Paenibacillus mucilaginosus JZ1 and application thereof

Abstract

The invention belongs to the field of microorganisms, and particularly discloses paenibacillus mucilaginosus JZ1 and application thereof. According to the invention, paenibacillus mucilaginosus with multifunctional characteristics is screened from biological crust. The paenibacillus mucilaginosus JZ1 strain obtained by the invention has multiple functions of dissolving phosphorus, dissolving potassium, fixing nitrogen, and producing IAA, ACC deaminase, siderophore and extracellular polymeric substance (ESP), is an environment-friendly biological material, and does not cause environmental pollution. The bacterial strain is combined with the crust algae for application, so that the effects of promoting plant growth and improving soil can be stably exerted. The method has a relatively great application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a mucilaginous Bacillus JZ1 and its applications. Background Technology

[0002] Land desertification refers to land degradation in arid, semi-arid, and sub-humid arid regions. Desertification is one of the main types of desertification. It occurs when human activities disrupt fragile ecosystems on sandy surfaces under arid and windy conditions, resulting in land degradation primarily driven by wind and sand activity. The harms of desertification are mainly manifested in the following aspects: damage to land resources and reduction of human living space; destruction of production and living facilities, seriously threatening the safety of villages, towns, transportation, water conservancy, industrial and mining facilities, and national defense bases, affecting industrial and agricultural production, and restricting economic development; and deterioration of biological quality and reduction in species abundance, posing a threat to biodiversity.

[0003] Today, after half a century of desertification control efforts, my country has achieved significant results in transforming and managing deserts. Summarizing my country's experience in desertification control over the past half-century, afforestation and grass planting have been the main methods and approaches, yielding substantial results in practice. However, not all deserts can be controlled through these methods. Studies have shown that the vegetation cover of vascular plants in arid desert areas is ≤30%, while the cover of biological crusts in desert areas is ≥70%. Therefore, biological crusts are absolutely dominant in arid desert regions, holding particular significance for desertification prevention and control.

[0004] Soil crusts can generally be divided into physical soil crusts and biological soil crusts. Physical soil crusts (simply called soil crusts) usually refer to a hard crust formed on the soil surface due to the blockage of pores caused by external forces such as rainwater and runoff. Biological soil crusts (simply called biological crusts) refer to a complex biological soil layer formed by a very thin layer of soil composed of organisms such as bacteria, fungi, actinomycetes, algae, lichens, and bryophytes.

[0005] Furthermore, bacteria, actinomycetes, and fungi are widely distributed in biocrusts, serving as important components and pioneer species and a reservoir of numerous functional microorganisms. Current research on microorganisms in biocrusts is still in its early stages. Most studies utilize high-throughput sequencing to understand the taxonomic information of microorganisms at different developmental stages, while research on screening for multifunctional beneficial microorganisms from biocrusts is rarely reported.

[0006] Due to the strong resistance of their spores, spore-producing bacteria have a broader prospect for development and application as microbial agents. In particular, *Bacillus mucilaginosus* is often added as a strain in microbial fertilizers. For example, invention patent (application number 201610341875.7) discloses the effect of *Bacillus mucilaginosus* strain N6 in promoting corn growth and development and apple coloring rate; invention patent (application number: 202210717763.2) discloses a *Bacillus mucilaginosus* strain HB-02 with phosphorus-solubilizing, potassium-solubilizing, nitrogen-fixing, and IAA-producing abilities, which can promote crop production and increase crop yield. *Bacillus mucilaginosus* also contains a large capsule, which has the potential to bind and stabilize the soil. However, studies on isolating *Bacillus mucilaginosus* from biocrusts and investigating its characteristics and effects are still rare. Furthermore, there are no reports on the combined application effects of *Bacillus mucilaginosus* and biocrust algae. Summary of the Invention

[0007] Based on the urgent need for desertification control in my country, and considering the widespread presence of biocrusts in deserts and the diversity of microorganisms within them, this invention screens out mucilaginous Bacillus species with multifunctional characteristics from biocrusts and applies them in combination with biocrust algae to obtain the optimal bacteria-algae combination for ecological restoration of degraded land, ultimately achieving soil improvement and promoting plant growth.

[0008] This invention provides a mucilaginosus-like bacterium JZ1, with accession number CGMCC No. 28359, which was deposited on September 5, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, postal code: 100101), and classified as Paenibacillus mucilaginosus.

[0009] The present invention also provides the application of the aforementioned gelatinous Bacillus JZ1 in phosphorus solubilization, potassium solubilization and / or nitrogen fixation.

[0010] This invention also provides the application of the aforementioned gelatinous Bacillus JZ1 in the production of IAA, siderophores, and / or ACC deaminases.

[0011] This invention further provides a method for increasing the viable count of the aforementioned Bacillus spp. JZ1, which is achieved by mixing the Bacillus spp. JZ1 bacterial solution with a green algae solution.

[0012] Preferably, the green algae is Coelastrella sp. or Scenedesmus sp.

[0013] The present invention also provides a method for promoting plant growth, which involves using the aforementioned Bacillus subtilis JZ1 and green algae in combination to treat the soil for plant cultivation.

[0014] Preferably, the green algae is Coelastrella sp. or Scenedesmus sp.; the growth promotion refers to increasing the seed germination rate or the total plant biomass.

[0015] Specifically, the Bacillus spp. JZ1 and green algae are mixed at a volume ratio of 1:0.5-2 in the culture solution and used to irrigate the soil for planting plants. Preferably, irrigation is carried out every 4-6 days, with regular watering on a daily basis. The substrate moisture content is measured with a soil moisture meter every week to ensure that the substrate moisture content is maintained at 60% field capacity.

[0016] The present invention also provides a method for improving soil, which involves using the aforementioned Bacillus subtilis JZ1 and green algae in combination to treat the soil to be improved; specifically, the improvement of soil refers to increasing the stability of soil aggregates and / or increasing nutrients, more specifically the nutrients refer to the content of available phosphorus, available potassium, alkaline nitrogen, organic matter and extracellular polysaccharides in the soil.

[0017] Preferably, the green algae is Coelastrella sp. or Scenedesmus sp.; the Bacillus spp. JZ1 and the green algae are mixed at a volume ratio of 1:0.5-2 in the culture medium and then irrigated onto the soil to be improved.

[0018] The gelatinous Bacillus JZ1 obtained in this invention has the following advantages: (1) The strain is derived from biological crusts and is a potential microorganism that promotes the formation of biological crusts; (2) The strain is derived from karst desertification areas and has a certain drought resistance, making it easy to play a role in arid desert areas; (3) The strain has multiple functions such as phosphorus solubilization, potassium solubilization, nitrogen fixation, production of IAA, ACC deaminase, siderophore, and extracellular polymer (ESP), and is an environmentally friendly biological material that will not cause environmental pollution; (4) The strain can stably exert the effects of promoting plant growth and improving soil when used in combination with crust algae. Attached Figure Description

[0019] Figure 1 Phylogenetic tree diagram of strain JZ1 based on 16S rDNA gene sequence.

[0020] Information on the preservation of biological materials:

[0021] The gelatinous Bacillus JZ1 of this invention was deposited on September 5, 2023, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101), with accession number CGMCCNo.28359, and classified as Paenibacillus mucilaginosus.

