Method for measuring phosphorus content of soil microbial biomass

Abstract

The invention discloses a method for measuring soil microbial biomass phosphorus content, which comprises the following steps: (1) collecting a plurality of fresh soil samples, respectively taking a proper amount of fresh soil samples from each fresh soil sample, and air-drying at room temperature to obtain a plurality of air-dried soil samples; (2) weighing 1 part of each air-dried soil sample and 2 parts of each fresh soil sample, taking one part of the fresh soil sample as a contrast without fumigation, and adding a phosphorus standard to the other part of the fresh soil sample to measure the recovery rate (Rip); and (3) respectively adding a sodium bicarbonate solution into the air-dried soil sample and the fresh soil sample in the step (2), oscillating, filtering, and extracting a proper amount of filtrate to determine the phosphorus content. The method has the advantages of simple detection steps, high efficiency, no toxicity, no harm and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of soil detection, and in particular to a method for measuring the phosphorus content of soil microbial biomass. Background Art

[0002] Soil microorganisms not only control the cycling of soil organic matter and major nutrients but also serve as a vital active nutrient reservoir, playing a crucial role in maintaining crop nutrient availability. Soil microbial biomass phosphorus (MBP) reflects, to a certain extent, the ecosystem's material cycling capacity, soil fertility, and plant productivity. It is highly sensitive to environmental responses and is often used as an indicator of changes in soil quality. Therefore, accurate measurement of MBP is crucial for studying phosphorus balance and cycling mechanisms in the context of global climate change, as well as the response of soil microbial biomass to environmental changes, soil fertility, and nutrient availability.

[0003] Currently, soil microbial biomass phosphorus (MBP) content is determined using the chloroform fumigation method. The chloroform used in this method is a toxic reagent, posing certain hazards to humans and the environment. To ensure the purity of the chloroform vapor during fumigation, it must be purified before use. Chloroform fumigation requires a vacuum pump and reagents for carbon dioxide absorption. For batch soil samples, a vacuum dessicator is also required. During vacuuming, the glass dessicator can shatter and injure personnel, posing a potential safety hazard. To ensure effective vacuuming, the glass dessicator must be sealed with sealing oil, a process that places high demands on personnel to prevent air leaks. The chloroform fumigation method is cumbersome, has numerous restrictions, and is inconvenient to operate. Furthermore, when determining soil MBP, the effectiveness of soil fumigation is often significantly affected by uncontrollable factors such as soil moisture and soil aggregate compaction. When soil moisture content is low, microbial cells lose water, resulting in reduced cell permeability. Soil aggregates are dense and compact, and the water trapped in the pores between soil particles hinders the full diffusion of fumigants within the soil pores. This prevents some microorganisms from fully contacting the fumigant, preventing the effective lysis of microbial cells. This severely affects the release of microbial phosphorus and significantly interferes with the determination of soil microbial biomass phosphorus. Soil types are diverse, and different soil types exhibit significant differences in physical and chemical properties and microbial community structure. This variability makes the accurate determination of soil microbial biomass phosphorus challenging.

[0004] At present, the laboratory method for determining soil phosphorus content is to take 50 fresh soil samples and weigh 3 parts each, each soil sample weighing 2g, 1 part of the soil is placed in a desiccator with chloroform and evacuated until the chloroform boils for 2-3 minutes, placed in the dark for 24 hours, and the lid is opened to remove all chloroform. This is used as a fumigated soil sample, 1 part of the unfumigated soil sample is used as a control, and 1 part of the unfumigated soil sample is added with 0.2-1ml of 100mg / L phosphorus standard to determine the recovery rate (Rip). 40mL of 0.5mol / L sodium bicarbonate solution is added to each of the three soil samples, shaken for 30 minutes, filtered, and an appropriate amount of filtrate is extracted to determine the phosphorus content. At the same time, an appropriate amount of soil is weighed, placed in a 105℃ oven and dried to constant weight, then weighed and recorded, and the moisture coefficient is calculated. The moisture coefficient refers to the moisture conversion coefficient of the soil sample used for determination converted to dried soil weight. Fresh soil moisture coefficient = dried soil weight / fresh soil weight. Microbial biomass phosphorus content mg·kg -1 = (fumigation soil sample result - control soil sample result) / 0.4 / inorganic phosphorus spike recovery (Rip). Inorganic phosphorus spike recovery (Rip) = (spiked sample result - control soil sample result) / spiked amount * 100%. This method takes into account that chloroform fumigation does not completely destroy microorganisms and releases insufficient phosphorus. Therefore, 0.4 is used in the calculation to correct for the fumigation extraction rate.

