A biological bacterial fertilizer and application thereof in adjusting microbial community structure in soil containing microplastics

Abstract

This invention discloses a bio-fertilizer and its application in adjusting the microbial community structure of soil containing microplastics. The bio-fertilizer's raw materials include microbial inoculants; the strains in the microbial inoculants include *Bacillus amyloliquefaciens*, *Bacillus belye*, and *Bacillus tropicalis*. This invention provides a method for adjusting the microbial community structure of microplastic-contaminated soil by adding organic bio-fertilizer to the soil. This method is environmentally friendly, sustainable, and highly efficient.

Description

Technical Field

[0001] This invention relates to the field of agricultural ecological technology, and more specifically, to a bio-fertilizer and its application in adjusting the microbial community structure in soil containing microplastics. Background Technology

[0002] Microplastics (MPs) generally refer to plastic fragments, particles, or fibers with a diameter or length of less than 5 mm. As a novel, persistent environmental pollutant posing a serious threat to soil ecosystems, they have gained increasing attention in recent years. Due to the complexity of soil media and the difficulty in separation and detection, current research on the impact of microplastics on soil ecosystems is limited, with discussions primarily focusing on aquatic systems such as oceans and lakes. However, due to the increasing use of landfills for waste disposal, various human activities, and the gradual development of agricultural and industrial technologies, the introduction of microplastics inevitably affects various functions of terrestrial ecosystems, while also impacting soil animals, plants, and microorganisms.

[0003] Soil microorganisms are the most important functional components of soil biota and a crucial group in soil biological communities. Microbial biomass and community structure are extremely sensitive to agricultural management practices, and the quantity and diversity of soil microorganisms are important biological indicators of soil fertility. Rhizosphere microorganisms refer to microorganisms that are tightly attached to soil particles in the rhizosphere. Their numbers are generally higher than those in non-rhizosphere soils. The composition and quantity of rhizosphere microorganisms vary among different plants. Microbial community structure refers to the biological community composed of fungi, bacteria, actinomycetes, and viruses in a certain area or volume of soil under the combined regulation of biotic and abiotic factors. Its floristic composition, population size, and biological activity are closely related to soil type, plant type, metabolism, and climate.

[0004] Beneficial microorganisms in soil are an important component of soil fertility, participating in the transformation of matter and energy, the formation and decomposition of organic matter, the release and fixation of nutrients, and biological nitrogen fixation. Therefore, the composition and activity of the soil microbial community are crucial for plant growth. Organic bio-fertilizers are a new type of biological "fertilizer" that has emerged in recent years. Because they can improve soil fertility, increase the number and activity of beneficial soil microorganisms, promote crop growth, and increase crop yield, their production and usage have increased significantly in recent years. Applying organic bio-fertilizers can promote the growth of fungi and bacteria; however, the effects of co-exposure to organic bio-fertilizers and microplastics on the structure of the plant rhizosphere microbial community have not been reported.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a bio-fertilizer and its application in adjusting the microbial community structure in soil containing microplastics. This bio-fertilizer can adjust the rhizosphere microbial community in soil containing microplastics, making the microbial community structure in the soil more rational.

[0007] This invention is implemented as follows:

[0008] On the one hand, the present invention provides a bio-fertilizer, the raw materials of which include microbial agents;

[0009] The strains in the microbial agent include Bacillus amyloliquefaciens, Bacillus belliesi, and Bacillus tropicalis; the viable count ratio of Bacillus amyloliquefaciens, Bacillus belliesi, and Bacillus tropicalis is 5-10:5-10:2-5.

[0010] In some embodiments, the total viable count of the above-mentioned bio-fertilizer is ≥2.0 × 10⁻⁶. 8 cfu / g.

[0011] In some embodiments, the strain number of the above-mentioned Bacillus amyloliquefaciens is ACCC 10225; the preservation number of the above-mentioned Bacillus belyss is CGMCC NO.20317; and the preservation number of the above-mentioned Bacillus tropicalis is CGMCC NO.24737.

[0012] In some embodiments, the raw materials of the above-mentioned bio-fertilizer also include humic acid powder, nitrogen fertilizer, phosphate fertilizer, potassium fertilizer, black gluten powder, and dry filler powder.

[0013] In some embodiments, the raw materials of the bio-fertilizer, by weight, include: 1-10 parts of microbial inoculant; 20-30 parts of humic acid raw powder; 1-15 parts of nitrogen fertilizer; 1-10 parts of phosphate fertilizer; 1-15 parts of potassium fertilizer; 1-10 parts of black viscous powder; and 20-30 parts of dry filler powder.

[0014] In some embodiments, the preparation method of the above-mentioned bio-fertilizer includes: crushing, mixing and granulating humic acid raw powder, nitrogen fertilizer, phosphorus fertilizer, potassium fertilizer, black gluten powder and filler dry powder in proportion, drying, adding microbial agent in proportion, and mixing to obtain bio-fertilizer.

[0015] In some embodiments, the preparation method of the above-mentioned microbial agent includes aerobic culture of Bacillus amyloliquefaciens, Bacillus belye, and Bacillus tropicalis at 25-35°C and pH 6.5-7.5 for 24-96 h. After activation and expansion, the microbial agent is coupled with an encapsulation material prepared from sodium alginate and calcium chloride. After coupling, the microbial agent is prepared by spray drying to form a composite microcapsule, which is the microbial agent. The encapsulation material includes sodium alginate and calcium chloride.

