A composite preparation containing nitrogen-fixing bacteria and application thereof in increasing yield of sugarcane

Abstract

This invention discloses a compound preparation containing nitrogen-fixing bacteria and its application in increasing sugarcane yield, belonging to the field of microbial technology. It includes a compound microbial agent and biochar loaded with the compound microbial agent. The compound microbial agent is composed of the following strains: *Azotobacter brasiliensis* ACCC 04138, *Bacillus mucilaginosus* ACCC 19749, and *Pseudomonas fluorescens* CICC 23919. The compound microbial agent prepared by combining *Azotobacter brasiliensis*, *Bacillus mucilaginosus*, and *Pseudomonas fluorescens*, and specifically combined with biochar prepared from sugarcane bagasse and mushroom residue, can significantly improve the structure of acidic soil and increase sugarcane yield and sugar content. This invention provides a new compound microbial agent for sugarcane cultivation and provides a scientific basis for improving sugarcane yield and quality and ameliorhynchophylla.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound preparation containing nitrogen-fixing bacteria and its application in increasing sugarcane yield. Background Technology

[0002] Sugarcane, as an important sugar and energy crop, has a long growth cycle and a high nitrogen requirement. Traditional cultivation relies on the application of chemical nitrogen fertilizers (such as urea), which not only leads to high production costs but also causes a series of problems: low nutrient utilization: the low pH value (4.5-5.5) and low cation exchange capacity of red soil in the south make it easy for chemical nitrogen fertilizers to be lost through leaching and volatilization, resulting in soil nitrogen surplus and the risk of eutrophication of water bodies; soil quality degradation: long-term application of chemical nitrogen fertilizers leads to imbalance of soil microbial community, soil compaction, and decline in sugarcane root vitality; decreased sugarcane quality and stress resistance: excessive chemical nitrogen fertilizers can reduce the sugar content of sugarcane and make it prone to lodging under adverse conditions such as drought and typhoons.

[0003] To address the aforementioned issues, existing technologies have attempted to apply nitrogen-fixing bacteria to sugarcane cultivation. Some have used single nitrogen-fixing bacteria as microbial agents. However, the application of single nitrogen-fixing bacteria to sugarcane cultivation has the following drawbacks: Low nitrogen fixation efficiency: Single nitrogen-fixing bacteria have a very low survival rate in the acidic environment of red soil and cannot form a stable symbiotic relationship with sugarcane roots. The cumulative nitrogen fixation during the growth period cannot meet the nitrogen requirements of sugarcane; Limited function: It only focuses on nitrogen supply, failing to address the sugarcane's needs for phosphorus and potassium, and lacks the ability to improve soil acidification; Unstable application effect: It does not consider the differences in nutrient requirements of sugarcane at different growth stages (seedling stage, tillering stage, and elongation stage). The single application method leads to nutrient deficiency in the later stages, resulting in a low yield increase that falls far short of production needs. There are also applications that use nitrogen-fixing bacteria mixed with other microorganisms, such as invention patent CN114467668A "A method for promoting sugarcane growth," which discloses the use of nitrogen-fixing bacteria, soil conditioners, and organic fertilizers in combination. However, this patent requires the use of multiple raw materials to make a soil conditioner (kaolin mixed with lime water to make a mixed slurry, sugarcane leaf powder, humic acid, and filter mud), four types of microorganisms to make a compound microbial agent (Cercospora chinensis, Cercospora aequalis, Bacillus subtilis, and Lactobacillus reuteri), and organic fertilizer (made from...). The technology involves the simultaneous use of multiple raw materials, resulting in complex raw material composition and cumbersome operation. Furthermore, the technology does not verify the use of compound microbial agents in improving soil conditions, only providing experimental data on yield, and lacks more detailed evidence on the effects of the raw materials.

