20min each time; the microbial colonies growing on the culture plates are counted by using a colony counter, the number of colonies is corrected by adopting the positive-hole method, and then the concentration and particle size distribution characteristics of the air microbial aerosol are obtained.

2. The method of claim 1, wherein the method is used to test the microbial aerosol concentration and particle size of a low-pressure sprinkler irrigation system using biogas slurry. The concentration of microorganisms in the biogas slurry in step one is measured by the formula: ； wherein: is the concentration of microorganisms in each unit of volume, unit: CFU / mL, is the number of colonies counted on the petri dish, unit: pieces, is the volume of liquid used when coating, unit: mL, is the dilution multiple; Plate spread each diluted sample, record the number of colonies on each petri dish , calculate the microbial concentration of each sample , and take the average of these values as the microbial concentration in the biogas slurry.

3. The method of claim 1 or 2, wherein the method is characterized by, The positive-hole method is adopted to correct the number of colonies in step five, which comprises: ； In the formula, Corrected number of colonies for each layer of agar plate, N Number of sampling holes for each layer of agar plate, Actual number of colonies for each layer of agar plate; According to the corrected number of colonies of each layer of agar culture plates, the sampling air flow and the sampling time, the concentration of each level of microbial aerosol in the air is obtained: ； wherein is the concentration of microbial aerosol at each level, CFU / m 3 , is the sampling air flow L / min, t is the sampling time, min.

4. The method of claim 1 or 2, wherein, The particle size is obtained by a laser particle size analyzer or a microbial collector, and the logarithmic normal distribution of the particle size distribution characteristics in step five is obtained by the following formula: ； wherein: the frequency of particle diameters of 0.1 pm, the log mean of particle diameters, the log standard deviation of particle diameters, and particle diameters pm; the particle size distribution is typically calculated by particle size analysis, and the specific method can be obtained by using an aerosol particle size analyzer to obtain the aerosol concentration at different particle sizes, and then drawing a particle size distribution graph.

5. The method of claim 1 or 2, wherein, The concentration distribution characteristics of the air microbial aerosol in step five are obtained by the following formula: ； where: CFU / m3is the aerosol concentration at the distance of the nozzle CFU / m3is the aerosol concentration near the nozzle, CFU / m3is the aerosol concentration near the nozzle, m is the distance from the nozzle to the nearest measuring point, is the concentration decay exponent, usually obtained from experience or experimental data.

6. The method of claim 1 or 2, wherein, Step four specifically comprises: 1) biogas slurry pretreatment: the biogas slurry is mixed with clean water at a volume ratio of 1:1, stirred uniformly to obtain homogeneous biogas slurry, the homogeneous biogas slurry is gradiently diluted, each gradient dilution liquid is added dropwise to a culture dish plate and uniformly coated, and the culture dish plate is cultured at 37 DEG C for 24 hours, the number of colonies is counted to determine the concentration of microorganisms in the biogas slurry; 2) sampling preparation: LB agar medium is placed in each impact chamber of the six-stage screen impact collection unit, the six-stage screen impact collection unit is connected with the FA-1H microbial collector, the FA-1H microbial collector is started and adjusted to the preset flow; 3) sprinkling and sampling: the homogeneous biogas slurry is pressurized by a pressurizing device, atomized and sprayed out through the low-pressure nozzle, the spraying lasts for 20 minutes, and the diffused microbial aerosol is collected by the FA-1H microbial collector during the spraying; 4) culture and analysis: after the spraying is completed, the culture dishes in the six-stage screen impact collection unit are taken out in a sterile operation mode, and are cultured at 37 DEG C for 24 hours, the number of microbial colonies on each culture dish is counted, the concentration and particle size distribution of the microbial aerosol are analyzed in combination with the corresponding particle size range of each level of the six-stage screen impact collection unit.

7. The method of claim 6, wherein the method is a method of testing the microbial aerosol concentration and particle size of a low-pressure sprinkler irrigation of biogas slurry, characterized in that, The gradient dilution in step 1) is specifically mixing to prepare 10-1, 10-2, 10-3, 10-4, and 10-5 dilution solutions. 4 and 10-5 dilution solutions. 5 dilution solutions. In step 1), the culture dishes with the colony number between 30 and 300 are selected for counting; In step 2), the preset flow of the FA-1H microbial collector is 28.3 L / min.

8. A device for testing the concentration and particle size of microbial aerosols in low-pressure sprinkling irrigation with biogas slurry, implementing the method according to any one of claims 1 to 7, characterized in that, The device comprises a fertilizer storage device (1), a pressure regulating and spraying device (2), and a FA-1H intelligent microbial sampling device (3); the fertilizer storage device (1) is used for storing and mixing biogas slurry, the pressure regulating and spraying device (2) is used for applying specific pressure to the biogas slurry and atomizing and spraying the biogas slurry, and the FA-1H intelligent microbial sampling device (3) is used for collecting the microbial aerosol escaping in the spraying process.

9. The biogas liquid low pressure sprinkling microbial aerosol concentration and particle size testing device according to claim 8, characterized in that, The fertilizer storage device (1) comprises a fertilizer storage barrel (12), a mixer (11), and a ball valve (13); the mixer (11) is installed on the top of the fertilizer storage barrel (12), and the stirring part of the mixer (11) extends into the inside of the fertilizer storage barrel (12); the ball valve (13) is installed at the liquid outlet of the fertilizer storage barrel (12), and is connected with the pressure regulating and spraying device (2) through a water pipe; the connection parts of the water pipe, the ball valve (13), and the pressure regulating and spraying device (2) are all connected by threads and wrapped with a sealing raw material belt.

10. The biogas liquid low pressure sprinkling irrigation microorganism gas aerosol concentration and particle size test device according to claim 8, characterized in that, The FA-1H intelligent microbial sampling device (3) comprises a FA-1H microbial collector (31), a rubber hose (32), a six-stage screen hole impact collection unit (33), and a tripod support frame (34); the six-stage screen hole impact collection unit (33) is detachably installed on the top of the tripod support frame (34); one end of the rubber hose (32) is sealingly connected with the air outlet of the six-stage screen hole impact collection unit (33), and the other end is sealingly connected with the air inlet of the FA-1H microbial collector (31); the connection parts all adopt sealing joints.

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