Bacillus amyloliquefaciens producing gamma-pga and coated controlled-release compound fertilizer

Abstract

A bacillus amyloliquefaciens for producing gamma-PGA and a coated controlled-release compound fertilizer, belong to the field of microbial fermentation technology, the classification name of the strain H-27 is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bacillus amyloliquefaciens ), which is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 38389, and the preservation date is April 27, 2026. The bacillus amyloliquefaciens H-27 has excellent lignocellulose degradation and high-efficiency toxicity resistance degradation capacity, and the yield of gamma-PGA synthesized by one-step solid-state fermentation of corn straw is as high as 171.47 g / kg. The average molecular weight of the synthesized specific gamma-PGA is 114.334 kDa, and the application to the film controlled-release compound fertilizer can more effectively improve the stability of the compound fertilizer compared with the commercially available gamma-PGA, and the effective controlled-release of the compound fertilizer can improve the fertilizer efficiency.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a Bacillus amyloliquefaciens strain capable of synthesizing γ-PGA in a one-step process and its application in coated controlled-release compound fertilizer. Background Technology

[0002] γ-Polyglutamic acid (γ-PGA) is a high-molecular-weight polymer formed by the polymerization of D-glutamic acid and L-glutamic acid in different proportions. It possesses properties such as high water absorption, biocompatibility, and metal ion chelation, and has broad application prospects in agriculture, food, medicine, cosmetics, and environmental protection. Corn stalks are rich in cellulose, which can be converted into monosaccharides through hydrolysis, serving as a carbon source for γ-PGA fermentation. However, while corn stalks are a promising low-cost raw material for γ-PGA production, traditional processes require steps such as stalk pretreatment, enzymatic hydrolysis and saccharification, and fermentation. These processes necessitate the separate preparation of pretreatment reaction solutions, enzymatic hydrolysates, and fermentation broths, making the operation relatively cumbersome. Therefore, to simplify the process, it is necessary to screen for strains that combine corn stalk degradation capabilities with high γ-PGA production characteristics. Using corn stalks as raw material, a one-step solid-state fermentation process can be employed to produce γ-PGA, simultaneously performing substrate enzymatic hydrolysis and product conversion, thus simplifying the process and achieving a cleaner γ-PGA production process.

[0003] Existing technologies have reported that Bacillus amyloliquefaciens JX-6, using straw and soybean meal in a 1:1 mass ratio as the fermentation substrate, achieved a maximum γ-PGA yield of 166.99 g / kg. However, this high γ-PGA yield was achieved because the substrate contained a large amount of soybean meal, and JX-6's utilization of soybean meal was higher than that of corn straw. Furthermore, corn straw, as a typical lignocellulose raw material, contains a large amount of lignin, hemicellulose, etc., which continuously generate complex antibacterial and toxic substances under the solid-state fermentation microenvironment. In contrast, soybean meal contains almost no lignin and other components, and no toxic degradation products are generated under the fermentation microenvironment. If corn stalks are used as the substrate, JX-6 not only has a reduced utilization effect on the substrate, but also suffers from a severe decrease in γ-PGA yield due to the strong inhibition of toxic degradation products on JX-6 caused by the large amount of these products. Existing technologies have also clearly reported that when straw is used as the sole fermentation substrate, JX-6's own degradation ability on straw is weak and insufficient to support the one-step synthesis of γ-polyglutamic acid by JX-6. Therefore, straw pretreatment and exogenous enzymatic hydrolysis for simultaneous saccharification are required.

[0004] Therefore, to obtain high yields of γ-PGA using corn stalks as the sole fermentation substrate, it is necessary to screen for multifunctional microorganisms that can efficiently utilize corn stalks, resist the inhibition of toxic degradation products, and efficiently synthesize γ-PGA. Summary of the Invention

[0005] The purpose of this invention is to provide a strain that produces high-yield γ-PGA through a one-step solid-state fermentation of corn straw. This strain simultaneously performs substrate enzymatic hydrolysis and product transformation, simplifying the process and thus achieving a cleaner γ-PGA preparation process.

[0006] Another objective of this invention is to further apply the synthesized γ-PGA to the preparation of coated controlled-release compound fertilizer.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A strain H-27 that produces high γ-PGA through solid-state fermentation of corn stalks is characterized by the following: the strain is classified as *Bacillus amyloliquefaciens* (…). Bacillus amyloliquefaciens The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38389 and deposit date of April 27, 2026.

[0009] This invention uses neutral red dye plate screening and rigid red dye plate screening to initially screen for strain H-27, which possesses cellulase activity and can synthesize γ-PGA. The strain H-27 obtained in the initial screening exhibits the best γ-PGA synthesis capacity, with a yield of 123.83 g / kg.

[0010] The colonies of strain H-27 have irregular edges and a slightly translucent, membranous, raised surface. After 24 hours of incubation, some colonies produce a viscous, sticky mucus in their center, which tends to droop. When picked up with an inoculation loop, it is clearly stringy. Under an optical microscope, strain H-27 cells appear as short rods, and after Gram staining, the cells show a typical blue-purple color.

[0011] Since strain H-27 has the ability to degrade lignocellulose, it can efficiently synthesize γ-PGA under the inhibition of toxic products generated by lignocellulose degradation. Therefore, strain H-27 can be effectively used in the one-step solid-state fermentation of corn straw to synthesize γ-PGA.

[0012] Furthermore, by optimizing the solid-state fermentation parameters, the yield of γ-PGA reached 171.47 g / kg when corn straw was fermented for 96 hours with 50% peptone, 20% L-glutamate, 7% H-27 inoculum, 7.5 initial pH, and 37℃.

[0013] The conformation of γ-PGA is easily affected by external conditions, including pH, polymer concentration, and ionic strength, all of which can lead to conformational changes. γ-Polyglutamic acid is composed of L-glutamic acid or D-glutamic acid, with molecules interconnected by amide bonds between γ-carboxyl and α-amino groups. Due to the numerous side-chain carboxyl groups on the γ-PGA molecular chain, hydrogen bonds can form within or between molecules, giving it extremely strong moisturizing and water-absorbing properties. However, because the content of D-glutamic acid and L-glutamic acid varies among different γ-PGA varieties, their molecular weights also differ, resulting in significant property differences between γ-PGA from different sources.

[0014] The γ-PGA synthesized in this invention through a one-step solid-state fermentation of corn straw using Bacillus amyloliquefaciens H-27 has an average molecular weight of 114.334 kDa. When applied to coated controlled-release compound fertilizers, this γ-PGA exhibits excellent properties. Through coating and controlled release, it improves the fertilizer efficiency of the compound fertilizer and regulates soil physicochemical indicators such as enzyme activity, ammonium nitrogen, nitrate nitrogen, available nitrogen, and cation exchange capacity.

[0015] The above-mentioned strain H-27 can also be used in the preparation of film-film controlled-release compound fertilizer.

[0016] A method for preparing a coated controlled-release compound fertilizer based on γ-PGA synthesized from corn straw by solid-state fermentation using strain H-27 is characterized by comprising the following steps: S1. Synthesis of γ-PGA from corn straw via one-step solid-state fermentation using Bacillus amyloliquefaciens H-27; S2. Prepare coated controlled-release compound fertilizer using γ-PGA as a fertilizer additive.

