Fungicide, coal gangue fungus composite bio-fertilizer and preparation method thereof
By combining specific strain ratios with modified coal gangue carriers, a stable strain interaction system was constructed, solving the problems of strain competition and carrier resource in existing compound microbial fertilizers, and achieving efficient and low-cost fertilizer retention and yield increase effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING UNIV
- Filing Date
- 2026-05-09
- Publication Date
- 2026-06-05
Abstract
Description
Technical Field
[0001] This invention belongs to the field of compound microbial agent technology, specifically relating to a fertilizer-preserving and yield-increasing microbial agent, a compound bio-fertilizer with bacteria-carrying coal gangue, and its preparation method. Background Technology
[0002] In current agricultural production, compound microbial fertilizers have been widely used to improve soil fertility, enhance the rhizosphere microenvironment of crops, and reduce the application of chemical fertilizers while increasing efficiency. However, existing compound microbial fertilizers generally adopt a loose physical mixing mode of different functional strains, without fully considering the ecological niche compatibility, metabolic complementarity, and signal communication mechanisms among the strains. This leads to nutrient competition among the strains after they are applied to the soil, resulting in low colonization rates and poor functional expression stability, making it difficult to fully exert the expected growth-promoting and yield-increasing effects. At the same time, existing microbial fertilizers mostly use peat, vermiculite, etc. as carriers, which have the disadvantages of non-renewable resources and high costs, limiting their large-scale promotion and application.
[0003] Existing compound microbial fertilizers have significant defects in terms of strain compounding logic, carrier preparation, and bacterial loading process. They have not formed a stable strain interaction system, nor can they achieve a stable combination of bacterial agents and carriers. This results in weak overall fertilizer retention capacity, short effective viable bacteria survival time, and poor adaptability in adverse soils such as saline-alkali land, making it difficult to meet the application needs of modern agricultural production for efficient, low-cost, and environmentally friendly fertilizers. Summary of the Invention
[0004] This specification provides one or more embodiments of a fertilizer-enhancing and yield-increasing microbial agent, wherein the microbial agent is: Bacillus belye. (Bacillus velezensis) Pseudomonas schrenckii (Pseudomonas stutzeri) and Aspergillus niger (Aspergillus niger) The cell number ratio of Bacillus belyi, Pseudomonas schrenckii, and Aspergillus niger is 1.5~2.2:1~1.6:0.5~1.2.
[0005] This specification also provides one or more embodiments of a method for preparing a coal gangue-borne microbial compound bio-fertilizer, comprising the following steps: S1. The raw coal gangue is crushed and then calcined at a constant temperature. After cooling, a modified porous coal gangue carrier is obtained. S2. Configure the corresponding strains according to the aforementioned cell number ratio, inoculate Aspergillus niger into the fermenter for culture, and after the primary hyphal network is formed, inoculate Bacillus belye and Pseudomonas schrenckii for sequential batch continuous co-culture to obtain a synergistic fermentation symbiotic culture. S3. The modified porous coal gangue carrier is placed under negative pressure, the synergistic fermentation symbiotic bacterial liquid is sprayed in, and after restoring normal pressure, it is dried to obtain the coal gangue-carried bacterial compound bio-fertilizer.
[0006] In some embodiments, in step S1, the particle size of the crushed raw coal gangue is 1~3 mm; the temperature of the constant temperature calcination is 700~800℃, and the calcination time is 15~25 min.
[0007] In some embodiments, in step S1, the total porosity of the modified porous coal gangue carrier is 52.0% to 56.5%, and it has a bimodal pore size distribution, wherein the volume fraction of pores with a diameter greater than 0.03 mm accounts for 20% to 25%, and the volume fraction of pores with a diameter between 0.001 mm and 0.03 mm accounts for 26% to 30%.
[0008] In some embodiments, in step S1, the isothermal calcination is carried out in a fluidized bed reactor, with the minimum fluidizing gas velocity controlled at 0.08 m / s, and the temperature is increased to 700-800°C at a heating rate of 4-5°C / min.
[0009] In some embodiments, in step S2, the total inoculum amount is 2%~5% (v / v); the initial culture time of Aspergillus niger is 24~36 h, and the sequential batch co-culture time after bacterial inoculation is 24~40 h.
[0010] In some embodiments, in step S2, the conditions for the sequential batch co-culture are: temperature controlled at 28~35℃, stirring speed at 160~200 rpm, and dissolved oxygen maintained at 30%~40%.
[0011] In some embodiments, in step S3, the relative pressure of the negative pressure is controlled between -0.06 MPa and -0.08 MPa, and the pressure holding time is 15 to 20 minutes; after the co-fermentation symbiotic bacterial liquid is sprayed in and the pressure is restored to normal, it is allowed to stand for 10 to 15 minutes for infiltration.
[0012] In some embodiments, in step S3, the drying is performed at 35°C using low-temperature fluidized bed drying until the moisture content of the coal gangue-carrying microbial compound fertilizer is less than 15%.
[0013] One or more embodiments of this specification also provide a coal gangue-borne microbial compound bio-fertilizer, which is prepared by the preparation method of the coal gangue-borne microbial compound bio-fertilizer described in any of the above embodiments.
[0014] Beneficial effects: 1. By combining different cell numbers, Bacillus belye, Pseudomonas schrenckii, and Aspergillus niger achieve mutualistic symbiosis and signal adaptation, ensuring the stable functioning of the synergistic effect of the microbial community.
[0015] 2. Through the metabolic complementarity between the two bacteria, Bacillus belye synthesizes more extracellular polymers and amyloid proteins, providing the bacterial community with a three-dimensional physical shelter structure to resist salt and osmotic pressure stress.
[0016] 3. Through the differentiation of microbial niches and the complementarity of asymmetric metabolism, the stepwise synthesis of siderophores and the early storage of nitrogen-fixing gene clusters were naturally induced, which significantly enhanced the ability of the complex microbial community to chelate ferric iron and its overall nitrogen-fixing potential.
[0017] 4. Through the synergy between Aspergillus niger and bacteria, the mycelium provides colonization sites and secretes organic acids to decompose insoluble phosphorus, forming a stable carbon, nitrogen, and phosphorus metabolic cycle within the microbial community.
[0018] 5. By constructing a bimodal pore size distribution, the mechanical strength of the carrier and the load space are taken into account. The large pores are used to promote material transport, while the small pores are used to adsorb and fix the bacterial solution and provide microenvironment protection.
[0019] 6. By using sequential batch continuous co-culture to obtain synergistic fermentation symbiotic bacterial broth, the synergistic metabolic pathways of the bacterial community are fully activated, so that the bacterial broth contains both highly active bacterial cells and key metabolites.
[0020] 7. By combining modified coal gangue carrier with composite microbial community, the synergistic effect of adsorption and fertilizer retention and biological yield enhancement is achieved, which can continuously provide nutrition for crops and improve the soil environment. Detailed Implementation
[0021] Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial sources. The strains used in the embodiments of this invention are examples of that strain, utilizing its conventional physiological and biochemical characteristics, and do not constitute a limitation on the scope of protection.
[0022] This specification provides one or more embodiments of a fertilizer-enhancing and yield-increasing microbial agent, wherein the microbial agent is: Bacillus belye. (Bacillus velezensis) Pseudomonas schrenckii (Pseudomonas stutzeri) and Aspergillus niger (Aspergillus niger) The cell number ratio of Bacillus belyi, Pseudomonas schrenckii, and Aspergillus niger is 1.5~2.2:1~1.6:0.5~1.2.