[0016] On the other hand, the present invention provides the application of the above-mentioned bio-fertilizer in adjusting the microbial community structure in soil containing microplastics.

[0017] In some embodiments, in soils containing microplastics, the proportion of microplastics in the soil is 0% w / w to 0.5% w / w (w / w is the dry weight ratio of microplastics to soil).

[0018] In some embodiments, the aforementioned microorganisms include bacteria and fungi.

[0019] In some embodiments, the bacteria mentioned above include *Kaistobacter*, *Pseudomonadaceae*, *Luteolibacter*, *Nitrospira*, *Flavisolibacter*, *Burkholderia*, *Bacillus*, *Rhodoplanes*, *Flavobacterium*, and *Candidatus*.

[0020] In some embodiments, the fungi mentioned above include Mortierella, Cunninghamella, Chaetomium, Amylomyces, Cercomonas, Spizellomyces, and Epipyxis.

[0021] In some embodiments, the microplastics described above include biodegradable microplastics.

[0022] In some embodiments, the materials of the biodegradable microplastics include at least one of polybutylene terephthalate (PBAT), polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate (PBSA), carbon dioxide copolymer (PPC), polylactic acid (PLA), polyhydroxyalkanoate (PHA), and starch plastics.

[0023] The present invention has the following beneficial effects:

[0024] (1) The bio-fertilizer of the present invention is a new organic bio-fertilizer prepared by screening specific microorganisms. This bio-fertilizer can not only provide the nutrients required for crop growth, but also reshape the soil rhizosphere microbial community. Compared with traditional organic fertilizers, the bio-fertilizer of the present invention has complete nutrients and low cost. The organic bio-fertilizer prepared has stable quality, high fertilizer efficiency and complete functions. Moreover, all the components contained are environmentally friendly products, which are in line with the concept of sustainable development.

[0025] (2) The bio-fertilizer of the present invention can adjust the soil microbial community and increase the proportion of beneficial bacteria in the soil. In particular, the bio-fertilizer of the present invention can be applied to soil containing microplastics and can resist the interference of low concentration of microplastics. This method has the characteristics of being environmentally friendly, sustainable and efficient. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The genus-level structure of soil bacterial communities under different levels of PBAT-MPs mixed with BF is shown; CK is the untreated control; BF is bio-fertilizer; MPs are PBAT-MPs.

[0028] Figure 2 The Chao1 index was used to compare bacterial α-diversity among different treatment groups; CK was the untreated control; BF was bio-fertilizer; MPs were PBAT-MPs.

[0029] Figure 3 The Shannon index was used to compare bacterial α-diversity among different treatment groups; CK was the untreated control; BF was bio-fertilizer; MPs were PBAT-MPs.

[0030] Figure 4 The nonmetric multidimensional scaling (NMDS) plot represents the corresponding stress values ​​at the OTU level for different bacterial groups; CK is the untreated control; BF is the bio-fertilizer; MPs are PBAT-MPs.

[0031] Figure 5 The genus-level structure of soil fungal communities under different levels of PBAT-MPs mixed with BF;

[0032] Figure 6 The Chao1 index is used to compare fungal α-diversity among different treatment groups;

[0033] Figure 7 The Shannon index is used to compare fungal α-diversity among different treatment groups;

[0034] Figure 8 Non-metric multidimensional scaling (NMDS) plots and corresponding stress values ​​at OTU levels for different fungal groups; CK is the untreated control; BF is bio-fertilizer; MPs are PBAT-MPs;

[0035] Figure 9 Redundancy analysis (RDA) was performed between the relative abundance of bacterial (a) and fungal (b) genera and soil physicochemical parameters, soil enzyme activities, and plant biomass; red arrows represent soil physicochemical parameters (TN, total nitrogen; TP, total phosphorus; TK, total potassium; AHN, alkaline hydrolyzable nitrogen; AP, available phosphorus; AK, available potassium; SOC, soil organic carbon; OM, organic matter); blue arrows represent soil enzyme activities (ACP, acid phosphatase; CAT, catalase; CE, cellulase; DEH, dehydrogenase; INE, sucrase; URE, urease); green arrows represent plant biomass (AGB, aboveground biomass; BGB, belowground biomass).

[0036] Figure 10 The correlations between dominant soil (a) bacteria and (b) fungi (genus level) and soil physicochemical parameters, soil enzyme activity, and plant biomass are shown; the insets to the right of (a) and (b) are color intervals for different Pearson's r-values; *, **, and *** indicate statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively, with abbreviations as described above. Figure 9 . Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038] This invention provides a bio-fertilizer, the raw materials of which include microbial agents, humic acid powder, nitrogen fertilizer, phosphate fertilizer, potassium fertilizer, black gluten powder and dry filler powder.

[0039] Specifically, by weight, the raw materials of bio-fertilizer include: 20-30 parts of humic acid raw powder; 1-10 parts of microbial agent; 1-15 parts of nitrogen fertilizer; 1-10 parts of phosphate fertilizer; 1-15 parts of potassium fertilizer; 1-10 parts of black gluten powder; and 20-30 parts of dry filler powder.