[0017] Furthermore, the solid-state fermentation is carried out using corn stalks as the fermentation substrate, with the addition of 48-52% nitrogen source, 12-22% L-glutamate sodium, and inoculation with Bacillus amyloliquefaciens H-27 at an inoculation amount of 7-9%, adjusting the initial pH to 7-7.5, and conducting solid-state fermentation at 37-42℃.

[0018] Furthermore, the method for preparing the coated controlled-release compound fertilizer in step S2 is characterized by comprising the following steps: (1) Take ethanol, preheat it to 45~55℃, then slowly add glycerol, and stir continuously to obtain mixed system a; (2) Add soybean oil to system a, stir continuously, add sorbitol and γ-PGA after stirring, sonicate, add glutaraldehyde after sonication to obtain system b; (3) Take γ-PGA and add pure water to prepare γ-PGA solution; (4) The compound fertilizer is coated by rotary drum spraying process: The compound fertilizer granules are placed in the turntable and after the rotation starts, 1 / 3 of the system b is sprayed, mixed evenly and dried. 1 / 2 of the γ-PGA solution is added, mixed evenly and dried. 1 / 3 of the system b is sprayed, mixed evenly and dried. The remaining γ-PGA solution is added, mixed evenly and dried. Finally, the remaining system b is sprayed, mixed evenly and dried to obtain γ-PGA coated controlled-release compound fertilizer.

[0019] Furthermore, the volume ratio of ethanol to glycerol in (1) is 4~6:1.

[0020] Furthermore, in step (2), the ratio of system a, soybean oil, sorbitol, γ-PGA and glutaraldehyde is 10~13mL:12~16mL:18~22mL:1.5~2.5g:4~6mL.

[0021] Furthermore, in step (3), the ratio of γ-PGA to pure water in the γ-PGA solution is 25~35 g:300 mL.

[0022] Most specifically, the method for preparing the coated controlled-release compound fertilizer in step S2 is characterized by comprising the following steps: (1) Take ethanol and preheat it to 45~55℃, then slowly add glycerol and stir continuously for 15 min. After mixing, a mixture system a is obtained, with the volume ratio of ethanol to glycerol being 4~6:1. (2) While maintaining the temperature, add soybean oil to system a and stir continuously. After stirring evenly, add 1 mol / L sorbitol and stir for 4-6 min. Then add γ-PGA and sonicate at 0.2-0.4 kW power for 25-35 min. After sonication, add glutaraldehyde to obtain system b. The ratio of the amount of system a, soybean oil, sorbitol, γ-PGA and glutaraldehyde is 10-13 mL: 12-16 mL: 18-22 mL: 1.5-2.5 g: 4-6 mL. (3) Take γ-PGA and add pure water to prepare γ-PGA solution. The ratio of γ-PGA to pure water is 25~35 g:300mL. (4) Coating compound fertilizer using a rotary drum spray coating process: Place 10 kg of compound fertilizer granules into a rotary drum, start rotating, spray one-third of system b, rotate for 10 min to mix evenly, and then air dry. Add half of the γ-PGA solution, continue rotating for 15-25 min, and then air dry. Spray one-third of system b, rotate for 8-12 min to mix evenly, and then air dry. Add the remaining γ-PGA solution, continue rotating for 15-25 min, and then air dry. Spray the remaining system b, rotate for 8-12 min to mix evenly, and then air dry to obtain γ-PGA coated controlled-release compound fertilizer.

[0023] The present invention has the following technical effects: The *Bacillus amyloliquefaciens* H-27 strain of this invention possesses excellent ability to degrade lignocellulose, along with highly efficient resistance to toxic degradation and efficient synthesis of γ-PGA. Through a one-step solid-state fermentation of corn straw, the yield of γ-PGA synthesized reaches as high as 171.47 g / kg (significantly higher than the current top-level strain JX-6, which cannot directly utilize pure corn straw as a fermentation substrate and requires pretreatment of the straw and simultaneous saccharification with exogenous enzymes). Furthermore, the specific γ-PGA synthesized using this strain H-27 has an average molecular weight of 114.334 kDa and possesses unique physicochemical properties. When applied to film-film controlled-release compound fertilizers, it more effectively improves the stability of the compound fertilizer compared to commercially available γ-PGA, thereby enhancing fertilizer efficiency through effective controlled release. Attached Figure Description

[0024] Figure 1 Strains that exhibit a clear zone on neutral red agar plates.

[0025] Figure 2 Strains that have just passed through the red agar plate and have a transparent zone.

[0026] Figure 3 Comparison of the relative activities of cellulase systems in solid-state fermentation products of different strains.

[0027] Figure 4 : Colony morphology and Gram staining results of strain H-27.

[0028] Figure 5 Phylogenetic tree of strain H-27.

[0029] Figure 6 Effects of strain H-27 on changes in substrate composition during corn straw fermentation.

[0030] Figure 7 Optimization of parameters for solid-state fermentation synthesis of γ-PGA by strain H-27.

[0031] Figure 8High-performance liquid chromatograms of γ-PGA standard and product of strain H-27 (A: γ-PGA standard; B: strain H-27).

[0032] Figure 9 GPC molecular weight distribution spectrum of γ-PGA synthesized by solid-state fermentation of strain H-27.

[0033] Figure 10 Results of hygroscopicity test of coated controlled-release compound fertilizer prepared with γ-PGA.

[0034] Figure 11 Stability test results of coated controlled-release compound fertilizer prepared with γ-PGA.

[0035] Figure 12 Primary and differential nutrient dissolution rates of coated controlled-release compound fertilizers prepared with the participation of γ-PGA.

[0036] Figure 13 Soil indicators in field trials of coated controlled-release compound fertilizer prepared with γ-PGA.

[0037] Figure 14 Plant indicators in field trials of coated controlled-release compound fertilizer prepared with γ-PGA. Detailed Implementation

[0038] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0039] Example 1 Strain screening, isolation and identification 1. Strains isolation and screening: Sixty-four strains with fast growth rates and high colony surface viscosity were screened from farm-grown fermented black beans.

[0040] (1) Neutral red dye plate screening: γ-PGA, as a negatively charged anionic polypeptide, binds to a positively charged neutral red dye via electrostatic interaction, forming an insoluble complex. This leads to the consumption or precipitation of dye around the colony, resulting in a clear zone. The size of the clear zone can preliminarily characterize the γ-PGA production level of the strain, and this colorimetric method is suitable for the rapid screening of high-γ-PGA-producing strains. Through neutral red identification plate screening, a total of 13 strains capable of forming clear zones were isolated, such as... Figure 1The strains shown are H-5, H-6, H-7, H-12, H-19, H-21, H-27, H-29, H-30, H-45, H-48, H-49, and H-53.