[0023] In this application, *Bacillus belyssae*, *Pseudomonas schrenckii*, and *Aspergillus niger* are all known strains that were commercially available to the public prior to the application date. These strains are not new biological materials newly screened, isolated, mutated, or created by the applicant and not available to the public. This application does not seek protection for these strains themselves as new biological materials, nor does it use the strain corresponding to a specific accession number as a limitation of the technical solution of this application.
[0024] For those skilled in the art, purchasing the same strain through publicly available commercial channels, or purchasing the same functional strain with corresponding functional characteristics, allows them to implement this application and solve the technical problems to be addressed by following the cultivation, compounding, loading, and preparation methods described in this application.
[0025] For example, those skilled in the art can purchase Bacillus belelibrissin, Pseudomonas schrenckii, and Aspergillus niger from the China General Microbiological Culture Collection Center (CGMCC), the China Center for Type Culture Collection (CCTCC), or other commercial supply channels that can provide the corresponding strains to the public (e.g., Weifang Ruichen Biotechnology Co., Ltd., Nuoan Gene Technology (Wuhan) Co., Ltd.).
[0026] When coexisting in the microenvironment, the three strains constructed a stable symbiotic community through niche differentiation and staggered resource utilization. *Bacillus belye*, as a potent rhizosphere colonizer, was naturally induced to produce a stress response in response to survival competition and mild microenvironmental stress during the initial stages of fermentation co-culture, converting carbon sources into large amounts of extracellular polymeric substances (EPS) and amyloid protein fibers. This dense EPS matrix not only provided the entire microbial community with a three-dimensional physical shelter against subsequent saline-alkali and osmotic stresses, but also, thanks to its macromolecular network structure and abundant hydration groups, significantly enhanced the microenvironment's ability to retain and fix water and soluble nutrients.
[0027] *Pseudomonas schlegelii* and *Bacillus belyssae* exhibit asymmetric complementarity in iron nutrition and nitrogen metabolism. *Pseudomonas schlegelii* possesses excellent nitrogen-fixing potential, completing the early induction and functional reserve of nitrogen-fixation-related gene clusters in the co-culture system. After fertilizer is applied to the soil microenvironment, creating localized micro-oxygen / hypoxic zones, its nitrogenase activity is naturally activated, slowly releasing ammonia nitrogen or short peptides into the system, alleviating the nitrogen source limitation of *Bacillus belyssae* during proliferation and maintenance of EPS structure. Simultaneously, both bacteria secrete siderophores with different affinities, broadening the overall iron chelation spectrum under complex soil pH and ion environments, enabling the stepwise activation and efficient utilization of insoluble ferric iron in the environment.
[0028] In this interactive network, *Aspergillus niger* plays the role of both a spatial pioneer and a phosphorus activator. Its well-developed hyphal network provides microscopic attachment sites for bacteria to overcome the limitations of carrier pores and extend into a wider substrate. Furthermore, *Aspergillus niger* utilizes residual trace amounts of humic acid components in the modified coal gangue carrier and small-molecule metabolic byproducts released during bacterial death and lysis as carbon sources, continuously secreting organic acids such as oxalic acid and citric acid. This effectively lowers the local pH of the microenvironment and dissolves insoluble phosphates fixed by calcium, iron, and aluminum in the soil. The released free phosphorus not only promotes the growth of *Aspergillus niger* itself but also provides essential energy synthesis raw materials for the energy-intensive biological nitrogen fixation process of *Pseudomonas schlegelii* and the rapid proliferation of *Bacillus belyssae*.
[0029] Combining the three strains in a cell ratio of (1.5~2.2):(1~1.6):(0.5~1.2) can ensure that the above-mentioned mutualistic symbiosis, signal transduction and cross-border synergy processes are in a more suitable state, reduce the imbalance of metabolic pathway matching caused by the excessively high or low proportion of a certain type of strain, and ensure the stable functioning of the synergistic function of the microbial community.
[0030] This specification also provides one or more embodiments of a method for preparing a coal gangue-borne microbial compound bio-fertilizer, comprising the following steps: S1, crushing raw coal gangue and calcining it at a constant temperature, then cooling it to obtain a modified porous coal gangue carrier; S2, configuring corresponding bacterial strains according to the aforementioned cell number ratio, first inoculating Aspergillus niger into a fermenter for cultivation, and after forming a primary mycelial network, then inoculating Bacillus belye and Pseudomonas schrenckii for sequential batch continuous co-culture to obtain a synergistic fermentation symbiotic bacterial solution; S3, placing the modified porous coal gangue carrier under negative pressure, spraying in the synergistic fermentation symbiotic bacterial solution, restoring normal pressure, and drying to obtain the coal gangue-borne microbial compound bio-fertilizer.
[0031] Raw coal gangue has a dense structure, low porosity, and insufficient surface activity, making it difficult to accommodate sufficient bacterial cells when used directly as a bacterial carrier, and the bacterial cells exhibit weak adhesion. Pulverizing the raw coal gangue and then subjecting it to isothermal calcination allows the organic matter and volatile components inside the gangue to decompose and escape upon heating, gradually forming a rich porous structure. Simultaneously, controlling the calcination conditions reduces the possibility of pore sintering and closure due to excessive heating. The resulting modified porous coal gangue carrier, after cooling, possesses suitable pore space, serving as a site for bacterial attachment and colonization, providing a structural basis for the subsequent bacterial loading process.
[0032] A sequential batch co-culture strategy was adopted, inoculating Aspergillus niger from the fertilizer-enhancing and yield-increasing bacterial agent first. After its spores fully germinated and formed a primary microscopic hyphal network, Bacillus belye and Pseudomonas schrenckii were then inoculated according to the corresponding cell number ratio. Compared with single-strain culture followed by mixing or simultaneous inoculation of all three, this sequential batch timeline staggered design effectively avoids the nutrient competition and exclusion at the bottom layer caused by the early exponential proliferation of bacteria, allowing the subsequently inoculated bacteria to directly attach to the already formed hyphal framework for spatial colonization.
[0033] The pores of modified porous coal gangue are originally filled with air. If conventional spraying or soaking methods are used to load the bacterial solution, the air inside the pores is difficult to expel, which hinders the bacterial solution from penetrating deep into the pores. As a result, the bacteria mostly adhere to the outer surface of the carrier and are easily detached during subsequent treatment and application, leading to insufficient survival stability. Placing the modified porous coal gangue carrier under a negative pressure environment can expel most of the air inside the pores. After spraying in the symbiotic bacterial solution for co-fermentation, the pressure is then restored to normal. The pressure difference created inside and outside the pores at this point can drive the bacterial solution to penetrate into the pores. Subsequently, drying treatment is carried out, which allows the bacteria to be more stably fixed in the porous structure. This not only reduces the stress of adverse external environments on the bacteria by relying on the protective effect of the pores, but also allows them to gradually release and exert their effects after application, ultimately yielding a coal gangue-loaded bacterial compound bio-fertilizer.
[0034] In some embodiments, in step S1, the particle size of the crushed raw coal gangue is 1~3 mm; the temperature of the constant temperature calcination is 700~800℃, and the calcination time is 15~25 min.