[0040] In this invention, the total number of viable bacteria in the bio-fertilizer is ≥2.0×10⁻⁶. 8 cfu / g.

[0041] The strains in the microbial agent are selected from Bacillus amyloliquefaciens, Bacillus belliesi, and Bacillus tropicalis; the ratio of viable bacteria of Bacillus amyloliquefaciens, Bacillus belliesi, and Bacillus tropicalis is 5-10:5-10:2-5.

[0042] Preferably, the strain number of Bacillus amyloliquefaciens is ACCC 10225; the preservation number of Bacillus belyss is CGMCC NO.20317; and the preservation number of Bacillus tropicalis is CGMCC NO.24737.

[0043] The aforementioned Bacillus amyloliquefaciens, Bacillus belyssus, and Bacillus tropicalis are all existing strains, disclosed in patents CN201210219470.8, CN202110072888.X, and CN202210964216.4 respectively, and can all be obtained through purchase or donation.

[0044] In addition, in order to provide sufficient nutrients for crops, the bio-fertilizer of the present invention also includes humic acid raw powder, nitrogen fertilizer, phosphorus fertilizer, potassium fertilizer, black glutinous powder and dry filler powder.

[0045] As for the type of nitrogen fertilizer, it can be ammonium nitrogen fertilizer, nitrate nitrogen fertilizer, urea nitrogen fertilizer, or other types of nitrogen fertilizer; the phosphate fertilizer can be monoammonium phosphate, diammonium phosphate, phosphate fertilizer, double-burned phosphate fertilizer, calcium magnesium phosphate fertilizer, superphosphate, superphosphate, granular phosphate fertilizer, enriched superphosphate, ammonium phosphate, white phosphate fertilizer, light calcium phosphate, or other types of phosphate fertilizer; the potassium fertilizer can be potassium nitrate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride, or other types of potassium fertilizer. The specific choice of nitrogen, phosphate, and potassium fertilizer can be made according to actual needs, and this invention does not limit this.

[0046] By preparing the aforementioned specific microbial strains into microbial agents, and then compounding them with humic acid powder, nitrogen fertilizer, phosphorus fertilizer, potassium fertilizer, black glutinous powder, and filler dry powder, bio-fertilizers can not only provide the nutrients needed for crop growth, but also reshape the soil rhizosphere microbial community, making the microbial community more rational.

[0047] As a general technical concept, the present invention also provides a method for preparing the above-mentioned bio-fertilizer, the specific steps of which are as follows:

[0048] (1) Preparation of microbial inoculants:

[0049] The selected Bacillus amyloliquefaciens, Bacillus belye, and Bacillus tropicalis were cultured aerobically, activated, and fermented. Then, they were coupled with the encapsulation material and prepared into composite microcapsules by spray drying for later use.

[0050] The aerobic culture conditions are as follows: culture at 25~35℃ and pH=6.5~7.5 for 24-96h; the embedding material is prepared from sodium alginate and calcium chloride. The reason for using sodium alginate and calcium chloride is that sodium alginate can quickly undergo ion exchange with calcium ions to form a gel.

[0051] (2) The humic acid raw powder, nitrogen fertilizer, phosphate fertilizer, potassium fertilizer, black gluten powder and filler dry powder are crushed and mixed in proportion, then granulated and dried. Then microbial agents are added in proportion and mixed in proportion to obtain bio-fertilizer.

[0052] Experiments have proven that the bio-fertilizer of this invention can adjust the microbial community structure of soil containing microplastics. The microorganisms in this invention include bacteria and fungi. The bacteria include *Kaistobacter*, *Pseudomonadaceae*, *Luteolibacter*, *Nitrospira*, *Flavisolibacter*, *Burkholderia*, *Bacillus*, *Rhodoplanes*, *Flavobacterium*, and *Candidatus*. The fungi include *Mortierella*, *Cunninghamella*, *Chaetomium*, *Amylomyces*, *Cercomonas*, *Spizellomyces*, and *Epipyxis*.

[0053] In soils containing microplastics, the proportion of microplastics in the soil is 0% w / w to 0.5% w / w (w / w is the dry weight ratio of microplastics to soil).

[0054] The material of the microplastic is selected from at least one of polybutylene terephthalate (PBAT), polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate (PBSA), carbon dioxide copolymer (PPC), polylactic acid (PLA), polyhydroxyalkanoate (PHA), and starch plastic.

[0055] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0056] The strain of *Bacillus amyloliquefaciens* used in this invention embodiment has the strain number ACCC 10225; the preservation number of *Bacillus belyssus* is CGMCC NO.20317; and the preservation number of *Bacillus tropicalis* is CGMCC NO.24737. The biodegradable plastic polybutylene adipate terephthalate (PBAT) was purchased from Xinjiang Lanshan Tunhe Polyester Co., Ltd.

[0057] Example 1

[0058] This embodiment provides a bio-fertilizer, the raw material composition of which is as follows:

[0059] 20 parts humic acid raw powder; 10 parts microbial inoculant; 15 parts nitrogen fertilizer; 10 parts phosphate fertilizer; 15 parts potassium fertilizer; 10 parts black viscous powder; 20 parts dry filler powder.