[0004] Therefore, developing a compound formulation that can adapt to the acidic environment of red soil, take into account the multiple functions of "nitrogen fixation, phosphorus release, soil improvement and stress resistance", and is suitable for the nutrient requirements of sugarcane during its growth period has become the key to solving the sugarcane production problem. Summary of the Invention

[0005] The purpose of this invention is to provide a compound preparation containing nitrogen-fixing bacteria and its application in increasing sugarcane yield, in order to solve the problems existing in the prior art. The compound bacterial agent is prepared by combining *Azotrophus brasiliensis*, *Bacillus mucilaginosus* and *Pseudomonas fluorescens*, and then specifically combined with biochar prepared from sugarcane bagasse and mushroom residue. This can significantly improve the structure of acidic soil and increase sugarcane yield and sugar content.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a compound preparation containing nitrogen-fixing bacteria, comprising a compound bacterial agent and biochar loaded with the compound bacterial agent. The compound bacterial agent is composed of the following strains: Azospirillum brasilense ACCC04138, Bacillus mucilaginosus Krassilnikov ACCC 19749, and Pseudomonas fluorescens CICC 23919.

[0008] Preferably, in the compound microbial agent, the mass ratio of *Azotobacter brasiliensis*, *Bacillus mucilaginosus* and *Pseudomonas fluorescens* is (1-2):(0.5-2):(1-2), and the mass ratio of *Azotobacter brasiliensis* to the biochar is 1:(5-8).

[0009] Preferably, the biochar comprises bagasse and mushroom residue, wherein the mass ratio of bagasse to mushroom residue is (1-2):(1-2).

[0010] The present invention also provides a method for preparing the aforementioned compound microbial agent, comprising the following steps:

[0011] The cultures of *Azotobacter brasiliensis*, *Bacillus mucilaginosus*, and *Pseudomonas fluorescens* were prepared by fermentation and culture, respectively.

[0012] Sugarcane bagasse and mushroom residue are mixed and pyrolyzed at high temperature to produce biochar;

[0013] After mixing the bacterial solutions of *Azotobacter brasiliensis* and *Bacillus mucilaginosus*, the mixture was sprayed onto the biochar and stirred intermittently for 1-2 hours. Then, *Pseudomonas fluorescens* bacterial solution was added and stirring was continued for 1-2 hours to obtain a compound bacterial agent.

[0014] Preferably, the fermentation medium used to prepare *Azotobacter brasiliensis* and *Pseudomonas fluorescens* bacterial cultures is a nutrient broth medium, and the fermentation medium used to prepare the nutrient broth medium is a silicate bacteria medium.

[0015] Preferably, the high-temperature pyrolysis temperature is 500°C and the time is 3 hours.

[0016] Preferably, the intermittent stirring conditions are as follows: after spraying the biochar with *Azotobacter brasiliensis* and *Bacillus mucilaginosus* solutions, stirring is performed at 50 rpm for 5 minutes, and then stirred under the same conditions every 20 minutes.

[0017] Preferably, the optimal mass ratio of the *Azotobacter brasiliensis* bacterial solution, *Bacillus mucilaginosus* bacterial solution, and *Pseudomonas fluorescens* bacterial solution is 1.8:1:1.8.

[0018] The optimal mass ratio of sugarcane bagasse to mushroom residue is 1.5:1;

[0019] The optimal mass ratio of the *Braziliana nigrostriatus* bacterial solution to biochar is 1:8.

[0020] The present invention also provides the application of the aforementioned compound formulation in improving the soil structure of sugarcane fields.

[0021] This invention also provides the application of the aforementioned compound formulation in increasing sugarcane yield and sugar content. Examples of this invention show that it can significantly increase sugarcane yield and sugar content while reducing nitrogen fertilizer application.

[0022] The present invention discloses the following technical effects:

[0023] This invention involves compounding three bacteria—*Azotobacter brasiliensis*, *Bacillus mucilaginosus*, and *Pseudomonas fluorescens*—in an optimized ratio, and then combining this with biochar prepared from sugarcane bagasse and mushroom residue through high-temperature pyrolysis to provide excellent nutritional and environmental conditions for the microorganisms, thus creating a composite formulation. Experiments have shown that compared to using single or two bacteria in combination, this significantly increases the number of viable bacteria in the coexisting system. Planting experiments have demonstrated that the combined use of these three bacteria significantly increases soil pH, organic matter content, and the number of nitrogen-fixing bacteria, improving soil structure and laying the foundation for increased sugarcane yield and sugar content. This invention provides a novel compound microbial agent for sugarcane cultivation and offers a scientific basis for improving sugarcane yield and quality, as well as for the improvement of acidic soils. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The bacterial strains involved in the following examples are: Azospirillum brasilense ACCC04138, purchased from the China Agricultural Microbial Culture Collection Center (ACCC); Bacillus mucilaginosus Krassilnikov ACCC 19749, purchased from the China Agricultural Microbial Culture Collection Center (ACCC); and Pseudomonas fluorescens CICC 23919, purchased from the China Industrial Microbial Culture Collection Center (CICC).