[0035] In this embodiment, the raw coal gangue is crushed and sieved to a particle size of 1-3 mm. Particles within this size range can achieve relatively uniform heating during subsequent calcination, effectively overcoming the problems of excessive loss of small particles with the processing airflow and insufficient decomposition of internal organic matter due to excessively large particles. Subsequently, it is calcined at a constant temperature of 700-800℃ for 15 minutes. This calcination condition allows the organic matter and volatile components inside the coal gangue to decompose and escape more fully, gradually forming a rich pore structure. At the same time, it can reduce the probability of sintering and closure of pores due to excessive heating or excessive calcination time. The resulting modified porous coal gangue carrier has suitable pore space, which can provide a stable structural basis for subsequent loading of bacterial solution and fixation of active bacteria.
[0036] In some embodiments, in step S1, the total porosity of the modified porous coal gangue carrier is 52.0% to 56.5%, and it has a bimodal pore size distribution, wherein the volume fraction of pores with a diameter greater than 0.03 mm accounts for 20% to 25%, and the volume fraction of pores with a diameter between 0.001 mm and 0.03 mm accounts for 26% to 30%.
[0037] In this embodiment, the total porosity of the modified porous coal gangue carrier is controlled within the above-mentioned range, which can provide sufficient space for subsequent bacterial liquid loading, and avoid the mechanical strength of the carrier particles from decreasing due to excessive porosity, thereby reducing the excessive fragmentation of the carrier during subsequent processing, transportation and application, and maintaining the relative stability of the carrier structure.
[0038] The pores, which account for 20% to 25% of the volume and have a diameter greater than 0.03 mm, provide ample growth space for the initial attachment of microbial cells and the subsequent reproduction of the microbial community. At the same time, these pores have good connectivity and can serve as channels for the transfer of external nutrients and water into the carrier and the release of metabolic products from the microbial community, providing the basic conditions for the microbial community inside the carrier to maintain normal metabolic activity.
[0039] Pores with a volume fraction of 26% to 30% and a diameter between 0.001 mm and 0.03 mm can adsorb and retain the bacterial solution through capillary action, reducing the loss of bacteria during subsequent drying and application. At the same time, these relatively small pores can provide a relatively isolated microenvironment for the bacteria, reducing the stress of adverse external environmental factors on the loaded bacterial community and improving the survival rate and duration of action of the bacteria.
[0040] It should be noted that the total porosity and volume fraction of different pore sizes of the modified porous coal gangue carrier described in this invention were measured using a mercury porosimeter under standard testing conditions. The specific measurement range of the bimodal pore size distribution covers pores from micrometers to millimeters to ensure the accuracy and repeatability of the physical structural parameters of the carrier.
[0041] In some embodiments, in step S1, the isothermal calcination is carried out in a fluidized bed reactor, with the minimum fluidizing gas velocity controlled at 0.08 m / s, and the temperature is increased to 700-800°C at a heating rate of 4-5°C / min.
[0042] In this embodiment, the isothermal calcination in step S1 is carried out in a fluidized bed reactor, with the minimum fluidizing gas velocity controlled at 0.08 m / s. This ensures that the pulverized coal gangue particles are in a uniform suspended fluidized state, reducing the problem of uneven heating caused by particle accumulation. Then, the temperature is increased to 700-800°C at a heating rate of 4-5°C / min. This heating rate promotes the full decomposition of the decomposable components inside the coal gangue to drive pore development. At the same time, it avoids the problems of excessive sintering and closure of micropores caused by excessive heating or insufficient pore development caused by excessive heating. This ensures that the final modified porous coal gangue carrier has a pore structure and stable particle properties that are suitable for subsequent bacterial liquid loading.
[0043] In some embodiments, in step S2, the total inoculum amount is 2%~5% (v / v); the initial culture time of Aspergillus niger is 24~36 h, and the sequential batch co-culture time after bacterial inoculation is 24~40 h.
[0044] In this embodiment, the inoculum size range is suitable for the initial growth requirements of the three strains. The initial culture of 24-36 hours ensures that Aspergillus niger completes spore germination and initially constructs a spatially extended hyphal network; the subsequent sequential batch co-culture of 24-40 hours ensures that the bacteria complete sufficient proliferation at the hyphal colonization sites and reach the quorum sensing trigger threshold, promoting the establishment of a substrate cross-feeding network, while avoiding the problems of extreme nutrient depletion and accumulation of toxic metabolic byproducts caused by excessively long co-culture time.
[0045] The synergistic control of the two can maintain the ratio of the three strains in the final synergistic fermentation broth within a preset range, while the overall activity of the microbial community and the accumulation of related metabolites are at an appropriate level, which can meet the process requirements of subsequent loading onto modified porous coal gangue carriers.
[0046] In some embodiments, in step S2, the conditions for the sequential batch co-culture are: temperature controlled at 28~35℃, stirring speed at 160~200 rpm, and dissolved oxygen maintained at 30%~40%.
[0047] In this embodiment, the temperature range of 28~35℃ is suitable for the growth and functional metabolic needs of the three strains, ensuring that the enzymes related to growth and metabolism within the strains maintain appropriate activity. This reduces the probability of enzyme inactivation and growth inhibition due to excessively high temperatures, or of metabolic rate slowing down and insufficient proliferation and functional substance synthesis efficiency due to excessively low temperatures. This temperature range also allows the growth rates of the three strains to be maintained at a relatively coordinated level, reducing the possibility of excessive proliferation or growth retardation of a single strain, and providing a suitable temperature environment for establishing metabolic complementarity among the strains.
[0048] A stirring speed of 160-200 rpm ensures a more uniform distribution of nutrient substrate, microorganisms, and gases within the fermentation system, reducing localized nutrient deficiencies or microbial sedimentation and ensuring that all strains receive sufficient growth substrate. The shear force generated within this speed range is relatively gentle, reducing the probability of excessive shearing and breakage of Aspergillus niger hyphae and disruption of the extracellular polymer structures synthesized by Bacillus belyssum. Simultaneously, it promotes interstrain contact and the uniform diffusion of metabolites and quorum sensing molecules, providing a homogeneous environmental basis for the construction of a synergistic microbial network.
[0049] A dissolved oxygen level of 30%–40% can fully meet the vigorous aerobic respiration requirements of the three aerobic strains during the co-culture stage. This provides sufficient ATP energy for energy-intensive processes such as rapid strain proliferation, large-scale synthesis of extracellular polymers, and early transcriptional expression of nitrogen-fixing gene clusters, effectively reducing the probability of proliferation inhibition due to excessively low dissolved oxygen. Simultaneously, this dissolved oxygen range avoids excessive oxygen free radical damage to the bacterial cells and allows *Pseudomonas stearothermiae* to complete the necessary biomass accumulation during fermentation, enabling it to rapidly initiate and efficiently perform nitrogen fixation after being introduced into a slightly hypoxic soil environment.
[0050] The synergistic regulation of the above three parameters can be adapted to other control conditions already described in the co-culture process, ensuring that the microbial community can successfully proliferate during the co-culture process, reach the quorum sensing trigger threshold, promote the stable establishment of synergistic mechanisms such as substrate cross-feeding and quorum sensing bidirectional communication, so that the ratio of the three strains in the final synergistic fermentation symbiotic bacterial solution can be maintained within the preset range, and the overall activity of the microbial community and the accumulation of related functional metabolites are at a relatively suitable level, which is compatible with the process requirements of subsequent loading onto modified porous coal gangue carriers.
