Preparation method and application of microbial agent loaded on basis of oyster shell powder

Abstract

The invention discloses a microbial agent based on oyster shell powder loading and a preparation method and application thereof, the microbial agent based on oyster shell powder takes calcined oyster shell powder as a carrier, and bacillus is loaded on the carrier. Natural oyster shells are used as a raw material, and the bacterial loading and adsorbing material with good adsorption effect and high stability is prepared in a calcining manner. The calcined oyster shell powder is used for loading beneficial bacteria, a new way is provided for relieving waste shell pollution and recycling waste shell resources, meanwhile, the bacterial agent loaded with the calcined oyster shell powder is prepared, and the bacterial agent can be used for bioremediation and improvement of coastal saline-alkali soil.

Description

Technical Field

[0001] This invention relates to the preparation of bacterial-loaded materials, and more particularly to a method for preparing and applying a microbial agent based on oyster shell powder. Background Technology

[0002] With advancements in shellfish farming technology and rising market demand for these products, shellfish production has increased significantly. Therefore, how to effectively develop and utilize shellfish waste to achieve economic benefits has become a key issue and a hot topic of research for scholars in related fields. Oyster shells are formed during oyster growth through biomineralization of organic matter, possessing a highly ordered multi-layered microstructure. Oyster shells come in various shapes, including triangular, oval, elongated, and fan-shaped, and under an electron microscope, they exhibit a plate-like structure, primarily composed of calcium carbonate. Oyster shells are rich in calcium and more than 20 trace elements, such as copper, iron, zinc, manganese, and strontium, usually existing in the form of oxides. Currently, the main methods for treating shellfish waste are open-air dumping and ordinary landfill. These methods not only occupy a large amount of land resources, but also, if shellfish waste left outdoors for a long time is not properly treated, the remaining shellfish meat on the shells can easily breed mosquitoes, flies, and microorganisms, causing environmental pollution.

[0003] With the development of modern agriculture, microbial fertilizers have gained significant development space and have become a hot topic in research and application both domestically and internationally. Plant growth-promoting bacteria are a class of microorganisms widely present in soil and around plant rhizospheres, possessing various growth-promoting functions. They are commonly referred to as PGPR (plant growth promoting rhizobacteria). Currently, several species of Bacillus have been found to have PGPR functions. These bacteria, as beneficial plant microorganisms, are widely used in agricultural production and soil remediation. However, both microbial inoculants and other microbial fertilizers face severe challenges related to strain activity and the environment, such as short-lasting fertilizer effects, poor fertilizer activity, and short shelf life of microbial inoculants. Microorganisms in ordinary fertilizers cannot survive for long periods in saline-alkali soils, which significantly reduces the effectiveness of the fertilizer and increases hidden costs. Finding a carrier for the target microorganisms, allowing them to be immobilized in a specific micro-region and receive the nutrients needed for initial growth, can effectively solve these problems. Simultaneously, the chosen carrier should be green, environmentally friendly, low-cost, and renewable.

[0004] To address the problems of severe salinization, poor permeability, nutrient deficiency, and low plant diversity in coastal wetland saline-alkali soils, researchers have explored various methods for soil improvement and ecological restoration. Among these, microbial inoculants, due to their positive environmental effects, have been widely used to improve soil by synergistically promoting plant growth. However, microbial inoculants also suffer from issues such as susceptibility to environmental influences and microbial cell inactivation. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, this invention provides a microbial agent based on oyster shell powder. This invention uses natural oyster shells as raw materials and prepares bacterial loads and adsorption materials with good adsorption effect and high stability through calcination, so as to more effectively use them for plant-microbe synergistic bioremediation of coastal saline-alkali land.

[0006] The present invention also provides a method for preparing and applying the microbial agent based on oyster shell powder.

[0007] Technical solution: In order to achieve the above objectives, the present invention provides a microbial agent based on oyster shell powder, wherein the microbial agent based on oyster shell powder uses calcined oyster shell powder as a carrier and loads Bacillus spores on it.

[0008] The Bacillus is either gelatinous Bacillus K02 or Belize Bacillus LB002.