[0030] Culture medium formulation:

[0031] LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 20g agar, 1000mL water, pH 7.0. Sterilize at 121℃ for 15min.

[0032] Nutrient broth culture medium: 5g peptone, 3g beef extract, 5g sodium chloride, 15g agar, 1000mL distilled water, pH 7.0. Sterilize at 121℃ for 15min.

[0033] Silicate bacterial culture medium: 0.3g yeast extract, 10g sucrose, 0.5g ammonium sulfate, 0.5g calcium carbonate, 0.5g magnesium sulfate heptahydrate, 1g dipotassium hydrogen phosphate, 1000mL distilled water, pH 7.2. Sterilize at 121℃ for 15min.

[0034] Example 1: A compound preparation containing nitrogen-fixing bacteria

[0035] (1) Culture of strains

[0036] *Azotomyces brasiliensis* (ACCC 04138) was first activated on LB solid medium, then inoculated into nutrient broth medium, and cultured at 30°C with shaking at 180 rpm for 48 hours to obtain *Azotomyces brasiliensis* bacterial suspension; the bacterial concentration was 10... 8 CFU / mL or higher.

[0037] Bacillus mucilaginosus (ACCC 19749) was first activated on LB solid medium, then inoculated onto silicate bacterial medium, and cultured at 30°C and 180 rpm for 48 hours with shaking to obtain Bacillus mucilaginosus bacterial suspension; the bacterial concentration was 10... 8 CFU / mL or higher.

[0038] Activated *Pseudomonas fluorescens* (CICC 23919) on LB solid medium, then inoculated into nutrient broth medium and cultured at 30°C with shaking at 180 rpm for 48 hours to obtain *Pseudomonas fluorescens* bacterial suspension; the bacterial concentration was 10... 8 CFU / mL or higher.

[0039] (2) Carrier preparation

[0040] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C for 3 hours to prepare biochar.

[0041] (3) Preparation of compound formulations

[0042] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 1:1, sprayed onto the biochar, and intermittently stirred (stirred at 50 rpm for 5 min after adding the bacterial strain, and then stirred once every 20 min; the stirring conditions for other examples and comparative examples were the same) for 1 h. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:0.5) and stirred for another 1 h to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:5.

[0043] Example 2: A compound preparation containing nitrogen-fixing bacteria

[0044] (1) Culture of strains

[0045] Same as step (1) in Example 1.

[0046] (2) Carrier preparation

[0047] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1:2, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C for 3 hours to prepare biochar.

[0048] (3) Preparation of compound formulations

[0049] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 1.5:1, sprayed onto the biochar, and intermittently stirred for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1.5:1) and stirred for another 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:6.

[0050] Example 3: A compound preparation containing nitrogen-fixing bacteria

[0051] (1) Culture of strains

[0052] Same as step (1) in Example 1.

[0053] (2) Carrier preparation

[0054] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550℃ for 3 hours to prepare biochar.

[0055] (3) Preparation of compound formulations

[0056] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 1.8:1, sprayed onto the biochar, and intermittently stirred for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:1) and stirring was continued for 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:8.

[0057] Example 4: A compound preparation containing nitrogen-fixing bacteria

[0058] (1) Culture of strains

[0059] Same as step (1) in Example 1.

[0060] (2) Carrier preparation

[0061] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 2:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550℃ for 3 hours to prepare biochar.

[0062] (3) Preparation of compound formulations

[0063] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 2:1, sprayed onto the biochar, and intermittently stirred for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:2) and stirring was continued for 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:6.

[0064] Example 5: A compound preparation containing nitrogen-fixing bacteria

[0065] (1) Culture of strains

[0066] Same as step (1) in Example 1.

[0067] (2) Carrier preparation

[0068] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550℃ for 3 hours to prepare biochar.

[0069] (3) Preparation of compound formulations

[0070] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 1:2, sprayed onto the biochar, and intermittently stirred for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:1) and stirring was continued for 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:6.