[0051] In step S2, the fermentation medium used in the sequential batch co-culture is a complex liquid medium, which, by mass parts, comprises: 15-20 parts sucrose, 5-8 parts peptone, 2-3 parts beef extract, 1-2 parts sodium nitrate, 3-5 parts sodium chloride, 1-1.5 parts dipotassium hydrogen phosphate, 0.5 parts magnesium sulfate heptahydrate, 0.5 parts potassium chloride, 0.01-0.05 parts ferrous sulfate tetrahydrate, and 1000 parts distilled water, with the initial pH adjusted to 6.8-7.0.
[0052] In some embodiments, in step S3, the relative pressure of the negative pressure is controlled between -0.06 MPa and -0.08 MPa, and the pressure holding time is 15 to 20 minutes; after the co-fermentation symbiotic bacterial liquid is sprayed in and the pressure is restored to normal, it is allowed to stand for 10 to 15 minutes for infiltration.
[0053] By controlling the relative pressure of the negative pressure to -0.06MPa to -0.08MPa and maintaining the pressure for 15 to 20 minutes, the air trapped in the pores of the modified porous coal gangue can be discharged relatively fully, making room for the subsequent bacterial solution to enter the pores. This level of negative pressure will not damage the original pore structure of the coal gangue and can maintain the preset bimodal pore size distribution characteristics of the carrier. The appropriate holding time can also ensure that the air in pores of different depths and sizes is discharged relatively fully, reducing the probability that residual air in the pores will hinder the penetration of the bacterial solution.
[0054] After injecting the symbiotic fermentation solution under negative pressure conditions and then restoring normal pressure, the capillary negative pressure difference formed inside and outside the pores can drive the bacterial solution to penetrate into the pores. In particular, it can force the bacterial solution with a certain viscosity into the tiny pores of 0.001 mm to 0.03 mm, reducing the situation where the bacterial solution only adheres to the outer surface of the coal gangue particles and improving the uniformity of the loading of the strain and related metabolites inside the carrier.
[0055] After restoring to normal pressure, allowing the solution to stand and infiltrate for 10-15 minutes allows the bacterial solution that has entered the pores to be fully dispersed, enabling the bacterial cells and extracellular metabolites in the solution to fully contact the inner wall of the pores. This reduces the possibility of the bacterial solution flowing back out of the pores after the pressure difference disappears, and at the same time provides sufficient contact time for the subsequent binding process of bacterial cells and metabolites to the pore wall, ensuring the effective loading of the bacterial solution in the carrier pores.
[0056] In some embodiments, in step S3, the drying is performed at 35°C using low-temperature fluidized bed drying until the moisture content of the coal gangue-carrying microbial compound fertilizer is less than 15%.
[0057] In some embodiments, a low-temperature fluidized bed drying method at 35°C is used to treat the coal gangue carrier loaded with bacterial solution. The temperature of 35°C is relatively mild, which can reduce the damage to the activity of the three functional strains caused by excessively high temperatures, and will not destroy the original structure of the extracellular polymers and active metabolites remaining in the symbiotic fermentation solution. The fluidized bed drying process can keep the coal gangue particles in a uniform suspension state, and the moisture on the surface of each particle and inside the pores can be lost synchronously and uniformly. It is not easy for local insufficient drying or excessive heating to occur. When the moisture content of the material is less than 15%, it can reduce the probability of the growth of miscellaneous bacteria, improve the storage stability of the finished product, and also promote the full combination of the polar groups of the extracellular polymers in the fermentation broth with the silica-alumina hydroxyl groups on the inner wall of the modified porous coal gangue pores. This can improve the binding strength of functional strains and active metabolites in the pores of the carrier and reduce the possibility of cell detachment and loss during subsequent storage, transportation and application.
[0058] One or more embodiments of this specification also provide a coal gangue-borne microbial compound bio-fertilizer, which is prepared by the preparation method of any of the preceding coal gangue-borne microbial compound bio-fertilizers.
[0059] The coal gangue-borne microbial compound bio-fertilizer obtained in this embodiment uses calcined and modified porous coal gangue as a carrier to load a complex microbial community of Bacillus belye, Pseudomonas schrenckii, and Aspergillus niger obtained through co-culture, along with related metabolites. The adapted porous structure of the modified coal gangue provides colonization space for the strains and can also adsorb free ammonium ions through its own cation exchange capacity. The three types of strains can achieve synergistic function through metabolic mutual nutrition. The branched-chain amino acids synthesized by Pseudomonas schrenckii can supplement the metabolic needs of Bacillus belye and support the synthesis of extracellular polymers by Bacillus belye to provide stress protection for the microbial community. The quorum sensing molecules of the two can mutually regulate each other, improving nitrogen fixation efficiency and siderophore synthesis level. Aspergillus niger can assist the spread of the strains through hyphae, and the secreted organic acids can release insoluble phosphorus in the environment, opening up the carbon, nitrogen, and phosphorus cycle within the microbial community. After being applied to the soil, it can reduce nitrogen loss and continuously provide crops with usable nutrients and growth-promoting substances, achieving the effect of fertilizer retention and yield increase.
[0060] The method of the present invention will now be described in detail with reference to embodiments, comparative examples and experimental data.
[0061] The *Bacillus belyssioides*, *Pseudomonas schrenckii*, and *Aspergillus niger* used in the following examples and comparative examples are all commonly known strains that were commercially available to the public before the application date. The use of specific strains in the examples is solely for illustrating the implementation of the technical solution of this application and does not imply that this application must rely on strains from specific sources, specific batches, or specific accession numbers to achieve its objectives. As long as the strains used are of the same species and can be compounded and co-cultured according to the cell number ratio described in this application, the technical effects of this application can be achieved.
[0062] Example 1 This embodiment is an example of the preparation of a compound bio-fertilizer with bacteria carried on coal gangue. The fertilizer-preserving and yield-increasing bacterial agent used is composed of Bacillus belysae, Pseudomonas schrenckii, and Aspergillus niger, with the cell number ratio of the three strains being 1.75:1.25:0.75.
[0063] S1. First, the raw coal gangue is crushed to a particle size of 2mm. Then, the crushed raw coal gangue is fed into a fluidized bed reactor for isothermal calcination. The minimum fluidizing gas velocity of the fluidized bed is controlled at 0.08m / s. The temperature is increased to 700℃ at a heating rate of 4.5℃ / min and calcined at the same temperature for 15min. After natural cooling, a modified porous coal gangue carrier is obtained. The total porosity of the obtained modified porous coal gangue carrier is 54.25%, and it has a bimodal pore size distribution. The volume fraction of pores with a diameter greater than 0.03mm accounts for 22.5%, and the volume fraction of pores with a diameter between 0.001mm and 0.03mm accounts for 28%.
[0064] S2. With a total inoculum of 3.5% (v / v), batch inoculation was carried out according to the aforementioned cell number ratio: First, the proportionally allocated *Aspergillus niger* was inoculated into the fermenter and cultured for 24 hours. Then, *Bacillus belye* and *Pseudomonas schrenckii* were inoculated in corresponding proportions for sequential batch co-culture for 30 hours. The fermentation temperature was controlled at 30℃, the stirring speed at 170 rpm, and the dissolved oxygen level maintained at 35%. After the culture was completed, the synergistic fermentation symbiotic culture was obtained.
[0065] S3. The obtained modified porous coal gangue carrier is placed under negative pressure, and the relative pressure of the negative pressure is controlled at -0.07MPa. After maintaining the pressure for 17.5min, the above-mentioned synergistic fermentation symbiotic bacterial liquid is sprayed into the system. Then, the pressure is restored to normal and allowed to stand for 12.5min for infiltration. After that, it is subjected to low-temperature fluidized drying at 35℃ until the moisture content of the obtained material is less than 15%, thus obtaining the coal gangue-carried bacterial compound bio-fertilizer.