[0005] Other existing documents also disclose other determination methods. For example, the existing patent document 201810303945.9 discloses a method for determining soil microbial biomass phosphorus. The determination method includes the following steps: dividing soil samples into two groups, wherein the first group of samples is fumigated with chloroform, and after removing the chloroform, sodium bicarbonate is added for extraction and the first filtrate is filtered; the second group of samples is directly added with sodium bicarbonate for extraction and the second filtrate is filtered. After adjusting the first and second filtrates to neutrality, potassium persulfate is added to both, and the mixture is reacted under high temperature and high pressure for 10-25 minutes. The phosphorus content of the first and second filtrates after the reaction is determined, and the difference between the phosphorus content of the first filtrate and the phosphorus content of the second filtrate is the value of microbial biomass phosphorus. This method for determining microbial biomass phosphorus is simple, the determination time is short, and the amount of phosphorus loss during the experimental process is reduced. The application only uses one leaching agent, but the different soil pH values have a significant impact on the extraction effect, and abnormal test results often occur. Patent document CN115060567A discloses a method for measuring soil microbial biomass phosphorus. During the process, the soil is classified according to its acidity and alkalinity. Different extraction agents are used according to the soil pH, improving the efficiency and accuracy of the test. Furthermore, a vacuum oven is used instead of a vacuum desiccator for fumigation, making it convenient and suitable for batch operations.

[0006] The disclosure of the above background technology content is only used to assist in understanding the concept and technical solution of the present invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above content has been disclosed on the filing date of this patent application, the above background technology should not be used to evaluate the novelty and creativity of this application. Summary of the Invention

[0007] The present invention (mainly) aims to provide a method for determining the phosphorus content of soil microbial biomass with simple detection steps, high efficiency and safety.

[0008] To this end, the present invention provides a method for determining the phosphorus content of soil microbial biomass.

[0009] Preferably, the present invention may also have the following technical features:

[0010] A method for determining soil microbial biomass phosphorus content comprises the following steps:

[0011] (1) Collecting several fresh soil samples, taking an appropriate amount of fresh soil sample from each fresh soil sample and placing it at room temperature to air-dry, thereby obtaining several air-dried soil samples;

[0012] (2) Weigh one portion of each air-dried soil sample and two portions of each fresh soil sample. Use one portion of the fresh soil sample without fumigation as a control, and use one portion of the fresh soil sample spiked with phosphorus for determination of recovery (Rip);

[0013] (3) Sodium bicarbonate solution was added to the air-dried soil sample and the fresh soil sample in step (2), respectively, and the mixture was shaken and then filtered. An appropriate amount of filtrate was collected to determine the phosphorus content.

[0014] Furthermore, 1 / 3 of the fresh soil sample was air-dried.

[0015] Furthermore, the air-drying environment is pollution-free and well-ventilated.

[0016] Furthermore, the soil sample was spread on a clean filter paper.

[0017] Furthermore, during the air-drying process, air is blown by a fan to accelerate the removal of moisture from soil particles.

[0018] Furthermore, the air-drying time is 1-3 days.

[0019] Furthermore, the air-drying time is 1-2 days.

[0020] Furthermore, when determining the recovery rate, 0.2-1 ml of 100 mg / L phosphorus standard was added.

[0021] Further, 40 mL of 0.5 mol / L sodium bicarbonate solution was added.

[0022] Further, shake for 25-35 minutes.

[0023] The technical principle of this invention is that rapid air-drying of soil causes rapid water loss, creating a high osmotic pressure environment outside microbial cells and causing continuous water exudation from the cells. Excessive dehydration causes cell shrinkage and deformation, separation of protoplasts from the cell wall, and rupture of the cell membrane and cell wall. This releases organic and inorganic phosphorus compounds, such as nucleic acids and phospholipids, previously fixed within the cells directly into the soil. The increased phosphorus content of air-dried soil compared to fresh soil represents the contribution of soil microorganisms, from which the soil microbial biomass phosphorus content can be calculated.

