Preparation of a biological nitrogen agent and method for detecting activity thereof

Abstract

The present application relates to the technical fields of microbial culture and microbial inoculum preparation, and discloses a method for preparing and detecting the activity of a biological nitrogen inoculum, wherein the biological nitrogen inoculum uses Azospirillum brasilense as a functional bacterium, and the bacterial cells are subjected to short-time ammonium nitrogen treatment after being induced for nitrogen fixation function, the ammonium nitrogen is removed, and a protective component is added when the nitrogen fixation function has not fully recovered, followed by carrierization and low-temperature dehydration; the obtained inoculum maintains normal nitrogen fixation inhibition reaction under continuous high-ammonium conditions, and can quickly restore the nitrogen fixation function after the low-level residual ammonium subsides or the short-time ammonium nitrogen effect ends; the activity detection method continuously detects the reduction and recovery of the nitrogen fixation function of the inoculum through continuous ammonium nitrogen treatment, short-time ammonium nitrogen treatment and continuous detection after ammonium removal, and can be used to characterize the dynamic nitrogen fixation activity of the biological nitrogen inoculum obtained through different preparation treatments.

Description

Technical Field

[0001] This invention relates to the field of microbial culture and inoculant preparation technology, and in particular to a method for preparing a biological nitrogen inoculant and detecting its activity. Background Technology

[0002] Nitrogen is a nutrient element required in large quantities during crop growth. In agricultural production, chemical nitrogen fertilizers such as urea and ammonium nitrogen fertilizers are usually used to supplement the nitrogen needed by crops. Chemical nitrogen fertilizers can quickly form a nitrogen source that can be absorbed and utilized by crops after application, but their actual nitrogen supply effect is limited due to the influence of nitrogen utilization efficiency and soil nitrogen conversion process. Biological nitrogen agents use nitrogen-fixing microorganisms to convert atmospheric molecular nitrogen into a usable nitrogen source, which can be used as a supplement to chemical nitrogen fertilizers for crop nitrogen supply. Since microbial nitrogen fixation is affected by factors such as the growth status of the microorganisms, soil environment, and crop rhizosphere conditions, a certain amount of fast-acting nitrogen fertilizer usually needs to be retained in the actual fertilization process to meet the nitrogen needs of crops in the early stage of growth. Therefore, the combined use of biological nitrogen agents and chemical nitrogen fertilizers has become a way of applying microbial fertilizers.

[0003] Existing technologies have focused on the impact of bound nitrogen on the nitrogen-fixing function of nitrogen-fixing bacteria. For example, CN108048384A discloses an ammonium-tolerant nitrogen-fixing microorganism with a glnA gene deletion, which maintains its nitrogen-fixing ability under high ammonium concentrations by altering the regulation of ammonium nitrogen by the bacterial cells. CN113321558A discloses a long-acting nitrogen fertilizer containing nitrogen-fixing microorganisms, which combines urea, nitrogen-fixing microorganisms, and additives with adsorption and water-retention functions to balance the supply of chemical nitrogen source and the action of microorganisms. In addition, existing technologies also include screening ammonium-tolerant nitrogen-fixing bacteria, using carriers to load microorganisms, and controlling nitrogen fertilizer release. These technologies aim to improve the nitrogen-fixing ability of nitrogen-fixing bacteria in nitrogen-containing environments, improve the survival conditions of the bacteria, or coordinate the coexistence of chemical nitrogen fertilizers and microbial agents.

[0004] In actual soil environments, the content of available nitrogen is not constant. After nitrogen fertilizers such as urea are applied to the soil, they undergo dissolution, hydrolysis, migration, transformation, and crop absorption, causing the local ammonium nitrogen to gradually decrease from a high level. During this process, short-term fluctuations in ammonium concentration may occur due to continued fertilizer transformation, changes in soil moisture, and nitrogen migration. When a high level of ammonium nitrogen is present, nitrogen-fixing bacteria reduce their nitrogen-fixing function to reduce unnecessary energy consumption. However, after the available nitrogen gradually decreases, the low level of residual ammonium and the short-term increase in ammonium concentration still inhibit nitrogen-fixing function. Therefore, the bacteria repeatedly experience the process of decreasing and recovering nitrogen-fixing function during the low-nitrogen transition phase. Existing biological nitrogen-fixing agents mainly evaluate the performance of the agents based on the number of effective viable bacteria, survival stability, or nitrogen-fixing activity after recovery under nitrogen-free conditions. This is difficult to reflect the actual nitrogen-fixing state of the agents under the above-mentioned nitrogen source change conditions. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that existing biological nitrogen-fixing agents, in the application environment where the available nitrogen level changes from high to low, suffer from low residual ammonium or short-term fluctuations in ammonium concentration that repeatedly inhibit nitrogen fixation function, thus affecting the continuous performance of biological nitrogen fixation. To address this, the present invention provides a biological nitrogen-fixing agent, its preparation method, and its activity detection method.

[0006] To achieve the above objectives, the present invention provides a biological nitrogen-fixing agent comprising *Azotrophus brasiliensis* cells, a carrier for loading the cells, and a protective component for the formulation of the cells.

[0007] Before formulation, the *Azotrophus brasiliensis* cells are induced to have nitrogen fixation function, so that the nitrogen fixation activity of the cells reaches a stable level. Then, ammonium nitrogen is used for short-term treatment to reduce the nitrogen fixation activity of the cells. After the exogenous ammonium nitrogen is removed, protective components are added before the nitrogen fixation activity of the cells recovers to the pre-treatment level. The cells are then carrier-based and dehydrated at low temperature to obtain a solid biological nitrogen agent.

[0008] The selected *Azospirobacter brasiliensis* strain is one that exhibits reduced nitrogen fixation activity under the influence of ammonium nitrogen and recovers its nitrogen fixation activity upon removal of ammonium nitrogen.

[0009] Short-term ammonium nitrogen treatment is performed after the nitrogen fixation activity of the bacteria has reached a stable level, so that the bacteria before formulation are in a state where the immediate nitrogen fixation activity is lower than the nitrogen fixation activity after recovery.

[0010] The carrier includes diatomaceous earth and humic acid. Based on a total carrier mass of 100 parts, diatomaceous earth accounts for 65-75 parts and humic acid accounts for 25-35 parts.

[0011] The protective components include soybean lecithin, trehalose, and monosodium glutamate. The amount of soybean lecithin is 2-4 parts, the amount of trehalose is 1-3 parts, and the amount of monosodium glutamate is 0.3-0.8 parts, relative to 100 parts of carrier. The protective components are mixed with the bacterial cells after the exogenous ammonium nitrogen is removed, and then carrier-based and dehydrated at low temperature.

[0012] The effective viable bacteria count of the biological nitrogen-containing agent is ≥2×10⁻⁶. 8 CFU / g, moisture content is 6-10wt%.

[0013] When the activity detection method provided by this invention is used to directly detect the activity after rehydration and cell standardization, the immediate nitrogen fixation activity of the cells is lower than that after full recovery under nitrogen-free conditions. The nitrogen fixation function can be restored after short-term ammonium nitrogen treatment and removal of ammonium nitrogen, but the nitrogen fixation activity is still inhibited under continuous ammonium nitrogen conditions.

[0014] In one embodiment, after short-term ammonium nitrogen treatment and removal of ammonium nitrogen according to the activity detection method provided by the present invention, the time required for the nitrogen fixation activity of the bacteria to recover to 80% of the ammonium-free baseline nitrogen fixation activity is ≤12min. Under the detection conditions of multiple consecutive short-term ammonium nitrogen treatments, the retention rate of the cumulative nitrogen fixation activity within the detection cycle relative to the cumulative nitrogen fixation activity under the ammonium-free baseline conditions within the same detection time is ≥80%.

[0015] This invention also provides a method for preparing the above-mentioned biological nitrogen-containing bacterial agent, the method comprising cell propagation, induction of nitrogen fixation function, short-term treatment with ammonium nitrogen, removal of exogenous ammonium nitrogen, pre-recovery protection, and carrier formation and low-temperature dehydration, as detailed below:

[0016] S1: Activate and seed the Brazilian nitrogen-fixing spirochete, and inoculate the resulting seed solution into a propagation culture system containing a fixed nitrogen source for bacterial growth. Propagation is terminated once the bacterial count reaches the level required for subsequent nitrogen fixation induction.