[0060] The viable bacteria count of the bio-fertilizer is 2.0 × 10⁻⁶. 8 cfu / g. Among them, the viable count ratio of Bacillus amyloliquefaciens, Bacillus belyssus, and Bacillus tropicalis was 10:10:5.

[0061] The above-mentioned bio-fertilizer is prepared by the following method:

[0062] S1. Selected Bacillus amyloliquefaciens, Bacillus belye, and Bacillus tropicalis were cultured aerobically for 48 hours at 25℃ and pH=6.5 to activate and expand the fermentation, thus producing useful strains for industrial fermentation. Then, they were coupled with sodium alginate and ion gel and spray-dried to prepare composite microcapsules for later use.

[0063] S2. Nitrogen fertilizer, phosphorus fertilizer, potassium fertilizer, black gluten powder, humic acid raw powder and filler powder are pulverized, mixed, granulated in a rotary drum, and dried at low temperature according to the proportion, and added to the complex microbial agent microcapsules obtained in S1. The mixture is thoroughly mixed and stirred evenly to obtain the organic biological microbial fertilizer.

[0064] Example 2

[0065] This embodiment provides a bio-fertilizer, the specific preparation method of which is the same as that in Embodiment 1, the difference being the different raw material composition of the bio-fertilizer, which is as follows:

[0066] 20 parts humic acid raw powder; 5 parts microbial inoculant; 15 parts nitrogen fertilizer; 10 parts phosphate fertilizer; 15 parts potassium fertilizer; 10 parts black viscous powder; 25 parts dry filler powder.

[0067] Example 3

[0068] This embodiment provides a bio-fertilizer, the specific preparation method of which is the same as that in Embodiment 1, the difference being the different raw material composition of the bio-fertilizer, which is as follows:

[0069] 20 parts humic acid raw powder; 1 part microbial agent; 15 parts nitrogen fertilizer; 10 parts phosphate fertilizer; 15 parts potassium fertilizer; 10 parts black viscous powder; 29 parts dry filler powder.

[0070] Example 4

[0071] This embodiment provides a bio-fertilizer. The specific preparation method is the same as in Example 1. The difference is that the ratio of viable bacteria of Bacillus amyloliquefaciens, Bacillus belyssus, and Bacillus tropicalis in the microbial agent is different. The ratio in this embodiment is 5:10:5.

[0072] Example 5

[0073] This embodiment provides a bio-fertilizer. The specific preparation method is the same as in Example 1. The difference is that the ratio of viable bacteria of Bacillus amyloliquefaciens, Bacillus belyssus, and Bacillus tropicalis in the microbial agent is different. The ratio in this embodiment is 10:5:5.

[0074] Comparative Example 1

[0075] The difference from Example 1 is that it does not contain microbial inoculants.

[0076] Experimental Example 1

[0077] Fertilizer efficacy tests were conducted on Examples 1-5 and Comparative Example 1, as detailed below:

[0078] The bio-fertilizers from Examples 1-5 and Comparative Example 1 were added to the soil at a rate of 0.5% w / w and mixed thoroughly. The experimental crop was Shanghai bok choy. The experiment was conducted using pot cultivation, with each pot measuring 230mm × 180mm and containing 6kg of soil. Six pots were used as replicates per group. Shanghai bok choy seeds were disinfected twice with 75% ethanol for 2 minutes each time, followed by washing in sterile distilled water for 2 minutes. After germination, three seedlings with uniform growth and robust root systems at the four-leaf-one-heart stage were selected for transplanting. Basic indicators such as chlorophyll content, plant height, and total dry weight were measured for each group after 28 days. Specific results are shown in Table 1 below. Table 1 also includes a blank control group, which represents the performance of Shanghai bok choy without fertilizer application.

[0079] Table 1. Comparison of chlorophyll content, plant height, total dry weight, and yield increase of Shanghai bok choy under different treatments.

[0080]

[0081] Note: Different lowercase letters in the same row indicate significant differences between different treatments (P<0.05).

[0082] As can be seen from Table 1, compared with Shanghai bok choy without bio-fertilizer, the chlorophyll, plant height and total dry weight of the bok choy treated with bio-fertilizers from Examples 1-5 were significantly increased. Compared with the control group, the chlorophyll, plant height and total dry weight of the bok choy treated with bio-fertilizers from Examples 1-5 were also increased. This indicates that the bio-fertilizers from Examples 1-5 have good fertilizer effects, among which Example 1 has the best fertilizer effect and is the optimal formula.

[0083] Experimental Example 2

[0084] I. Verification of adjusting the rhizosphere microbial community by combining the bio-fertilizer of Example 1 with microplastics, the steps are as follows:

[0085] 1. Soil Treatment: The test soil was selected from farmland soil in Guangming District, where plastic products have never been used. The experiment used pot cultivation, with a size of 230mm × 180mm, and 6kg of soil per pot (the specific amount may vary depending on actual conditions). Basic physicochemical properties of the soil (total nitrogen, phosphorus, potassium, pH, organic matter, etc.) were confirmed. Three treatment groups were used: BF (0.5% w / w) and PBAT-MPs (0.1% w / w), BF (0.5% w / w) and PBAT-MPs (0.5% w / w), and BF (0.5% w / w) and PBAT-MPs (2.5% w / w). Only BF (0.5% w / w) was applied. Each group was replicated in 6 pots. BF was the microbial fertilizer corresponding to Example 1, and CK was the control group.