[0071] Example 6: A compound preparation containing nitrogen-fixing bacteria

[0072] (1) Culture of strains

[0073] Same as step (1) in Example 1.

[0074] (2) Carrier preparation

[0075] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550℃ for 3 hours to prepare biochar.

[0076] (3) Preparation of compound formulations

[0077] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 3:1, sprayed onto the biochar, and intermittently stirred for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 2:1) and stirring was continued for 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:8.

[0078] Comparative Example 1

[0079] Compared to Example 3, the difference is that no carrier is used to prepare the composite formulation. The preparation steps are as follows:

[0080] (1) Culture of strains

[0081] Same as step (1) in Example 1.

[0082] (2) Mix the *Azotobacter brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) at a mass ratio of 1.8:1, stir intermittently for 1 hour, then add *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotobacter brasiliensis* bacterial solution and *Pseudomonas fluorescens* bacterial solution is 1:1) and continue stirring for 1 hour to obtain the composite preparation.

[0083] Comparative Example 2

[0084] Compared to Example 3, the difference lies in that the three bacterial cultures are simultaneously mixed and added to the carrier. The preparation steps are as follows:

[0085] (1) Culture of strains

[0086] Same as step (1) in Example 1.

[0087] (2) Carrier preparation

[0088] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C to prepare biochar.

[0089] (3) Preparation of compound formulations

[0090] The *Azotomyces brasiliensis* bacterial solution, *Bacillus mucilaginosus* bacterial solution, and *Pseudomonas fluorescens* bacterial solution prepared in step (1) were mixed and sprayed onto the biochar. The mixture was stirred intermittently for 2 hours to obtain a composite preparation. The mass ratio of *Azotomyces brasiliensis* bacterial solution to *Bacillus mucilaginosus* bacterial solution was 1.8:1, the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:1, and the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:8.

[0091] Comparative Example 3

[0092] Compared to Example 3, the difference is that only *Azotomyces brasiliensis* and *Bacillus mucilaginosus* bacterial solutions were added. The preparation steps are as follows:

[0093] (1) Culture of strains

[0094] The same method as step (1) of Example 1 for preparing the bacterial suspension of *Azotobacter brasiliensis* and *Bacillus mucilaginosus*.

[0095] (2) Carrier preparation

[0096] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C to prepare biochar.

[0097] (3) Preparation of compound formulations

[0098] The bacterial solutions of *Azotoxinus brasiliensis* and *Bacillus mucilaginosus* prepared in step (1) were mixed at a mass ratio of 1.8:1, sprayed onto the biochar, and intermittently stirred for 1 hour to obtain a composite preparation; the mass ratio of *Azotoxinus brasiliensis* bacterial solution to biochar was 1:8.

[0099] Comparative Example 4

[0100] Compared to Example 3, the difference is that only *Azotrophus brasiliensis* and *Pseudomonas fluorescens* bacterial suspensions were added. The preparation steps are as follows:

[0101] (1) Culture of strains

[0102] The same method as step (1) of Example 1 for preparing the bacterial suspensions of *Azotobacter brasiliensis* and *Pseudomonas fluorescens*.

[0103] (2) Carrier preparation

[0104] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C to prepare biochar.

[0105] (3) Preparation of compound formulations

[0106] The bacterial solutions of *Azotospira brasiliensis* and *Pseudomonas fluorescens* prepared in step (1) were mixed at a mass ratio of 1:1, sprayed onto the biochar, and intermittently stirred for 1 hour to obtain a composite preparation; the mass ratio of *Azotospira brasiliensis* bacterial solution to biochar was 1:8.

[0107] Comparative Example 5

[0108] Compared to Example 3, the difference is that only *Bacillus mucilaginosus* and *Pseudomonas fluorescens* bacterial suspensions were added. The preparation steps are as follows:

[0109] (1) Culture of strains

[0110] The same method as step (1) of Example 1 for preparing Bacillus mucilaginosus and Pseudomonas fluorescens bacterial suspensions.

[0111] (2) Carrier preparation

[0112] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C to prepare biochar.