[0017] S2: The propagated *Azotrophus brasiliensis* was transferred into a nitrogen fixation induction culture system without an external nitrogen source. Nitrogen fixation function was induced under microaerophilic conditions to gradually increase the nitrogen fixation activity of the bacteria. The induction was terminated when the nitrogen fixation activity reached a stable level. The change in the nitrogen fixation activity of the bacteria was used as the basis for judgment during the induction process.

[0018] Furthermore, the nitrogen fixation function is induced at 28-32℃ and pH 6.5-7.2 for 2-18 hours.

[0019] In one embodiment, the nitrogen fixation activity of the bacteria is continuously sampled and measured. When the nitrogen fixation activity reaches more than 85% of the stable nitrogen fixation activity of the batch of bacteria and the change in nitrogen fixation activity at adjacent sampling points does not exceed 10%, the induction of nitrogen fixation function is terminated.

[0020] S3: Add ammonium nitrogen to the bacterial culture system that has completed nitrogen fixation induction for a short time to reduce the nitrogen fixation activity of the bacteria. By controlling the concentration of ammonium nitrogen and the treatment time, the bacteria can restore nitrogen fixation activity after the ammonium nitrogen is removed.

[0021] Furthermore, the ammonium nitrogen is provided by an ammonium salt, preferably NH4Cl, during the treatment process. + The concentration is 0.05-1.00 mmol / L, the treatment time is 3-20 min, and NH4 is preferred. + The concentration was 0.10-0.50 mmol / L, and the treatment time was 3-10 min. The NH4... + The concentration and treatment time should be selected within the ranges mentioned above.

[0022] In one embodiment, the treatment conditions are determined based on the following criteria: the immediate nitrogen fixation activity of the bacteria at the end of ammonium nitrogen treatment is 10-30% of the nitrogen fixation activity before treatment, and the bacteria can recover to more than 80% of the nitrogen fixation activity before treatment within 20 minutes after the removal of ammonium nitrogen. This reduces the nitrogen fixation activity of the bacteria after short-term ammonium nitrogen treatment while retaining the recovery ability after the removal of ammonium.

[0023] S4: Immediately after the ammonium nitrogen treatment, the cell temperature is lowered and the exogenous ammonium nitrogen in the culture system is removed. The exogenous ammonium nitrogen is removed by low-temperature centrifugation and washing. During the treatment, the cells are washed or filtered with an isotonic solution without fixed nitrogen.

[0024] Furthermore, the ammonium nitrogen removal process is carried out at 4-10°C to remove residual NH4 in the in vitro environment of the bacteria. + Exogenous ammonium nitrogen removal is completed when the concentration decreases to below 0.02 mmol / L, to achieve the aforementioned residual NH4+. + The concentration time is used as the starting point for subsequent protection treatment.

[0025] S5: After removing exogenous ammonium nitrogen, keep the bacterial cells at 4-10℃. Before the bacterial cells’ nitrogen fixation activity has recovered to the level before ammonium nitrogen treatment, add the pre-cooled protective components and mix them. After the protection treatment is completed, do not carry out nitrogen-free culture to fully restore the bacterial cells’ nitrogen fixation activity, but directly carry out carrier treatment.

[0026] Furthermore, the protective component is added within 0-15 minutes after the removal of exogenous ammonium nitrogen, preferably within 5-10 minutes. The specific addition time is controlled based on the fact that the immediate nitrogen fixation activity of the bacteria is still lower than the nitrogen fixation activity before ammonium nitrogen treatment.

[0027] S6: The protected bacterial cells are mixed with a carrier composed of diatomaceous earth and humic acid, so that the bacterial cells are loaded in the carrier, and then dehydrated at low temperature to obtain the biological nitrogen-containing bacterial agent.

[0028] Furthermore, the wet material after mixing the bacteria and the carrier has a moisture content of 20-30 wt%, and the material temperature during the dehydration process does not exceed 35℃. The dehydration process ends when the moisture content of the obtained bacterial agent is reduced to 6-10 wt%.

[0029] In the above preparation method, nitrogen fixation function induction is carried out before short-term ammonium nitrogen treatment, so that ammonium nitrogen acts on the bacteria whose nitrogen fixation activity has reached a stable level. After the ammonium nitrogen treatment, the exogenous ammonium nitrogen is removed first, and then the protective component is added before the nitrogen fixation activity of the bacteria has recovered to the pre-treatment level. Subsequently, carrier formation and low-temperature dehydration are carried out, so that the obtained biological nitrogen-containing bacterial agent is different from the bacterial agent obtained by direct formulation after propagation, direct formulation after nitrogen fixation induction, and formulation after ammonium nitrogen removal and nitrogen-free recovery treatment.

[0030] The present invention also provides a method for detecting the activity of the above-mentioned biological nitrogen-fixing agent. The method involves rehydrating, separating, and standardizing the bacterial cell count of the agent before direct detection. It does not include a pre-culture step without fixed nitrogen to fully restore the nitrogen-fixing activity of the bacteria. The method measures the changes in nitrogen-fixing activity of the bacteria under ammonium-free conditions, continuous ammonium nitrogen conditions, and short-term ammonium nitrogen treatment followed by removal of ammonium nitrogen, in order to evaluate the nitrogen-fixing response of the biological nitrogen-fixing agent under different ammonium nitrogen conditions.

[0031] The activity detection method includes adding the test biological nitrogen agent to a sterile isotonic rehydration solution for rehydration, allowing the bacteria in the carrier to enter the rehydration solution, removing the carrier particles and collecting the bacteria, adjusting the bacterial concentration of different test samples according to the OD600 of the bacterial suspension to keep the bacterial amount between different samples within a preset deviation range, completing the bacterial standardization and directly using it for subsequent nitrogen fixation activity detection, and normalizing the nitrogen fixation activity detection results according to the bacterial protein content.

[0032] Standardized bacterial cells were used for ammonium-free baseline nitrogen fixation activity detection, continuous ammonium nitrogen detection, and recovery detection after short-term ammonium nitrogen treatment. The ammonium-free baseline nitrogen fixation activity detection was conducted in a detection culture system without an external fixed nitrogen source and without the addition of external ammonium nitrogen. After the nitrogen fixation activity reached a stable stage, the corresponding detection results were taken as the ammonium-free baseline nitrogen fixation activity. The continuous ammonium nitrogen detection maintained a set ammonium nitrogen concentration during the detection period to determine the decrease in bacterial nitrogen fixation activity under continuous ammonium nitrogen treatment. The short-term ammonium nitrogen detection subjected the bacterial cells to ammonium nitrogen treatment for a limited time. After the treatment, the external ammonium nitrogen was removed, and the recovery process of bacterial nitrogen fixation activity was continuously measured.

[0033] Furthermore, during the continuous detection of ammonium nitrogen, NH4 + The concentration was 1-5 mmol / L, and NH4 was monitored during the detection period. + Concentration, kept within the set range; NH4 during short-term ammonium nitrogen detection. + The concentration was 0.05-0.20 mmol / L, the treatment time was 3-10 min, and the residual NH4 in the external environment after treatment was removed. + The concentration was reduced to below 0.02 mmol / L, to achieve the above-mentioned residual NH4. + The time of concentration is used as the starting point for the recovery detection.

[0034] After short-term removal of ammonium nitrogen, the nitrogen fixation activity of the bacteria was measured at set time intervals. The recovery endpoint was 80% of the nitrogen fixation activity without ammonium. The time required for the nitrogen fixation activity of the bacteria to recover to the recovery endpoint was recorded as T80.

[0035] Furthermore, the standardized bacterial cells were subjected to two or more short-term ammonium nitrogen treatments. After each treatment, the exogenous ammonium nitrogen was removed and a recovery phase was set up. The nitrogen fixation activity was measured at the same time interval throughout the entire detection cycle. Based on the nitrogen fixation activity and corresponding time at each detection time point, the cumulative nitrogen fixation activity between adjacent detection time points was calculated using the trapezoidal integral method and summed to obtain the cumulative nitrogen fixation activity throughout the entire detection cycle. The cumulative nitrogen fixation activity under repeated short-term ammonium nitrogen treatment conditions was compared with the cumulative nitrogen fixation activity under ammonium-free baseline conditions within the same detection time, and the obtained ratio was used as the dynamic cumulative activity retention rate.