[0086] 2. Test crop: Shanghai bok choy, a common economic crop with a growth period of about 20-30 days; Shanghai bok choy seeds were disinfected twice with 75% ethanol for 2 minutes each time, and then washed in sterile distilled water for 2 minutes. After germination, 3 seedlings with uniform growth and strong root system at the four-leaf-one-heart stage were selected for transplanting.

[0087] 3. The microplastic is a biodegradable plastic, polybutylene adipate terephthalate (PBAT).

[0088] 4. Microplastic treatment: The biodegradable plastic polybutylene adipate terephthalate (PBAT) microplastics are crushed in a pulverizer, and the crushed microplastics are sieved through a 100-mesh sieve to obtain microplastic powder.

[0089] 5. Cultivation cycle: The cultivation cycle is 28 days. During this process, the soil moisture content is maintained at 60% to 80% of the field capacity of the soil using the weighing method.

[0090] 6. DNA Extraction from Rhizosphere Microorganisms: After maturity, the plants were removed from the soil, and the roots were separated from the attached soil. Loose soil adhering to the root surface was shaken off, leaving approximately 1 mm of soil on the root surface as the rhizosphere soil. The plant root zone sample was placed in a 50 mL tube containing 30 mL of sterile phosphate-buffered saline (PBS) solution and vigorously stirred to remove all rhizosphere soil from the root surface. The rhizosphere soil was concentrated by centrifugation at 10000g for 30 seconds. The plant root zone sample without rhizosphere soil was washed twice again with sterile PBS solution to remove any visible attached soil. Any large matrix particles attached to the roots were manually removed using sterile forceps. Soil samples were collected from six pots for each treatment (n = 6). DNA was extracted from the rhizosphere soil using the FastDNA™ Spin Kit according to the manufacturer's instructions and eluted with 50 μL of DES. DNA was extracted from the plant root zone samples using the CTAB method. The DNA concentration and mass of the samples were measured using a Nanodrop spectrophotometer.

[0091] II. Analysis of Test Results:

[0092] 1. High-throughput sequencing data analysis

[0093] High-throughput sequencing was performed on the V3-V4 regions of the 16S rRNA gene and the V4 region of the 18S rRNA gene of *Clerodendrum chinense* rhizosphere bacteria. After quality control and sequence filtering, a total of 2,615,647 16S rRNA sequences and 2,505,903 18S rRNA sequences were recorded. After optimization of the original sequences based on 97% similarity, they were clustered into 17,607 bacterial OTUs and 6,658 fungal OTUs, respectively. Shannon and Chao1 indices were used to characterize the diversity and richness of the microbial community, respectively. Non-metric multidimensional scaling (NMDS) was used to analyze whether there were significant differences in the microbial community structure and composition among the treatment groups. Redundancy analysis (RDA) was used to reveal potential associations between the microbial community and related environmental factors.

[0094] 2. Results of bacterial community analysis

[0095] (1) Analysis of the effects of applying the microbial fertilizer corresponding to Example 1 alone and co-treatment with microbial fertilizer and PBAT-MPs on the phylum-level community of rhizosphere bacteria in Shanghai bok choy.

[0096] Table 1. Rhizosphere soil bacterial phylum horizontal community composition under different treatments

[0097]

[0098] After applying the microbial fertilizer corresponding to Example 1 (Table 1), compared with the control (CK), the dominant bacterial communities at the rhizosphere soil bacterial level of *Boletus edulis* increased by 1.09%, 10.91%, 48.66%, 21.17%, and 5.57%, respectively, for Proteobacteria, Bacteroidetes, Verrucomicrobia, TM7 (glycobacteria), and Actinobacteria. Among these, Verrucomicrobia and TM7 became the dominant bacteria. Some Verrucomicrobia phyla made significant contributions to the degradation of polysaccharides and xylan. Their genomes encode various glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases, and esterases, possessing mechanisms for hydrolyzing various polysaccharides. This demonstrates that the microbial fertilizer of this invention shaped the rhizosphere soil bacterial community of *Boletus edulis*.

[0099] Different proportions of PBAT-MPs in the soil have varying effects on the rhizosphere bacterial community mediated by microbial fertilizer. Compared to applying microbial fertilizer alone, the addition of 0.1% w / w PBAT-MPs increased the relative abundance of Gemmatimonadetes, Verrucomicrobia, and Actinobacteria by 10.37%, 37.80%, and 33.37%, respectively, while decreasing the relative abundance of Chloroflexi by 17.44%. Verrucomicrobia and Actinobacteria are the dominant bacteria; this indicates that the addition of low concentrations of PBAT-MPs does not affect the dominant position of Verrucomicrobia and has no significant impact on the composition of the rhizosphere soil bacterial community shaped by microbial fertilizer. The 0.5% w / w PBAT-MPs treatment mainly increased the relative abundance of TM7 saccharobacteria by 81.42%, making TM7 the dominant bacteria. This demonstrates that the presence of 0.1% w / w PBAT-MPs in the soil did not significantly alter the dominant bacterial community in the rhizosphere of Shanghai bok choy. The application of the microbial fertilizer corresponding to Example 1 can still effectively adjust the microbial structure in the soil containing 0.1% w / w PBAT-MPs.