[0009] The method for preparing the microbial agent based on oyster shell powder according to the present invention includes the following steps:

[0010] (1) After cleaning and drying the oyster shells, crush them and then calcine the crushed oyster shell powder to obtain an oyster shell powder carrier.

[0011] (2) Take the prepared oyster shell powder carrier, put it into the culture medium for sterilization, add Bacillus seed liquid, carry out shake flask culture and loading, after culture, let it settle naturally, remove the supernatant, centrifuge and wash to collect the precipitate, and obtain oyster shell powder loaded with microbial agent.

[0012] In step (1), the oyster shells are scrubbed in water and soaked overnight to remove salt, biomass and other surface residues. They are then rinsed in deionized water, dried, and then thoroughly ground with a grinder, sieved and stored for later use.

[0013] In step (1), the calcination temperature is 100℃-600℃ and the calcination time is 3-4h.

[0014] In step (2), the mass-to-volume ratio of oyster shell powder carrier, Bacillus seed solution, and culture medium is 1:1-2:20 mL, and the OD of the Bacillus seed solution is... 600 It is 1-2.

[0015] In step (2), the culture time for the load is 8-12h, the culture temperature is 30-37℃, and the rotation speed is 150-200rpm.

[0016] The present invention relates to the application of oyster shell powder-loaded microbial agents in the remediation of coastal saline-alkali soils and the promotion of the growth of the salt-tolerant plant Suaeda glauca.

[0017] Among them, the application of microbial agents based on oyster shell powder combined with the salt-tolerant plant Suaeda glauca in the remediation of coastal silty saline-alkali sandy soil.

[0018] In this invention, oyster shell powder is obtained from processed oyster shells. Using oyster shell powder and calcined modified oyster shell powder as a carrier, the calcination modification provides more binding sites and pores, stabilizing and enhancing the bacterial adsorption effect. This overcomes the limitations of weak adaptability and low activity of bacteria participating in bioremediation. This invention adsorbs Bacillus lentigines and Bacillus belyeis onto oyster shell powder to prepare a composite microbial agent. The structural changes of the carrier material were analyzed using X-ray diffraction and thermogravimetric analysis, and the effects of bacterial inoculum size and loading time on the number of viable bacteria were investigated to screen for optimal preparation conditions.

[0019] In this invention, the calcined oyster shells possess a larger porous structure, providing more loading sites and conditions. Thermogravimetric analysis shows that the oyster shells undergo a phase transition at approximately 700℃. At excessively high temperatures, the oyster shells transform into CaO. Considering the high cost of higher temperatures and the alkalinity of CaO, this invention does not set the calcination temperature above 700℃; 400℃ achieves good results. Furthermore, through shaking culture, *Bacillus spp. K02* and *Bacillus belye* LB002 were successfully combined with modified oyster shell powder via physical adsorption. Investigating the number of viable bacteria loaded on the carrier under different experimental conditions revealed that culture time and culture method significantly affected the total number of viable bacteria. This is presumably because microorganisms exhibit better growth and reproduction tendencies under suitable environments. Simultaneously, the altered physical properties of the carrier, with its increased porosity and larger specific surface area, provide bacteria with more attachment sites. Adsorption-related studies indicate that electrostatic interaction is a crucial component of the microscopic adsorption process; the bacterial cell walls carry a negative charge and adsorb onto the carrier through electrostatic interactions. Related studies have shown that the carrier and microorganisms can bind firmly because the microorganisms produce a large amount of extracellular polymers (EPS), which promote better microbial adhesion.

[0020] The extracellular capsule of the gelatinous Bacillus used in this invention is mainly composed of polysaccharides. The presence of extracellular polysaccharides may provide some conditions for the adsorption of bacteria onto the oyster shell surface. Scanning electron microscopy revealed the adhesion between the bacterial strain and broken oyster shell powder. Furthermore, after loading with *Bacillus belye*, spherical objects were observed around the strain, presumably aragonite induced by *Bacillus belye*. During the strain loading process, the oyster shell powder surface came into contact with acidic substances produced by the bacteria, leading to the dissolution of some surface calcium carbonate, an increase in calcium ion concentration, and bacterial-induced precipitation of trace amounts of aragonite.