[0036] In one embodiment, when evaluating the aforementioned biological nitrogen agent T80 and its dynamic cumulative activity retention rate, short-term ammonium nitrogen treatment is performed using 0.20 mmol / L NH4+. + After 5 minutes of treatment, the residual NH4 was removed. + The concentration was reduced to below 0.02 mmol / L; the dynamic cumulative activity retention rate was detected by performing three consecutive short-term ammonium nitrogen treatment cycles, with a 15-minute recovery phase after each removal of ammonium nitrogen.

[0037] The nitrogen fixation activity was determined using the acetylene reduction method. Standardized bacterial cells were placed in a nitrogen-free detection culture system, and the cells were brought into contact with acetylene within a specified reaction time. The amount of ethylene produced was measured, and the results were normalized according to the amount of bacterial protein. The T80 and dynamic cumulative activity retention rate were calculated based on the nitrogen fixation activity measured by the acetylene reduction method.

[0038] The above-mentioned biological nitrogen-fixing agent induces the nitrogen-fixing activity of the bacteria to reach a stable level through nitrogen fixation function, followed by short-term ammonium nitrogen treatment and removal of exogenous ammonium nitrogen. Protective components are added before the nitrogen-fixing activity of the bacteria recovers to the pre-treatment level, and then carrier-based and low-temperature dehydration is carried out.

[0039] The activity detection method evaluates the dynamic nitrogen fixation activity of biological nitrogen-fixing agents prepared under different treatments by assessing the decrease in nitrogen fixation activity under continuous ammonium nitrogen conditions, the recovery rate of nitrogen fixation activity after short-term removal of ammonium nitrogen, and the cumulative nitrogen fixation activity.

[0040] The technical effects and advantages of this invention are as follows:

[0041] In this invention, nitrogen fixation function of *Azotrophus brasiliensis* is induced first, enabling the bacteria to establish stable nitrogen fixation. Then, a short-term ammonium nitrogen treatment is performed. Protective components are added before the bacteria's nitrogen fixation activity recovers to pre-treatment levels. Subsequently, carrier formation and low-temperature dehydration are carried out, resulting in a bacterial agent that maintains low immediate nitrogen fixation activity and rapid subsequent recovery. Compared to methods such as direct formulation after propagation, direct formulation after nitrogen fixation induction, or formulation after ammonium withdrawal followed by nitrogen-free recovery treatment, this treatment sequence allows the resulting bacterial agent to maintain nitrogen fixation inhibition under continuous high ammonium conditions, while exhibiting rapid recovery of nitrogen fixation activity after a decrease in low-level ammonium nitrogen or the removal of short-term ammonium nitrogen. It also maintains high cumulative nitrogen fixation activity under repeated short-term ammonium nitrogen treatment. Furthermore, by combining dynamic activity detection after continuous and short-term ammonium nitrogen treatments, the reduction and recovery of nitrogen fixation function in the resulting bacterial agent can be evaluated. Attached Figure Description

[0042] The accompanying drawings are used to illustrate the technical solutions and corresponding experimental results of the present invention, and do not constitute a limitation on the scope of protection of the present invention. Figure 1 NH4 + Standard curve for the colorimetric determination of indophenol blue; Figure 2 A gas chromatographic standard curve for ethylene quantification; Figure 3 Gas chromatograms of acetylene reduction reactions in bacteria under different nitrogen-fixing states to produce ethylene; Figure 4 The graph shows the change in bacterial nitrogen fixation activity over induction time during the nitrogen fixation function induction process. Figure 5 The graph shows the changes in nitrogen fixation activity of bacteria immediately after removal of exogenous ammonium nitrogen and the changes in nitrogen fixation activity over time after 10 minutes of recovery. Figure 6 A comparison chart of T80 and dynamic cumulative activity retention rate of biological nitrogen-containing agents obtained by different treatment methods; Figure 7 This is a comparison graph of conventional nitrogen fixation activity evaluation and dynamic nitrogen fixation activity evaluation for different treatment agents. Detailed Implementation

[0043] Unless otherwise stated, all reagents used below are commercially available analytical grade products, the water used for culture and cell treatment is sterile purified water, the culture medium is sterilized before use, all experiments use the same source of Azotobacter brasiliensis, different treatment groups in the same experiment use the same batch of propagated bacterial solution, and except for the explicitly set variables, all other treatment conditions are kept consistent.

[0044] The functional bacteria used was Azospirillum brasilense Sp7, strain number ATCC29145.

[0045] The propagation medium, per liter, includes 5.0 g sodium malate, 5.0 g glucose, 1.0 g yeast extract, 0.50 g NH4Cl, 0.50 g K2HPO4, 0.20 g KH2PO4, 0.20 g MgSO4·7H2O, 0.10 g NaCl, 0.02 g CaCl2, 0.005 g FeCl3, and 0.002 g Na2MoO4·2H2O, with a pH of 6.8-7.0.

[0046] The nitrogen fixation induction medium, per liter, includes 5.0 g sodium malate, 0.50 g K₂HPO₄, 0.20 g KH₂PO₄, 0.20 g MgSO₄·7H₂O, 0.10 g NaCl, 0.02 g CaCl₂, 0.005 g FeCl₃, and 0.002 g Na₂MoO₄·2H₂O, without any external nitrogen source, and has a pH of 6.8-7.0. The above-mentioned malate culture system without any external nitrogen source was used as the detection medium for nitrogen fixation activity determination.

[0047] The bacterial cells were washed with a 0.85wt% sterile NaCl aqueous solution, which was cooled to 4-10℃ before use. The bacterial agent carrier was composed of diatomaceous earth and humic acid. Based on a total carrier mass of 100 parts, diatomaceous earth was 65-75 parts and humic acid was 25-35 parts.

[0048] The protective components include 2-4 parts soybean lecithin, 1-3 parts trehalose, and 0.3-0.8 parts monosodium glutamate. Before use, the carrier is treated at 80-90℃ for 30-60 minutes. Soybean lecithin is dispersed in sterile water at 40-45℃, and then trehalose and monosodium glutamate are added. The mixture is then cooled to 8-10℃ to obtain the protective solution.

[0049] The effective viable count was determined by plate counting, and the results are expressed as CFU / mL or CFU / g. NH4 + The concentration was determined using the indophenol blue colorimetric method, and different concentrations of NH4 were prepared using NH4Cl. + The absorbance of the standard solution was measured at 630 nm to establish a standard curve, and the resulting NH4 was obtained. + The standard curve of concentration versus absorbance is as follows: Figure 1 As shown, for NH4 + For samples with concentrations exceeding the standard curve's measurement range, dilution should be performed before measurement, and the NH4 content in the original sample should be calculated based on the dilution factor. + The concentration and bacterial protein were determined using the Bradford method. A standard curve was established using bovine serum albumin, and the absorbance was measured at 595 nm.

[0050] Nitrogen fixation activity was determined using the acetylene reduction method. 1 mL of the bacterial suspension and test culture medium were added to a 100 mL serum bottle to make a total liquid volume of 20 mL. The acetylene reduction reaction was carried out under set conditions. Ethylene in the gas phase of the reaction system was determined by gas chromatography. A quantitative standard curve was established using ethylene standard gases of different concentrations versus corresponding chromatographic peak areas. Figure 2 As shown, the concentration of gaseous ethylene is calculated from the standard curve based on the peak area of ​​the ethylene in the sample to be tested. Then, the amount of ethylene generated is converted based on the gas phase volume of the reaction system. Finally, the nitrogen fixation activity is calculated according to the reaction time and the amount of bacterial protein in the corresponding sample.

[0051] The acetylene reduction reaction time for routine nitrogen fixation activity testing was 10 min. For dynamic nitrogen fixation activity testing, the gas phase of the serum vial contained 10 vol% acetylene, 0.5 vol% O2, and the remainder Ar. The acetylene reduction reaction time was 2 min. Independent parallel samples were used for each detection time point. All results were converted to bacterial protein content and expressed as nmol C2H4 / (min·mg bacterial protein). A0 and Aoff values ​​before and after short-term ammonium nitrogen treatment, as well as data used to evaluate the recovery process of nitrogen fixation activity after removal of exogenous ammonium nitrogen, were measured under dynamic nitrogen fixation activity testing conditions. When measuring Aoff, the NH4+ in the detection system was... + The concentration should remain consistent with the concentration at the end of the corresponding ammonium nitrogen treatment.