[0022] The LC8 strain of *Coelastrella* was deposited on July 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 41199, and classified as *Coelastrella* sp.

[0023] The *Scenedesmus* EMC7 strain was deposited on July 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China). The accession number for EMC7 is CGMCCNo.41200, and the classification name is *Scenedesmus* sp. Detailed Implementation

[0024] Example 1: Isolation and screening of spore-producing strains

[0025] (1) Source of crusted soil samples

[0026] Samples were collected from Jianshui County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province. Within each study area, five sampling points were selected based on factors such as minimal human interference, consistent topography, and stable biological crust development to collect sub-soil from the crust. The samples were transported back to the laboratory as soon as possible and stored in a refrigerator at 4°C for microbial isolation and screening.

[0027] (2) Isolation of spore-forming strains

[0028] Weigh 10g of soil sample from the subcrust layer stored at 4℃ and add it to a sterilized Erlenmeyer flask containing 90mL of deionized water and glass beads. Shake at 28℃ and 180r / min for 30min, then place in a 90℃ water bath for 30min to kill all microorganisms except spores. Dilute the soil suspension to 10⁻⁶ using a serial dilution method. -2 -10 -7 Diluted bacterial suspension, take 10 -3 -10 -6Four concentrations of soil suspension, 100 μL each, were injected into beef extract peptone solid medium. The suspensions were then evenly spread onto medium plates using the dilution and plating method. The plates were incubated at 30°C for 3-5 days, and colony growth was observed. Based on bacterial growth, strains with different appearances, colors, and morphologies were selected, and a total of 50 strains were picked and numbered (JP1-JP50). After multiple streak plating cultures to confirm that the strains were pure cultures, single colonies were picked and incubated in beef extract peptone liquid medium at 30°C and 180 rpm for 24 hours. The mixture was then mixed with 30% glycerol at a 1:1 ratio and stored at -20°C for subsequent functional analysis.

[0029] Example 2: Screening and quantitative determination of microorganisms with phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing properties.

[0030] (1) Screening and quantitative determination of phosphorus-solubilizing microorganisms

[0031] 1) Qualitative screening of phosphate-solubilizing microorganisms

[0032] The bacterial strains stored at -20℃ were removed and, after thawing, inoculated at a rate of 1% into beef extract peptone liquid medium. The medium was incubated at 30℃ and 150 rpm for 24 hours. 6 μl of the activated bacterial solution was then inoculated onto inorganic phosphorus solid plates. The medium formulation was as follows: glucose 10 g / L, FeSO4·7H2O 0.03 g / L, NaCl 0.3 g / L, KCl 0.3 g / L, MnSO4·4H2O 0.03 g / L, MgSO4·7H2O 0.3 g / L, (NH4)2SO4 0.5 g / L, CaPO4 10 g / L, agar 18 g / L, natural pH, sterilized at 121℃ for 30 minutes. Each strain was incubated at 30℃ for 5-7 days after inoculation, and the presence or absence of a phosphorus-solubilizing zone was used to determine whether the strain possessed phosphorus-solubilizing function.

[0033] 2) Quantitative determination of microorganisms with phosphorus-solubilizing function

[0034] The relevant reagents for quantitative detection are prepared as follows:

[0035] 5 mg / L Phosphorus Standard Solution: Dissolve 0.4394 g of dried KH2PO4 in 100 ml of water, add 5 ml of concentrated sulfuric acid for preservation, and dilute to 1 L with water to obtain a 100 mg / L phosphorus solution. This solution can be stored for a long time (in a refrigerator). Take 10 ml of the above solution and dilute to 5 mg / L in a 200 ml volumetric flask. This solution should not be stored for a long time.

[0036] 5g / L Potassium Antimony Tartrate: Dissolve 0.5g of potassium antimony tartrate in 100ml of water.

[0037] Molybdenum-antimony anti-chromic reagent: First, prepare a stock solution of molybdenum-antimony sulfate. Slowly add 126 ml of concentrated sulfuric acid to 400 ml of water. Then, dissolve 10 g of ammonium molybdate in 300 ml of water at 60°C. After cooling, slowly pour the H₂SO₄ solution into the ammonium molybdate solution. Add 100 ml of 5 g / L potassium antimony tartrate. After cooling, bring the volume to 1000 ml and store in a brown bottle. Then, dissolve 1.5 g of ascorbic acid in 100 ml of the prepared molybdenum-antimony sulfate stock solution. This is the molybdenum-antimony anti-chromic reagent and should be prepared and used immediately.

[0038] Accurately pipette 0 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, and 10 ml of 5 mg / L phosphorus standard solution into 50 ml volumetric flasks. Add 2 drops of 2,6-dinitrophenol as an indicator (replace with a pH meter for adjustment). Adjust the solution to a pale yellow color with dilute sulfuric acid and 4 mol / L NaOH solution. Add 5.0 ml of molybdenum antimony reagent (this process should be done as slowly as possible to prevent the solution from overflowing with air bubbles), mix well, and dilute to volume. This yields phosphorus standard solutions with phosphorus contents of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg / L, respectively. Incubate at room temperature (above 15°C) for 30 min. Measure the absorbance of the solution at a wavelength of 700 nm. Plot a standard curve with absorbance as the ordinate and phosphorus concentration (mg / L) as the abscissa.

[0039] Microorganisms with phosphorus-solubilizing function were obtained through qualitative screening and inoculated into inorganic phosphorus liquid culture medium at a 1% inoculum, at 30℃ and 180 rpm. -1 After 72 hours of shaking culture, the bacterial suspension was centrifuged at 10,000 rpm for 15 minutes at 4°C. 2 ml of the supernatant was collected and used as a blank reference, with sterile culture medium as the control. 1 ml of the sample was transferred to a 25 ml volumetric flask, diluted with water to approximately 15 ml, and 2-3 drops of dinitrophenol indicator were added. The solution was adjusted to a slightly yellow color with 4 mol / L NaOH solution. 2.5 ml of molybdenum antimony anti-chromic reagent was accurately added, the mixture was shaken well, and water was added to bring the volume to a final depth. The solution was incubated at room temperature (above 15°C) for 30 minutes. The colorimetric determination process was the same as that for the phosphorus standard solution. The corresponding phosphorus content (μg / ml) was calculated from the standard curve.

[0040] Calculation formula: Available phosphorus content of bacterial suspension (μg / ml) = ρ·V·n / V0

[0041] In the formula: ρ—the mass concentration of available phosphorus (μg / ml) obtained from the standard curve.

[0042] V—Volume of colorimetric solution after final volume adjustment

[0043] n—the multiple to be distributed

[0044] V0 — Total volume of bacterial suspension (ml).

[0045] (2) Screening and quantitative determination of potassium-solubilizing microorganisms

[0046] 1) Qualitative screening of potassium-solubilizing microorganisms

[0047] Take 6 μL of the activated bacterial culture and inoculate it onto a potassium-solubilizing solid medium plate. The medium formula is as follows: sucrose 5g, Na2HPO4 2g, MgSO4·7H2O 0.5g, FeCl3 0.005g, CaCO3 0.1g, potassium feldspar 1.0g, agar 18g, distilled water 1000mL, natural pH, 121℃, steam sterilized for 30min. After inoculation, each strain is incubated at 30℃ for 5-7 days to observe the results. The presence or absence of a potassium-solubilizing zone indicates whether the strain possesses potassium-solubilizing function.