[0041] (2) Plate screening of rigid red dyes: Congo red specifically binds to cellulosic substrates (such as CMC) to form a red complex. When cellulase secreted by microorganisms hydrolyzes the substrate, the Congo red-substrate complex dissociates, forming a clear zone around the colony. The size of this clear zone can visually reflect the strength of cellulase activity. Using 13 functional strains initially screened on neutral red agar plates as material, further screening was conducted using Congo red identification plates. The results are as follows: Figure 2 As shown, the results indicated that 10 strains could form obvious hydrolytic clear zones, confirming that these 10 strains had cellulase activity and the ability to degrade cellulose.

[0042] Enzyme activities of cellulose-degrading enzymes in 10 bacterial strains (H-5, H-6, H-7, H-12, H-19, H-27, H-29, H-30, H-45, and H-48) were measured under solid-state fermentation conditions. The average enzyme activity of strain H-27 was set as 100%, and the differences in enzyme activity among the strains were visually compared using relative enzyme activities. Results are as follows: Figure 3 As shown, strain H-27 possesses a complete cellulase system with balanced activity and excellent synergistic effect. Strain H-27 exhibits balanced performance in the detection of four types of cellulase activities, with high endonuclease activity. It can efficiently hydrolyze the β-1,4 glycosidic bonds inside the cellulose molecule, breaking the macrocellulose molecule into oligosaccharide chains, and has application potential in one-step bioconversion of straw.

[0043] (3) Screening of solid-state fermentation yield using perforated plates and shake flasks Ten strains obtained from the initial screening were activated and subjected to solid-state fermentation experiments in well plates. The results showed that... Strain H-27 exhibited the best γ-PGA synthesis capacity, with a yield of up to 123.83 g / kg.

[0044] 2. Strain identification (1) Identification of colony and cell morphology: After activation, the high-yielding strains obtained through screening were serially diluted appropriately. 100 μL of each dilution was plated onto neutral red selection plates and incubated at 37°C for 24 h. Colony morphology, appearance characteristics, and mucus adhesion ability were observed. Results are as follows: Figure 4As shown in Figure A, the colonies of strain H-27 have irregular edges and a slightly translucent, membranous, raised shape on the surface. After 24 hours of cultivation, some colonies produce a viscous mucus in the center, which is sticky and tends to droop. When picked up with an inoculation loop, obvious stringiness can be seen.

[0045] (2) Gram staining: Take an appropriate amount of bacterial suspension and drop it onto a glass slide. After allowing it to air dry, fix it quickly with a flame 2-3 times. Add crystal violet staining solution and stain for 2 minutes. Rinse with water until colorless, then add iodine solution as mordant for 1 minute. Rinse with sterile water. Decolorize with 95% ethanol, rinse to remove ethanol, and finally counterstain with safranin for 2 minutes. Rinse with sterile water, air dry, and examine under a microscope. Results are as follows: Figure 4 As shown in Figure B, under an optical microscope, strain H-27 appears as short rods. After Gram staining, the cells turn a typical blue-purple color, indicating that strain H-27 is a Gram-positive bacterium.

[0046] (3) Molecular identification: DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit. PCR amplification and sequencing were performed using universal 16S rDNA primers. A phylogenetic tree was constructed and analyzed by BLAST sequence alignment. The results showed that the 16S rDNA sequence alignment of strain H-27 was consistent with... Bacillus amyloliquefaciens NBRC15535 has the highest similarity, and the phylogenetic tree of strain H-27 is as follows: Figure 5 As shown, this strain is identified as Bacillus amyloliquefaciens. B. amyloliquefaciens It was named Bacillus amyloliquefaciens (B. amyloliquefaciens) Bacillusammyloliquefaciens H-27.

[0047] The strain H-27 was preserved; the strain is classified as Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38389 and deposit date of April 27, 2026.

[0048] Example 2 Effects of strain H-27 on compositional changes of corn straw solid substrate The degradation effect of strain H-27 on corn straw was verified by analyzing the changes in its composition during fermentation. The changes in lignocellulose content were measured before fermentation and during the fermentation process (0h, 24h, 36h, 48h, 72h, 96h). The results are shown below. Figure 6As shown, the contents of cellulose, hemicellulose, and lignin in straw all decreased significantly within a 96-hour fermentation cycle. Among them, cellulose showed the fastest degradation rate within 48 hours, indicating that the cellulase system of the strain had high catalytic activity in the early stage of fermentation. Hemicellulose was degraded simultaneously with cellulose, effectively disrupting the dense physical structure of straw, exposing more usable substrate, and further improving the overall degradation efficiency.

[0049] Example 3 Optimization of solid-state fermentation parameters By studying the strains B. amyloliquefaciens The optimization of H-27 solid-state fermentation conditions was investigated to explore the impact of key influencing factors on γ-PGA yield. For example... Figure 7 As shown in Figures A and B, in solid-state fermentation, the highest γ-PGA yield (157.59 ± 10.79 g / kg) was achieved when peptone addition was 50% and L-glutamate addition was 20%. However, as the addition of these two components continued to increase, the γ-PGA yield decreased. Excessive nutrient supply can induce excessive cell growth and reproduction, leading to a preferential flow of metabolic substrates to cell biomass synthesis, resulting in a metabolic diversion effect and thus inhibiting the accumulation of γ-PGA. As shown in Figure 7C, when the inoculum concentration was 7%, the yield was 159.73 ± 5.99 g / kg. The inoculum concentration has a significant regulatory effect on the fermentation process. A suitable initial inoculum concentration can effectively shorten the lag phase of bacterial growth, accelerate cell proliferation, and significantly shorten the fermentation cycle, thereby promoting the rapid synthesis and accumulation of γ-PGA. During solid-state fermentation, temperature and initial pH can affect the catalytic activity of enzymes related to cellulose hydrolysis and γ-PGA synthesis, thus affecting the synthesis and accumulation of γ-PGA. Depend on Figure 7 As shown in Figures D and E, the γ-PGA yield reached 166.16±3.85 g / kg at a fermentation temperature of 37℃. When the initial pH of the system was 7.5, the γ-PGA yield was 168.41±7.67 g / kg. Figure 7F shows the change in γ-PGA yield at different fermentation times. The γ-PGA yield reached its peak at 96 h of fermentation, at 171.47±3.14 g / kg. With prolonged fermentation time, the γ-PGA yield decreased, indicating that the cells entered the death phase. The activity of enzymes related to γ-PGA synthesis significantly decreased, and intracellular hydrolases were induced, degrading the already generated γ-PGA. Furthermore, the continuous accumulation of metabolic byproducts such as organic acids caused pH shifts and microenvironment deterioration, further inhibiting cell metabolic activity and ultimately leading to a decline in γ-PGA yield.

[0050] In this invention, strain H-27 uses corn stalks as a fermentation substrate to achieve a high yield of γ-polyglutamic acid (171.47±3.14 g / kg) through a one-step process (self-degradation of lignocellulose in corn stalks and simultaneous synthesis of γ-polyglutamic acid). This is significantly superior to strain JX-6, which currently uses solid-state fermentation to produce γ-polyglutamic acid. Compared with JX-6, H-27 has the following advantages: it uses corn stalks as a fermentation substrate and achieves simultaneous saccharification and fermentation of γ-polyglutamic acid in one step. JX-6 cannot use pure corn stalks as a fermentation substrate, but instead uses soybean meal and corn stalks in equal proportions, which are more readily available. When using pure corn stalks as a fermentation substrate, JX-6's self-degradation of lignocellulose is insufficient to produce γ-polyglutamic acid through simultaneous saccharification and fermentation in one step. It requires organic acid pretreatment of the stalks and the addition of exogenous enzymes for saccharification to achieve the synthesis of γ-polyglutamic acid using corn stalks as a fermentation substrate.