[0021] This invention utilizes oyster shells to prepare modified oyster shell powder as a carrier for target bacteria. Characterization and analysis of its basic properties, surface functional groups, and morphological structure reveal that this carrier is not only rich in elements such as carbon and oxygen but also possesses abundant porous structures and surface functional groups such as -OH, -CH, and -CO, which are more conducive to bacterial attachment and survival. Microbial inoculants were successfully prepared using a physical adsorption method. Single-factor experiments showed that calcining oyster shell powder significantly increased the bacterial loading capacity; at a calcination temperature of 400℃, the viable bacterial loading capacity of oyster shell powder for both bacteria was significantly improved. Furthermore, loading time, loading method, and initial bacterial dosage significantly affected the loading effect of oyster shell powder on the two bacteria. Under oscillating adsorption conditions, calcined oyster shell powder exhibited superior bacterial loading capacity. Analysis results showed that the two bacteria were effectively loaded onto the surface and pores of the oyster shell powder carrier. Using modified oyster shell powder to prepare bacterial carriers has advantages such as low cost, high utilization rate, no pollution, stability, and environmental friendliness.

[0022] This invention utilizes oyster shell powder (K02-COYS, LB002-COYS) successfully loaded with plant growth-promoting bacteria in combination with the salt-tolerant plant Suaeda glauca for the remediation of coastal silty saline-alkali sandy soil. By measuring the physiological indicators of Suaeda glauca, soil salinity, soil elements, soil nutrients and mineral composition in pot experiments, the invention explores the improvement and remediation effects of K02-COYS and LB002-COYS on saline-alkali soil and their growth-promoting effect on the salt-tolerant plant Suaeda glauca.

[0023] In this invention, calcination of oyster shell powder significantly increases bacterial loading. Loading time, loading method, and initial bacterial dosage all significantly affect the bacterial loading effect of oyster shell powder. With increasing calcination temperature, the viable bacterial loading of oyster shell powder for both types of bacteria increases, showing a significant improvement at 400℃. Compared to static adsorption, the ability of the carrier to load bacteria is significantly enhanced under oscillating adsorption conditions. The results indicate that applying these two microbial agents can promote the growth of Suaeda salsa, significantly increasing its aboveground plant height and biomass; it can effectively reduce soil pH and EC values, decrease soil salinity, and increase the content of available nutrients and organic matter in the soil.

[0024] This invention relates to the study of loading microbial agents onto calcined oyster shell powder. Two microbial agents, K02-COYS and LB002-COYS, were prepared by loading strains K02 and LB002 onto calcined oyster shell powder using a physical adsorption method. The results showed that K02-COYS had the highest effective viable count at a calcination temperature of 400℃, a loading time of 12 h, and an initial inoculum size of 1.5 mL (20 mL culture system), with a maximum viable count of 4.35 × 10⁻⁶. 6 CFU / g; Under the conditions of calcination temperature of 400℃, loading time of 8h, and initial inoculum of 10%, LB002-COYS had the highest effective viable count, with a maximum viable count of 10.7×10⁻⁶ CFU / g. 8 CFU / g.