[0066] Example 2 This embodiment is an example of the preparation of a compound bio-fertilizer with bacteria carried on coal gangue. The fertilizer-preserving and yield-increasing strains used are composed of Bacillus belyssae, Pseudomonas schrenckii, and Aspergillus niger, with a cell number ratio of 1.5:1:0.5.
[0067] S1. First, the raw coal gangue is crushed to a particle size of 1 mm. Then, the crushed raw coal gangue is fed into a fluidized bed reactor for isothermal calcination. The minimum fluidizing gas velocity of the fluidized bed is controlled at 0.08 m / s. The temperature is increased to 750℃ at a heating rate of 4℃ / min and calcined at the same temperature for 25 min. After natural cooling, a modified porous coal gangue carrier is obtained. The total porosity of the obtained modified porous coal gangue carrier is 52.5%, and it has a bimodal pore size distribution. The pore volume fraction with a diameter greater than 0.03 mm accounts for 21%, and the pore volume fraction with a diameter between 0.001 mm and 0.03 mm accounts for 26.5%.
[0068] S2. With a total inoculum of 2% (v / v), batch inoculation was performed according to the cell number ratio described above: First, the proportionally allocated *Aspergillus niger* was inoculated into the fermenter and cultured for 24 hours. Then, *Bacillus belye* and *Pseudomonas schrenckii* were inoculated in the corresponding proportions for sequential batch co-culture for 24 hours. The fermentation temperature was controlled at 28℃, the stirring speed at 160 rpm, and the dissolved oxygen level maintained at 30%. After the culture was completed, the synergistic fermentation symbiotic culture was obtained.
[0069] S3. The obtained modified porous coal gangue carrier is placed in a negative pressure environment, and the relative pressure of the negative pressure is controlled at -0.06MPa. After maintaining the pressure for 15 minutes, the above-mentioned synergistic fermentation symbiotic bacterial liquid is sprayed into the system. Then, the pressure is restored to normal and allowed to stand for 10 minutes for infiltration. After that, it is subjected to low-temperature fluidized drying at 35℃ until the moisture content of the obtained material is less than 15%, thus obtaining the coal gangue-carried bacterial compound bio-fertilizer.
[0070] Example 3 This embodiment is an example of the preparation of a compound bio-fertilizer with bacteria carried on coal gangue. The fertilizer-preserving and yield-increasing bacterial agent used is composed of Bacillus belysae, Pseudomonas schrenckii, and Aspergillus niger, with the cell number ratio of the three strains being 2:1.5:1.
[0071] S1. First, the raw coal gangue is crushed to a particle size of 3mm. Then, the crushed raw coal gangue is fed into a fluidized bed reactor for isothermal calcination. The minimum fluidizing gas velocity of the fluidized bed is controlled at 0.1m / s. The temperature is increased to 800℃ at a heating rate of 5℃ / min and calcined at the same temperature for 20min. After natural cooling, a modified porous coal gangue carrier is obtained. The total porosity of the obtained modified porous coal gangue carrier is 56.0%, and it has a bimodal pore size distribution. The pore volume fraction with a diameter greater than 0.03mm accounts for 25%, and the pore volume fraction with a diameter between 0.001mm and 0.03mm accounts for 31%.
[0072] S2. With a total inoculum of 4% (v / v), batch inoculation was performed according to the aforementioned cell number ratio: First, the proportionally allocated *Aspergillus niger* was inoculated into the fermenter and cultured for 30 hours. Then, *Bacillus belye* and *Pseudomonas schrenckii* were inoculated in corresponding proportions for sequential batch co-culture for 36 hours. The fermentation temperature was controlled at 35℃, the stirring speed at 200 rpm, and the dissolved oxygen level maintained at 40%. After the culture was completed, the synergistic fermentation symbiotic culture was obtained.
[0073] S3. The obtained modified porous coal gangue carrier is placed under negative pressure, and the relative pressure of the negative pressure is controlled at -0.08MPa. After maintaining the pressure for 20 minutes, the above-mentioned synergistic fermentation symbiotic bacterial liquid is sprayed into the system. Then, the pressure is restored to normal and allowed to stand for 15 minutes for infiltration. After that, it is subjected to low-temperature fluidized drying at 35℃ until the moisture content of the obtained material is less than 15%, thus obtaining the coal gangue-carried bacterial compound bio-fertilizer.
[0074] Comparative Example 1 This comparative example provides a method for preparing a compound bio-fertilizer containing bacteria from coal gangue. The difference from Example 1 is that the fertilizer-preserving and yield-increasing bacterial agent used consists only of Bacillus belyssus and Pseudomonas schrenckii, without the addition of Aspergillus niger, and the cell number ratio of the two strains is 1.75:1.25. Due to the lack of Aspergillus niger, no prior culture is performed in step S2. Bacillus belyssus and Pseudomonas schrenckii are directly inoculated according to the set inoculation amount and ratio, and co-cultured continuously for 30 hours. The remaining steps and parameters are the same as in Example 1.
[0075] Comparative Example 2 This comparative example provides a method for preparing a compound bio-fertilizer containing bacteria from coal gangue. The difference between this method and Example 1 is that the fertilizer-preserving and yield-increasing bacterial agent used consists only of Bacillus belye and Aspergillus niger, without the addition of Pseudomonas stearothermiae. The cell number ratio of the two strains is 1.75:0.75. All other steps and parameters are the same as in Example 1.
[0076] Comparative Example 3 This comparative example provides a method for preparing a compound bio-fertilizer containing bacteria from coal gangue. The difference between this method and Example 1 is that the fertilizer-preserving and yield-increasing bacterial agent used consists only of Pseudomonas schlegelii and Aspergillus niger, without the addition of Bacillus belysinus. The cell number ratio of the two strains is 1.25:0.75. All other steps and parameters are the same as in Example 1.
[0077] Comparative Example 4 This comparative example provides a method for preparing a compound bio-fertilizer containing bacteria from coal gangue. The difference from Example 1 is that the cell ratio of Bacillus vesiculosus, Pseudomonas schrenckii, and Aspergillus niger in the fertilizer-preserving and yield-increasing bacterial agent used is 1:2:1.5; the remaining steps and parameters are the same as in Example 1.
[0078] Comparative Example 5 This comparative example provides a method for preparing a coal gangue-borne microbial compound fertilizer. The difference between this method and Example 1 is that in step S1, the raw coal gangue is crushed and not calcined, but used directly as a carrier; the remaining steps and parameters are the same as in Example 1.
[0079] Comparative Example 6 This comparative example provides a method for preparing a coal gangue-borne microbial compound fertilizer. The difference between this method and Example 1 is that in step S3, the modified porous coal gangue carrier is directly sprayed with synergistic fermentation symbiotic bacterial liquid without undergoing negative pressure treatment; the remaining steps and parameters are the same as in Example 1.
[0080] Comparative Example 7 This comparative example provides a method for preparing a coal gangue-borne microbial compound fertilizer. The difference between this method and Example 1 is that in step S1, the raw coal gangue is crushed to a particle size of 4 mm; the remaining steps and parameters are the same as in Example 1.
[0081] Comparative Example 8 This comparative example provides a method for preparing a coal gangue-borne microbial compound fertilizer. The difference between this method and Example 1 is that the constant temperature calcination temperature in step S1 is 650℃; the remaining steps and parameters are the same as in Example 1.