[0051] Molecular weight determination and product identification of γ-PGA: The fermentation products of strain H-27 were qualitatively identified using HPLC. The results are as follows: Figure 8 As shown in Figure A, the chromatographic peak time of the γ-PGA standard is 1.832 min, and the corresponding peak time of the fermentation product of strain H-27 is 1.807 min. Figure 8 As shown in Figure B, this indicates that the fermentation product of strain H-27 contains γ-PGA. The molecular weight of γ-PGA in the fermentation product of strain H-27, as determined by GPC, is 114.334 kDa. Figure 9 As shown.

[0052] Example 4 Preparation of γ-PGA controlled-release coated compound fertilizer: The fertilizer used in this invention is high-tower granulated compound fertilizer (produced by Jilin Longyuan Agricultural Service Co., Ltd., with a total nutrient content ≥48.0% and N-P2O5-K2O of 26-10-12).

[0053] γ-PGA: The γ-PGA used in this experiment has an average molecular weight of 114.334 kDa and was prepared by solid-state fermentation of corn straw by strain H-27 in Example 3.

[0054] The specific preparation steps are as follows: (1) Take 10 mL of ethanol and preheat it to 50°C. Then slowly add 2 mL of glycerol and stir continuously for 15 min. After mixing, the mixture a is obtained. (2) Keep at 50℃. Add 15 mL of soybean oil to system a and stir continuously. After stirring, add 20 mL of 1 mol / L sorbitol and stir for 5 min. Then add 2 g of γ-PGA and sonicate at 0.3 kW power for 30 min. After sonication, add 5 mL of glutaraldehyde to obtain system b (divided into three equal parts, each about 13 mL). (3) Take 30 g γ-PGA and add 300 mL of pure water to prepare a solution. Divide the solution into two equal parts, each 150 ml. (4) Coating compound fertilizer using a rotary drum spray coating process: Place 10 kg of compound fertilizer granules into a rotary drum, start rotating, spray one-third of system b, rotate for 10 min to mix evenly, and then air dry. Add half of the γ-PGA solution, continue rotating for 20 min, and then air dry. Spray one-third of system b, rotate for 10 min to mix evenly, and then air dry. Add the remaining γ-PGA solution, continue rotating for 20 min, and then air dry. Spray the remaining system b, rotate for 10 min to mix evenly, and then air dry to obtain γ-PGA coated controlled-release compound fertilizer, denoted as 0.3% Z-PGA-NPK.

[0055] By adjusting the amount of γ-PGA in step (3) to 10g, 20g, 40g, 60g and 80g, γ-PGA coated controlled-release compound fertilizers with different contents were prepared, which were recorded as 0.1% Z-PGA-NPK, 0.2% Z-PGA-NPK, 0.4% Z-PGA-NPK, 0.6% Z-PGA-NPK and 0.8% Z-PGA-NPK, respectively.

[0056] Physicochemical properties and slow-release performance tests of coated controlled-release compound fertilizer: The above six different concentrations of γ-PGA coated controlled-release compound fertilizers were tested for hygroscopicity, fertilizer stability, and nutrient release.

[0057] 1. Moisture absorption test Weigh three 20 g (0.01) fertilizer samples and place them in uncovered petri dishes. Place the fertilizer and petri dishes on top of a desiccator containing distilled water, cover the desiccator, and weigh the fertilizer after 14 days.

[0058] 2. Fertilizer stability Each fertilizer treatment group was set up with 3 replicates, each replicate containing 200 g. The samples were placed in 250 mL Erlenmeyer flasks, and the mouths of the flasks were covered with a breathable and water-retaining Parafilm membrane. The fertilizer samples were then placed in a constant temperature incubator at 25℃. The precipitation phenomenon was observed and samples were taken at 0.25 days, 0.5 days, 1 day, 2 days, 3 days, 7 days, and 14 days. Three replicates of each treatment were taken each time, with 10 g from each replicate. The γ-PGA content was determined by the CTAB method.

[0059] 3. Nutrient release measurement Accurately weigh 5.00 g of controlled-release fertilizer sample, using iso-NPK as a control, and place it in an Erlenmeyer flask. Add 100 ml of distilled water to the Erlenmeyer flask (fertilizer-to-water ratio of 1:20), and let it stand at room temperature for 24 h. Measure the content of dissolved N, P, and K, and calculate the primary dissolution rate. The primary dissolution rate is the percentage of dissolved nutrients relative to the total nutrients in the fertilizer.

[0060] 5.00 g of fertilizer sample was placed under the same fertilizer-to-water ratio and conditions for 7 days. The content of dissolved N, P and K was measured, and the differential dissolution rate was calculated. Differential dissolution rate (%) = [(cumulative nutrient dissolution amount × 100 / nutrient content in sample) - primary dissolution rate] × [1 / (number of days of placement - 1)].

[0061] Methods for determining N, P, and K: N element: Sulfuric acid digestion and distillation method 1) Digestion: Accurately weigh 2.5–5.0 g of the sample into a Kjeldahl flask, add 4 g of catalyst, then add 30 ml of concentrated sulfuric acid, shake well, and let stand overnight. During digestion, begin with a gentle heat, observing carefully. If excessive bubbles appear, stop heating and allow to cool before resuming slow heating, taking care to prevent the sample from overflowing from the Kjeldahl flask. When the contents of the Kjeldahl flask become gelatinous and emit white fumes, gradually increase the heat and continue digestion. Once the solution in the Kjeldahl flask turns green, heat for another 15 minutes. When the contents turn white, digestion is complete.

[0062] 2) Distillation: Dilute the digestion solution with distilled water to a final volume of 250 ml, then take a certain amount of the solution (to make it contain about 10~25 mg of N) and distill it according to the Kjeldahl method for total nitrogen content in the soil.

[0063] 3) Titration: Titrate the ammonia solution absorbed by boric acid with standard hydrochloric acid until the solution color changes abruptly from blue to wine red, which is the endpoint. Perform a blank test simultaneously. Calculate the nitrogen content of the sample based on the amount of standard hydrochloric acid consumed in the titration.

[0064] 4) Calculation: N%=(VVo )×C×0.014×ts×100 / m In the formula: V: the amount of standard hydrochloric acid consumed in the titration of the sample (ml).

[0065] Vo: The amount of standard hydrochloric acid consumed in the blank test (ml).

[0066] C: Concentration of standard hydrochloric acid solution (mol / L).

[0067] 0.014: millimolecular mass of nitrogen atom, g / mmol.

[0068] m: Mass of fertilizer sample (g).

[0069] ts: Dispensing ratio of the test solution.