[0100] The highest addition (2.5% w / w PBAT-MPs) treatment significantly affected the rhizosphere soil microenvironment shaped by microbial fertilizer, with decreases of 44.66%, 40.54%, 32.89%, 28.92%, 12.82%, and 9.68% in the following phyla: Gemmatimonadetes, Chloroflexi, Nitrospirae, Bacteroidetes, Acidobacteria, and Actinobacteria, respectively. This demonstrates that 2.5% w / w PBAT-MPs in the soil affects the microbial community shaped by microbial fertilizer, resulting in significant changes in community composition.

[0101] (2) Analysis of the effects of applying the microbial fertilizer corresponding to Example 1 alone and co-treatment with microbial fertilizer and PBAT-MPs on the microbial community of Shanghai bok choy rhizosphere.

[0102] Shanghai green rhizosphere soil bacteria at the genus level ( Figure 1 After applying microbial fertilizer, the dominant bacterial colonies were: Luteolibacter Bacteria genus, Bacillus genus Bacillus , Ramlibacter genus *Bacteria*, genus *Flavobacterium* Flavobacterium Among them, Bacillus spp. Bacillus The significant increase in the content (135.06%) proves the successful colonization of the microbial fertilizer; while Luteolibacter Fungi can participate in the decomposition and utilization of organic matter by plants, and can also have antagonistic effects against certain fungal diseases of plants; while Ramlibacter This genus is also classified as a common beneficial group of plants, and studies have shown through isolation and culture that... Ramlibacter These bacteria are important degrading bacteria.

[0103] If the soil contains 0.1% w / w PBAT-MPs, after adding the microbial fertilizer from Example 1, Luteolibacter Fungi and Ramlibacter The dominant bacterial genus is *Bacteria*; however, if the soil contains 0.5% w / w PBAT-MPs, Ramlibacter Fungi Flavisolibacter Fungi Candidatus_Koribacter The dominant bacterial genus was observed, and the dominant community changed somewhat compared to the minimum addition ratio, but... Ramlibacter The proportion of microorganisms remains relatively large. This indicates that the microbial fertilizer of Example 1 can resist microplastic interference at a concentration of 0.1% - 0.5% w / w PBAT-MPs and can still regulate the bacterial structure in the soil.

[0104] The dominant colonies in the 2.5% w / w PBAT-MPs treatment group were Kaistobacter Genus: Pseudomonas Pseudomonadaceae_Pseudomonas, Luteolibacter The dominant bacterial species showed significant changes compared to other addition ratios. This indicates that a high addition ratio of PBAT-MPs significantly affected the dominant bacterial community in the rhizosphere soil of *Agrocybe aizoon* shaped by microbial fertilizer.

[0105] (3) Analysis of α-diversity of microbial community in rhizosphere bacteria of Shanghai bok choy under the conditions of applying the microbial fertilizer corresponding to Example 1 alone and co-treatment with microbial fertilizer and PBAT-MPs.

[0106] Alpha diversity of the control group (CK), the microbial fertilizer treatment group (BF), and different proportions of PBAT-MPs co-existing with microbial fertilizer (BF+0.1% MPs, BF+0.5% MPs, BF+2.5% MPs) was analyzed based on 97% sequence similarity using OTUs. The Chao1 and Shannon indices of rhizosphere bacteria diversity in each treatment were also assessed. The results showed that ( Figure 2 , Figure 3 Under low-dose PBAT-MPs treatment (<2.5% w / w), the bacterial diversity and species richness of the rhizosphere soil in Shanghai bok choy showed no significant changes. This also verifies that microbial fertilizers can adjust the rhizosphere soil bacterial community even with low PBAT-MPs concentrations.

[0107] However, the Chao1 and Shannon indices of the treatment with 2.5% w / w PBAT-MPs co-added with microbial fertilizer were significantly lower than those of other treatment groups, indicating that a high proportion of PBAT-MPs can significantly reduce bacterial diversity and species richness in the rhizosphere soil of Shanghai green plants, while also affecting the effectiveness of microbial fertilizer.

[0108] (4) β-diversity analysis of the rhizosphere bacterial community of Shanghai bok choy under the following conditions: application of the microbial fertilizer corresponding to Example 1 alone and co-treatment with the microbial fertilizer and PBAT-MPs.

[0109] For β diversity, NMDS analysis showed relatively clear clustering patterns of bacterial communities in each treatment group. Figure 4 The communities belonging to the same treatment group had similar compositions and clustered into one group (PERMANOVA, F = 5.3715, P = 0.001). It should be noted that the confidence ellipse of the 2.5% w / w PBAT-MPs coexisting with the microbial fertilizer did not overlap with other treatment groups, indicating that the bacterial community under this treatment was relatively independent.

[0110] 3. Results of fungal community analysis

[0111] (1) Analysis of the effects of applying the microbial fertilizer corresponding to Example 1 alone and co-treatment with microbial fertilizer and PBAT-MPs on the phylum-level community of rhizosphere fungi in Shanghai bok choy.