[0048] 2) Quantitative determination of microorganisms with potassium-solubilizing function

[0049] The test strain was inoculated at a rate of 1% into a 100mL Erlenmeyer flask containing 30mL of potassium-solubilizing liquid medium. The flask was incubated at 28℃ and 180rpm for 7 days. An equal volume of potassium-solubilizing liquid medium without inoculation was used as a blank control. The fermentation broth after 7 days was collected and digested with 30% H₂O₂ until clear and transparent, approximately 30-60 minutes. The broth was then centrifuged at 6000rpm for 10 minutes. The supernatant was transferred to a 50mL volumetric flask and brought to volume. The soluble potassium content was determined using a flame spectrophotometer.

[0050] (3) Screening of nitrogen-fixing microorganisms and determination of nitrogenase activity

[0051] 1) Qualitative screening of nitrogen-fixing microorganisms

[0052] Using a sterile toothpick, the activated bacterial solution was inoculated onto Ashby nitrogen-free solid medium using the streak method. The Ashby nitrogen-free medium formula is as follows: glucose 10 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaSO4·2H2O 0.2 g / L, CaCO3 5 g / L, agar 18 g / L, pH 7.2. After 6 subcultures, good growth indicates nitrogen fixation ability; failure to grow indicates lack of nitrogen fixation ability. To further verify the nitrogen fixation ability of the screened strain, the nitrogenase gene nifH was amplified using PCR. The primers for nifH gene amplification were:

[0053] PolyF (TGCAYCCSAARGCBGACTC) and PoyR (ATSGCCATCATYTCRCCGGA) were used to detect the PCR amplification products by 1% agarose gel electrophoresis to further verify whether the strain has nitrogen-fixing properties.

[0054] 2) Quantitative determination of nitrogen-fixing capacity of nitrogen-fixing microorganisms

[0055] The test strain was inoculated at a 1% inoculum into a 100mL Erlenmeyer flask containing 30mL Ashby nitrogen-free liquid medium. The flask was incubated at 30℃ and 180rpm for 24h in a shaker, centrifuged at 6000r / min for 10min, and the supernatant was filtered through a 0.25μm filter for sterilization before use. Nitrogenase activity was used to evaluate the nitrogen-fixing capacity of the microorganisms. Nitrogenase activity was measured using a nitrogenase (NITS) enzyme-linked immunosorbent assay kit (Jiangsu Meijing Biotechnology Co., Ltd., JM-13057O2-48T). Blank wells (blank control wells without sample and enzyme-labeled reagent, all other steps were the same), standard wells, and test sample wells were prepared. The specific procedures are as follows: Accurately add 50 μL of standard to the enzyme-labeled plate. Add 40 μL of sample diluent to the sample wells, then add 10 μL of the sample to be tested (final sample dilution is 5-fold) and gently shake to mix. Seal the plate with sealing film and incubate at 37°C for 30 min, then discard the diluted supernatant. Dilute the washing buffer provided with the kit 30 times and fill each sample well. Let stand for 30 seconds and discard. Repeat this process 5 times, then pat dry. Except for the blank control wells, add 50 μL of enzyme-labeled reagent to each well and incubate again at 37°C for 30 min. Repeat the aforementioned washing steps. Then, add 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B provided with the kit to each well sequentially, gently shake to mix, and incubate at 37°C in the dark for 10 min. Add 50 μL of stop solution to each well to terminate the reaction. Set the temperature to 0 with the blank wells and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm using an enzyme-labeled plate reader. The nitrogenase activity of the supernatant of each strain was calculated by substituting the concentration of the standard solution into the formula.

[0056] Experimental Results: Qualitative analysis of the phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing functions of 50 purified spore-forming bacterial strains was conducted. The results showed that 9 strains possessed phosphorus-solubilizing function, 11 strains possessed potassium-solubilizing function, and 10 strains possessed nitrogen-fixing function (see Table 1 for details). The phosphorus-solubilizing capacity, potassium-solubilizing capacity, and nitrogenase activity of these functional bacteria were measured. The results showed that the phosphorus-solubilizing capacity of each strain ranged from 22.87 to 86.73 mg / L, the potassium-solubilizing capacity ranged from 1.13 to 3.47 mg / L, and the nitrogenase activity ranged from 71.56 to 158.73 ng / L. Strains possessing all three functions were identified as JP9, JP11, JP18, and JP22 (see Tables 1 and 2 for details).

[0057] Table 1 Qualitative results of microorganisms exhibiting phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing properties.

[0058] strain number Phosphorus dissolution Potassium solubilization Nitrogen fixation strain number Phosphorus dissolution Potassium solubilization Nitrogen fixation JP5 - + + JP 22 + + + JP7 - + + JP 28 + + - JP 8 - - + JP 31 + - + JP 9 + + + JP 34 + - + JP 11 + - + JP 38 - + - JP 16 + - - JP 40 - + + JP 18 + + + JP 41 - + - JP 21 + - - JP 47 + + -

[0059] Note: "+" indicates that this function is available, and "-" indicates that this function is not available.

[0060] Table 2. Quantitative determination of microorganisms with phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing properties.

[0061] strain number Soluble phosphorus Potassium solubility Nitrogenase activity strain number Phosphorus dissolution Potassium solubilization Nitrogenase activity JP5 -- 3.03 141.64 JP 22 51.65 3.47 141.29 JP7 -- 2.71 156.56 JP 28 22.87 2.16 -- JP 8 -- -- 116.75 JP 31 34.72 -- 125.03 JP 9 33.54 1.31 158.73 JP 34 36.70 -- 71.56 JP 11 34.55 1.13 133.09 JP 38 -- 1.95 -- JP 16 30.45 -- -- JP 40 -- 1.92 82.31 JP 18 86.73 1.91 121.16 JP 41 -- 1.94 -- JP 21 49.98 -- -- JP 47 24.66 2.97 --

[0062] Note: Phosphorus and potassium solubilization are measured in mg / L, and nitrogenase activity is measured in ng / L.

[0063] Example 3: Screening and quantitative determination of microorganisms producing IAA, siderophores, and ACC deaminase

[0064] Microorganisms with strong phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing abilities were selected, and their ability to produce IAA, siderophores, and ACC deaminase was further screened and quantitatively determined in order to screen out highly efficient and multifunctional strains with multiple functions.

[0065] (1) Screening of IAA-producing functional microorganisms

[0066] 1) Qualitative screening of IAA-producing functional microorganisms

[0067] After culturing each strain in beef extract peptone liquid medium containing tryptophan (100 mg / L) for 3 days, 2 mL of the bacterial culture was transferred to an EP tube and centrifuged at 12,000 rpm for 10 min. 1 mL of the supernatant was then mixed with 1 mL of Salkowski chromogenic reagent (chromogenic reagent formula: 12 g FeCl3, 430 mL 98% H2SO4, 570 mL H2O). The mixture was reacted at 25°C in the dark for 30 min. If the mixture turned pink, it indicated that the strain produced IAA.