[0070] K element: Flame photometry method 1) Preparation of test solution of compound fertilizer: Weigh 0.300 g of sample into a 50 ml beaker, add 6 or 7 drops of concentrated hydrochloric acid, then add 20 ml of distilled water, boil at low temperature for 10 min, cool and transfer to a 100 ml volumetric flask with distilled water to make up to the mark, let it stand to clarify or filter with dry filter paper.

[0071] 2) Determination of the test solution: Pipette 5 ml of the clear liquid or filtrate of the above test solution (equivalent to 250~2500 micrograms of K2O) into a 50 ml volumetric flask, add water to make up to the mark, shake well, and then measure directly on a flame photometer and read the reading on the galvanometer.

[0072] 3) Construction of standard curve: Take 0, 5, 10, 15, 25 and 35 ml of 100 mg / L standard solution into 50 ml volumetric flasks, dilute to volume with distilled water and shake well to obtain a standard series with K content of 0, 10, 20, 30, 50 and 70 mg / L. Then measure the K concentration using a flame photometer and read the galvanometer reading. Plot the galvanometer reading on graph paper as the ordinate and the K concentration (mg / L) as the abscissa to construct the standard curve.

[0073] Calculation result: K2O%=C×V×ts×100×1.2046 / (m×10) In the formula: C: mg / L of the test solution obtained from the standard curve.

[0074] V: Measured volume.

[0075] ts: Divide into multiples.

[0076] 1.2046: The coefficient for converting K to K2O.

[0077] m: Mass of the sample (g).

[0078] P element: Potassium persulfate oxidation-molybdenum blue colorimetric method 1) Weigh 0.200 g of the sample that has passed through a 100-mesh sieve into a 100 ml Erlenmeyer flask. After moistening it with a small amount of water, add 10-15 ml of 1:1 nitric acid. Place a small funnel at the mouth of the flask and heat it slowly on a hot plate for 20 minutes to evaporate the solution in the flask to a paste-like consistency (do not evaporate it to dryness). Then add 20 ml of boiling distilled water and heat it to a gentle boil. Finally, filter the solution through dense, phosphorus-free filter paper into a 100 ml volumetric flask. Rinse the filter paper several times with hot distilled water, make up to volume, and shake well before use.

[0079] 2) Pipette 2-10 ml of the above test solution into a 50 ml volumetric flask (P content 0.05-2 mg), add 2 drops of dinitrophenol indicator, neutralize with 6 mol / L NaOH until a light yellow color just appears, add water to about 35 ml, accurately add 10 ml of ammonium vanadate colorimetric reagent, make up to volume, let stand for 30 minutes, and then read the value at 490 nm wavelength using an ELISA reader.

[0080] 3) Preparation of the standard curve: Pipette 100 mg / L P2O5 standard solution at concentrations of 0, 2.5, 5.0, 7.5, 12.5, and 15.0 mg / L into separate 50 ml volumetric flasks. Add water to a final volume of 35 ml, accurately add 10 ml of ammonium vanadate colorimetric reagent, and dilute to volume to obtain a series of P2O5 concentrations of 0, 5.0, 10.0, 15.0, 25.0, and 30.0 mg / L. Read the values ​​using a microplate reader at 490 nm after 15–20 min. Plot the standard curve on graph paper with the absorbance rate on the ordinate and the phosphorus pentoxide concentration (mg / L) on the abscissa.

[0081] Result calculation: P2O5% = A × colorimetric volume × fractionation factor / m × 10 6 ×100 In the formula: A: The concentration of P2O5 in the test solution (mg / L) obtained from the standard curve.

[0082] m: Sample mass (g).

[0083] 10 6 Convert mg to g and colorimetric volume mL to L.

[0084] 100: Converted to percentage content.

[0085] Reagents: ①Ammonium vanadate colorimetric reagent: Weigh 12.5 g of (NH4)6Mo7O 24• Dissolve 4H₂O (ammonium molybdate) in approximately 200 ml of water. Separately, dissolve 0.625 g of NH₄VO₃ (ammonium metavanadate) in 150 ml of boiling water, cool, add 125 ml of concentrated nitric acid, and then cool to room temperature. Then slowly pour the ammonium molybdate solution into the ammonium metavanadate nitric acid solution while stirring, and finally dilute with water to 500 ml.

[0086] ②6 mol / L NaOH solution: Weigh 24 g of NaOH, dissolve it in water, and dilute to 100 ml.

[0087] ③ 2,6- or 2,4-dinitrophenol indicator: 0.25 g of dinitrophenol dissolved in 100 ml of water (saturated).

[0088] ④ P2O5 standard solution (each milliliter is equivalent to 500 micrograms of P2O5, i.e., 500 mg / L P2O5): Weigh 0.9587 g of potassium dihydrogen phosphate that has been dried at 45°C for 3 h, dissolve it in a small amount of water, and then transfer and dilute to a 1000 ml volumetric flask. Pipette 100 ml of the above standard solution into a 500 ml volumetric flask, and dilute with water to the mark to obtain a 100 mg / L P2O5 standard solution, which will be used as the working solution.

[0089] Moisture absorption test results are as follows Figure 10 As shown, six different concentrations of γ-PGA-coated controlled-release compound fertilizers exhibited varying degrees of moisture absorption after 14 days. The control (CK) group, the uncoated compound fertilizer, had a water absorption rate of 12.03%. The moisture absorption of all six γ-PGA-coated controlled-release compound fertilizers was lower than that of the CK group, indicating that the compound fertilizer is water-soluble, and the γ-PGA coating slows down the dissolution rate, achieving a slow-release effect. The best coating quality was achieved with 0.1% Z-PGA-NPK (9.26%), which had a soft texture, weak adhesion, and was not easily detached. The second best was 0.3% Z-PGA-NPK.

[0090] Fertilizer stability results are as follows Figure 11 As shown, under normal temperature conditions, the γ-PGA content of 0.1% Z-PGA-NPK, 0.2% Z-PGA-NPK, 0.3% Z-PGA-NPK and 0.4% Z-PGA-NPK did not change significantly over time in six different concentrations of γ-PGA-coated controlled-release compound fertilizers, while the γ-PGA content of 0.6% Z-PGA-NPK and 0.8% Z-PGA-NPK decreased slowly over time.

[0091] The nutrient dissolution rate of γ-PGA-coated controlled-release compound fertilizer was determined by the 7-day static incubation method, and the results are as follows: Figure 12As shown, the primary dissolution rate reflects the proportion of nutrients rapidly released in the initial stage of fertilizer application, while the differential dissolution rate represents the sustained release capacity during the slow-release stage. Combining these two metrics allows for a comprehensive evaluation of the release characteristics of controlled-release fertilizers. Comparing six γ-PGA-coated controlled-release compound fertilizers, the dissolution behavior of three nutrients showed a common characteristic: the differential dissolution rate was significantly higher than the primary dissolution rate, proving that this γ-PGA-coated compound fertilizer possesses controlled-release characteristics. The low initial nutrient release effectively avoids the risk of crop burn, while the continuous and stable release in the later stages can match the nutrient requirements of crops throughout their entire growth cycle. From a cost-saving perspective, 0.3% Z-PGA-NPK is the optimal choice.