[0052] The relative content of NifH was determined by Western blot. The relative content of other treated samples was calculated using the bacterial cell samples after nitrogen fixation induction as a reference.

[0053] During the induction of nitrogen fixation function, the nitrogen fixation activity of the bacteria was measured at regular intervals. The induction was terminated when the nitrogen fixation activity reached more than 85% of the stable nitrogen fixation activity of the batch of bacteria and the change in nitrogen fixation activity at adjacent sampling points did not exceed 10%.

[0054] After nitrogen fixation induction was completed, a portion of the bacterial culture from the same batch was taken to measure the nitrogen fixation activity before short-term ammonium nitrogen treatment and recorded as A0. After the short-term ammonium nitrogen treatment ended, the bacterial nitrogen fixation activity was measured and recorded as Aoff. After the removal of exogenous ammonium nitrogen, the recovery process of bacterial nitrogen fixation activity was measured. The short-term ammonium nitrogen treatment conditions were determined based on Aoff being 10-30% of A0 and the nitrogen fixation activity recovering to more than 80% of A0 within 20 minutes after ammonium removal.

[0055] After short-term ammonium nitrogen treatment, exogenous ammonium nitrogen is removed, and residual NH4 in the bacterial environment is eliminated. +Recovery time was calculated when the concentration dropped below 0.02 mmol / L. The time required for the bacterial nitrogen fixation activity to recover to 80% of the ammonium-free baseline nitrogen fixation activity was taken as T80. When the 80% recovery level was between two adjacent detection time points, a linear calculation was performed based on the time and nitrogen fixation activity of the adjacent detection points.

[0056] The preparation of biological nitrogen-containing agents includes the following steps:

[0057] S1: Activated *Azotobacter brasiliensis* was seed cultured, and the resulting seed culture was inoculated into propagation medium at an inoculum rate of 5-10 vol%. The culture was then incubated at 28-32℃ and pH 6.5-7.2 until the effective viable count reached 1×10⁻⁶. 9 Propagation was stopped once the concentration of CFU / mL reached a certain level.

[0058] S2: Centrifuge the propagated bacterial culture to collect the bacterial cells, resuspend them in nitrogen-fixing induction medium, and culture them at 28-32℃ and pH 6.5-7.2. The dissolved oxygen is controlled at 0.2-0.8 mg / L, and the induction time is 2-18 h. During this period, nitrogen-fixing activity is measured every 2-4 h. After the above-mentioned conditions for the termination of nitrogen-fixing function induction are met, proceed to the next step.

[0059] S3: Add sterile NH4Cl solution to the bacterial culture that has completed nitrogen fixation induction, so that NH4Cl... + The concentration was 0.05-1.00 mmol / L, and the treatment time was 3-20 min. + The concentration and treatment time were selected in combination, and the aforementioned Aoff and the recovery conditions of nitrogen fixation activity after ammonium withdrawal were used as the basis for determining the treatment conditions.

[0060] S4: Immediately after the ammonium nitrogen treatment, the bacterial culture should be cooled to 4-10℃. Exogenous ammonium nitrogen should be removed by low-temperature centrifugation or membrane concentration combined with filtration. Wait until residual NH4+ is reached. + Ammonium withdrawal was completed and timing began when the concentration was below 0.02 mmol / L.

[0061] S5: Keep the bacterial cells after ammonium withdrawal at 4-10℃, add the pre-cooled protective solution within 0-15 min and mix for 5-10 min, preferably within 5-10 min after ammonium withdrawal, and directly perform carrier processing after protection is completed.

[0062] S6: Mix the protected bacterial cells with the carrier to achieve a wet material moisture content of 20-30 wt%. Then, perform low-temperature vacuum drying, ensuring the material temperature does not exceed 35℃. Stop drying when the moisture content of the bacterial agent decreases to 6-10 wt%, yielding an effective viable bacterial count ≥ 2 × 10⁻⁶. 8 CFU / g of biological nitrogen-containing bacteria agent.

[0063] For activity testing, 5.00 g of the test bacterial agent was added to 45 mL of a rehydration solution composed of 0.85 wt% NaCl and 0.02 wt% Tween-80. The solution was shaken at 10℃ and 120 rpm for 10 min. The carrier particles were removed by centrifugation at 500 g for 3 min. The supernatant was then centrifuged at 6000 g and 8℃ for 8 min to collect the bacterial cells. After washing and resuspending, the bacterial cell concentration for different samples was adjusted according to OD600 to achieve the desired OD600 for each sample. 600 The deviation is controlled within 5%. After the bacterial cells are standardized, they are directly used for detection without pre-culture without fixed nitrogen.

[0064] No NH4 is added during ammonium-free standard testing. + The nitrogen fixation activity of the bacteria was measured at set time intervals. When the change in nitrogen fixation activity between two consecutive adjacent detection time points did not exceed 10%, the nitrogen fixation activity was considered to have entered a stable phase. The nitrogen fixation activity in this stable phase was used as the ammonium-free baseline nitrogen fixation activity. During continuous ammonium nitrogen detection, NH4+ was used to ensure that the nitrogen fixation activity was within a certain range. + The concentration was maintained at 1-5 mmol / L, and 0.05-0.20 mmol / L NH4+ was used for short-term ammonium nitrogen detection. + Treat for 3-10 minutes, then remove exogenous ammonium nitrogen to obtain residual NH4+. + When the concentration is below 0.02 mmol / L, it is considered as 0 min of ammonium withdrawal. Samples are taken at 0, 2, 5, 10, 15, 20 and 30 min to measure nitrogen fixation activity. For samples whose nitrogen fixation activity still does not reach 80% of the ammonium-free baseline nitrogen fixation activity after 30 min of ammonium withdrawal, the recovery detection time is extended until the recovery endpoint is reached. T80 is calculated based on the recovery curve.

[0065] Repeated ammonium nitrogen treatment is defined as a cycle consisting of one short-term ammonium nitrogen treatment and the recovery process after ammonium withdrawal. The cumulative nitrogen fixation activity within the detection cycle is calculated based on the nitrogen fixation activity at each detection time point. The ratio of the cumulative nitrogen fixation activity under repeated ammonium nitrogen treatment conditions to the cumulative nitrogen fixation activity under ammonium-free conditions within the same detection time is used as the dynamic cumulative activity retention rate. In one embodiment, 0.20 mmol / L NH4 is used. + The treatment lasted 5 minutes, followed by a 15-minute recovery period after ammonium withdrawal, constituting one cycle. This process was repeated for three consecutive cycles.

[0066] Example 1

[0067] This embodiment prepares a biological nitrogen-containing agent according to the aforementioned preparation method.

[0068] S1: Activated *Azotobacter brasiliensis* was inoculated into 100 mL of propagation medium and cultured at 30℃ and 180 r / min for 18 h. The resulting seed culture was then inoculated into 1.5 L of propagation medium at an inoculation rate of 8 vol% and cultured at 30℃ and pH 6.8 for 18 h. During the culture, the stirring speed was 250 r / min and the aeration rate was 0.8 vvm. After the culture was completed, the effective viable count of the bacterial solution was measured to be 1.2 × 10⁻⁶. 9 CFU / mL.

[0069] S2: Centrifuge the propagated bacterial culture at 6000g for 8 min, collect the bacterial cells and resuspend them in 1.5L of nitrogen fixation induction medium. Induce nitrogen fixation function at 30℃ and pH 6.8 with a stirring speed of 150r / min and dissolved oxygen in the culture medium maintained at 0.4-0.6mg / L. During the induction process, nitrogen fixation activity was measured at regular intervals. After 8 hours of induction, the aforementioned conditions for ending nitrogen fixation function induction were met. At this time, the nitrogen fixation activity of the bacterial cells was measured to be 14.8nmol C2H4 / (min·mg bacterial protein).

[0070] S3: Add sterile NH4Cl solution to the bacterial culture that has completed nitrogen fixation induction, so that NH4Cl... + The concentration reached 0.20 mmol / L, and the mixture was treated at 30℃ and 150 r / min for 5 min. After the treatment, Aoff was measured. A portion of the bacterial culture was taken to remove exogenous ammonium nitrogen and then tested for recovery. The test results showed that the treatment conditions met the aforementioned short-term ammonium nitrogen treatment conditions.