[0113] (3) Preparation of compound formulations

[0114] The Bacillus mucilaginosus and Pseudomonas fluorescens bacterial solutions prepared in step (1) were mixed at a mass ratio of 1:1.8, sprayed onto the biochar, and intermittently stirred for 1 hour to obtain a composite preparation; the mass ratio of Pseudomonas fluorescens bacterial solution to biochar was 1:8.

[0115] Comparative Example 6

[0116] Compared to Example 3, the difference is that only *Azotomyces brasiliensis* bacterial culture was added. The preparation steps are as follows:

[0117] (1) Culture of strains

[0118] The same method for preparing the *Azotobacter brasiliensis* bacterial solution as in step (1) of Example 1.

[0119] (2) Carrier preparation

[0120] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C to prepare biochar.

[0121] (3) Preparation of compound formulations

[0122] The *Azotoxinus brasiliensis* bacterial solution prepared in step (1) was sprayed onto the biochar and intermittently stirred and mixed for 1 hour to obtain a composite preparation; the mass ratio of *Azotoxinus brasiliensis* bacterial solution to biochar was 1:8.

[0123] Comparative Example 7

[0124] Compared to Example 3, the difference is that intermittent stirring is not performed. The preparation steps are as follows:

[0125] (1) Culture of strains

[0126] Same as step (1) in Example 1.

[0127] (2) Carrier preparation

[0128] Sugarcane bagasse and mushroom residue were screened, impurities removed, and dried separately. Then, they were mixed at a mass ratio of 1.5:1, wrapped in double-layer aluminum foil, and placed in a muffle furnace for pyrolysis at 550°C to prepare biochar.

[0129] (3) Preparation of compound formulations

[0130] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 1.8:1, sprayed onto the biochar, mixed evenly, and left to stand for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:1) were added, mixed evenly, and left to stand for another 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:8.

[0131] Comparative Example 8

[0132] Compared to Example 3, the difference is that only sugarcane bagasse is used as the raw material for biochar. The preparation steps are as follows:

[0133] (1) Culture of strains

[0134] Same as step (1) in Example 1.

[0135] (2) Carrier preparation

[0136] Sugarcane bagasse is screened, impurities are removed and dried, wrapped in double-layer aluminum foil and placed in a muffle furnace for pyrolysis at a high temperature of 550℃ to prepare biochar.

[0137] (3) Preparation of compound formulations

[0138] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 1.8:1, sprayed onto the biochar, and intermittently stirred for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:1) and stirring was continued for 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:8.

[0139] Comparative Example 9

[0140] Compared to Example 3, the difference is that only mushroom residue is used as the raw material for biochar. The preparation steps are as follows:

[0141] (1) Culture of strains

[0142] Same as step (1) in Example 1.

[0143] (2) Carrier preparation

[0144] The mushroom residue was sieved, impurities were removed and dried. It was then wrapped in double-layer aluminum foil and placed in a muffle furnace for pyrolysis at a high temperature of 550℃ to prepare biochar.

[0145] (3) Preparation of compound formulations

[0146] The *Azotomyces brasiliensis* bacterial solution and *Bacillus mucilaginosus* bacterial solution prepared in step (1) were mixed at a mass ratio of 1.8:1, sprayed onto the biochar, and intermittently stirred for 1 hour. Then, *Pseudomonas fluorescens* bacterial solution (the mass ratio of *Azotomyces brasiliensis* bacterial solution to *Pseudomonas fluorescens* bacterial solution was 1:1) and stirring was continued for 1 hour to obtain the composite preparation; the mass ratio of *Azotomyces brasiliensis* bacterial solution to biochar was 1:8.

[0147] The viable bacterial count of the compound preparations prepared in Examples 1-6 and Comparative Examples 1-9 was determined using the plate count method. The results are shown in Table 1.