[0068] 2) Quantitative determination of IAA production by the strain

[0069] Prepare standard IAA gradient solutions of 0, 10, 20, 30, 40, and 50 mg / L. Mix 1 mL of each concentration of IAA solution with 1 mL of Salkowski chromogenic reagent, react at 25°C in the dark for 30 min, and then measure their OD530 to construct a standard curve. For the qualitative experiments, mix the supernatant of the IAA-producing strains with Salkowski chromogenic reagent at a 1:1 ratio, react at 25°C in the dark for 30 min, and measure the absorbance (OD530) at 530 nm. Substitute the absorbance values ​​of each strain sample into the standard curve to calculate the IAA concentration in each bacterial culture medium.

[0070] (2) Screening of functional microorganisms that produce siderophores

[0071] 1) Qualitative screening of siderogenic functional microorganisms

[0072] Aspirate 6 μL of the activated bacterial solution and inoculate it onto a siderophore detection synthetic medium plate (the medium formulation is as follows: 10.87 g of Chromium Azurite S (CAS) synthetic medium powder, which needs further optimization; add 1 mL of 1 mmol / L CaCl2, 20 mL of 1 mmol / L MgSO4·7H2O, 30 mL of 10% casein amino acids, and 20 mL of 20% glucose solution, natural pH, sterilized at 115℃ for 30 min). Incubate at 30℃ for 5-7 days and observe the results. The presence or absence of an orange-yellow transparent zone around the colony indicates the strain's siderophore-producing ability; strains exhibiting this ability are considered siderophore-producing.

[0073] 2) Quantitative determination of siderophore produced by the strain

[0074] The activated iron-producing bacteria suspension was inoculated at a rate of 1% into SA iron-limited medium (medium formula as follows: sucrose, 20.0g; L-asparagine, 2.0g; K2HPO4, 0.5g; FeSO4·7H2O, 0.5g; 8-hydroxyquinoline added for iron removal; 1L distilled water, natural pH, sterilized at 115℃ for 30min). The medium was then incubated in a shaker incubator for 48h with shaking parameters of 150r / min and 28℃. An uninoculated SA iron-limited medium was used as a control. The bacterial suspension grown for 48 hours was transferred to a sterilized 10 mL centrifuge tube. The centrifuge parameters were adjusted to 13000 r / min and centrifuged for 15 min. The supernatant was transferred to a small test tube treated with HCl. A certain amount of freshly prepared CAS detection solution was added to make the volume ratio of supernatant to detection solution 1:1. After thorough mixing, the mixture was allowed to stand for 1 hour. The absorbance value (As) at a wavelength of 630 nm was measured. Double-distilled water was used as a control for zeroing. The absorbance value (Ar) at a wavelength of 630 nm of the supernatant of uninoculated SA iron-limited medium after centrifugation and reaction with an equal volume of detection solution was used as the reference value. The siderophore activity unit was expressed by the following formula: [(Ar-As) / Ar]*100.

[0075] (3) Screening of microorganisms producing ACC deaminase

[0076] 1) Qualitative screening of ACC-producing deaminase functional microorganisms

[0077] DF medium was prepared (formula as follows: 6g disodium hydrogen phosphate; 4g potassium dihydrogen phosphate; 0.2g ferric sulfate heptahydrate; 0.2g magnesium sulfate heptahydrate; 2g ammonium sulfate; 2g glucose; 2mL gluconic acid; 2g citric acid; distilled water to a final volume of 1L, pH 7.2, sterilized at 121℃ for 20min). 6μL of the activated bacterial culture of the test strain was inoculated into DF medium and incubated at 30℃ for 24h. The strain was then transferred to nitrogen-free ADF medium containing ACC (formula as follows: 3mmol / L ACC replacing (NH4)2SO4 in DF medium as the sole nitrogen source), and incubated at 30℃ for another 24h. The results were then observed. Strains that could grow on ADF medium were preliminarily determined to have the ability to produce ACC deaminase. To further verify the ability of the screened strains to produce ACC deaminase, the gene encoding ACC deaminase was amplified using PCR. The candidate strains were inoculated onto beef extract peptone agar plates. Single colonies were picked up with sterile toothpicks and transferred to PCR tubes containing 20 μL of sterile water. After mixing, the tubes were heated at 95°C for 30 min to rupture the bacteria, which were then used directly as DNA templates. The gene encoding ACC deaminase is the acdS gene. PCR amplification of the ACC deaminase gene was performed using a designed specific primer pair. The reaction mixture consisted of 25 μL of DNA template, 1 μL each of forward and reverse primers, 8.5 μL of dd H2O, and 12.5 μL of 2×Taq PCR Super Mix. The PCR amplification program was as follows: 94°C pre-denaturation for 10 min; 94°C denaturation for 40 s, 65°C annealing for 50 s, 72°C extension for 1 min, 30 cycles; and a final extension at 72°C for 10 min. The amplified products were examined by 1% agarose gel electrophoresis. The presence of a bright band indicated that the strain possessed the gene encoding ACC deaminase.

[0078] 2) Quantitative determination of ACC deaminase activity produced by the strain

[0079] The tested bacterial strain was inoculated into beef extract peptone liquid medium and cultured for 24 h. Cells were collected by centrifugation at 4 °C and 6000 rpm. After two washes and centrifugations with DF stock solution, the cells were resuspended in ADF medium and cultured at 28 °C and 200 rpm for 48 h. Cells were then collected to induce ACC deaminase production. Cells were collected by centrifugation at 4 °C, washed twice with 0.1 mol / L Tris-HCl buffer (pH 7.6), and resuspended in 600 μL of 0.1 mol / L Tris-HCl buffer (pH 8.5). 30 μL of toluene was added, and the cells were rapidly shaken for 30 s to lyse the cells. 200 μL of the mixture was transferred to 20 μL of 0.5 MACC solution, mixed thoroughly, and incubated at 30 °C for 15 min. Add 1 mL of 0.56 M HCl, mix well, centrifuge at 14000 rpm for 5 min, and transfer 1 mL of the supernatant to a 7 mL centrifuge tube. Add 0.15 mL of 0.1% 2,4-dinitrophenylhydrazine (dissolved in 2 M HCl), react at 30 °C for 15-30 min, and then add 1 mL of 2NNaOH. Perform the same reaction with 1 mL of Tris-HCl buffer (pH 8.5) as a blank tube to zero the sample. Use 1 mL of α-butanone at concentrations of 0, 0.1, 0.5, 1.0, 2.0, and 3.0 mM as standard solutions and measure the absorbance at 540 nm.

[0080] Total bacterial protein content was determined using the Coomassie Brilliant Blue method. Bovine serum albumin at concentrations of 0, 20, 40, 60, 80, and 100 mg / L was used as a standard solution. 5 mL of Coomassie Brilliant Blue G250 staining solution was added, and the reaction was carried out for 5 min before measuring the absorbance at 595 nm. ACC deaminase activity was expressed as the amount of α-butanone formed per milligram of protein per minute in the enzyme assay system, expressed in μmol / L α-butanone / mgprotein / min.