[0025] The microbial inoculant prepared by this invention and loaded with oyster shell powder is used to promote the growth of Suaeda salsa and improve coastal saline-alkali soil. In pot experiments, the effects of two microbial inoculants (K02-COYS and LB002-COYS) on the plant height, root length, biomass, and other physiological indicators of Suaeda salsa growth were analyzed. The results showed that both inoculants significantly increased the aboveground plant height, underground root length, and biomass of Suaeda salsa. Compared with the control group, the application of K02-COYS alone and the application of LB002-COYS alone increased the aboveground plant height by 31.16% and 52.71%, the root length by 78.90% and 64.14%, and the biomass by 330.78% and 392.31%, respectively. The combined application of K02-COYS and LB002-COYS increased the height of Suaeda salsa by 76.10%, root length by 65.93%, and biomass by 430.77%. Soil physicochemical parameters showed that, compared to the control group, the application of K02-COYS and LB002-COYS decreased soil pH by 0.17 and 0.20, respectively; decreased electrical conductivity by 637.67 μS / cm and 834.00 μS / cm, respectively; and increased water content by 1.08% and 2.15%, respectively. Soil water-soluble sodium ion content decreased by 75.40 mg / kg and 62.82 mg / kg, respectively. The water-soluble magnesium ion content decreased by 109.76 mg / kg and 114.59 mg / kg, respectively; the water-soluble calcium ion content decreased by 1161.82 mg / kg and 1239.93 mg / kg, respectively; the water-soluble potassium ion content decreased by 172.58 mg / kg and 149.13 mg / kg, respectively; the soil carbonate content increased by 0.90% and 0.68%, respectively; the soil organic matter increased by 2.04% and 1.99%, respectively; the total carbon in the soil increased by 0.42‰ and 0.78‰, respectively; and the total sulfur increased by 0.17‰ and decreased by 0.078‰, respectively.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0027] 1. This invention utilizes calcined oyster shell powder loaded with beneficial bacteria, providing a new approach to alleviate pollution from discarded shellfish and to recycle waste shell resources.

[0028] 2. This invention prepares a bacterial agent loaded with calcined oyster shell powder, which can be used to improve coastal saline-alkali soil and promote the growth of Suaeda salsa. The application of the bacterial agent loaded with oyster shell powder significantly increases the content of available phosphorus and organic matter in coastal saline-alkali soil, significantly reduces the content of soluble salts, significantly improves soil nutrients, accelerates the growth rate of Suaeda salsa, and improves the quality of saline-alkali soil. This invention provides a new method and material for solving the pollution and reuse problems of waste shellfish resources, and analyzes its effects and mechanisms. Attached Figure Description

[0029] Figure 1 XRD patterns of oyster shell powder before calcination (a) and after calcination at different temperatures (b).

[0030] Figure 2 This is the FTIR image of oyster shell powder.

[0031] Figure 3 The TG-DTG curves and DTA curves of oyster shell powder are shown in (a) and (b).

[0032] Figure 4 Morphological characterization and elemental composition of oyster shells (ac) and calcined oyster shells (ef).

[0033] Figure 5 The bacterial load of calcined oyster shell powder under different loading times.

[0034] Figure 6 The bacterial load of calcined oyster shell powder under different inoculation amounts.

[0035] Figure 7 The bacterial load of oyster shell powder at different calcination temperatures.

[0036] Figure 8 The effect of different loading methods on the bacterial load.

[0037] Figure 9 XRD analysis (a) and infrared analysis (b) of two bacterial inoculants loaded with powder.

[0038] Figure 10 The structural characteristics of K02-COYS and LB002-COYS are shown.

[0039] Figure 11 Morphological characteristics and elemental composition analysis of K02-COYS(ac) and LB002-COYS(df).

[0040] Figure 12The growth of Suaeda salsa in different treatment groups.

[0041] Figure 13 The chlorophyll content of Suaeda salsa in different treatment groups.

[0042] Figure 14 The soluble sugar content of Suaeda salsa in different treatment groups.

[0043] Figure 15 The malondialdehyde content of Suaeda salsa in different treatment groups.

[0044] Figure 16 The free proline content of Suaeda salsa in different treatment groups.

[0045] Figure 17 Soil salinity and alkalinity for different treatment groups.

[0046] Figure 18 Soil moisture content (a) and available potassium and available phosphorus content (b) for different treatment groups.

[0047] Figure 19 Soil organic matter content in different treatment groups.

[0048] Figure 20 XRD analysis of soil powder from different treatment groups.

[0049] Figure 21 The values ​​represent the soil carbonate content in different treatment groups.

[0050] Figure 22 Correlation analysis of soil physicochemical properties (a) and its correlation analysis with plant physiological indicators (b). Detailed Implementation

[0051] The present invention will be further explained below with reference to the embodiments and accompanying drawings.

[0052] Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0053] The soil matrix was taken from the saline-alkali soil in the coastal area of ​​Lianyun New City, Lianyungang City (119°27′E, 34°76′N).