[0082] Comparative Example 9 This comparative example provides a method for preparing a coal gangue-borne microbial compound bio-fertilizer. The difference from Example 1 is that in step S2, the total inoculation amount of the mixed inoculation is 1% (v / v); the remaining steps and parameters are the same as in Example 1.
[0083] Comparative Example 10 This comparative example provides a method for preparing a coal gangue-borne microbial compound fertilizer. The difference between this method and Example 1 is that in step S3, the relative pressure of the negative pressure is controlled to be -0.04 MPa; the remaining steps and parameters are the same as in Example 1.
[0084] Comparative Example 11 This comparative example provides a method for preparing a coal gangue-borne microbial compound bio-fertilizer. The difference between this method and Example 1 is that in step S2, the three strains are cultured separately and then physically mixed, without performing a sequential batch co-culture step; the remaining steps and parameters are the same as in Example 1.
[0085] Comparative Example 12 This comparative example provides a method for preparing a coal gangue-borne microbial compound fertilizer. The difference from Example 1 is that in step S3, the modified porous coal gangue carrier is replaced with an equal mass of conventional commercially available peat soil carrier (passed through a 2mm sieve); the remaining steps and parameters are the same as in Example 1.
[0086] Comparative Example 13 This comparative example provides a method for preparing a coal gangue-borne microbial composite bio-fertilizer. The difference from Example 1 is that in step S1, the heating rate and time of isothermal calcination are changed, so that the total porosity of the obtained modified porous coal gangue carrier is 53.5%, but it mainly exhibits a single-peak large-pore distribution. The volume fraction of pores with a diameter greater than 0.03 mm accounts for 48%, and the volume fraction of pores with a diameter between 0.001 mm and 0.03 mm accounts for only 4%. The remaining steps and parameters are the same as in Example 1.
[0087] The products corresponding to the above embodiments and comparative examples were tested using the following methods: Fertilizer moisture holding capacity: Weigh 5.00g (accurate to 0.01g) of air-dried fertilizer sample that has passed through a 2mm sieve, place it in a pre-weighed Buchner funnel lined with two layers of quantitative filter paper, add excess deionized water to completely submerge the fertilizer, let it stand and soak for 2 hours, then turn on the vacuum pump to filter until no water droplets drip continuously. Immediately weigh the total mass of the Buchner funnel, filter paper, and wet fertilizer. Then remove the wet fertilizer and dry it in a 105℃ oven until constant weight, weigh the dry fertilizer, and calculate the moisture holding capacity: Moisture holding capacity (%) = ((total mass of wet fertilizer - mass of Buchner funnel - mass of filter paper) - mass of dry fertilizer) / mass of dry fertilizer × 100%; Crop seed germination rate: 400 plump, undamaged, and disease-free seeds of the tested crop were randomly selected and divided into 4 replicates of 100 seeds each. The seeds were evenly placed in 9cm diameter petri dishes lined with two layers of moist qualitative filter paper and placed in an artificial climate chamber. Temperature and light conditions were set according to the appropriate germination parameters for the corresponding crop, and the relative humidity was controlled at 80%. An appropriate amount of deionized water was added daily to keep the filter paper moist. Germination was judged when the radicle broke through the seed coat by 1mm. The number of normally germinated seeds was recorded for 7 consecutive days, and the germination rate was calculated as follows: Germination rate (%) = (Total number of normally germinated seeds / Total number of tested seeds) × 100%; Crop seedling height growth rate: Select seeds of the test crop with uniform grain weight and sprouted white after germination. When the seedlings grow to the two-leaf-one-heart stage, randomly select 30 seedlings of uniform growth from each treatment. Measure the initial plant height of each plant with a ruler (vertical height from the root-stem junction to the top of the highest leaf, accurate to 0.1 cm). After 14 days of normal management according to the preset water and fertilizer plan, measure the plant height of the corresponding plants again and calculate the plant height growth rate: Plant height growth rate (%) = (average plant height at the end of the measurement period - initial average plant height) / initial average plant height × 100%; Fresh weight yield increase rate of crop seedlings: A treatment group applying the tested fertilizer and a control group with identical planting and management conditions except for not applying the tested fertilizer were set up. Each group was set up with 3 biological replicates. Twenty seedlings of the tested crop with uniform growth at the two-leaf-one-heart stage were selected from each replicate and transplanted. When the seedlings reached the four-leaf-one-heart stage, the whole seedlings were removed with roots, the substrate attached to the roots was rinsed with deionized water, the free water on the surface of the plants was dried with absorbent paper, and the fresh weight of the whole plant was weighed (accurate to 0.01g). The average fresh weight per plant in the treatment group and the control group were calculated respectively, and the fresh weight yield increase rate was calculated: Fresh weight yield increase rate (%) = (average fresh weight per plant in the treatment group - average fresh weight per plant in the control group) / average fresh weight per plant in the control group × 100%.
[0088] Germination rate of crops under salt and alkali stress: The test method is the same as the aforementioned “germination rate of crop seeds”, except that the daily replenishment of deionized water is replaced with a mixed salt and alkali stress solution containing 0.3% NaCl and 0.1% Na2CO3. The number of normally germinated seeds is recorded continuously for 7 days, and the germination rate under salt and alkali stress is calculated.
[0089] Soil available phosphorus enhancement rate: Test substrate soils lacking available phosphorus content were selected and divided into a treatment group (applied with the tested fertilizer) and a control group (not applied). After 30 days of cultivation at room temperature according to a pre-set water and fertilizer program, the available phosphorus content (mg / kg) in each soil group was determined using the sodium bicarbonate extraction-molybdenum antimony colorimetric method. The available phosphorus enhancement rate was calculated as follows: Available phosphorus enhancement rate (%) = (available phosphorus content in the treatment group - available phosphorus content in the control group) / available phosphorus content in the control group × 100%.
[0090] Soil available iron enhancement rate: Slightly alkaline test substrate soils with low available iron content were selected, and the grouping and culture conditions were the same as those for soil available phosphorus determination. After 30 days of culture, the available iron content (mg / kg) in each group of soil was determined by DTPA (diethylenetriaminepentaacetic acid) extraction-atomic absorption spectrophotometry. The available iron enhancement rate was calculated as follows: Available iron enhancement rate (%) = (available iron content in treatment group - available iron content in control group) / available iron content in control group × 100%.