[0081] Experimental Results: Qualitative detection of IAA production, siderophore production, and ACC deaminase activity was performed on spore-forming strains with phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing abilities. The results showed that 6 strains produced IAA, 2 produced siderophores, and 6 produced ACC deaminase (see Table 3 for details). Quantitative determination of IAA, siderophore, and ACC deaminase activity was also performed. The results showed that the IAA production ranged from 8.93 to 31.79 mg / L, the siderophore production ranged from 54.01% to 64.16%, and the ACC deaminase activity ranged from 0.11 to 0.56 U / mg. Only JP18 and JP38 possessed two or more of these functions (see Tables 3 and 4 for details).

[0082] Table 3 Qualitative results of strains producing IAA, siderophores, and ACC deaminase.

[0083] strain number IAA Ferrite carrier ACC deaminase strain number IAA Ferrite carrier ACC deaminase JP5 - - - JP 22 + - - JP7 - - + JP 28 + - + JP 8 - - - JP 31 + - - JP 9 - - - JP 34 - - - JP 11 + - - JP 38 + + + JP 16 - - + JP 40 + - - JP 18 - + - JP 41 - - - JP 21 - - + JP 47 - - +

[0084] Note: "+" indicates that this function is available, and "-" indicates that this function is not available.

[0085] Table 4. Quantitative determination of IAA-, siderophore-, and ACC-deaminase-producing strains.

[0086] strain number IAA Ferrite carrier ACC deaminase strain number IAA Ferrite carrier ACC deaminase JP5 -- -- -- JP 22 25.83 -- -- JP7 -- -- 0.11 JP 28 9.21 -- 0.32 JP 8 -- -- -- JP 31 24.03 -- -- JP 9 -- -- -- JP 34 -- -- -- JP 11 31.79 -- -- JP 38 9.81 64.16 0.56 JP 16 -- -- 0.38 JP 40 8.93 -- -- JP 18 -- 54.01 -- JP 41 -- -- -- JP 21 -- -- 0.44 JP 47 -- -- 0.16

[0087] Note: IAA unit: mg / L, siderophore unit: %, ACC deaminase activity unit: U / mg.

[0088] Example 4: Determination of the strain's ability to produce extracellular polysaccharides (ESP)

[0089] Microorganisms with strong phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing abilities were selected, and strains capable of producing two or more of these enzymes, as well as IAA, siderophores, and ACC deaminases, were selected to test the strains' ability to produce ESP.

[0090] (1) Qualitative test for ESP production by the strain

[0091] Prepare a solid culture medium for screening EPS-producing bacteria (formula as follows: 5.0g yeast extract, 5.0g peptone, 5.0g acid hydrolysate, 5.0g glucose, 5.0g soluble starch, 3.0g sodium pyruvate, 3.0g K₂HPO₄, 0.5g MgSO₄, 15.0g agar powder, pH 7.0-7.5, 1L distilled water, sterilized at 121℃ for 20min). Spot 6μL of the activated bacterial solution onto a solid plate and incubate at 30℃. Observe colony growth daily, selecting strains with a viscous texture. A toothpick can be used to assist in selection; the strains with a viscous, stringy consistency are the EPS-producing strains obtained in the initial screening.

[0092] (2) Quantitative determination of ESP production by the strain

[0093] Based on the qualitative results above, strains producing ESP were selected, activated, and then inoculated into 10% beef extract peptone liquid medium, incubated at 30°C and 180 rpm. -1 Seed culture was obtained after culturing for 24 hours under the specified conditions; it was then inoculated into the basal medium at an inoculum rate of 4% (V / V) and incubated at 30℃ and 180 rpm. -1 Cultured for 48 hours at 4000 r·min -1 Centrifuge for 15 min to remove bacterial cells and obtain filtrate. Add 3 volumes of anhydrous ethanol to the filtrate and incubate overnight at 4°C. -1Centrifuge for 15 min to obtain a precipitate. Add an appropriate amount of distilled water to the precipitate to reconstitute it and obtain the crude EPS sample solution. Prepare two standard curves. (1) Plot glucose standard curve using phenol-sulfuric acid method (for calculating total sugar in the sample): Dry glucose at 105℃ to constant weight and prepare concentrations of 0, 10, 20, 30, 40, 50, and 60 μg·mL. -1 Glucose standard solution. Take 1 ml and add 0.5 ml of 6% phenol and 2.5 ml of concentrated sulfuric acid respectively, mix and shake well. After cooling to room temperature, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 490 nm. Plot the glucose standard curve with the glucose solution concentration as the abscissa and the absorbance as the ordinate. (2) DNS method to plot glucose standard curve (used to calculate reducing sugar in sample): Dry glucose at 105℃ to constant weight, and prepare concentrations of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg·mL. -1 A standard glucose solution was prepared. 1 mL of the solution was added to 1 mL of DNS reagent and reacted in a boiling water bath for 5 minutes, then immediately cooled with ice water. After standing for 10 minutes, 8 mL of distilled water was added, and the mixture was shaken well. The absorbance was measured at 490 nm using a microplate reader. A glucose standard curve was plotted with the glucose solution concentration on the x-axis and the absorbance on the y-axis. The total sugar content of the test strain sample solutions was determined using the phenol-sulfuric acid method, and the reducing sugar content was determined using the DNS method. The difference between the total sugar content and the reducing sugar content was the EPS content.

[0094] Experimental Results: Qualitative detection of ESP production was performed on spore-forming strains with phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing abilities. The results showed that four strains exhibited ESP production ability: JP16, JP22, JP38, and JP47. The quantitative determination of ESP production by these strains is shown in Table 5. The results indicated that JP22 produced the highest ESP amount at 24.61 mg / L, followed by JP38 at 22.52 mg / L.

[0095] Table 5 Quantitative determination of ESP production by strains

[0096]

[0097] Example 5: Molecular identification of the strain's 16S rDNA

[0098] Based on the determination of phosphorus solubility, potassium solubility, nitrogen fixation, IAA production, siderophore activity, ACC deaminase activity, and ESP activity, strain JP22 not only possesses the soil nutrient-enhancing properties of phosphorus solubility, potassium solubility, and nitrogen fixation, but also exhibits high IAA and EPS production capabilities. Compared with other strains, its overall performance is superior, with phosphorus solubility, potassium solubility, nitrogen fixation, IAA production, and EPS production at 51.65 mg / L, 3.47 mg / L, 141.29 ng / L, 25.83 mg / L, and 24.61 mg / L, respectively. Therefore, this strain was selected for 16S rDNA full-length sequencing, and its taxonomic position was determined by BLAST sequence alignment using the NCBI website. The candidate strain was streaked onto beef extract peptone agar plates. Single colonies were picked up with sterile toothpicks and transferred to PCR tubes containing 20 μl of sterile water. After thorough mixing, the toothpicks were removed and discarded. The tubes were then labeled accordingly and heated at 95℃ for 30 min to rupture the bacteria, allowing them to be used directly as DNA templates. PCR amplification was performed using universal primers. The PCR reaction mixture consisted of 25 μl of DNA template, 1 μl each of forward and reverse primers, 8.5 μl of dd H2O, and 12.5 μl of 2×TaqPCR Super Mix. The PCR amplification program was as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 52℃ annealing for 1 min, 72℃ extension for 1 min 30 s, for 30 cycles; and a final extension at 72℃ for 10 min. After 1% agarose gel electrophoresis, the amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared and analyzed on the NCBI website, and a phylogenetic tree was constructed using MEGA X software.