[0092] Practical application effect test: The field trial was conducted from April to October 2025 at the experimental field of Jilin Agricultural University, with maize as the test crop. The lateral distance between the fertilizer furrow and the seedling strip was 8 cm, and the fertilizer application depth was 12 cm. All fertilizers were applied as basal fertilizer in a single application. Except for the tested fertilizer, all other field management practices followed the conventional planting habits of local farmers.

[0093] The field trial consisted of four treatments, each replicated four times, for a total of 16 plots, each plot measuring 35m. 2 Each cell was divided into 4.5 m × 1 m protective rows. The four treatments were: no fertilizer (CK); Zone 2: compound fertilizer (NPK); Zone 3: 0.3% (prepared using commercially available agricultural γ-PGA with an average molecular weight of 110 kDa, according to the same process as in Example 4) γ-PGA coated controlled-release compound fertilizer (0.3% S-PGA-NPK); Zone 4: 0.3% (self-made γ-PGA, prepared according to the process in Example 4) γ-PGA coated controlled-release compound fertilizer (0.3% Z-PGA-NPK).

[0094] Soil sample collection: Sampling was conducted in an "S" pattern at the seedling stage, small trumpet stage, large trumpet stage, tasseling stage, grain filling stage, milk stage, and full maturity stage of maize growth. Five points were randomly selected in the experimental plot, and soil samples from the top 10 cm of the cultivated layer were collected, mixed, and quickly placed in an ultra-low temperature freezer at -80℃ for storage.

[0095] Soil-related index determination: testing soil samples for ammonium nitrogen, nitrate nitrogen, available nitrogen, and cation exchange capacity.

[0096] Determination of soil ammonium nitrogen and nitrate nitrogen: Weigh 10 g of soil sample that has passed through a 40-mesh sieve, extract with 100 mL of 1 mol / L KCl solution, and determine soil ammonium nitrogen (NH4+) using an AA3-HR fully automated continuous flow analyzer. + -N) and nitrate nitrogen (NO3) - -N content.

[0097] Determination of soil available nitrogen: 1) Weigh 2 g of soil sample (accurate to 0.001 g) that has passed through a 40-mesh sieve and 1 g of ferrous sulfate powder. Lay the sample flat in the outer chamber of the diffusion dish and gently rotate the dish to ensure the sample is spread evenly.

[0098] 2) Add 2 ml of 2% boric acid solution to the inner chamber of the diffusion dish, add 2 drops of nitrogen mixed indicator, and then slowly rotate it to one side to make a narrow slit appear in the diffusion dish. Immediately add 10 mL of 1.8 mol / L sodium hydroxide to the outer chamber of the dish with a pipette, and immediately tighten it.

[0099] 3) Gently rotate the diffusion dish horizontally to ensure the alkaline solution is thoroughly mixed with the soil, then place it in a 40℃ container. After 24 hours in a constant temperature incubator, the solution was taken out and titrated with a 0.01 mol / L HCl standard solution (before titration, observe whether there are air bubbles at the bottom of the burette and remove the air bubbles). The solution was titrated until it changed from blue to slightly red. The amount of hydrochloric acid used was recorded as V. The amount of hydrochloric acid used in the blank titration was recorded as V0.

[0100] Result calculation: Hydrolyzable nitrogen (mg / 100g) = N × (V - V0) × 14 / m × 100 Where: N – the molar concentration of standard hydrochloric acid; V – The volume of hydrochloric acid in milliliters used to titrate the sample; V0 – Volume of hydrochloric acid in milliliters for blank test; 14 – The molar mass of one nitrogen atom is mg / mol; m – Mass of the dried soil sample; 100 – the amount of nitrogen in milligrams per 100 grams of sample.

[0101] The results are as follows Figure 13 As shown. Soil cation exchange capacity (CEC) reflects soil nutrient retention capacity. The CEC of each treatment group remained at a high level from the seedling stage to the small tasseling stage, and then showed a slow downward trend. At the small tasseling stage, the CEC of the 0.3% S-PGA-NPK treatment was 31.5 cmol / kg, higher than CK (28.8 cmol / kg) and NPK (27.8 cmol / kg). After the tasseling stage, the CEC of the 0.3% Z-PGA-NPK treatment remained relatively stable, significantly higher than the other treatments. These results indicate that the γ-PGA-coated compound fertilizer synthesized by strain H-27 of this invention can effectively increase soil cation exchange capacity, enhance soil nutrient retention capacity, and help reduce nutrient leaching loss. Soil alkaline-available nitrogen is an important component of soil available nitrogen, such as... Figure 13 As shown in Figure B, the 0.3% Z-PGA-NPK treatment achieved an available nitrogen content of 141.2 mg / kg during the grain-filling stage, significantly higher than CK (68.8 mg / kg), NPK (66.5 mg / kg), and 0.3% S-PGA-NPK (67.4 mg / kg). The available nitrogen content in the NPK treatment decreased rapidly after the tasseling stage and was significantly lower than the other treatments at maturity, indicating that γ-PGA-coated compound fertilizer can effectively delay soil nitrogen release and prolong the nutrient supply cycle, with the γ-PGA synthesized in this invention showing even more significant effects. Figure 13 As shown in C, ammonium nitrogen (NH4) + The ammonium nitrogen content reached its peak during the seedling stage and then rapidly declined as maize grew. The 0.3% Z-PGA-NPK treatment group had an ammonium nitrogen content of 19.22 mg / kg during the grain-filling stage, significantly higher than the NPK treatment group (6.93 mg / kg). Although the ammonium nitrogen content in the 0.3% Z-PGA-NPK treatment group was slightly lower than that in the NPK treatment in the early stages, the decline was more gradual, indicating that it could delay the release of ammonium nitrogen and reduce nitrogen loss in the early stages. Figure 13 As shown in Figure D, the nitrate nitrogen (NO3) in the 0.3% Z-PGA-NPK treatment group - The nitrate nitrogen content increases slowly with the growth period, reaching a peak during the grain-filling stage. The 0.3% Z-PGA-NPK treatment showed a nitrate nitrogen content of 57.59 mg / kg during the grain-filling stage, significantly higher than the NPK treatment group (12.84 mg / kg). This indicates that the use of γ-PGA-coated compound fertilizer can ensure stable transformation and continuous supply of nitrogen in the soil.

[0102] Soil enzyme activity assay: Soil samples were collected during the seedling, tasseling, and macrocephala stages of maize growth. The activities of urease and sucrase were measured using a kit. The assays were performed by the Science Compass service platform.

[0103] (1) Soil urease is a key hydrolytic enzyme involved in soil nitrogen transformation, and its activity level can directly reflect the efficiency of soil nitrogen cycling and nitrogen supply capacity. Using the seedling stage, large trumpet stage and tasseling stage of maize as observation nodes, the effects of four fertilization treatments (CK, NPK, 0.3% S-PGA-NPK and 0.3% Z-PGA-NPK) on soil urease activity were investigated. The results are shown in Table 1.