[0054] Oyster shells: purchased from the seafood market.

[0055] Bacillus velezensis LB002, provided by Nanjing Normal University (NCBI accession number NZ-CP037417.1, see Li XF, et al., RSC Advances, 2025, 15(11), 8795-8808; Wang XX, et al., Water research, 2024, 250:121087);

[0056] Paenibacillus mucilaginosus K02 (strain preservation number CCTCC AB 2021090).

[0057] Nitrogenous medium: sucrose 10 g / L, yeast extract 0.3 g / L, (NH4)2SO4 0.2 g / L, KH2PO4 1 g / L, MgSO4 1 g / L, pH adjusted to 7.0–7.5.

[0058] LB medium (Luria-Bertani medium): sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, natural pH.

[0059] Microbial agent viable count:

[0060] (1) Gelatinous Bacillus-like loading group: The obtained bacterial agent was transferred to a sterile centrifuge tube, sterile distilled water was added, and the mixture was vortexed for 20 min to prepare a bacterial suspension. A serial dilution of 1:10 was performed to obtain 1×10⁶ cells / mL. -4 1×10 -5 1×10 -6 Diluted bacterial suspension. Accurately pipette 100 μL, set up 3 replicates, and spread it evenly onto nitrogenous solid medium in a petri dish using a sterile spreader. Incubate the solid medium plates in a constant temperature incubator (30℃, 16 h). After incubation, observe the colony morphology on the plates and count the number of viable bacteria.

[0061] (2) Bacillus belyssus loading group: The obtained bacterial agent was transferred to a sterile centrifuge tube, sterile distilled water was added, and the mixture was vortexed for 20 min to prepare a bacterial suspension. A serial dilution of 1:10 was performed to obtain 1×10⁻⁶ cells / mL. -5 1×10 -6 1×10 -7 Diluted bacterial suspension. Accurately pipette 100 μL, set up 3 replicates, and spread it evenly onto LB solid medium in a petri dish using a sterile spreader. Incubate the solid medium plates in a constant temperature incubator (37℃, 10 h). After incubation, observe the colony morphology on the plates and count the number of viable bacteria.

[0062] (3) Blank control group: Weigh 1g of the prepared calcined oyster shells, place them in an Erlenmeyer flask, add liquid culture medium, autoclave for 20min (121℃), and cool for later use. Add the sterilized seed liquid, mix well, and place in a constant temperature shaking incubator for culture. The sampling and culture and counting methods are the same as above.

[0063] Test soil: The test soil used in the pot experiment was coastal soil from the coastal area of ​​Lianyun New City, Lianyungang City, Jiangsu Province (119°27′E, 34°76′N). The climate zone is a humid monsoon climate zone that is transitional between warm temperate and subtropical, and the soil type is typical coastal silty saline-alkali soil.

[0064] Potting setup and test plants: The containers used for the potted experiments were cylindrical plastic flowerpots (14cm top diameter × 11cm bottom diameter × 14cm height). A plastic tray was placed at the bottom. The test plants were Suaeda salsa, with plump and uniformly sized seeds selected, washed clean with water, and then dried in a ventilated place.

[0065] Example 1

[0066] Preparation and analysis of oyster shell powder carrier

[0067] Oyster shells were scrubbed and soaked overnight in tap water to remove salt, biomass, and other surface residues. They were then rinsed with deionized water, dried at 60°C for 24 hours, thoroughly ground, and passed through a 100-mesh sieve for later use. The prepared oyster shell powder was calcined at 400°C for 3 hours in a muffle furnace and used for subsequent experiments.

[0068] The prepared oyster shell powder and calcined oyster shell powder samples were characterized and analyzed using instruments such as X-ray diffractometer (XRD-BTX using Co-Kα, Olympus, USA), Fourier transform infrared spectrometer (FTIR, Hyperion 2000, DEU), simultaneous thermal analyzer (TGA-DTA using Diamond DMA, Perkin-Elmer, USA), pore analyzer (BET, Autosorb-iQ), scanning electron microscope and energy dispersive spectroscopy (SEM-EDS, Zeiss Gemini Sigma 300VP, SEM-Oxford Xplore 30, DEU).