[0099] Experimental Results: The results showed that strain JP22 shared 99.72% or higher homology with several Paenibacillus mucilaginosus strains on the website. Therefore, this strain was renumbered as Paenibacillus mucilaginosus JZ1. This strain was deposited on September 5, 2023, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101), with accession number CGMCC No. 28359. The phylogenetic tree of strain JZ1 based on the 16S rDNA gene sequence is shown below. Figure 1 As shown.

[0100] Example 6: The effect of mixing Bacillus subtilis JZ1 with Algae cereus on promoting the number of viable bacteria.

[0101] The cultured Bacillus subtilis JZ1 bacterial suspension was diluted with sterile culture medium to a concentration of 10 mg / ml. 8The number of viable bacteria was determined. Four cultured green algae strains were diluted with sterile culture medium to a concentration of 10⁶ cells / ml of algal solution. 4 Viable bacterial count (all 4 green algae strains were isolated from biological crusts in our laboratory; their strain numbers and classification information are shown in Table 6). 300ml of *Bacillus subtilis* JZ1 bacterial suspension and 300ml of each of the 4 green algae suspensions were mixed at a 1:1 (V / V) ratio and sprayed onto the sterilized soil surface. The soil was sourced from bare soil from the spoil heap of the Heidaigou open-pit mine in Ordos City, Inner Mongolia, and was sterilized twice using high-pressure steam. Each flowerpot contained 5kg (flowerpot height 17.5cm, diameter 25.5cm). Simultaneously, two control treatments were set up: one containing only Bacillus subtilis JZ1 bacterial solution and the other containing a 1:1 mixture of Bacillus subtilis JZ1 bacterial solution and four types of green algae (pre-mixed in a 1:1:1:1 ratio). A total of six treatments were set up, numbered JZ1 (containing only Bacillus subtilis JZ1 bacterial solution), JZ1+CC1 (containing Bacillus subtilis JZ1 bacterial solution and CC1 algae solution), JZ1+LC8 (containing Bacillus subtilis JZ1 bacterial solution and LC8 algae solution), JZ1+EMC7 (containing Bacillus subtilis JZ1 bacterial solution and EMC7 algae solution), JZ1+AMC5 (containing Bacillus subtilis JZ1 bacterial solution and AMC5 algae solution), and JZ1+4 algae (containing Bacillus subtilis JZ1 bacterial solution and 4 algae solution). Each treatment had 10 replicates, for a total of 60 replicates. For the first two weeks after treatment, the soil was weighed and watered regularly every day to ensure consistent humidity in each pot. Watering was stopped starting from the third week. The soil was then taken out twice, in the fourth and twelfth weeks, to determine the number of Bacillus subtilis surviving in each treatment.

[0102] Table 6. Species information of four green algae.

[0103] Algal strain number Latin name CC1 Chlorella sp. LC8 Coelastrella sp. EMC7 Scenedesmus sp. AMC5 Scenedesmus sp.

[0104] The results (see Table 7) showed that the number of viable Bacillus subtilis JZ1 bacteria in the soil was significantly higher in the treatment group containing algae solution than in the treatment group without algae solution at both 4 and 12 weeks of application. The application of a mixture of four algae solutions did not show a higher number of viable bacteria than the application of a mixture of single algae solutions. Four weeks after application to the soil, the viable number of JZ1 bacteria was 0.45 × 10⁻⁶. 7 CFU / g, while the soil treated with LC8 algal solution had the highest number of JZ1 viable bacteria, at 1.12 × 10⁻⁶. 7 CFU / g; After 12 weeks of application to the soil, the viable bacterial count in the JZ1-treated soil was 0.11 × 10⁻⁶. 7 The CFU / g concentration of the soil treated with LC8 algal solution was 0.73 × 10⁻⁶ CFU / g. 7CFU / g. The number of viable Bacillus subtilis in the soil of the JZ1+4 algae treatment group was lower than that of the JZ1+LC8 and JZ1+EMC7 treatment groups. This result indicates that the addition of algal solution is beneficial to the growth and survival of Bacillus subtilis, possibly because the presence of these algae provides nutrients for Bacillus subtilis JZ1, thus enhancing its survival ability. The application of single algae is comparable to that of the mixed application of 4 algae, and even the application of single algae in LC8 and EMC7 is more effective than the mixed application of 4 algae. Furthermore, previous research by the inventors revealed that LC8 and EMC7 exhibited superior performance in terms of growth rate, extracellular polysaccharide production, and soluble polysaccharide production. Both algae were deposited on July 5, 2024, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101), abbreviated as CGMCC. The accession number of LC8 is CGMCC No. 41199, and its classification name is Coelastrella sp. The accession number of EMC7 is CGMCC No. 41200, and its classification name is Scenedesmus sp.

[0105] Table 7. Effects of Bacillus inoculation ratio and storage time on viable bacterial count (unit: CFU / g)

[0106]

[0107] Example 7: Study on the effect of combined application of Bacillus subtilis JZ1 and Algae spp. on plant growth promotion

[0108] The test plant was tall fescue (Festuca elata), and the soil was bare soil from the spoil heap of the Heidaigou open-pit mine in Ordos City, Inner Mongolia. Healthy, plump tall fescue seeds were selected and sown evenly in plastic pots (outer diameter 10.5 cm, height 8 cm, bottom diameter 8 cm) filled with sandy substrate. Each pot contained 500 g of substrate, and 50 seeds were sown per pot. After sowing, a thin layer of soil was placed on the surface of the seeds. The following treatments were set up: JZ1 (containing only Bacillus subtilis JZ1 bacterial solution), JZ1+LC8 (containing Bacillus subtilis JZ1 bacterial solution and LC8 algal solution in a 1:1 mixture), JZ1+EMC7 (containing Bacillus subtilis JZ1 bacterial solution and EMC7 algal solution in a 1:1 mixture), JZ2 (containing a strain of Bacillus subtilis JZ2 from bare soil in the karst rocky desertification area of ​​Jianshui, Yunnan, isolated and preserved in our laboratory), and a control with the same amount of sterile water. Each treatment was replicated in 5 pots.

[0109] Bacillus subtilis JZ1 and ZJ2 were inoculated into beef extract peptone liquid medium and cultured at 30℃ with shaking for 36-72 h. Two algal strains, LC8 and EMC7, were inoculated into BG11 liquid medium (formula as follows: 1.5g NaNO3, 0.04g K2HPO3·3H2O, 0.075g MgSO4·7H2O, 0.036g CaCl2·2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g EDTA-Na2, 0.02g Na2CO3, 1mL trace elements, 1L distilled water, pH adjusted to 7.0-7.2) and cultured for 3 weeks at 27℃ and 2700 lx light intensity. The bacteria and algae involved in each treatment were diluted to ensure a viable count of 10-1 for Bacillus subtilis. 8 CFU / mL, viable cell count in algal solution 10 4 Algal cells / mL. Each treatment was applied at a rate of 80 mL, irrigated every 5 days, with regular routine watering. Substrate moisture was measured weekly using a soil moisture meter to ensure it remained at 60% field capacity. Two months after tall fescue began growth, plant height and aboveground height were recorded for each treatment. Aboveground dry weight, underground length, and dry weight were measured after harvest.