[0112] Table 2. Rhizosphere soil fungal community composition under different treatments

[0113]

[0114] After applying the microbial fertilizer corresponding to Example 1 (Table 2), compared with the control (CK), the dominant fungal communities at the rhizosphere soil level in Shanghai green plants increased by 53.33%, 62.17%, and 81.61% for Cercozoa, Diatomea, and Annelida, respectively. Cercozoa, Diatomea, and Annelida became the dominant fungi.

[0115] Different proportions of PBAT-MPs in the soil have varying effects on the rhizosphere bacterial community mediated by microbial fertilizers. With the addition of 0.1% w / w PBAT-MPs, compared to applying microbial fertilizer alone, Mucoromycota, Ciliophora, and Cercozoa became the dominant bacteria. The 0.5% w / w PBAT-MPs treatment primarily increased the relative abundance of Ciliophora by 75.97%, making Ciliophora the dominant bacterial group.

[0116] The highest addition (2.5% w / w PBAT-MPs) treatment significantly affected the soil environment improved by the microbial fertilizer, with the relative abundance of Ascomycota, Chytridiomycota, Diatomae, and Cnidaria decreasing by 19.81%, 30.04%, 66.63%, and 13.44%, respectively. Conversely, the abundance of Mucoromycota, Ciliophora, Annelida, and Ochrophyta increased by 42.16%, 66.99%, 188.70%, and 26.45%, respectively.

[0117] (2) Analysis of the effects of applying the microbial fertilizer corresponding to Example 1 alone and co-treatment with microbial fertilizer and PBAT-MPs on the microbial community of Shanghai bok choy rhizosphere fungi.

[0118] After applying microbial fertilizer, the dominant fungal colonies in the rhizosphere soil of Shanghai cypress were significantly different from those in control (CK). Figure 5 ), manifested as Chaetomium genus Chaetomium Increased by 96.21%, *Ceratophyllum* genus Cercomonas The content increased by 54.50%. Chaetomium has potential biocontrol effects against many plant pathogens and is considered to play an important role in regulating soil nutrient balance. The significant increase in Chaetomium content with the application of microbial fertilizer indicates that the fertilizer increased the content of related beneficial microorganisms.

[0119] If the soil contains 0.1% w / w PBAT-MPs, *Cannabis* spp. Cunninghamella Starch molds Amylomyces The dominant colony. If the soil contains 0.1% w / w PBAT-MPs, *Cannabis* spp. Cunninghamella The relative abundance was further increased, making it the dominant bacteria in this treatment; and this bacteria has a promoting effect on the effective prevention and control of plant and fungal soil-borne diseases; thus proving that the microbial fertilizer of the present invention can resist low concentrations of microplastic interference and still regulate the fungal structure in the soil.

[0120] In the 2.5% w / w PBAT-MPs treatment group, the dominant fungal colonies in the rhizosphere soil changed significantly, with the order Moniliales (palpitates) becoming more prominent. Haplotaxida , Pseudoplatyophrya Fungi became the dominant community. It is worth noting that... Mortierella The relative abundance of this fungus was significantly lower than that of other PBAT-MPs-added groups. This fungus, as an oil-producing humic fungus, has various agricultural benefits, such as promoting crop growth and inhibiting soil-borne pathogens. This indicates that a high proportion of PBAT-MPs added has a significant impact on beneficial fungal colonies in the rhizosphere.

[0121] (3) Analysis of α-diversity of microbial community in rhizosphere fungi of Shanghai bok choy under the conditions of applying the microbial fertilizer corresponding to Example 1 alone and co-treatment with microbial fertilizer and PBAT-MPs.

[0122] Alpha diversity of the control group (CK), the microbial fertilizer treatment group (BF), and different proportions of PBAT-MPs co-existing with microbial fertilizer (BF+0.1% MPs, BF+0.5% MPs, BF+2.5% MPs) was analyzed based on 97% sequence similarity using OTUs. The Chao1 and Shannon indices of rhizosphere fungi diversity in each treatment were also assessed. The results showed that ( Figure 6 , Figure 7 Under the microbial fertilizer treatment group and the low-dose PBAT-MPs treatment (<2.5% w / w), there were no significant changes in bacterial diversity and species richness in the rhizosphere soil of *Agropyron chinense*. Only the Chao1 index of the treatment with 2.5% w / w PBAT-MPs and microbial fertilizer was significantly higher than that of the control group, indicating that the high proportion of PBAT-MPs and microbial fertilizer can significantly increase the fungal diversity of the rhizosphere soil of *Agropyron chinense*.

[0123] (4) β-diversity analysis of the microbial community of Shanghai bok choy rhizosphere fungi under the conditions of applying the microbial fertilizer corresponding to Example 1 alone and co-treatment with microbial fertilizer and PBAT-MPs.

[0124] For β-diversity, NMDS analysis showed that the confidence ellipses under the five treatment groups almost completely overlapped, indicating that the fungal communities under these treatments might be similar (p<0.01; Figure 8). However, PERMANOVA results revealed significant differences in β-diversity among the five treatments (PERMANOVA, F = 1.499, P<0.05). The results indicate that the differences within treatment groups were greater than the differences between treatment groups.