[0071] S4: Immediately cool the remaining bacterial culture to 8℃, centrifuge at 6000g and 8℃ for 5 min to collect the bacterial cells, add 500mL of pre-cooled (8℃) bacterial washing buffer to resuspend, centrifuge again, and wash twice using the same method. After the final wash, resuspend the bacterial cells in 100mL of pre-cooled bacterial washing buffer and measure the NH4+ in the bacterial culture. + The concentration was 0.014 mmol / L, and the timing was started from the moment resuspension was completed.

[0072] S5: Prepare a protective solution for 1000g of carrier, including 30g of soybean lecithin, 20g of trehalose, and 5g of monosodium glutamate. Disperse the soybean lecithin with some sterile water at 42℃, add trehalose and monosodium glutamate, and then add sterile water to make the total mass of the protective solution 250g. After mixing, cool to 8℃, remove ammonium for 5min, and then add the protective solution to the bacterial suspension. Mix at 8℃ for 5min.

[0073] S6: Take 700g of diatomaceous earth and 300g of humic acid, mix them, treat them at 85℃ for 45min and cool them to 8℃. Mix the protected bacterial mixture obtained in S5 with the carrier for 10min. The moisture content of the resulting wet material is 22.3wt%. Then, perform low-temperature vacuum drying, keeping the material temperature at 32±2℃. After drying to a moisture content of 7.9wt%, cool and sieve to obtain the biological nitrogen-containing bacterial agent.

[0074] The effective viable count of the obtained biological nitrogen-containing bacteria agent was 5.6 × 10⁶. 8 CFU / g, sealed and stored for subsequent validation tests.

[0075] Experimental Example 1

[0076] Take the same batch of *Azotrophus brasiliensis* bacterial culture that has completed propagation, mix them thoroughly, and divide them into several equal-volume samples. Centrifuge at 6000g for 8 min to collect the bacterial cells, and resuspend them in an equal volume of nitrogen-fixing induction medium to ensure that the initial bacterial cell concentration of each group is consistent. Nitrogen fixation function is induced at 30℃, pH 6.8, and 150 r / min. The dissolved oxygen in the culture medium is maintained at 0.4-0.6 mg / L by adjusting the aeration rate. Samples are taken at 0h, 2h, 4h, 6h, 8h, and 12h of induction, and an additional sample is taken at 18h to observe the changes in nitrogen fixation activity after it enters the stable stage.

[0077] Nitrogen fixation activity, viable bacterial count, and relative NifH content were measured at each time point. The NifH content was normalized to 100% using the results of the sample induced for 12 hours. Another batch of propagated bacterial culture was cultured in the propagation medium for 8 hours as a nitrogen fixation control group. Each treatment was performed independently three times. The results are shown in Table 1.

[0078] Table 1. Changes in nitrogen fixation activity of bacteria during nitrogen fixation induction.

[0079] Based on the nitrogen fixation activity test results in Table 1, a curve showing the change in bacterial nitrogen fixation activity with induction time was plotted, as follows: Figure 4 As shown, the nitrogen fixation activity of the bacteria gradually increased with the extension of induction time, and entered a relatively stable stage after 8 hours of induction. Combined with the results of the effective viable cell count detection in Table 1, it can be seen that the effective viable cell count of the bacterial solution reached 1×10⁻⁶ at the end of the propagation. 9 The number of viable cells was above CFU / mL, but the nitrogen fixation activity was only 1.1 nmol C2H4 / (min·mg bacterial protein). After being transferred to nitrogen fixation induction medium, the number of viable cells changed little, while the nitrogen fixation activity gradually increased. This indicates that obtaining a high number of cells through propagation does not mean that the cells have reached the nitrogen fixation functional state required for subsequent short-term ammonium nitrogen treatment.

[0080] The nitrogen fixation activities at 8h, 12h, and 18h ​​of induction were 14.8, 15.2, and 14.9 nmol C2H4 / (min·mg cell protein), respectively. Figure 4 The consistent trend indicates that the nitrogen fixation activity of the bacteria has entered a stable stage after 8 hours of induction. The nitrogen fixation activity at 8 hours of induction has reached more than 85% of the stable nitrogen fixation activity of this batch of bacteria. Moreover, the change in nitrogen fixation activity between two adjacent sampling points from 6 hours to 8 hours does not exceed 10%, which meets the aforementioned conditions for the termination of nitrogen fixation function induction. Therefore, 8 hours was used as the representative induction time in subsequent experiments.

[0081] Experiment Example 2

[0082] Take the same batch of bacterial suspensions that underwent 8 hours of nitrogen fixation induction under the conditions of Experiment Example 1, mix thoroughly, and first take a portion of the bacterial suspension to determine A0. Dispense the remaining bacterial suspensions into 100 mL portions for each group. OD values ​​for each group of bacterial suspensions are then determined. 600 To control the deviation within 5%, equal volumes of sterile NH4Cl working solution of different concentrations were added to each solution to ensure the NH4Cl concentration was within acceptable limits. + The concentrations were 0, 0.05 mmol / L, 0.20 mmol / L, 0.50 mmol / L and 1.00 mmol / L, respectively.

[0083] After adding and mixing NH4Cl, timing began. Each group was treated at 30℃ and 150 r / min for 5 min. Immediately after treatment, independent parallel samples were taken to determine Aoff. During the detection process, the NH4Cl content of the corresponding treatment group was maintained. + Concentration, the remaining bacterial suspension was immediately cooled to 8°C, centrifuged at 6000g for 5 min, washed twice with 100 mL of pre-cooled isotonic buffer without fixed nitrogen, and resuspended until residual NH4 in the external solution was removed. + When the concentration was below 0.02 mmol / L, it was recorded as 0 min of ammonium withdrawal. Subsequently, it was restored under nitrogen-free conditions at 30℃, and independent parallel samples were taken at 0 min, 2 min, 5 min, 10 min, 15 min, 20 min and 30 min to determine nitrogen fixation activity.

[0084] The ratio of Aoff to A0 at the end of the treatment was used to represent the immediate nitrogen fixation activity level, and the ratio of nitrogen fixation activity to A0 at 20 min after ammonium withdrawal was used as the recovery rate at 20 min after ammonium withdrawal. In addition, the nitrogen fixation activity of the same batch of bacteria that had completed 8 h of nitrogen fixation function induction and had not been treated with ammonium nitrogen was continuously measured under ammonium-free conditions. The detection results after the nitrogen fixation activity entered the stable stage were used as the ammonium-free baseline nitrogen fixation activity, and T80 was calculated according to the recovery curve. Each treatment was performed independently 3 times, and the results are shown in Table 2.

[0085] Table 2 Different NH4 + Nitrogen fixation status after 5 min of concentration treatment

[0086] After nitrogen fixation induction was completed, bacterial cells from the same batch were selected and treated with 0.20 mmol / L NH4+. + Samples obtained after 5 minutes of treatment and after removal of exogenous ammonium nitrogen and recovery were subjected to gas chromatography analysis. The resulting ethylene gas chromatogram is shown below. Figure 3 As shown.

[0087] like Figure 3 As shown, ethylene peaks were detected in all three states of bacterial samples after acetylene reduction. Combined with the quantitative detection results of the corresponding samples, it can be seen that the bacterial cells after nitrogen fixation induction have a high ethylene production. The ethylene production decreased after short-term ammonium nitrogen treatment, and the ethylene production increased again after the removal of exogenous ammonium nitrogen and restoration, which corresponds to the changes in nitrogen fixation activity shown in Table 2.

[0088] With NH4 + With increasing concentration, Aoff gradually decreased. At 0.05 mmol / L, Aoff remained at 61.8% of A0, failing to meet the aforementioned short-term ammonium nitrogen treatment conditions. At 0.20 mmol / L and 0.50 mmol / L, Aoff was 18.3% and 12.1% of A0, respectively, and the recovery rate after 20 minutes of ammonium withdrawal was above 80%. + When the concentration was increased to 1.00 mmol / L, Aoff decreased to 8.0%, while T80 was extended to 15.4 min, indicating that although increasing the concentration of ammonium nitrogen treatment can further reduce immediate nitrogen fixation activity, it will prolong the recovery time of nitrogen fixation function after ammonium withdrawal.