[0110] Experimental Results: The growth indicators of tall fescue under different treatments are shown in Table 8. Table 8 shows that the treatment with JZ1 bacterial solution significantly improved plant dry weight, underground dry weight, aboveground dry weight, total plant length, underground length, and aboveground length compared to the treatment with JZ2 bacterial solution, indicating that the *Bacillus mucilaginosus* of this invention is superior in promoting plant growth. The two treatment groups, JZ1+LC8 and JZ1+EMC7, showed significantly higher indicators than the JZ1 bacterial solution treatment group, indicating that the combined application of LC8 and EMC7 algae solutions with JZ1 bacterial solution is more beneficial for promoting plant growth, significantly promoting the root and leaf growth and development of tall fescue, and increasing root and leaf biomass. The treatment with the best effect was JZ1+LC8, with plant dry weight, underground dry weight, aboveground dry weight, total plant length, underground length, and aboveground length of 2.48g, 1.08g, 1.40g, 23.4cm, 9.8cm, and 13.6cm, respectively. The above indicators were significantly higher in the treatments with bacterial solutions (JZ1, JZ2) and combined bacterial-algae treatments (JZ1+LC8, JZ1+EMC7) than in the treatments with only water.

[0111] Table 8. The effect of combined application of Bacillus subtilis JZ1 and Algae spp. on plant growth.

[0112] deal with water JZ1 JZ1+LC8 JZ1+EMC7 JZ2 Germination rate (%) 78 84 86 85 83 Dry weight of the plant (g) 1.50 2.14 2.48 2.29 1.83 Dry weight of underground part (g) 0.62 0.87 1.08 1.05 0.77 Dry weight of aboveground parts (g) 0.88 1.27 1.40 1.24 1.06 Total plant length (cm) 17.4 20.2 23.4 21.8 18.7 Underground Minister (cm) 7.5 8.7 9.8 9.3 7.9 Length above ground (cm) 9.9 11.5 13.6 12.5 10.8

[0113] Example 8: Soil improvement effect of combined application of Bacillus subtilis JZ1 and Algae spp.

[0114] After harvesting the seedlings, the substrate from the tall fescue root system was thoroughly shaken off and mixed with the substrate in the pots to serve as a sample for determining the physicochemical properties of the substrate. The sample was passed through a 1-2 mm sieve and used to determine pH, electrical conductivity (EC), organic matter (OM), available phosphorus (AP), available potassium (AK), alkaline nitrogen (AN), soil exopolysaccharides, and soil aggregate stability.

[0115] pH and conductivity determination: Weigh 5g of each 2mm sieved, air-dried sample into an Erlenmeyer flask, add 50mL of distilled water at a ratio of 1:10, shake on a shaker for 20min, let stand for 30min, then continue stirring for 5-10min. After standing, use the supernatant for pH and conductivity determination. Available phosphorus (AP) determination: Weigh 2.5g of air-dried sample (passed through a 1mm sieve) into a 100mL plastic bottle, add a small spoonful of phosphorus-free activated carbon and 100mL of 0.5mol / L NaHCO3 extract, shake on a shaker for 30min, remove and immediately filter using a drying funnel and phosphorus-free filter paper, collect the filtrate; pipette 10mL of the filtrate into a 50mL volumetric flask, add 5mL of molybdenum-antimony mixed colorimetric reagent, shake carefully, and after 30min, measure the OD880 value on an ELISA reader, and calculate the available phosphorus content of the sample according to the phosphorus standard curve. Determination of available potassium (AK): Weigh 5g of air-dried sample that has passed through a 1mm sieve into a 100mL plastic bottle, add 50mL of 1mol / L neutral ammonium acetate solution, shake in a shaker for 30min, filter, and directly measure the potassium content of the filtrate on a flame photometer. Calculate the available potassium content of the sample according to the potassium standard curve. Determination of alkaline nitrogen (AN): Weigh 2g of air-dried sample that has passed through a 0.25mm sieve, place it in the outer chamber of a clean diffusion dish, gently rotate the diffusion dish to evenly spread the sample, and add H3BO4 to the inner chamber of the diffusion dish. -Add 2 mL of methyl red-bromocresol green mixed indicator solution, then coat the edge of the outer chamber of the diffusion dish with alkaline glue, cover with frosted glass leaving a slit in the outer chamber, and quickly add 10 mL of NaOH solution. Immediately seal the dish tightly, gently rotate the diffusion dish to cover all samples with the alkaline solution, place it in a 40℃ incubator for 48 hours, and then titrate with 0.01 mol / L (1 / 2 H2SO4) standard solution. Record the volume of standard solution used during titration, and calculate the alkaline nitrogen content in the sample according to the formula. Organic matter (OM) determination: Pass the sample through a 0.25 mm sieve. Weigh 0.1-0.5 g and place it in a test tube. Add 5 mL of 0.8 M potassium dichromate solution and 5 mL of concentrated sulfuric acid. Shake well and place in an oil bath. Adjust the temperature to 175℃ and boil for 5 minutes. Remove the test tube and allow it to cool. Pour the solution into a 200 mL beaker, add water to maintain a total volume of 60-70 mL, add approximately 3-4 drops of o-phenanthroline indicator, and titrate with 0.2 M ferrous sulfate solution. The titration is complete when the solution changes from yellow through green, then emerald green, and finally brownish-red. Record the volume of ferrous sulfate used in the titration and calculate the organic matter content using the formula. Extracellular polysaccharide (EPS) determination: Weigh 1g of the sample after passing it through a 0.25mm sieve, add 10mL of 2mol / L H2SO4, boil in a water bath for 2 hours, and then filter while hot. Wash the digestion tube several times with hot deionized water, and collect the filtrate and washings into a new digestion tube. Add 1g of CaCO3, and after no more gas is produced, filter again. Collect the filtrate and dilute to 20mL to obtain the sample solution to be tested. Pipette 2mL of the test solution into a clean, dry 10mL centrifuge tube, immediately add 6mL of anthrone reagent, shake to mix, and heat in a boiling water bath for 15min. Quickly remove and immerse in an ice-water bath to cool for 15min, and measure the absorbance at 620nm. Calculate the sugar concentration in each sample solution and its extracellular polysaccharide content based on the glucose content scaling curve. Soil aggregate stability determination: Weigh 200g of soil sample and place it on a sieve with apertures of 2mm, 1mm, and 0.25mm to separate aggregates >2mm, 1-2mm, 0.25-1mm, and <0.25mm. Weigh the mass of soil aggregates of each particle size and calculate the mass percentage of each particle size. The mean weight diameter (MWD) and geometric mean diameter (GMD) are used to represent the stability of soil aggregates. The higher the MWD and GMD values, the greater the aggregate stability. Therefore, both indicators are calculated simultaneously. The specific formulas are as follows:

[0116]

[0117] Where: MWD is the average weight diameter; w i x represents the mass percentage of the i-th particle size; i The average diameter of the aggregate.