[0125] 4. Correlation analysis between rhizosphere microbiota and environmental factors in Shanghai juvenile deciduous vine.

[0126] RDA analysis revealed the relationship between bacterial and fungal community structure and soil properties, enzyme activity, and plant biomass. The results of bacterial community analysis showed that the RDA had a first axis value of 0.15 and a second axis value of 0.14, indicating that these two axes could explain approximately 29% of the total variation in the soil bacterial community (Figure 9a). For soil fungi, the first two axes of the RDA explained 12.9% and 10.6% of the total variation, respectively (Figure 9b). Among environmental factors, pH, total nitrogen (TN), soil organic carbon (SOC), organic matter (OM), acid phosphatase (ACP), sucrase (INE), cellulase (CE), and plant underground biomass (BGB) were closely correlated with the microbial community, based on axis length and angle (p<0.05).

[0127] Spearman correlation heatmaps further assessed the relationship between abundant bacterial genera and environmental factors. For bacterial communities, soil organic carbon, organic matter, and sucrase were the three major environmental factors with significant influence, and they were significantly correlated with most dominant genera (Fig. 10a, p < 0.01). Other genera, such as Bacillus, were also significantly correlated. Bacillus It is mainly affected by a significant negative correlation with soil organic carbon, organic matter, sucrase, and pH. Meanwhile, the genus *Rhodotorula*... Rhodoplanes It was also negatively correlated with cellulase, but significantly positively correlated with plant underground biomass (p<0.01). Furthermore, Ramlibacter Fungal genera were significantly positively correlated with three major environmental factors, as well as pH, total nitrogen, and cellulase, and significantly negatively correlated with plant underground biomass (p<0.01). For fungal communities, the correlation between microorganisms and their environmental factors was mainly positive (Fig. 10b). Soil organic carbon and organic matter were the main environmental factors affecting the relative abundance of fungal communities, with only a few fungal genera showing no correlation. The relative abundance of other fungal taxa, such as... Bromeliothrix The fungal genus was also positively correlated with sucrase (p<0.05), but significantly negatively correlated with underground biomass (p<0.001). Furthermore, it was also found that... Mycamoeba The content of alkaline nitrogen (AHN) was significantly negatively correlated with the content of bacterial genus (p<0.05).

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a bio-fertilizer in adjusting the microbial community structure in soil containing microplastics, characterized in that, The raw materials of the bio-fertilizer, by weight, include: 1-10 parts of microbial agent; 20-30 parts of humic acid raw powder; 1-15 parts of nitrogen fertilizer; 1-10 parts of phosphate fertilizer; 1-15 parts of potassium fertilizer; 1-10 parts of black viscous powder; and 20-30 parts of dry filler powder. The strains in the microbial agent include Bacillus amyloliquefaciens, Bacillus belyssus, and Bacillus tropicalis; the viable count ratio of Bacillus amyloliquefaciens, Bacillus belyssus, and Bacillus tropicalis is 5-10:5-10:2-5; The strain number of Bacillus amyloliquefaciens in the microbial agent is ACCC 10225; the preservation number of Bacillus belyss is CGMCC NO.20317; and the preservation number of Bacillus tropicalis is CGMCC NO.24737. The microorganisms include bacteria and fungi; The bacteria include *Kaistobacter*, *Pseudomonadaceae*, *Luteolibacter*, *Nitrospira*, *Flavisolibacter*, *Burkholderia*, *Bacillus*, *Rhodoplanes*, *Flavobacterium*, and *Candidatus*. The fungi include Mortierella, Cunninghamella, Chaetomium, Amylomyces, Cercomonas, Spizellomyces, and Epipyxis.

2. The application according to claim 1, characterized in that, The total number of viable bacteria in the bio-fertilizer is ≥2.0×10⁸ cfu / g.

3. The application according to claim 1, characterized in that, The preparation method of the bio-fertilizer includes: crushing, mixing and granulating the humic acid raw powder, nitrogen fertilizer, phosphorus fertilizer, potassium fertilizer, black gluten powder and filler dry powder in proportion, drying and then adding the microbial agent in proportion, and mixing well to obtain the bio-fertilizer.

4. The application according to claim 1, characterized in that, The preparation method of the microbial agent includes aerobic culture of Bacillus amyloliquefaciens, Bacillus belye, and Bacillus tropicalis at 25-35°C and pH 6.5-7.5 for 24-96 hours. After activation and expansion, the microbial agent is coupled with an embedding material prepared from sodium alginate and calcium chloride. After coupling, the microbial agent is prepared into a composite microcapsule by spray drying, which is the microbial agent.

5. The application according to claim 1, characterized in that, In the soil containing microplastics, the proportion of microplastics in the soil is 0% w / w to 0.5% w / w (w / w is the dry weight ratio of microplastics to soil).

6. The application according to claim 5, characterized in that, The microplastics are biodegradable microplastics.

7. The application according to claim 6, characterized in that, The biodegradable microplastics are made of at least one of the following materials: polybutylene terephthalate (PBAT), polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate (PBSA), carbon dioxide copolymer (PPC), polylactic acid (PLA), polyhydroxyalkanoate (PHA), and starch plastics.

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