[0148] Table 1. Results of viable cell count

[0149]

[0150] As can be seen from the experimental data in Table 1, for the production of compound preparations, the type and amount of the compounded microbial strains, the method of adding the strains, the treatment method during the compounding process, and the selection of carrier raw materials all have a direct impact on the viable count content in the compound microbial agent. The selection and amount of strains affect the coexistence state and the rate of formation of dominant bacteria. The method of adding the strains and the treatment method during compounding (adding *Pseudomonas fluorescens* and the other two strains separately, and intermittent stirring after each addition of strains) can ensure that strains with relatively consistent nutritional and environmental requirements avoid competition, satisfying the needs of one strain first, and then, because after mixing *Azotobacter brasiliensis* and *Bacillus mucilaginosus* bacterial solutions with the carrier, based on the porous nature of biochar... Environmental conditions and nutrient composition promote the synergistic coexistence of two bacteria, making them the dominant species. This also generates secondary metabolites. Consequently, when *Pseudomonas fluorescens* is added later, there are more nutrients available, preventing competition with *Azospirillum brasiliensis* for nutrients and thus hindering their growth. Furthermore, to prevent *Azospirillum brasiliensis* from becoming the dominant species and gaining greater nutritional competitiveness, the amount of *Pseudomonas fluorescens* added needs to be carefully controlled to ensure the synergistic coexistence of the three bacteria. Intermittent stirring is necessary after adding the bacteria, partly to meet aerobic requirements and partly to prevent the rapid proliferation of either bacterium within a short period, which could disrupt the coexistence of the three species, when adding a large quantity of one or two bacteria from the biochar. Based on years of research experience in microbial strains and agriculture, the inventors deduced that sugarcane bagasse and mushroom residue contain dietary fiber, various amino acids, vitamins, calcium, zinc, and other trace elements, providing nutrition. After high-temperature treatment, the density difference between the two can create a porous structure with varying pore sizes. In particular, the mycelium in mushroom residue not only provides a carrier for the process but also, due to its rich organic matter and enzymes, is significant for improving soil structure and increasing crop yield. However, the biochar prepared from sugarcane bagasse alone can only meet the needs of the carrier, and the nutrients it contains are insufficient to support the stable coexistence of the three types of bacteria. Mixing it with mushroom residue in a specific ratio to prepare biochar solves the needs of both carrier and nutrition, allowing the three types of bacteria to develop and coexist in a balanced manner.

[0151] Application examples

[0152] Experiment location: A farmer in Jiangzhou District, Chongzuo City, Guangxi Province.

[0153] Experiment period: March-December 2024.

[0154] Experimental crop: sugarcane (Guitang 42).

[0155] Experimental grouping: Ten acres of sugarcane from each farmer's land were randomly divided into equal-sized plots. Fertilization experiments were conducted using the compound preparations prepared in Examples 1-6 and Comparative Examples 1-9, with a blank control consisting of the compound preparations not used in any of the above groups. Three parallel replicate experiments were set up using the same compound microbial agent.

[0156] Application method: Apply compound microbial agent at a rate of 1.5 kg / mu as a base fertilizer 20 days before planting; apply it to the roots once during the tillering stage and once 100 days after planting, at a rate of 1.0 kg / mu each time. Other fertilization and management should be carried out in accordance with conventional sugarcane planting management methods.

[0157] Soil pH, organic matter content, and nitrogen-fixing bacteria count were measured before planting. Six months after planting, soil pH, organic matter content, and nitrogen-fixing bacteria count were measured (using the conventional dilution plating method, with nitrogen-free medium). The results are shown in Table 2.

[0158] Table 2 Experimental Results

[0159]

[0160] Organic matter content growth rate (%) = (soil organic matter content before planting - soil organic matter content 6 months after planting) / soil organic matter content before planting × 100.

[0161] Nitrogen-fixing bacteria growth rate (%) = (Number of nitrogen-fixing bacteria in the soil before planting - Number of nitrogen-fixing bacteria in the soil 6 months after planting) / Number of nitrogen-fixing bacteria in the soil before planting × 100.

[0162] As can be seen from the data in Table 2 above, the compound formulations of Examples 1-6 of this invention, compared with Comparative Examples 1-9 and the blank control group, can increase the organic matter content and nitrogen-fixing bacteria count in the soil, while the pH value does not change significantly, thus significantly improving the soil structure. In the blank control group, the pH value, organic matter content, and nitrogen-fixing bacteria count remained almost unchanged, and there was no significant improvement in soil structure. Although the comparative examples were beneficial to the increase of organic matter content and nitrogen-fixing bacteria count to some extent, the effect was not as good as that of Examples 1-6, and the effect of Example 3 was the most outstanding.