[0104] Table 1:

[0105] Soil urease activity exhibited stage-specific differentiation throughout maize's growth process, with significant differences in the regulatory effects of different fertilization treatments. During the seedling stage, urease activity in all treatment groups was at a moderate level with small differences between groups. The NPK group and the 0.3% Z-PGA-NPK group showed slightly higher activity than the CK group, while the 0.3% S-PGA-NPK group was close to the CK group. At the large tasseling stage, the urease activity in the CK group reached a peak (approximately 490.56 μg / d / g), while the activities in the other fertilization treatments remained relatively stable. At the tasseling stage, the urease activity in the 0.3% Z-PGA-NPK group reached a peak of 560.44 μg / d / g, the highest value throughout the entire growth period, significantly higher than other treatment groups. Studies by Wang Taohu et al. have shown that in studies using γ-PGA to enhance the effectiveness of urea, the increase in urease activity in the rhizosphere soil increased with the increase in the proportion of γ-PGA added to urea. In wheat and maize planting systems, the addition of 0.5% γ-PGA-enhanced urea maintained the highest level of rhizosphere soil urease activity.

[0106] Soil sucrase is a core hydrolytic enzyme involved in soil carbon cycling, and its activity can reflect the efficiency of soil organic matter transformation and the activity of microbial metabolism. Table 2 shows the soil sucrase activities in different treatment groups.

[0107] Table 2:

[0108] As maize growth progresses, soil sucrase activity generally exhibits a pattern of initial increase followed by stabilization. Significant differences in enzyme activity were observed among the treatment groups during the seedling stage. The 0.3% S-PGA-NPK treatment group reached its highest activity of 33.77 mg / d / g during the seedling stage, significantly higher than the CK group (26.81 mg / d / g), the NPK group (29.63 mg / d / g), and the 0.3% Z-PGA-NPK treatment group (26.71 mg / d / g). The 0.3% S-PGA-NPK treatment group rapidly dissolved and released small-molecule organic carbon in the early stages, strongly stimulating microbial metabolism in a short period, resulting in a significant increase in sucrase activity. The self-made γ-PGA in this invention has a more stable structure and degrades more slowly, thus releasing only a small amount of organic carbon during the seedling stage. Simultaneously, maize root exudates are low during the seedling stage, resulting in limited rhizosphere carbon substrate supply; therefore, the sucrase activity in this treatment was low during the seedling stage. The large trumpet stage is the peak stage for overall enzyme activity. The activity in the CK group rose to 31.56 mg / d / g, and in the NPK group it also rose to 32.24 mg / d / g. The activity in the 0.3% Z-PGA-NPK treatment group increased to 31.24 mg / d / g, a 16.96% increase compared to the seedling stage, while the 0.3% S-PGA-NPK treatment showed a declining trend. The 0.3% Z-PGA-NPK treatment group reached its peak activity of 32.06 mg / d / g at the tasseling stage, significantly higher than other groups. The soil sucrase activity in this treatment group gradually increased with the progress of maize growth, which is related to the slow-release characteristics of the synthesized γ-PGA in this invention and the nutrient metabolism patterns during the maize growth period.

[0109] Corn growth indicators and yield measurement: At the milk stage, 10 maize plants were selected from each treatment plot and harvested whole from the root. Plant height (measured with a tape measure from the base of the maize stalk to the growing point), diameter (measured with calipers approximately 1 mm above the cotyledon node), and leaf chlorophyll content (SPAD value) were measured. Fresh weight of the plants was also measured, and dry weight was measured after drying. At full maturity, 20 ears of maize were randomly selected from each treatment plot, and ear fresh weight (weighed after hulling), kernel fresh weight, and kernel dry weight were measured. Maize yield, nitrogen fertilizer partial productivity, nitrogen fertilizer agronomic efficiency, nitrogen biological efficiency, and nitrogen use efficiency were calculated.

[0110] The effects of different fertilization treatments on field crop growth indicators, including plant height, stem diameter, chlorophyll content (SPAD value), and plant water content. The growth of maize plants is as follows: Figure 14As shown, all indicators in the CK treatment group were at the lowest levels, with a plant height of approximately 2.38 m, stem diameter of 18.59 mm, chlorophyll SPAD value of 35.75, and water content of 58.22%. The indicators in the conventional NPK treatment group were improved compared to the control group, with plant height increasing to 2.65 m, stem diameter to 23.11 mm, chlorophyll SPAD value to 41.38, and water content to 57.52%. In the 0.3% S-PGA-NPK treatment group, plant height, stem diameter, and chlorophyll content reached 2.47 m, 23.70 mm, and 41.45, respectively, with a water content of 55.06%. The stem diameter in the 0.3% Z-PGA-NPK treatment group was 28.33 mm, an increase of 22.59% compared to the NPK group. The water content of the 0.3% Z-PGA-NPK treatment group was 53.45%, which was 7.08% lower than that of the NPK group. The decrease in plant water content was not caused by water shortage stress, but by the dilution effect caused by the increase in dry matter accumulation. This indicates that γ-PGA-encapsulated compound fertilizer can significantly improve the biomass accumulation efficiency of maize, help enhance the mechanical strength of the stem, and improve the plant's resistance to lodging.

[0111] The effects of different fertilizer treatments on maize yield are shown in Table 3.

[0112] Table 3:

[0113] The maize yields showed a clear gradient among the treatment groups. The 0.3% Z-PGA-NPK treatment group had the highest yield (11892.77 kg / ha), followed by the 0.3% S-PGA-NPK treatment group (11020.64 kg / ha) and the NPK treatment group (10217.22 kg / ha). The 0.3% Z-PGA-NPK treatment group showed a 16.4% yield increase compared to the NPK treatment group, and the 0.3% S-PGA-NPK treatment group showed a 7.86% yield increase compared to the NPK treatment group. This result indicates that the application of γ-PGA-encapsulated controlled-release compound fertilizer can significantly increase maize yield, and the γ-PGA synthesized in this invention has a more pronounced effect.

[0114] The effects of different fertilization treatments on nitrogen fertilizer utilization rate in maize are shown in Table 4.

[0115] Table 4:

[0116] Significant differences were observed in nitrogen use efficiency (NPFP) of maize under γ-PGA-encapsulated controlled-release compound fertilizer application. Nitrogen partial productivity (NPFP) was highest in the 0.3% Z-PGA-NPK treatment group, reaching 79.29 kg / kg, a 16.42% increase compared to the conventional NPK treatment (68.11 kg / kg). Similarly, nitrogen agronomic efficiency (NAE) was highest in the 0.3% Z-PGA-NPK treatment group, at 25.27 kg / kg, a 79.22% increase compared to the NPK treatment group (14.10 kg / kg). Nitrogen physiological efficiency (PNUE) showed the opposite trend: the NPK treatment group had the highest NPNUE (113.64 kg / kg), the 0.3% Z-PGA-NPK treatment group had the lowest (50.65 kg / kg), and the 0.3% S-PGA-NPK treatment reached 78.41 kg / kg, with significant differences among the treatments. Nitrogen fertilizer use efficiency (NUPE) showed a significant trend, reaching 34.85% in the 0.3% Z-PGA-NPK treatment group, which is 3.21 times that of the NPK treatment (10.87%).