[0024] 1. Detailed explanation of the measurement method steps

[0025] (1) Soil sample collection and pretreatment

[0026] 1. Collection and Air Drying Initial Steps

[0027] First, collect several fresh soil samples, which are the basic materials for the entire determination. Fresh soil samples can reflect the immediate situation of microbial biomass phosphorus in the soil under natural conditions to the greatest extent. They are biologically active and contain complete phosphorus forms. Take an appropriate amount of fresh soil sample from each fresh soil sample and air-dry at room temperature. The reason for choosing room temperature air-drying instead of other methods such as high-temperature drying is that high temperature may destroy the soil microbial structure, leading to the loss or conversion of phosphorus in microorganisms, and distorting the determination results. Obtain several air-dried soil samples. The air-drying process aims to reduce the soil moisture content, so that the microbial biomass phosphorus is fully released, and at the same time facilitate subsequent operations, such as improving the accuracy of weighing, and some physical and chemical properties are more stable in the air-dried state for analysis.

[0028] Furthermore, the fresh soil sample is spread out flat, and four diagonal samples are taken for air drying. This ratio has been verified by a large number of experiments. It can not only ensure that there are enough air-dried soil samples for subsequent parallel experiments, but also retain most of the fresh samples for fresh sample-related testing, and mutually verify the accuracy of the results. The air-drying environment requires to be pollution-free and well-ventilated. Protective nets are installed on doors and windows to prevent external impurities from mixing into the soil samples and changing the original background value of the soil's phosphorus content. Good ventilation accelerates the dissipation of water vapor, which promotes a uniform and efficient air-drying process. The soil sample can be evenly spread out in a small box or small plate with clean filter paper to form a thin layer with a thickness of no more than about 0.5 cm. For the large soil aggregates, they are carefully broken into small particles with a particle size of about 2-3 mm using tools or manual operations. This treatment method greatly increases the surface area of the soil particles and effectively accelerates the evaporation rate of water in the soil particles. Choosing filter paper as the carrying medium, on the one hand, prevents the soil from coming into contact with other unclean surfaces and adsorbing impurities; on the other hand, the filter paper can absorb a small amount of seeping moisture and increase the air-drying speed; during the air-drying process, a fan is used to blow air to accelerate air flow, thereby accelerating the removal of moisture from soil particles. The air-drying time can be greatly shortened from the possible 5-7 days under natural conditions to 1-3 days, or even optimized to 1-2 days. This significantly improves work efficiency while ensuring sample quality and reduces the uncertainty factors that may be introduced by long-term exposure of soil to the air.

[0029] (2) Sample grouping and processing preparation

[0030] 1. Grouping and spiking operations

[0031] One portion of each air-dried soil sample and two portions of each fresh soil sample were weighed and carefully grouped for processing. One fresh soil sample was not fumigated as a control. This control sample is of great significance as it represents the original microbial biomass phosphorus state of the soil and is not disturbed by fumigation or other treatments, providing a baseline for subsequent calculations of microbial biomass phosphorus changes. Another fresh soil sample was spiked with a phosphorus standard for determination of recovery (Rip). A known amount of phosphorus standard was added (when determining the recovery, 0.2-1 ml of 100 mg / L phosphorus standard was added. This concentration and volume range is a precise range determined through repeated experiments, which ensures that the amount of phosphorus standard is sufficient to accurately calculate the recovery without masking the response signal of the soil's own microbial biomass phosphorus due to excessive addition). The final phosphorus content of the spiked sample was measured, combined with the theoretical addition amount, and based on the recovery calculation formula, it was used to evaluate the strength of the soil's adsorption and fixation of phosphorus, and to make reasonable corrections to the measurement results.

[0032] (III) Core steps of phosphorus content determination

[0033] 1. Extraction and filtration

[0034] 40mL of 0.5mol / L sodium bicarbonate solution was added to the air-dried soil sample and fresh soil sample in step (2) respectively. The sodium bicarbonate solution was selected as the extraction agent because it has good selective extraction ability for soil microbial biomass phosphorus. It can effectively dissolve the phosphorus in the soil microbial cells and adsorbed state under relatively mild conditions, and minimize the excessive dissolution of other phosphorus forms such as soil mineral phosphorus, ensuring that the extracted phosphorus mainly comes from microbial biomass phosphorus. Adding 40mL of this volume can fully soak the soil sample to achieve efficient extraction, and will not affect the subsequent detection accuracy due to excessive dilution of phosphorus concentration due to excessive solution volume; the concentration of 0.5mol / L is the best choice for balancing the extraction ability and the disturbance to the soil system. After the soil particles are dispersed, their specific surface area increases, and the contact area with the extract increases. The phosphorus wrapped inside the soil particles is more easily contacted and dissolved by the extract, thereby improving the extraction effect of phosphorus. Oscillation is performed for 25-35 minutes. This oscillation simulates mechanical stirring, ensuring full contact between the sodium bicarbonate solution and soil particles, accelerating phosphorus dissolution. This optimal timeframe is crucial for ensuring sufficient phosphorus dissolution while avoiding prolonged oscillation that could rupture microbial cells, release intracellular interfering substances, or cause re-adsorption and precipitation of dissolved phosphorus. The optimal timeframe was determined through multiple comparative experiments. Filtration then removes soil particle residue, resulting in a clarified filtrate. This provides a pure sample for subsequent, accurate phosphorus measurement, preventing soil particles from clogging phosphorus measurement instruments (such as spectrophotometers) and interfering with scattered light, ensuring reliable results.