[0089] Further fixation of NH4 + The concentration was 0.20 mmol / L, and the treatment time was set to 3 min, 5 min, 10 min, and 20 min, respectively. Aoff determination, ammonium withdrawal, and recovery detection after treatment were all performed according to the above method. Additionally, a 10 mmol / L NH4+ concentration was used. + The long-term high ammonium group was treated for 120 min. After the treatment, the exogenous ammonium nitrogen was removed using the same method and the recovery detection was extended to 60 min. Each treatment was performed independently 3 times. The results are shown in Table 3.

[0090] Table 3 NH4 + The effect of treatment time on nitrogen fixation recovery

[0091] Using 0.20 mmol / L NH4 +At 3 min of treatment, Aoff was still 34.0% of A0, which did not reach the aforementioned immediate nitrogen fixation activity control range. At 5 min and 10 min of treatment, Aoff was 18.3% and 12.7% of A0, respectively. The recovery rate was higher than 90% at 20 min after ammonium withdrawal. After further extending the treatment to 20 min, Aoff decreased to 6.9%, and T80 was extended to 18.8 min. After long-term high ammonium treatment, the nitrogen fixation activity did not recover to 80% of the ammonium-free baseline at 60 min after ammonium withdrawal.

[0092] The results showed that short-term ammonium nitrogen treatment requires simultaneous control of the decrease in nitrogen fixation activity and the recovery capacity after ammonium withdrawal; simply increasing NH4+ is insufficient. + Neither increasing the concentration nor extending the treatment time resulted in a faster subsequent recovery; therefore, subsequent experiments used 0.20 mmol / L NH4+. + The processing time was 5 minutes, which was used as a representative processing condition.

[0093] Experimental Example 3

[0094] Take the bacterial culture that has undergone 8 hours of nitrogen fixation induction under the conditions of Experiment Example 1. First, take a portion of the bacterial culture to determine A0, and then treat the remaining bacterial culture with 0.20 mmol / L NH4+. + After processing for 5 min, the cells were immediately cooled to 8°C and centrifuged at 6000g for 5 min to collect the cells. The cells were then washed twice with an equal volume of pre-cooled cell washing buffer and resuspended. The NH4+ content in the extracellular fluid was then determined. + Concentration, when residual NH4 + When the concentration is below 0.02 mmol / L, it is recorded as ammonium withdrawal 0 min.

[0095] The obtained bacterial suspension was kept at 8℃, and independent parallel samples were taken at 0 min, 2 min, 5 min, 10 min, 15 min, 20 min and 30 min after ammonium withdrawal. One sample was used to immediately measure nitrogen fixation activity under dynamic nitrogen fixation activity detection conditions and recorded as Ai. The other sample was used to recover for 10 min under nitrogen-free conditions at 30℃ and then measured nitrogen fixation activity under the same detection conditions and recorded as Ar. Each treatment was performed independently 3 times. The results are shown in Table 4.

[0096] Table 4. Immediate nitrogen fixation activity and recovery nitrogen fixation activity at different times after ammonium withdrawal.

[0097] Based on the detection results in Table 4, plot the curves showing the changes in the immediate nitrogen fixation activity Ai of the bacteria after ammonium withdrawal and the nitrogen fixation activity Ar of the samples after 10 min of recovery at each ammonium withdrawal time point over time. Figure 5As shown, after the removal of exogenous ammonium nitrogen, Ai gradually increased with the extension of the ammonium removal time. Within 0-15 min of ammonium removal, Ai increased from 2.7 nmol C2H4 / (min·mg bacterial protein) to 5.6 nmol C2H4 / (min·mg bacterial protein), and continued to increase after 15 min, reaching 12.8 nmol C2H4 / (min·mg bacterial protein) at 30 min.

[0098] After 10 min of recovery under nitrogen-free conditions at 30℃, samples obtained at each ammonium withdrawal time point maintained Ar values ​​within the range of 13.4-14.0 nmol C2H4 / (min·mg bacterial protein), indicating that even with low immediate nitrogen fixation activity in the early stages of ammonium withdrawal, the bacteria still retained the ability to recover subsequently. Figure 5 The variation of Ai with ammonium withdrawal time was investigated. The 0-15 min after ammonium withdrawal can be regarded as the operation stage of the pre-recovery protection treatment. Subsequent experiments used 5-10 min after ammonium withdrawal as the representative operation time.

[0099] Experiment Example 4

[0100] Take the same batch of propagation bacterial culture, mix thoroughly, and divide into six equal groups (AF). Each group should have the same initial bacterial culture volume and cell concentration, and be treated as follows:

[0101] Group A: After propagation, the mixture was directly cooled to 8°C, a protective solution was added, and the bacterial agent was prepared according to the carrier preparation and drying conditions of Example 1.

[0102] Group B: Nitrogen fixation function was induced for 8 hours according to the conditions of Example 1, followed by cooling to 8°C and formulation, without short-term ammonium nitrogen treatment;

[0103] Group C: Nitrogen fixation induction was performed sequentially for 8 hours using the method described in Example 1, followed by 0.20 mmol / L NH4+. + After 5 minutes of treatment and removal of exogenous ammonium nitrogen, a protective solution was added and formulation was carried out 5 minutes after ammonium removal.

[0104] Group D: After propagation, the bacterial cells were collected by centrifugation, washed and resuspended in a nitrogen-free culture system until residual NH4 in the bacterial culture medium was removed. + Add NH4Cl after the concentration is below 0.02 mmol / L, so that NH4... + The concentration was 0.20 mmol / L and the treatment lasted for 5 min. Then the exogenous ammonium nitrogen was removed and the medium was transferred to nitrogen fixation induction medium for 8 h of nitrogen fixation function induction. After the induction was completed, the medium was formulated.

[0105] Group E: Nitrogen fixation induction was performed for 8 hours using the method described in Group C, followed by 0.20 mmol / L NH4+. +After 5 min of treatment and removal of exogenous ammonium nitrogen, the mixture was restored at 30 °C without fixed nitrogen for 30 min, then cooled to 8 °C and formulated.

[0106] Group F: After 8 hours of nitrogen fixation induction, 10 mmol / L NH4+ was used. + The treatment lasted 120 minutes. After the treatment, the exogenous ammonium nitrogen was removed using the same method as in Group C. Five minutes after the ammonium removal, a protective solution was added for formulation.

[0107] The composition and dosage of the protective solution, the ratio of diatomaceous earth and humic acid, the moisture content of the wet material, the drying temperature and the drying endpoint of each group were controlled according to Example 1. The final moisture content of each group of bacterial agents was 7.5-8.3 wt%, and each group was prepared independently in 3 batches.

[0108] After the bacterial agent was prepared, the number of viable bacteria was determined. Another sample of the bacterial agent was rehydrated using the aforementioned method and the bacterial cells were standardized. One sample was tested for immediate nitrogen fixation activity under the aforementioned dynamic nitrogen fixation activity detection conditions. The other sample was tested for fully recovered nitrogen fixation activity after being restored for 2 hours at 30℃ under nitrogen-free conditions. The T80 and dynamic cumulative activity retention rate were also measured for each group. The dynamic cumulative activity was detected using 0.20 mmol / L NH4+. + A cycle consisted of 5 minutes of treatment followed by 15 minutes of recovery after ammonium withdrawal. Three cycles were performed consecutively, and the results are shown in Table 5.

[0109] Table 5. Detection results of microbial agents obtained from different treatment methods

[0110] Table 5 lists the effective viable count, immediate nitrogen fixation activity, nitrogen fixation activity after 2 hours of recovery, T80, and dynamic cumulative activity retention rate of the bacterial agents obtained by different treatment methods. The comparison results of T80 and dynamic cumulative activity retention rate for each group of bacterial agents are as follows: Figure 6 As shown, the effective viable count in group AE was 5.1 × 10⁻⁶. 8 -5.6×10 8 The nitrogen fixation activity after 2 hours of recovery was 14.1-14.6 nmol C2H4 / (min·mg bacterial protein). The two indicators were similar across groups, indicating that the shorter T80 and higher dynamic cumulative activity retention rate of group C could not be explained by the effective viable count or the increase in nitrogen fixation activity after 2 hours of recovery.