[0118]

[0119] In the formula: GMD is the geometric mean diameter; m i For the mass (g) of aggregates of different particle sizes; m ’ The average diameter of the aggregate.

[0120] Experimental Results: As shown in Table 9, under the treatment conditions of applying bacterial solutions (JZ1 and JZ2) and combined bacterial-algae solutions (JZ1+LC8 and JZ1+EMC7), the pH, electrical conductivity, and nutrient content of the soil in the open-pit mine spoil heap planted with tall fescue were all higher than those treated with water alone. Compared with JZ2, Bacillus subtilis JZ1 significantly increased the content of soil organic matter (OM), available phosphorus (AP), available potassium (AK), alkaline nitrogen (AN), and soil exopolysaccharides (EPS), which were 2.96%, 7.64 mg / kg, 48.73 mg / kg, 163.33 mg / kg, and 0.44 mg / g, respectively, indicating that the strain JZ1 of this invention has the effect of improving soil nutrients. The combined application of bacterial-algae solutions (JZ1+LC8 and JZ1+EMC7) further enhanced the improvement of soil nutrients in both treatment groups. Compared with the treatment group receiving only JZ1 bacterial solution, the combined application of bacterial-algae solutions was more effective, with JZ1+LC8 showing the most significant improvement. The contents of available phosphorus (AP), available potassium (AK), available nitrogen (AN), organic matter (OM), and extracellular polysaccharides (ESP) in the soil increased by 37.83%, 15.73%, 13.02%, 56.42%, and 77.27%, respectively, compared with the treatment receiving JZ1 bacterial solution. This indicates that the addition of Bacillus subtilis can improve soil nutrients, and the combined application of bacterial-algae solutions can have a synergistic effect. Different strains showed different effects, with the JZ1+LC8 bacterial-algae combination showing the best results.

[0121] Table 9. Effects of combined application of Bacillus colloidus JZ1 and Algae spp. on soil physicochemical properties.

[0122] deal with water JZ1 JZ1+LC8 JZ1+EMC7 JZ2 pH 7.84 7.68 7.65 7.64 7.70 EC (μS / cm) 108.53 133.90 142.51 137.53 131.97 AP (mg / kg) 2.28 7.64 10.53 9.72 6.12 AK (mg / kg) 33.20 48.71 56.37 52.16 41.07 AN (mg / kg) 51.33 163.10 184.33 177.32 88.76 OM (%) 1.34 2.96 4.63 4.28 2.47 EPS (mg / g) 0.10 0.44 0.78 0.65 0.29

[0123] Soil aggregates are an important component of soil, influencing various physicochemical properties and significantly impacting soil erosion. They are a crucial indicator for evaluating soil resistance to erosion. Aggregates are classified by size into macroaggregates (diameter > 0.25 mm) and microaggregates (< 0.25 mm). The size of aggregates significantly affects their stability; soils with a higher content of microaggregates tend to have poorer structure and weaker resistance to erosion. Weight-mean diameter (MWD) and geometric mean diameter (GMD) are used to evaluate soil aggregates, reflecting their size distribution and serving as important indicators of aggregate distribution and stability. Good soil structure and high aggregate stability are crucial for improving soil fertility, increasing agronomic productivity, enhancing soil porosity, and reducing soil erosion. As shown in Table 10, compared with the treatment with only water, the application of bacterial solution (JZ1 and JZ2) and the combined bacterial-algae solution (JZ1+LC8 and JZ1+EMC7) both increased the proportion of large aggregates. Among them, the JZ1+LC8 treatment had the highest proportion of large aggregates >2mm, at 50.49%. The proportions of micro-aggregates (<0.25mm) in the soil treated with bacterial solution (JZ1 and JZ2) and the combined bacterial-algae solution (JZ1+LC8 and JZ1+EMC7) were 9.17%, 7.15%, 8.08%, and 10.08%, respectively. The MWD and GMD values ​​also showed that the JZ1+LC8 treatment had the highest values, at 1.60mm and 1.19mm, respectively, which were higher than the other three treatments and the water treatment.

[0124] Table 10 Soil aggregate composition and stability after combined application of Bacillus colloidus JZ1 and Algae spp.

[0125] Aggregate index water JZ1 JZ1+LC8 JZ1+EMC7 JZ2 >2mm (%) 35.65 45.46 50.49 48.12 39.32 1-2mm (%) 6.84 7.43 7.21 7.54 7.11 0.25-1mm (%) 41.27 37.93 35.15 36.26 43.49 <0.25mm(%) 16.23 9.17 7.15 8.08 10.08 MWD(mm) 1.27 1.49 1.60 1.55 1.38 GMD(mm) 0.84 1.08 1.19 1.14 1.04

[0126] Note: MWD is the mass mean diameter, and GMD is the geometric mean diameter.

Claims

1. A type of gelatinous Bacillus JZ1 ( Paenibacillus mucilaginosus Its characteristics are as follows: Its accession number is CGMCC No.28359.

2. The application of the gelatinous Bacillus JZ1 as described in claim 1 in phosphorus solubilization, potassium solubilization and / or nitrogen fixation.

3. The application of the gelatinous Bacillus JZ1 as described in claim 1 in the production of IAA, siderophores and / or ACC deaminases.

4. A method for increasing the viable count of Bacillus subtilis JZ1 as described in claim 1, characterized in that, It is achieved by mixing the bacterial solution of Bacillus subtilis JZ1 with the solution of green algae.

5. The method as described in claim 4, characterized in that, The green algae mentioned is *Starry Sky Algae* ( Coelastrella sp.) or Scenedesmus (sp.) Scenedesmus sp.).

6. A method for promoting plant growth, characterized in that, The gelatinous Bacillus JZ1 as described in claim 1 and green algae are used in combination to treat soil for plant cultivation.

7. The method as described in claim 6, characterized in that, The green algae mentioned is *Starry Sky Algae* ( Coelastrella sp.) or Scenedesmus (sp.) Scenedesmus (sp.); the term "growth promotion" refers to increasing seed germination rate or total plant biomass.

8. The method as described in claim 6, characterized in that, The Bacillus spp. JZ1 and green algae are mixed at a volume ratio of 1:0.5-2 in the culture solution and used to irrigate the soil for planting plants. Preferably, irrigation is carried out every 4-6 days, with regular watering on a daily basis. The substrate moisture content is measured with a soil moisture meter every week to ensure that the substrate moisture content is maintained at 60% field capacity.

9. A method for improving soil, characterized in that, The gelatinous Bacillus JZ1 as described in claim 1 and green algae are used in combination to treat the soil to be improved; specifically, the soil improvement refers to increasing the stability of soil aggregates and / or increasing nutrients, more specifically the nutrients refer to the content of available phosphorus, available potassium, alkaline nitrogen, organic matter and extracellular polysaccharides in the soil.

10. The method as described in claim 9, characterized in that, The green algae mentioned is *Starry Sky Algae* ( Coelastrella sp.) or Scenedesmus (sp.) Scenedesmus sp.); The gelatinous Bacillus JZ1 and green algae were mixed at a volume ratio of 1:0.5-2 of the culture solution and then applied to the soil to be improved.

Images