[0035] 2. The necessity and importance of non-interchangeable steps and process parameter optimization

[0036] (1) Irreplaceability of steps

[0037] 1. If fumigation or spike treatment is performed first and then the soil is air-dried, the fumigant residue and spiked phosphorus may change their original distribution and content in the soil due to volatilization, adsorption changes and other factors during the air-drying process, making it impossible for subsequent measurement results to truly reflect the initial state of soil microbial biomass phosphorus, and the setting of control samples and spiked samples will become meaningless.

[0038] 2. If the extraction operation is performed before the grouping of fresh soil samples and air-dried soil samples is completed and before spiking, the changes in phosphorus content in soil samples in different states (control fresh samples, spiked fresh samples, and air-dried samples) cannot be accurately traced, the recovery rate cannot be effectively calculated, and the original value of microbial biomass phosphorus cannot be evaluated. The entire measurement logic is chaotic, and the data loses comparability and credibility.

[0039] (2) Necessity and importance of process parameter optimization

[0040] 1. For example, the air-drying time can be optimized from a wide range of natural air-drying times to 1-3 days or even 1-2 days, which greatly shortens the experimental cycle and reduces time costs. At the same time, it avoids impurity contamination caused by long-term air-drying and the potential interference of the natural succession of microbial communities on phosphorus content, ensuring efficient experiments and accurate results.

[0041] 2. The amount of phosphorus standard added in the recovery rate determination should be accurate to 0.2-1 ml of 100 mg / L phosphorus standard. If the amount added is too small, the detection signal will be easily submerged by background noise in a complex soil system, making it difficult to accurately calculate the recovery rate. If the amount added is too large, the soil phosphorus system will be excessively changed, obscuring the true situation of microbial biomass phosphorus, distorting the recovery rate calculation, and making it impossible to effectively evaluate the accuracy of the determination.

[0042] 3. Precise optimization of sodium bicarbonate solution concentration, volume, and oscillation time. Each parameter change is directly related to the extraction efficiency, purity, and stability of phosphorus. Incorrect concentration may lead to incomplete extraction or excessive extraction of impurity phosphorus, inaccurate volume may affect extraction balance, and deviations in oscillation time may lead to insufficient phosphorus dissolution or introduce interference. Only with the coordination of these finely optimized parameters can the accurate extraction and determination of microbial biomass phosphorus from complex soil environments be guaranteed, providing solid data support for soil science research and practical applications, and achieving high-accuracy and high-reliability technical results that cannot be achieved by traditional crude methods.

[0043] In summary, this method for determining soil microbial biomass phosphorus content, through rigorous step design and sophisticated process parameter optimization, forms a complete, accurate, and efficient determination system, providing a powerful tool for the field of soil microbial biomass phosphorus research.

[0044] Compared with the prior art, the present invention has the following technical advantages:

[0045] 1. No toxic reagents or complicated equipment required, safer and more environmentally friendly

[0046] Existing techniques (such as chloroform fumigation) require the use of the highly toxic reagent chloroform and a vacuum filtration device, which poses high toxicity risks and equipment dependence. However, this invention replaces chemical fumigation with natural air drying at room temperature, completely eliminating toxic reagents and eliminating the need for vacuum operation. This avoids the harm of toxic substances to the human body and pollution to the environment, while also reducing experimental costs and equipment requirements, in line with the trend of green analytical chemistry.

[0047] 2. Microbial phosphorus release is more thorough and the test results are more realistic

[0048] Traditional methods rely on chemical fumigation to destroy microbial cells, but this can lead to problems such as incomplete cell rupture and unstable fumigation effects (due to differences in vacuum level, fumigation time, soil structure, and moisture content). This results in phosphorus release being susceptible to human manipulation and objective soil conditions. This new method, however, uses air drying to dehydrate the soil, gradually rupturing microbial cells due to osmotic pressure imbalance, leading to a continuous and full release of phosphorus. This avoids the occasional interference of chemical methods, directly reflects the true content of soil microbial biomass phosphorus, and significantly improves data stability and accuracy.