[0111] The immediate nitrogen fixation activity of group A was 1.8 nmol C2H4 / (min·mg bacterial protein), which was lower than that of group C (3.1 nmol C2H4 / (min·mg bacterial protein). However, the T80 of group A was 26.8 min, and the dynamic cumulative activity retention rate was only 55.2%. This indicates that the low immediate nitrogen fixation activity of the bacterial agent after preparation is not enough to obtain a fast recovery ability after ammonium withdrawal.

[0112] Group B underwent only nitrogen fixation induction, with a T80 of 15.6 min and a dynamic cumulative activity retention rate of 69.4%. Group D had short-term ammonium nitrogen treatment before nitrogen fixation induction, with a T80 of 18.3 min and a dynamic cumulative activity retention rate of 66.8%. Neither group achieved the 7.9 min and 87.1% retention rates of Group C, indicating that the order of nitrogen fixation induction and short-term ammonium nitrogen treatment is related to the dynamic nitrogen fixation characteristics of the resulting bacterial agent.

[0113] After undergoing the same nitrogen fixation induction, short-term ammonium nitrogen treatment, and ammonium withdrawal process as Group C, Group E received an additional 30-minute nitrogen fixation recovery treatment without fixed nitrogen. Its T80 increased to 14.9 minutes, and the dynamic cumulative activity retention rate decreased to 68.6%. This indicates that the recovery and re-formulation after ammonium withdrawal failed to obtain the dynamic nitrogen fixation characteristics exhibited by Group C, which corresponds to the nitrogen fixation activity changes in the pre-recovery stage in Experiment Example 3.

[0114] After long-term high ammonium treatment, the T80 of group F was extended to 37.6 min, and the nitrogen fixation activity was fully restored, decreasing to 12.0 nmol C2H4 / (min·mg bacterial protein). The dynamic cumulative activity retention rate was 42.7%, indicating that the sustained strong inhibition formed by increasing the ammonium nitrogen concentration and extending the treatment time cannot replace the short-term ammonium nitrogen treatment used in this invention.

[0115] Experimental Example 5

[0116] Select the bacterial agents from groups A, B, and C in Experiment Example 4. Take samples from the same batch and rehydrate, remove the carrier, and standardize the bacterial cells according to the aforementioned method. For each bacterial agent, set up an ammonium-free group, a continuous high ammonium group, a stepwise decreasing low ammonium group, and a repeated short-term ammonium nitrogen group. Each treatment was performed independently 3 times, and the nitrogen fixation activity was measured according to the aforementioned dynamic nitrogen fixation activity detection conditions.

[0117] Ammonium-free group does not add NH4 + The nitrogen fixation activity was tested at 30℃ for 60 minutes, and the test results after the nitrogen fixation activity entered the stable stage were taken as the nitrogen fixation activity without ammonium.

[0118] Continuous addition of NH4Cl to the high ammonium group makes NH4 + The concentration was 5.0 mmol / L, and the NH4 in the external liquid was monitored within a 60-minute detection period. + Concentration, when NH4 +When the concentration is below 4.5 mmol / L, add NH4Cl to bring the concentration to 5.0 mmol / L to achieve the measured NH4 concentration. + The concentration was maintained at 4.6-5.1 mmol / L. The average value was calculated based on the nitrogen fixation activity during the detection period, and the percentage decrease in nitrogen fixation activity relative to that of the same group without ammonium was used as the sustained high ammonium inhibition rate.

[0119] The stepwise decrease in low ammonium group first adds NH4Cl to make NH4 + The concentration was 0.10 mmol / L. After treatment for 10 min, the bacterial cells were collected using the aforementioned low-temperature ammonium removal method and resuspended in a solution containing 0.05 mmol / L NH4. + The samples were further treated in the detection medium for 10 minutes, then the exogenous ammonium nitrogen was removed again and the samples were transferred to the detection medium without an external fixed nitrogen source for another 40 minutes. The total detection time was 60 minutes. Nitrogen fixation activity was determined using independent parallel samples at each time point. The cumulative activity retention rate under the step-decreasing low ammonium condition was calculated based on the ratio of the cumulative nitrogen fixation activity during the detection period to the cumulative nitrogen fixation activity under the same detection time under the same ammonium-free condition in the same group.

[0120] The repeated short-term ammonium nitrogen group used 0.20 mmol / L NH4+. + After 5 minutes of treatment, the residual NH4 was removed. + When the concentration was reduced to below 0.02 mmol / L, it was recovered for 15 min at 30℃ without fixed nitrogen. This was taken as a cycle and three cycles were performed continuously. Nitrogen fixation activity was determined using independent parallel samples at each detection time point. The dynamic cumulative activity retention rate was calculated based on the entire detection cycle, and T80 was calculated based on the recovery curve after the first short-term ammonium nitrogen treatment and ammonium withdrawal. The results are shown in Table 6.

[0121] Table 6. Nitrogen fixation activity of various bacterial agents under different ammonium nitrogen conditions.

[0122] The stable nitrogen-fixing activities of groups A, B, and C under ammonium-free conditions were 14.0 nmol C2H4 / (min·mg bacterial protein), 14.4 nmol C2H4 / (min·mg bacterial protein), and 14.3 nmol C2H4 / (min·mg bacterial protein), respectively, under sustained conditions of 5.0 mmol / L NH4. + Under the conditions, the nitrogen fixation inhibition rate of all three groups exceeded 90%, indicating that group C did not change the response of the bacteria to the inhibition of nitrogen fixation activity under continuous high ammonium conditions.

[0123] Under the stepwise decrease of low ammonium nitrogen, the cumulative activity retention rate of group C was 78.4%, which was higher than that of group A (48.6%) and group B (60.1%). After three consecutive short-term ammonium nitrogen treatments, the T80 of group C was 7.9 min, and the dynamic cumulative activity retention rate was 87.1%. The T80s of groups A and B were 26.8 min and 15.6 min, respectively, and the dynamic cumulative activity retention rates were 55.2% and 69.4%, respectively. This indicates that the difference in group C is mainly reflected in the gradual decrease of low-level ammonium nitrogen and the recovery stage of nitrogen fixation activity after the end of short-term ammonium nitrogen treatment.

[0124] The above results indicate that the bacterial agent prepared according to the method of the present invention maintains nitrogen fixation inhibition under continuous high ammonium conditions, but has a faster recovery rate of nitrogen fixation activity after the reduction of low-level ammonium nitrogen or the removal of short-term ammonium nitrogen, and can maintain a high cumulative nitrogen fixation activity during repeated short-term ammonium nitrogen treatment.

[0125] Experimental Example 6

[0126] The bacterial agents A, B, C and E prepared in Experiment Example 4 were selected, and samples from the same batch of each group were used to detect the effective viable count, nitrogen fixation activity after recovery from nitrogen fixation, and dynamic nitrogen fixation activity.

[0127] The effective viable count was determined according to the aforementioned plate counting method. When testing nitrogen fixation activity after nitrogen fixation was restored, each bacterial agent was rehydrated, the carrier was removed, and the bacterial cells were standardized according to the aforementioned method. After pre-culturing at 30°C under nitrogen-free conditions for 2 hours, the nitrogen fixation activity was determined by the acetylene reduction method.

[0128] Dynamic nitrogen fixation activity assays were performed without the aforementioned 2-hour pre-culture without fixed nitrogen. After cell rehydration and standardization, the assays were performed directly according to the aforementioned dynamic assay method. The nitrogen fixation inhibition rate under continuous high ammonium conditions (5.0 mmol / L) and the nitrogen fixation inhibition rate under 0.20 mmol / L NH4+ conditions were measured. + The dynamic cumulative activity retention rate of T80 after 5 min of treatment and after three consecutive short-term ammonium nitrogen treatments were measured independently three times. The results are shown in Table 7.

[0129] Table 7. Detection results obtained from different activity evaluation methods

[0130] The results were compared based on the detection results of nitrogen fixation activity, T80, and dynamic cumulative activity retention rate after 2 hours of recovery without fixed nitrogen in Table 7. The results are as follows: Figure 7As shown, the nitrogen fixation activities of groups A, B, C, and E after 2 hours of pre-culture without fixed nitrogen were 14.0, 14.4, 14.3, and 14.5 nmol C2H4 / (min·mg bacterial protein), respectively, with little difference among groups. However, there were significant differences in T80 and dynamic cumulative activity retention rate among the groups. Specifically, the T80 of group C was 7.9 min, and the dynamic cumulative activity retention rate was 87.1%. The T80s of groups A, B, and E were 26.8 min, 15.6 min, and 14.9 min, respectively, and the dynamic cumulative activity retention rates were 55.2%, 69.4%, and 68.6%, respectively.