[0163] To further demonstrate the function of the compound formulation of this invention, in addition to the above experiments, considering the excessive application of nitrogen fertilizer in conventional sugarcane cultivation methods in my country, a treatment with reduced nitrogen fertilizer application was also set up as a comparison. Treatment I: The total amount of nitrogen fertilizer applied throughout the growth period was halved, and all other aspects were the same as the blank control; Treatment II: The total amount of conventional fertilization applied throughout the growth period was halved, plus the compound formulation prepared in Example 3, and all other aspects were the same as in Example 3. The yield and sugar content of sugarcane after harvest were measured after different treatments, and the results are shown in Table 3.

[0164] Table 3 Results of yield and sugar content determination

[0165]

[0166] As can be seen from the data in Table 3, the compound formulations of Examples 1-6 of this invention significantly increased sugarcane yield and sugar content compared to the blank control group and the comparative group, with Example 3 showing the best effect, consistent with the data in Tables 1 and 2. Even after halving the nitrogen fertilizer and mixing it with the compound formulation, the effect of significantly increasing sugarcane yield and sugar content was still achieved, and the effect was comparable to that of Example 3. This indicates that the compound formulation prepared by this invention can significantly reduce the amount of nitrogen fertilizer used without affecting sugarcane yield and sugar content, which is of great significance for mitigating the negative effects of long-term excessive use of nitrogen fertilizer.

[0167] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound preparation containing nitrogen-fixing bacteria, characterized in that, The mixture includes a compound microbial agent and biochar loaded with the compound microbial agent, wherein the compound microbial agent is composed of the following strains: *Azotobacter brasiliensis* (… Azospirillum brasilense ACCC04138, Bacillus mucilaginosus ( Bacillus mucilaginosus Krassilnikov ACCC 19749 and Pseudomonas fluorescens ( Pseudomonas fluorescens CICC 23919; In the compound microbial agent, the mass ratio of *Azotobacter brasiliensis*, *Bacillus mucilaginosus* and *Pseudomonas fluorescens* is (1-2):(0.5-2):(1-2), and the mass ratio of *Azotobacter brasiliensis* to the biochar is 1:(5-8).

2. The compound formulation as described in claim 1, characterized in that, The biochar includes bagasse and mushroom residue, and the mass ratio of bagasse to mushroom residue is (1-2):(1-2).

3. A method for preparing the compound microbial agent according to claim 1 or 2, characterized in that, Includes the following steps: The cultures of *Azotobacter brasiliensis*, *Bacillus mucilaginosus*, and *Pseudomonas fluorescens* were prepared by fermentation and culture, respectively. Sugarcane bagasse and mushroom residue are mixed and pyrolyzed at high temperature to produce biochar; After mixing the bacterial solutions of *Azotobacter brasiliensis* and *Bacillus mucilaginosus*, the mixture was sprayed onto the biochar and stirred intermittently for 1-2 hours. Then, *Pseudomonas fluorescens* bacterial solution was added and stirring was continued for 1-2 hours to obtain a compound bacterial agent.

4. The preparation method according to claim 3, characterized in that, The fermentation medium used to prepare *Azotobacter brasiliensis* and *Pseudomonas fluorescens* bacterial cultures was nutrient broth medium, while the fermentation medium used to prepare the nutrient broth medium was silicate bacteria medium.

5. The preparation method according to claim 3, characterized in that, The high-temperature pyrolysis temperature is 500℃ and the time is 3 hours.

6. The preparation method according to claim 3, characterized in that, The intermittent stirring conditions are as follows: after spraying the biochar with *Braziliana zoiformis* and *Bacillus mucilaginosus* solutions, stir at 50 rpm for 5 minutes, and then stir under the same conditions once every 20 minutes.

7. The preparation method according to claim 3, characterized in that, The optimal mass ratio of the *Azotobacter brasiliensis* bacterial suspension, *Bacillus mucilaginosus* bacterial suspension, and *Pseudomonas fluorescens* bacterial suspension is 1.8:1:1.

8. The optimal mass ratio of sugarcane bagasse to mushroom residue is 1.5:1; The optimal mass ratio of the *Braziliana nigrostriatus* bacterial solution to biochar is 1:

8.

8. The application of the compound formulation as described in claim 1 or 2 in improving the soil structure of sugarcane fields.

9. The application of the compound formulation as described in claim 1 or 2 in increasing sugarcane yield and sugar content.

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