[0049] 3. The operation process is extremely simple to reduce human errors

[0050] The traditional method needs to go through multiple steps such as fumigation, extraction, and determination. The process is cumbersome (such as chloroform fumigation requires more than 24 hours), and there are many manual operation links (such as soil moisture adjustment, vacuum control, fumigant treatment and residual control, etc.), which are easy to introduce errors. The present invention integrates air drying, cell rupture, and phosphorus release into a single natural process, omitting steps such as fumigation, filtration, pressure maintenance, and complex pre-treatment, and reducing the number of operating steps by more than 50%. Only the key pre-treatment needs to be completed by controlling the air drying conditions, with less human intervention, which significantly improves the repeatability and popularity of the method (especially suitable for grassroots laboratories to determine the microbial biomass phosphorus content of batch soil samples). BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the microbial biomass phosphorus content of the fresh soil sample and the six samples taken during the air-drying process in the present invention.

[0052] Figure 2 It is a schematic diagram comparing the phosphorus difference between the room temperature air-dried sample and the control sample, and the phosphorus difference between the fumigated sample and the control sample.

[0053] Figure 3 The graphs show the measurement results of the three methods. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be emphasized that the following description is merely illustrative and is not intended to limit the scope of the present invention and its application.

[0055] Non-limiting and non-exclusive embodiments will be described with reference to the following figures, wherein like reference numerals refer to like parts unless otherwise specifically specified.

[0056] A method for determining soil microbial biomass phosphorus content comprises the following steps:

[0057] (1) Collect several fresh soil samples. Take an appropriate amount of fresh soil sample from each fresh soil sample and air-dry it at room temperature to obtain several air-dried soil samples. Preferably, take 1 / 3 of the fresh soil sample for air-drying. The moisture coefficient of the air-dried soil sample should be ≥ 0.95. The moisture coefficient of the air-dried soil sample should be ≥ 0.95, which is an important indicator of the end of air-drying. At this time, the number of surviving soil microorganisms is extremely low, the released microbial biomass phosphorus tends to be stable, and the test results also tend to be stable.

[0058] Air-drying should be done in a pollution-free, well-ventilated environment. Spread the soil sample flat on clean filter paper. To quickly disperse moisture from the soil particles, turn on a fan to increase air flow and accelerate the removal of moisture from the soil particles. For the air-drying process, only the sample needed for testing (a small amount) should be spread out in a thin layer and air-dried. Turn on a fan to accelerate air flow. Dry for 1-2 days until the moisture coefficient is ≥0.95. Once air-dried, bag the sample for testing. Soil drying is faster, but it can cause some active phosphorus compounds to volatilize or be destroyed, resulting in lower test results.

[0059] No other reagents need to be added to rupture the cells during the air-drying process. The loss of water caused by air-drying at room temperature will reduce the fluidity of the cell membrane, and the membrane lipids may undergo a phase transition from a liquid crystal state to a gel state. This change will destroy the structural integrity of the cell membrane, causing cracks or holes in the cell membrane, leading to leakage of cell contents and cell rupture. At the same time, the proteins on the cell membrane may also denature due to dehydration, further affecting the function of the cell membrane and increasing the risk of cell rupture. From the analysis of the test results, it can be seen that the microbial biomass phosphorus in air-dried soil is greater than that in fresh soil. The moisture coefficient of air-dried soil is ≥0.95, and the measured microbial biomass phosphorus content tends to be stable, indicating that the soil microbial biomass phosphorus is fully released.

[0060] Specifically, 50 fresh soil samples were collected (50 soils were collected in Jilin, Changchun, Shanghai, Guangxi, Guangdong, etc., including various types such as clay loam, loam, silt clay, sandy loam, etc. The soil appearance is obviously different, such as some soil particles are sticky, some particles are loose, and some contain more gravel. Secondly, the dryness and wetness of each fresh soil are also different, that is, the moisture coefficient is 0.60-0.98), 1 / 4 to 1 / 2 of the fresh soil sample was taken from each fresh soil sample and air-dried at room temperature, and then air-dried under the same conditions, and finally 50 air-dried soil samples were obtained, and each fresh soil sample corresponds to an air-dried soil sample.

[0061] Different moisture coefficients have a significant impact on the test results, as can be seen in Figure 1 and Table 1, Figure 1 and sample numbers 1 to 6 in Table 1 are sample numbers 1 to 6 in Table 3.