[0069] X-ray diffraction is commonly used for phase analysis, crystallinity determination, and precise determination of lattice parameters. This experiment used X-ray diffraction to analyze oyster shell powder and calcined oyster shell powder, and the results are as follows: Figure 1 As shown. Analysis revealed that calcite-type calcium carbonate is the main mineral component of oyster shells (ICDD No. 89-1305). After calcination at various temperatures, it was found that the mineral phase of the oyster shell powder did not change significantly, and all were calcite-type calcium carbonate. This may be because the phase transition temperature of calcium carbonate is higher than the highest temperature set in the experiment, so the mineral change process from calcium carbonate to calcium oxide was not shown.

[0070] According to FTIR spectra ( Figure 2 The display shows 1477cm. -1 1083cm -1 856cm -1 713cm -1 It is CO3 2- Characteristic absorption peaks

[121] Among them, 1477cm -1 CO3 2- antisymmetric stretching vibration absorption peak, 856 cm⁻¹ -1 The out-of-plane bending absorption peak is 713 cm⁻¹. -1 This is an in-plane bending absorption peak. Besides the characteristic peaks of calcite, 3425 cm⁻¹ is also present. -1 2920cm -1 2518cm -1 The characteristic peaks at this location are those at the positions of -OH, CH, and NH / C=O.

[0071] TG-DTG thermal analysis results indicate that the mass loss of oyster shells mainly occurs in two stages. Figure 3 a). The first stage of mass loss (2.3 wt%) occurred in the temperature range of 25 °C to 647.3 °C, presumably caused by the decomposition and volatilization of trace organic matter in the oyster shells. The second stage of mass loss (97.7 wt%) occurred in the temperature range of 647.3–1000 °C, and according to the DTA curve analysis, there was a significant endothermic peak at 731.2 °C. Figure 3 (b) This is due to the mass loss caused by the decomposition of CaCO3 in the oyster shell into CaO and CO2, accompanied by an endothermic reaction. Based on the above results, we can infer that the oyster shell is an organic-inorganic mineral complex, with a CaCO3 content of approximately 97.7 wt% and an organic matter content of approximately 2.3 wt%.

[0072] Table 1 shows the comparative analysis results of oyster shell powder before and after calcination. The results show that the initial average pore size of uncalcined oyster shell powder (OYS) was 24.20 nm. After calcination, the pore size of oyster shell powder (COYS) increased significantly, with an average pore size of 39.35 nm. The results indicate that the pore size of oyster shell powder was significantly increased after calcination, providing larger adsorption sites, which is beneficial for the adsorption of microorganisms.

[0073] Table 1 Surface structural characteristics of different oyster shell powders

[0074]

[0075] The microstructure of oyster shells was analyzed using scanning electron microscopy. Figure 4 ). Figure 4 c shows that the main elements of oyster shells are C, O, and Ca, while after calcination at 400℃ ( Figure 4 f) The main elements and their content in oyster shells did not change significantly, indicating that their main components remained unchanged. From Figure 4 b shows that the uncalcined oyster shell has a rough surface. Figure 4 The surfaces of a and b) have a small number of irregular porous structures. The oyster shell fragments are large and accumulate in a sheet-like structure, while the outer shells of oyster shells that have undergone high-temperature calcination ( Figure 4 d and e) are fragmented, resulting in a significant increase in the number of surface pores with a pore size of approximately 40 μm. This significantly increases the specific surface area and forms a honeycomb-like porous structure. This porous structure not only enhances the adsorption and slow-release capacity of oyster shell powder for bacteria but may also improve soil aeration and water and fertilizer retention capacity.

[0076] Example 2

[0077] Microbial culture and preparation of microbial agents

[0078] 1. Cultivation of Bacillus jellyoides K02 strain: The K02 strain stored at -80℃ was revived by a warm water bath, streaked in nitrogenous solid medium, and then incubated statically at 30℃ for 24 hours. Single colonies were picked and the culture was repeated once. Using an inoculation loop, 1-2 loops of a single colony were picked and cultured in nitrogenous liquid medium (100 mL) at a constant temperature with shaking to obtain Bacillus jellyoides K02 seed culture (culture conditions: 180 r / min, 30℃, culture time 48 h).