[0131] Table 7 also shows that the effective viable counts of groups A, B, C, and E are all 5.2 × 10⁻⁶. 8 -5.6×10 8 CFU / g, at a sustained 5.0 mmol / L NH4 + The nitrogen fixation inhibition rate under the conditions was around 90%, indicating that there were little difference among the groups in terms of the number of culturable cells and the nitrogen fixation inhibition response under continuous high ammonium conditions.

[0132] Both groups C and E underwent nitrogen fixation induction, short-term ammonium nitrogen treatment, and removal of exogenous ammonium nitrogen. Group E, in addition to this, underwent a nitrogen-free recovery treatment after ammonium removal. The nitrogen fixation activities of the two groups were similar after 2 hours of nitrogen-free pre-culture, but there were still differences in T80 and dynamic cumulative activity retention rate. This indicates that the single nitrogen fixation activity after full recovery cannot reflect the recovery differences of bacterial agents obtained from different preparations after the end of short-term ammonium nitrogen treatment.

[0133] Therefore, the number of effective viable bacteria and the single nitrogen fixation activity after recovery from nitrogen fixation can evaluate the number of culturable bacteria in the bacterial agent and the nitrogen fixation capacity after recovery, but it is difficult to reflect the nitrogen fixation function recovery process under ammonium nitrogen change conditions. The dynamic nitrogen fixation characteristics of biological nitrogen bacterial agents obtained from different preparation treatments can be further evaluated by T80 and dynamic cumulative activity retention rate.

[0134] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A method for preparing a biological nitrogen-containing bactericidal agent, characterized in that, Includes the following steps: S1: Activate and seed culture the Brazilian azospira, and inoculate the resulting seed culture into a propagation culture system containing a fixed nitrogen source for propagation culture; S2: The propagated Azotospira brasiliensis was transferred into a nitrogen fixation induction culture system without an external nitrogen fixation source, and nitrogen fixation function was induced under microaerophilic conditions until the nitrogen fixation activity of the bacteria reached a stable level. S3: Add ammonium salt to the bacterial culture system that has completed nitrogen fixation induction, so that NH4+... + A concentration of 0.05-1.00 mmol / L and a treatment time of 3-20 min reduced the nitrogen fixation activity of the bacteria. S4: Immediately after the ammonium nitrogen treatment, lower the cell temperature to 4-10℃ to remove exogenous ammonium nitrogen until the residual NH4 in the cell environment is removed. + Concentration below 0.02 mmol / L; S5: After removing exogenous ammonium nitrogen, maintain the bacterial cells at 4-10℃ to allow residual NH4 in the external environment to escape. + When the concentration is below 0.02 mmol / L, add the pre-cooled protective component within 0-15 min, before the nitrogen fixation activity of the bacteria has recovered to the level before the ammonium nitrogen treatment; S6: After the protection treatment is completed, the obtained bacterial cells are directly mixed with the carrier for carrier formation, and then dehydrated. During the dehydration process, the material temperature does not exceed 35℃ to obtain biological nitrogen-containing bacterial agent.

2. The method for preparing the biological nitrogen-containing agent according to claim 1, characterized in that: In S2, nitrogen fixation induction was carried out at 28-32℃ and pH 6.5-7.2, with dissolved oxygen in the culture system at 0.2-0.8 mg / L. The induction time was 2-18 h. During the induction process, samples were taken every 2-4 h to measure the nitrogen fixation activity of the cells. Nitrogen fixation induction was terminated when the nitrogen fixation activity of the cells reached more than 85% of the nitrogen fixation activity of the stable phase of the batch of cells, and the change in nitrogen fixation activity at adjacent sampling points did not exceed 10%.

3. The method for preparing the biological nitrogen-containing agent according to claim 1, characterized in that: In S3, NH4 + The concentration was 0.10-0.50 mmol / L, and the treatment time was 3-10 min. The NH4... + The concentration and treatment time were selected in combination, with the immediate nitrogen fixation activity of the bacteria at the end of the ammonium nitrogen treatment being 10-30% of the nitrogen fixation activity before treatment, and the nitrogen fixation activity of the bacteria recovering to more than 80% of the nitrogen fixation activity before treatment within 20 minutes after the removal of exogenous ammonium nitrogen as the basis for determining the treatment conditions.

4. The method for preparing the biological nitrogen-containing agent according to claim 1, characterized in that: In S4, exogenous ammonium nitrogen is removed by low-temperature centrifugation washing, and the bacterial cells are washed with an isotonic solution without fixed nitrogen during the washing process.

5. The method for preparing the biological nitrogen-containing agent according to claim 1, characterized in that: In S5, the protective component is added within 5-10 minutes after the removal of exogenous ammonium nitrogen.

6. The method for preparing the biological nitrogen-containing agent according to claim 1, characterized in that: In S6, the wet material after the protected bacterial cells and carrier are mixed has a moisture content of 20-30 wt%. It is then dried under low temperature and reduced pressure until the moisture content of the resulting biological nitrogen-containing agent is reduced to 6-10 wt%.

7. A biological nitrogen-producing agent prepared by the preparation method according to any one of claims 1-6, characterized in that, The biological nitrogen-fixing agent includes *Azotrophus brasiliensis* cells, a carrier, and a protective component; After the biological nitrogen-containing bacterial agent is rehydrated and the bacterial cells are standardized, 0.20 mmol / L NH4+ is used. + The bacterial cells were treated for 5 minutes. After treatment, the exogenous ammonium nitrogen was removed, and the residual NH4 in the bacterial environment was allowed to dissipate. + When the concentration is below 0.02 mmol / L, the time required for the nitrogen fixation activity of the bacteria to recover to 80% of the nitrogen fixation activity without ammonium is ≤12 min.

8. The biological nitrogen-containing agent according to claim 7, characterized in that: The carrier includes diatomaceous earth and humic acid. Based on a total carrier mass of 100 parts, diatomaceous earth accounts for 65-75 parts and humic acid accounts for 25-35 parts. The protective components include soybean lecithin, trehalose, and monosodium glutamate, with soybean lecithin comprising 2-4 parts, trehalose comprising 1-3 parts, and monosodium glutamate comprising 0.3-0.8 parts relative to 100 parts of carrier.

9. A method for detecting the activity of a biological nitrogen-containing agent as described in claim 7 or 8, characterized in that, Includes the following steps: C1: The biological nitrogen-containing agent to be tested is rehydrated, the carrier particles are removed and the bacterial cells are collected. The bacterial cell count is standardized, and the standardized bacterial cells are divided into several portions. No nitrogen-free pre-culture is performed, and each portion is used for subsequent testing. C2: Take a standardized bacterial cell and test it under the condition of no external fixed nitrogen source and no external ammonium nitrogen. The test result after the nitrogen fixation activity enters the stable stage is used as the ammonium-free benchmark nitrogen fixation activity. C3: Take a standardized bacterial cell and test it under continuous ammonium nitrogen conditions to determine the decrease in nitrogen fixation activity of the bacterial cells under continuous ammonium nitrogen conditions; C4: Take a standardized bacterial cell and treat it with ammonium nitrogen for a short time. After the treatment, remove the exogenous ammonium nitrogen and continuously measure the recovery process of the bacterial cell nitrogen fixation activity. C5: Evaluate the dynamic nitrogen fixation activity of the biological nitrogen agent based on the decrease in nitrogen fixation activity of the bacteria under continuous ammonium nitrogen and the recovery of nitrogen fixation activity after the removal of exogenous ammonium nitrogen.

10. The method for detecting the activity of the biological nitrogen-containing agent according to claim 9, characterized in that: In C4, 0.20 mmol / L of NH4 is used. + After 5 minutes of treatment, residual NH4 in the bacterial environment was removed. + The concentration was reduced to below 0.02 mmol / L, to achieve residual NH4+. + The time required to restore the concentration is used as the starting point for the recovery test. The time required for the bacterial nitrogen fixation activity to recover to 80% of the ammonium-free baseline nitrogen fixation activity is recorded as T80.

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