[0091] Table 1 Test Results Group Water holding capacity (%) Crop seed germination rate (%) Crop seedling height growth rate (%) Fresh weight gain rate of crops during the seedling stage (%) Germination rate (%) under salt-alkali stress Increase rate of available phosphorus in soil (%) Increase in available iron in soil (%) Example 1 68.3 97.2 78.5 62.4 85.4 42.6 55.3 Example 2 67.9 96.8 77.9 61.8 84.9 41.8 54.6 Example 3 72.6 98.1 82.3 68.7 88.6 46.5 61.2 Comparative Example 1 52.1 82.5 51.2 32.7 56.2 15.4 38.5 Comparative Example 2 48.7 80.3 47.6 28.9 61.5 31.2 22.4 Comparative Example 3 45.2 77.8 42.3 24.6 42.8 35.6 20.1 Comparative Example 4 56.8 86.4 58.7 38.5 68.3 28.5 41.2 Comparative Example 5 22.4 65.7 26.3 11.2 35.4 18.2 15.6 Comparative Example 6 59.3 88.2 62.4 42.1 71.5 32.4 44.8 Comparative Example 7 54.6 85.7 57.9 37.8 66.8 29.6 40.5 Comparative Example 8 51.2 83.6 53.1 34.2 63.4 26.3 36.7 Comparative Example 9 57.8 87.1 59.6 39.4 70.1 30.5 42.6 Comparative Example 10 60.2 89.5 64.7 44.6 73.6 34.2 46.8 Comparative Example 11 64.5 85.3 60.2 40.5 58.7 24.5 31.2 Comparative Example 12 55.4 86.1 58.4 38.6 65.2 28.7 38.4 Comparative Example 13 58.2 84.8 56.5 36.3 61.4 25.6 35.1 Analysis of Experimental Results The coal gangue-borne microbial compound bio-fertilizer prepared in Example 1 had a water retention rate of 68.3%, a crop seed germination rate of 97.2%, a seedling height growth rate of 78.5%, and a seedling fresh weight yield increase rate of 62.4%. In the tests of stress resistance and microenvironmental physicochemical indicators, the germination rate under salt-alkali stress reached 85.4%, and the increases in available phosphorus and available iron in the soil reached 42.6% and 55.3%, respectively. These data indicate that under the conditions of the three-strain ratio, sequential batch co-culture, modified porous coal gangue carrier, and negative pressure microbial loading used in Example 1, the obtained coal gangue-borne microbial compound bio-fertilizer has good effects on fertilizer retention, growth promotion, stress resistance, and soil microenvironment improvement.
[0092] The coal gangue-borne microbial compound bio-fertilizer prepared in Example 2 had a water retention rate of 67.9%, a normal germination rate of 96.8%, a plant height growth rate of 77.9%, and a fresh weight yield increase rate of 61.8%, respectively. Under salt-alkali stress, the germination rate was 84.9%, and the increases in available phosphorus and available iron were 41.8% and 54.6%, respectively. All performance indicators were similar to those in Example 1, indicating that the preparation process has good stability.
[0093] The fertilizer prepared in Example 3 showed the best performance in all indicators, with a water holding capacity of 72.6%, germination rates of 98.1% and 88.6% under normal and saline-alkali stress conditions, respectively, plant height and fresh weight increases of 82.3% and 68.7%, respectively, and increases in available phosphorus and available iron of 46.5% and 61.2%, respectively. This indicates that under the parameter settings of this example, the pore structure of the modified coal gangue and the loading of the co-cultured bacterial solution achieved a better match, resulting in a more significant overall fertilizer retention and yield increase effect.
[0094] Comparative Example 1 used an inoculant without Aspergillus niger, consisting only of Bacillus belyssus and Pseudomonas schrenckii. The resulting fertilizer exhibited a significantly reduced normal germination rate of 82.5%, and a marked decrease in plant height growth rate (51.2%) and fresh weight yield increase rate (32.7%); particularly, the soil available phosphorus enhancement rate dropped to 15.4%. This indicates that the organic acids secreted by Aspergillus niger play a crucial role in decomposing insoluble phosphorus in the microenvironment, and their absence hinders phosphorus metabolism within the microbial community.
[0095] Comparative Example 2, without the addition of *Pseudomonas schlegelii*, showed a decrease in water holding capacity to 48.7%, fresh weight gain to 28.9%, and soil available iron enhancement to 22.4%. This indicates that *Pseudomonas schlegelii* plays a crucial role in early nitrogen fixation and asymmetric iron nutrient complementarity. The absence of this strain leads to a break in readily available nitrogen sources in the microenvironment, weakens the cascade iron chelation capacity, and consequently significantly reduces the stress resistance and growth-promoting capacity of the entire interaction system.
[0096] Comparative Example 3 did not include *Bacillus belyssus*. This group had a water holding capacity of only 45.2%, a germination rate that dropped significantly to 42.8% under salt-alkali stress, and an increase in available iron of only 20.1%. This indicates that *Bacillus belyssus* is not only indispensable for iron chelation, but its synthesized extracellular polymers also provide necessary physical space protection for the bacterial community. The lack of this component leads to a significant reduction in the survival rate of the remaining strains under salt-alkali stress.
[0097] Comparative Example 4 deviated from the cell number ratio of the present invention. Its water holding capacity was 56.8%, fresh weight yield increase was 38.5%, and soil available phosphorus and available iron increase rates were 28.5% and 41.2%, respectively. All performance indicators were significantly lower than those of the Example, indicating that deviating from the preset ratio leads to an imbalance in the microbial metabolic network and weakens the synergistic effect between the combined strains.
[0098] Comparative Example 5 used raw coal gangue directly as a carrier without calcination treatment. Its performance was the lowest among all test groups, with a water holding capacity of only 22.4% and a germination rate under salt and alkali stress of 35.4%. This demonstrates that unmodified coal gangue has a dense structure and low porosity, failing to provide effective attachment space for bacteria. Calcination modification is a prerequisite for achieving bacterial carrier functionality.
[0099] The modified carrier of Comparative Example 6 was not subjected to negative pressure treatment and was directly sprayed with fermentation broth under normal pressure. Its water holding capacity was 59.3%, and its germination rate under salt-alkali stress was 71.5%. This indicates that under normal pressure conditions, the viscous broth is difficult to overcome the air resistance inside the pores to enter the micropores, and mostly adheres to the shallow surface, resulting in insufficient actual loading inside the carrier and easy loss during application under adverse conditions.
[0100] Comparative Example 7 increased the particle size of raw coal gangue to 4 mm. The resulting fertilizer's water retention rate decreased to 54.6%, and the fresh weight yield dropped to 37.8%. This is because the excessively large particle size makes it difficult for the internal organic matter to be fully decomposed and released during the calcination stage, resulting in insufficient pore formation and thus reducing the carrier's loading capacity and water retention effect.
[0101] In Comparative Example 8, the isothermal calcination temperature was controlled at 650℃. The resulting fertilizer had a water retention rate of 51.2%, a salt-alkali germination rate of 63.4%, and an effective phosphorus enhancement rate of only 26.3%. This indicates that excessively low calcination temperatures cannot promote the full decomposition of the internal components of coal gangue, resulting in an unsuitable pore structure that limits the spatial colonization of the bacterial solution and the efficiency of subsequent nutrient transport to the microenvironment.
[0102] Comparative Example 9 reduced the mixed inoculum size to 1% (v / v). Its normal germination rate was 87.1%, and the fresh weight yield increase was 39.4%. The low inoculum size resulted in insufficient initial cell density, making it difficult to quickly establish effective quorum sensing communication in the early stage of co-culture, ultimately limiting the overall biological activity of the fermentation broth.
[0103] The negative pressure relative pressure of Comparative Example 10 was controlled at -0.04 MPa. Its water holding capacity was 60.2%, and its performance was slightly lower than that of the Example. This indicates that insufficient negative pressure leads to incomplete air removal from the micropores, and the residual air hinders the deep penetration of the bacterial solution at the microscale level.
[0104] Comparative Example 11 involved culturing the three strains separately before physical mixing, without a sequential batch co-culture step. Under salt-alkali stress, the germination rate dropped to 58.7%, and the increases in available phosphorus and available iron were only 24.5% and 31.2%, respectively. This comparative data demonstrates that physical mixing after individual culturing cannot effectively stimulate niche differentiation and resource utilization among the strains. Without mild co-culture stress and substrate cross-feeding, the synthesis of large amounts of extracellular polymers and the early accumulation of functional genes such as nitrogenase cannot be naturally induced; sequential batch co-culture is a key technological step in constructing this symbiotic network.