[0117] In summary, the application of the coated controlled-release compound fertilizer containing γ-PGA synthesized by strain H-27 of this invention can significantly improve the nitrogen fertilizer partial productivity, nitrogen fertilizer agronomic efficiency, and nitrogen fertilizer use efficiency of maize; its nitrogen synergistic effect is the best, indicating that the γ-PGA synthesized by this invention is an effective synergist for improving nitrogen use efficiency in maize fields.

[0118] Example 5 Preparation of γ-PGA controlled-release coated compound fertilizer: The fertilizer used in this invention is high-tower granulated compound fertilizer (produced by Jilin Longyuan Agricultural Service Co., Ltd., with a total nutrient content ≥48.0% and N-P2O5-K2O of 26-10-12).

[0119] γ-PGA: The γ-PGA used in this experiment has an average molecular weight of 114.334 kDa and was prepared by solid-state fermentation of corn straw by strain H-27 in Example 3.

[0120] The preparation steps are as follows: (1) Take ethanol and preheat it to 45°C, then slowly add glycerol and stir continuously for 15 min. After mixing, a mixed system a is obtained, with a volume ratio of ethanol to glycerol of 4:1. (2) While maintaining the temperature, add soybean oil to system a and stir continuously. After stirring evenly, add 1 mol / L sorbitol and stir for 4 min. Then add γ-PGA and sonicate at 0.2 kW power for 25 min. After sonication, add glutaraldehyde to obtain system b. The ratio of the amount of system a, soybean oil, sorbitol, γ-PGA and glutaraldehyde is 13 mL: 16 mL: 22 mL: 2.5 g: 6 mL. (3) Take γ-PGA and add pure water to prepare γ-PGA solution. The ratio of γ-PGA to pure water is 35 g: 300 mL. (4) Coating compound fertilizer using a rotary drum spray coating process: Place 10 kg of compound fertilizer granules into a rotary drum, start rotating, spray one-third of system b, rotate for 10 min to mix evenly, and then air dry. Add half of the γ-PGA solution, continue rotating for 15 min, and then air dry. Spray one-third of system b, rotate for 8 min to mix evenly, and then air dry. Add the remaining γ-PGA solution, continue rotating for 15 min, and then air dry. Spray the remaining system b, rotate for 8 min to mix evenly, and then air dry to obtain γ-PGA coated controlled-release compound fertilizer.

[0121] Example 6 Preparation of γ-PGA controlled-release coated compound fertilizer: The fertilizer used in this invention is high-tower granulated compound fertilizer (produced by Jilin Longyuan Agricultural Service Co., Ltd., with a total nutrient content ≥48.0% and N-P2O5-K2O of 26-10-12).

[0122] γ-PGA: The γ-PGA used in this experiment has an average molecular weight of 114.334 kDa and was prepared by solid-state fermentation of corn straw by strain H-27 in Example 3.

[0123] The preparation steps are as follows: (1) Take ethanol and preheat it to 55°C, then slowly add glycerol and stir continuously for 15 min. After mixing, a mixed system a is obtained, with a volume ratio of ethanol to glycerol of 6:1. (2) While maintaining the temperature, add soybean oil to system a and stir continuously. After stirring evenly, add 1 mol / L sorbitol and stir for 6 min. Then add γ-PGA and sonicate at 0.4 kW power for 35 min. After sonication, add glutaraldehyde to obtain system b. The ratio of the amount of system a, soybean oil, sorbitol, γ-PGA and glutaraldehyde is 12 mL: 14 mL: 18 mL: 1.5 g: 4 mL. (3) Take γ-PGA and add pure water to prepare γ-PGA solution. The ratio of γ-PGA to pure water is 25 g: 300 mL. (4) Coating compound fertilizer using a rotary drum spray coating process: Place 10 kg of compound fertilizer granules into a rotary drum, start rotating, spray one-third of system b, rotate for 10 min to mix evenly, and then air dry. Add half of the γ-PGA solution, continue rotating for 25 min, and then air dry. Spray one-third of system b, rotate for 12 min to mix evenly, and then air dry. Add the remaining γ-PGA solution, continue rotating for 25 min, and then air dry. Spray the remaining system b, rotate for 12 min to mix evenly, and then air dry to obtain γ-PGA coated controlled-release compound fertilizer.

Claims

1. A strain H-27 for producing γ-PGA through solid-state fermentation of corn straw, characterized by: The strain is classified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus amyloliquefaciens The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 38389 and deposit date of April 27, 2026.

2. The application of strain H-27 as described in claim 1 in the one-step solid-state fermentation of corn straw to synthesize γ-PGA.

3. The application of strain H-27 as described in claim 1 in the preparation of coated controlled-release compound fertilizer, characterized in that: γ-PGA synthesized by solid-state fermentation using strain H-27 was used to prepare coated controlled-release compound fertilizer.

4. A method for preparing a coated controlled-release compound fertilizer based on strain-H27 as described in claim 1, characterized in that, Includes the following steps: S1. Synthesis of γ-PGA from corn straw via one-step solid-state fermentation using Bacillus amyloliquefaciens H-27; S2. Prepare coated controlled-release compound fertilizer using γ-PGA as the coating material for compound fertilizer.

5. The method for preparing a coated controlled-release compound fertilizer as described in claim 4, characterized in that: The solid-state fermentation is carried out using corn stalks as the fermentation substrate, with 48-52% peptone and 18-22% L-glutamate added based on the mass of the fermentation substrate. Bacillus amyloliquefaciens H-27 is inoculated at an inoculation amount of 7-9%, and the initial pH is adjusted to 7-7.

5. Solid-state fermentation is carried out at 37-42℃.

6. The method for preparing a coated controlled-release compound fertilizer as described in claim 4 or 5, characterized in that: The specific steps for preparing the coated controlled-release compound fertilizer in step S2 are as follows: (1) Take ethanol and preheat it to 45~55℃, then slowly add glycerol and stir continuously to obtain mixed system a; (2) Add soybean oil to system a, stir continuously, add sorbitol and γ-PGA after stirring, sonicate, add glutaraldehyde after sonication to obtain system b; (3) Take γ-PGA and add pure water to prepare γ-PGA solution; (4) The compound fertilizer is coated by rotary drum spraying process: The compound fertilizer granules are placed in the turntable and after the rotation starts, 1 / 3 of the system b is sprayed, mixed evenly and dried. 1 / 2 of the γ-PGA solution is added, mixed evenly and dried. 1 / 3 of the system b is sprayed, mixed evenly and dried. The remaining γ-PGA solution is added, mixed evenly and dried. Finally, the remaining system b is sprayed, mixed evenly and dried to obtain γ-PGA coated controlled-release compound fertilizer.

7. The method for preparing a coated controlled-release compound fertilizer as described in claim 6, characterized in that: The volume ratio of ethanol to glycerol in (1) is 4~6:

1.

8. A method for preparing a coated controlled-release compound fertilizer as described in claim 7, characterized in that: In step (2), the ratio of system a, soybean oil, sorbitol, γ-PGA and glutaraldehyde is 10~13mL: 12~16mL: 18~22mL: 1.5~2.5g: 4~6mL.

9. A method for preparing a coated controlled-release compound fertilizer as described in claim 8, characterized in that: In step (3), the ratio of γ-PGA to pure water in the γ-PGA solution is 25~35 g:300 mL.

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