[0062] Table 1 shows the moisture coefficient of the same fresh soil sample taken 6 times during the air-drying process

[0063]

[0064]

[0065] As can be seen from Table 1, the moisture coefficient of the fresh soil sample increases with the extension of air-drying time. Air-drying 1 represents the moisture coefficient after 4 hours of air-drying, air-drying 2 represents the moisture coefficient after 8 hours of air-drying, air-drying 3 represents the moisture coefficient after 20 hours of air-drying, air-drying 4 represents the moisture coefficient after 28 hours of air-drying, air-drying 5 represents the moisture coefficient after 32 hours of air-drying, and air-drying 6 represents the moisture coefficient after 40 hours of air-drying.

[0066] Experiment on changes in microbial biomass phosphorus in fresh soil samples and at six time points during air-drying:

[0067] 6 fresh soil samples were taken to determine the initial moisture coefficient (weigh a certain amount of fresh soil, dry it and weigh it, dry soil weight / fresh soil weight = moisture coefficient). At the same time, three samples were weighed as required, one for fumigation treatment and one for non-fumigation as a control sample, and one for measuring the inorganic phosphorus recovery rate and the microbial biomass phosphorus content of the fresh soil; the room temperature was not lower than 15°C in a well-ventilated room free of chemical pollution. Place a portion of fresh soil on clean, dry filter paper in a small dish or box. Lay it flat and air-dry for 4 hours. Then, mix the soil sample on the filter paper and remove a portion for the first test. The moisture coefficient of this portion is determined using the initial moisture coefficient determination method (see Air Drying 1 in the table). After 8 hours, mix the soil sample on the filter paper and remove a portion for the second test, following the same procedure. After 20 hours, mix the soil sample on the filter paper and remove a portion for the third test, following the same procedure. After 28 hours, mix the soil sample on the filter paper and remove a portion for the fourth test, following the same procedure. After 32 hours, mix the soil sample on the filter paper and remove a portion for the fifth test, following the same procedure. After 40 hours, mix the soil sample on the filter paper and remove a portion for the sixth test, following the same procedure. As shown in Table 1, the moisture coefficients of the six samples gradually increase during the air-drying process, indicating that the soil samples are losing moisture. After 32 hours of air-drying, the moisture coefficients of all soil samples are greater than 0.95.

[0068] Depend on Figure 1 It can be seen that with the increase of soil moisture coefficient, that is, the decrease of soil water content, the microbial biomass phosphorus content gradually increased. The microbial biomass phosphorus content after air drying for 32 hours and 40 hours tended to be consistent.

[0069] This analysis shows that as the soil continues to lose water, the phosphorus released by soil microbial cells increases. After 32 hours and 40 hours of air drying, when the moisture coefficient is greater than 0.95, the phosphorus release rate of soil microbial cells almost stops, and the soil microbial biomass phosphorus content tends to be stable.

[0070] (2) Weigh one portion of each air-dried soil sample and two portions of each fresh soil sample. Use one portion of the fresh soil sample as a control without fumigation, and add 0.2-1 ml of 100 mg / L phosphorus standard to the other portion of the fresh soil sample for determination of recovery rate (Rip).

[0071] The air-dried soil sample and fresh soil sample selected in step (2) were weighed 2 g each.

[0072] (3) Sodium bicarbonate solution was added to the air-dried soil sample and the fresh soil sample in step (2), respectively, and the mixture was shaken for 30 min, filtered, and an appropriate amount of filtrate was collected to determine the phosphorus content.

[0073] In step (3), 40 mL of 0.5 mol / L sodium bicarbonate solution was added.

[0074] Weigh appropriate amounts of fresh soil samples and air-dried soil samples from step (1), dry them in an oven at 105°C to constant weight, weigh and record them, and calculate the moisture coefficients of the fresh and air-dried soil samples respectively.

[0075] in,

[0076] Fresh sample moisture coefficient = oven-dried soil weight / fresh soil weight;

[0077] Moisture coefficient of air-dried soil sample = oven-dried soil weight / air-dried soil weight.

[0078] Microbial biomass phosphorus content mg·kg-1 = (measurement result of air-dried soil sample - measurement result of control soil sample) / 0.90 / inorganic phosphorus spike recovery rate Rip;

[0079] Inorganic phosphorus spike recovery (Rip) = (specified sample result - control result) / spiked amount * 100%.

[0080] 0.90 is the correction factor. Since there are still a small amount of microorganisms in the air-dried soil samples, they cannot be completely destroyed and release intracellular phosphorus substances. The calculation results are corrected based on this.