[0105] Comparative Example 12 replaced the modified porous coal gangue with conventional peat soil as the carrier. Its fresh weight yield increased by 38.6%, and the germination rate under salt-alkali stress was 65.2%. This indicates that conventional peat soil cannot provide targeted microporous protection and cation adsorption in its physical structure, and the survival of the complex microbial community is easily affected in complex soil stresses, confirming the advantages of the specific modified coal gangue carrier in stabilizing the microbial community.
[0106] The total porosity of the carrier in Comparative Example 13 was within the preset range, but it mainly exhibited a single-peak large-pore distribution, lacking micropores with diameters ranging from 0.001 mm to 0.03 mm. The resulting fertilizer's water holding capacity decreased to 58.2%, and its germination rate under salt-alkali stress decreased to 61.4%. This data confirms the importance of a bimodal pore size distribution for maintaining product performance: the lack of micropores reduces the capillary retention of the bacterial solution by the carrier, making the loaded microbial community more susceptible to external high osmotic pressure stress, thus leading to a decrease in the fertilizer's actual nutrient retention and stress resistance.
[0107] The differences between Example 1 and the comparative examples show that, in Comparative Example 1 without the addition of *Aspergillus niger*, the soil available phosphorus enhancement rate decreased from 42.6% in Example 1 to 15.4%, indicating that *Aspergillus niger* plays an important role in the activation of insoluble phosphorus and phosphorus cycling. In Comparative Example 2 without the addition of *Pseudomonas schlegelii*, the soil available iron enhancement rate decreased from 55.3% in Example 1 to 22.4%, and the fresh weight yield increase rate decreased from 62.4% to 28.9%, indicating that *Pseudomonas schlegelii* makes a significant contribution to iron nutrient activation and growth promotion. In Comparative Example 3 without the addition of *Bacillus belyssus*, the water holding capacity decreased from 68.3% in Example 1 to 45.2%, and the germination rate under salt-alkali stress decreased from... The percentage decreased from 85.4% to 42.8%, indicating that *Bacillus belyssiensis* and its extracellular polymeric components have a significant impact on stress resistance and microenvironment maintenance. Although Comparative Example 4 contained three strains, its cell count ratio deviated from the range defined in this application, and all indicators were lower than in Example 1, indicating that the three strains cannot achieve the same effect by arbitrarily mixing them, but need to be within the appropriate ratio range. Comparative Example 11, where the three strains were cultured separately and then physically mixed without sequential batch co-culture, showed significantly lower germination rate, available phosphorus enhancement rate, and available iron enhancement rate under salt-alkali stress than in Example 1, indicating that sequential batch co-culture is beneficial for establishing interactions between strains. From the above comparisons, it can be seen that in this application, the combination of *Bacillus belyssiensis*, *Pseudomonas schrenckii*, and *Aspergillus niger* can exert a synergistic effect of promoting growth, resisting stress, and improving the microenvironment in modified porous coal gangue carriers.
[0108] In summary, using a predetermined ratio of Bacillus belye, Pseudomonas schlegelii, and Aspergillus niger for sequential batch co-culture, combined with modified porous coal gangue with a specific bimodal pore size distribution and a negative pressure-carrying bacterial process, can significantly improve the fertilizer's nutrient retention, yield-increasing effect, and stress resistance. The omission of any single strain, deviation from the ratio, alteration of the carrier pore structure, or omission of key process steps will weaken the aforementioned synergistic effect, thereby reducing the overall performance of the fertilizer.
[0109] The applicant declares that the detailed process flow of this invention is illustrated by the above embodiments, but this invention is not limited to the above detailed process flow, that is, it does not mean that this invention must rely on the above detailed process flow to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.
Claims
1. A fertilizer-preserving and yield-increasing microbial agent, characterized in that, The bacterial agent is: Bacillus belye (Bacillus) velezensis) Pseudomonas schrenckii (Pseudomonas stutzeri) and Aspergillus niger (Aspergillus niger) The cell number ratio of Bacillus belyi, Pseudomonas schrenckii, and Aspergillus niger is 1.5~2.2:1~1.6:0.5~1.
2.
2. A method for preparing a coal gangue-supported microbial compound bio-fertilizer, characterized in that, Includes the following steps: S1. After crushing the raw coal gangue, it is calcined at a constant temperature and then cooled to obtain a modified porous coal gangue carrier. S2. Configure the corresponding bacterial strains according to the cell number ratio described in claim 1, inoculate Aspergillus niger into a fermenter for culture, and after the formation of a primary mycelial network, inoculate Bacillus belye and Pseudomonas schrenckii for sequential batch continuous co-culture to obtain a synergistic fermentation symbiotic bacterial solution. S3. The modified porous coal gangue carrier is placed under negative pressure, the synergistic fermentation symbiotic bacterial liquid is sprayed in, and after restoring normal pressure, it is dried to obtain the coal gangue bacterial composite bio-fertilizer.
3. The method for preparing a coal gangue-supported microbial compound bio-fertilizer according to claim 2, characterized in that, In step S1, the particle size of the crushed raw coal gangue is 1~3 mm; the temperature of the constant temperature calcination is 700~800℃, and the calcination time is 15~25 min.
4. The method for preparing a coal gangue-supported microbial compound bio-fertilizer according to claim 2, characterized in that, In step S1, the total porosity of the modified porous coal gangue carrier is 52.0%~56.5%, and it has a bimodal pore size distribution, wherein the volume fraction of pores with a diameter greater than 0.03 mm accounts for 20%~25%, and the volume fraction of pores with a diameter between 0.001 mm and 0.03 mm accounts for 26%~30%.
5. The method for preparing a coal gangue-supported microbial compound bio-fertilizer according to claim 2, characterized in that, In step S1, the isothermal calcination is carried out in a fluidized bed reactor, with the minimum fluidizing gas velocity controlled at 0.08 m / s, and the temperature is increased to 700-800℃ at a heating rate of 4-5℃ / min.
6. The method for preparing a coal gangue-supported microbial compound bio-fertilizer according to claim 2, characterized in that, In step S2, the total inoculum amount is 2%~5% (v / v); the initial culture time of Aspergillus niger is 24~36 h, and the sequential batch co-culture time after bacterial inoculation is 24~40 h.
7. The method for preparing a coal gangue-supported microbial compound bio-fertilizer according to claim 2, characterized in that, In step S2, the conditions for the sequential batch co-culture are: temperature controlled at 28~35℃, stirring speed at 160~200 rpm, and dissolved oxygen maintained at 30%~40%.
8. The method for preparing a coal gangue-supported microbial compound bio-fertilizer according to claim 2, characterized in that, In step S3, the relative pressure of the negative pressure is controlled between -0.06 MPa and -0.08 MPa, and the pressure holding time is 15 to 20 minutes; after the co-fermentation symbiotic bacterial liquid is sprayed in and the pressure is restored to normal, it is allowed to stand and permeate for 10 to 15 minutes.
9. The method for preparing a coal gangue-supported microbial compound bio-fertilizer according to claim 2, characterized in that, In step S3, the drying is carried out at 35°C using low-temperature fluidized bed drying until the moisture content of the coal gangue-carrying microbial compound fertilizer is less than 15%.
10. A coal gangue-based microbial compound fertilizer, characterized in that, It is prepared by the method of preparing coal gangue-borne bacteria compound bio-fertilizer according to any one of claims 2-9.