[0081] The results of the determination of 50 soil samples using the original method (fumigation method) and the method of the present invention (room temperature air drying method) (raw data are shown in Table 3) were analyzed by t-test: paired two-sample mean analysis, P (T <= t) two-tailed > 0.05, indicating that there was no significant difference in the observed values of the results measured by the two methods, indicating that the method of the present invention has no effect on the determination of soil microbial biomass phosphorus. As shown in Table 2, for the data of 50 samples, P (T <= t) two-tailed = 0.7428 > 0.05.

[0082] The results of the determination of 50 soil samples using the original method (fumigation method) and the method of the present invention (room temperature air drying method) were compared. As shown in Table 3, the soil microbial biomass phosphorus content of samples 9, 12, 28, 29, 31, 32, 40-48 measured by the room temperature air drying method was lower than that of the fumigation method, and the soil microbial biomass phosphorus content of the other 35 samples measured by the room temperature air drying method was higher than that of the fumigation method. The phosphorus difference between the 50 soil samples at room temperature air drying and the control samples was greater than or close to the phosphorus difference between the fumigation samples and the control samples, as shown in Table 3. Figure 2As shown. The phosphorus difference between the room temperature air-dried sample and the control sample, and the phosphorus difference between the fumigated sample and the control sample were not included in the correction coefficient, which directly represents the effect of the two methods in determining microbial biomass phosphorus. The phosphorus difference between the room temperature air-dried sample and the control sample = soil microbial biomass phosphorus measured by the room temperature air-drying method * 0.90, which represents the release of microbial biomass phosphorus from the air-dried soil. The phosphorus difference between the fumigated sample and the control sample = soil microbial biomass phosphorus * 0.40, which represents the release of microbial biomass phosphorus from the fumigated soil. The above shows that the release of microbial biomass phosphorus from the air-dried soil tends to be greater than that from the fumigated soil. This is mainly because the room temperature air-drying method is different from the fumigation method. The room temperature air-drying method is not limited by objective and human factors such as soil particles, moisture, and fumigation effect. The release of soil microbial biomass phosphorus is more complete, and the measured results are more representative and can better reflect the actual status of soil microbial biomass phosphorus. Comprehensive analysis shows that the results measured by the room temperature air-drying method are representative and better than the fumigation method.

[0083] Table 2

[0084]

[0085] Table 3 Determination results of two methods

[0086]

[0087]

[0088] Note: The difference in phosphorus between the room temperature air-dried sample and the control sample = phosphorus content of microbial biomass by room temperature air-dried method * 0.90

[0089] The difference between the phosphorus content of the fumigated sample and the control sample = the phosphorus content of the fumigated microbial biomass * 0.40

[0090] The method of the present invention (room temperature air drying method) comprises the following steps: (1) collecting 50 fresh soil samples, taking an appropriate amount of fresh soil sample from each fresh soil sample and air drying at room temperature for 32 hours to obtain a plurality of air-dried soil samples with an air-dried soil moisture coefficient of ≥0.95;

[0091] (2) Weigh one portion of each air-dried soil sample and two portions of each fresh soil sample, each weighing 2 g. Use one portion of the fresh soil sample as a control without fumigation, and add 0.2-1 ml of 100 mg / L phosphorus standard to the other portion for recovery determination (Rip);

[0092] (3) Add 40 mL of 0.5 mol / L sodium bicarbonate solution to the air-dried soil sample and the fresh soil sample in step (2), shake for 30 min, filter, and take an appropriate amount of filtrate to determine the phosphorus content.

[0093] The original method (fumigation method) is as follows: take 50 fresh soil samples and weigh 3 parts each, weigh 2g of each fresh soil sample, put 1 part of soil into a dryer with chloroform and evacuate until the chloroform boils violently for 2.5 minutes, place it in the dark for 24 hours, open the lid to remove all chloroform, and use it as a fumigated soil sample. 1 part of the unfumigated soil sample is used as a control, and 1 part of the unfumigated soil sample is added with 0.2-1ml of 100mg / L phosphorus standard to determine the recovery rate (Rip); add 40mL of 0.5mol / L sodium bicarbonate solution to each of the three soil samples, shake for 30 minutes, filter, and extract an appropriate amount of filtrate to determine the phosphorus content. Weigh an appropriate amount of soil from the 50 fresh soil samples, put it in a 105℃ oven and dry it to constant weight, then weigh and record it, and calculate the moisture coefficient. Moisture coefficient = dried soil weight / fresh soil weight. Microbial biomass phosphorus content mg·kg -1 = (fumigation soil sample result - control soil sample result) / 0.4 / inorganic phosphorus spike recovery (Rip). Inorganic phosphorus spike recovery (Rip) = (spiked sample result - control soil sample result) / spiked amount * 100%. This method takes into account that chloroform fumigation does not completely destroy microorganisms and releases insufficient phosphorus. Therefore, 0.4 is used in the calculation to correct for the fumigation extraction rate.

[0094] 60°C drying method: Take a portion of fresh soil and place it in a clean aluminum box. Dry it in a 60°C oven for 4-6 hours until the soil moisture coefficient is ≥0.95. Then, treat the dried soil sample according to steps (2) and (3) of the method of the present invention (greenhouse air drying method). In other words, the difference between the 60°C drying method and the method of the present invention is that drying is used instead of air drying.

[0095] As can be seen from Table 3, the following parallel samples:

[0096] Comparing the results of the three methods, the room temperature air-drying method produced results greater than those of the fumigation method and the 60°C drying method, indicating more complete release of microbial biomass phosphorus. The mean CV values for the fumigation and 60°C drying methods were 16.48% and 29.21%, respectively, while the mean CV for the room temperature air-drying method was 6.78%, significantly lower than those for the fumigation and 60°C drying methods. This suggests that the room temperature air-drying method is not affected by objective factors such as soil particles and moisture, resulting in sufficient and stable release of soil microbial biomass phosphorus. Therefore, the results are less variable, more precise, and more representative. In contrast, the fumigation method is subject to objective factors such as particles and moisture, resulting in greater variability and lower precision. During the 60°C drying process, the soil microbial biomass phosphorus undergoes some uncertain changes due to the high temperature, leading to unstable and poorly precise results.

[0097] Table 4 Comparison of the determination results of three methods

[0098]

[0099] The five samples 35 to 39 in Table 4 correspond to samples 35 to 39 in Table 3. Figure 3 As can be seen, the room temperature air-drying method consistently produced greater results than the fumigation method, while the 60°C oven-drying method produced less. One soil showed a greater result than the fumigation method, while the others showed less. This suggests that the room temperature air-drying method, through its gentle treatment, fully promotes phosphorus release from microorganisms, while the oven-drying method severely damages microorganisms, resulting in a loss of active phosphorus in some soils and causing lower results.

[0100] Those skilled in the art will recognize that numerous variations to the foregoing description are possible, and that the examples and figures are intended only to describe one or more specific implementations.

[0101] Although what is considered to be exemplary embodiments of the present invention has been described and illustrated, it will be understood by those skilled in the art that various changes and substitutions may be made thereto without departing from the spirit of the present invention. In addition, many modifications may be made to adapt a particular situation to the teachings of the present invention without departing from the central concept of the invention described herein. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but rather encompasses all embodiments and their equivalents falling within the scope of the present invention.

Claims

1. A method for determining soil microbial biomass phosphorus content, characterized in that: The steps include: (1) Collecting several fresh soil samples, taking an appropriate amount of fresh soil sample from each fresh soil sample and placing it at room temperature to air-dry, thereby obtaining several air-dried soil samples; (2) Weigh one portion of each air-dried soil sample and two portions of each fresh soil sample. Use one portion of the fresh soil sample without fumigation as a control, and use one portion of the fresh soil sample spiked with phosphorus for recovery determination. (3) Sodium bicarbonate solution was added to the air-dried soil sample and the fresh soil sample in step (2), respectively, and the mixture was shaken and then filtered. An appropriate amount of filtrate was collected to determine the phosphorus content.

2. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: Take 1 / 3 of the fresh soil sample and air-dry it.

3. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: The air drying environment should be pollution-free and well ventilated.

4. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: Spread the soil sample evenly on clean filter paper.

5. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: During the air-drying process, fans are used to blow air to accelerate the removal of moisture from soil particles.

6. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: The air drying time is 1-3 days.

7. The method for determining soil microbial biomass phosphorus content according to claim 6, wherein: The air-drying time is 1-2 days, and the moisture coefficient of the air-dried soil is ≥0.

95.

8. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: When determining the recovery rate, add 0.2-1 ml of 100 mg / L phosphorus standard.

9. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: Add 40 mL of 0.5 mol / L sodium bicarbonate solution.

10. The method for determining soil microbial biomass phosphorus content according to claim 1, wherein: Oscillate for 25-35 minutes.

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