[0079] Cultivation of Bacillus belyssae LB002: The LB002 strain stored at -80℃ was revived by a warm water bath, streaked in LB solid medium, and then incubated statically at 37℃ for 8 hours. Single colonies were picked and the culture was repeated once. Using an inoculation loop, 1-2 loops of a single colony were picked and cultured in 100mL of LB liquid medium at a constant temperature with shaking to obtain Bacillus belyssae LB002 seed culture (culture conditions: 180r / min, 37℃, culture time 8h).

[0080] 2. Wash and soak oyster shells in tap water overnight to remove salt, biomass, and other surface residues. Rinse with deionized water, dry the washed shells at 60°C for 24 hours, then grind them thoroughly using a grinder and pass them through a 100-mesh sieve for later use. Calcinate the prepared oyster shell powder in a muffle furnace at 400°C for 3 hours. The calcined oyster shell powder will be used for subsequent experiments.

[0081] 3. Weigh 1g of the prepared oyster shell powder carrier and place it in a 50mL Erlenmeyer flask. Add 18.5mL of nitrogenous liquid culture medium, then autoclave at 121℃ for 20min. Cool to room temperature and add Bacillus subtilis K02 seed culture (OD200) that has been grown for 24h. 600Approximately 1.6-1.5 mL of the mixture was shaken and incubated in a constant temperature shaking incubator (170 rpm, 30°C). After 12 hours of loading, the mixture was removed and allowed to settle naturally for 3 minutes. The supernatant was removed, and the mixture was washed with sterile water. The mixture was then centrifuged for 5 minutes (4500 rpm) in a high-speed centrifuge. After washing twice, the precipitate was collected to obtain the microbial inoculum K02-COYS, which was oyster shell powder loaded with Bacillus cereus K02.

[0082] Weigh 1 g of the prepared oyster shell powder carrier and place it in a 50 mL Erlenmeyer flask. Add 18 mL of LB liquid culture medium, then autoclave at 121 °C for 20 min and cool to room temperature. Add Bacillus belye seed culture (OD) that has been inoculated and cultured for 8 h. 600 Approximately 1.32 mL was added, shaken to mix, and then placed in a constant temperature shaking incubator (37℃, 170 rpm). After 8 hours of loading, the mixture was removed, allowed to settle naturally for 3 minutes, the supernatant was removed, and the mixture was washed with sterile water and centrifuged for 5 minutes (4500 rpm) in a high-speed centrifuge. The washing process was repeated twice, and the precipitate was collected. This yielded the oyster shell-loaded Bacillus belye microbial agent LB002-COYS.

[0083] Example 3

[0084] Determining the optimal load time

[0085] According to the method of Example 2, in step 3:

[0086] (1) Samples were taken from the gelatinous Bacillus spp. K02 loading group at loading times of 4h, 8h, 12h, 16h, 20h, and 24h. The number of viable bacteria loaded was measured to determine the optimal loading time.

[0087] (2) Samples were taken from the Bacillus belyss LB002 loading group at loading times of 2h, 4h, 6h, 8h, 10h, 12h, 16h, and 20h. The number of viable bacteria loaded was measured to determine the optimal loading time.

[0088] The bacterial load of calcined oyster shell powder under different loading times is shown in the figure. Figure 5 .Depend on Figure 5 As can be seen, the adsorption capacity of the carrier for *Bacillus spp.* K02 was low from 4 to 8 hours, but increased significantly from 12 to 16 hours with increasing adsorption time, and the average viable bacterial load was 3.8 × 10⁻⁶. 6 CFU / g and there was no significant difference between the two. With increasing adsorption time, the load on the support gradually decreased, and a significant decrease in adsorption was observed at loading times of 20 h and 24 h; according to Figure 5b indicates that the adsorption capacity of the carrier for Bacillus belye was low in the first 6 hours, but increased significantly from 8 to 10 hours with increasing adsorption time. The average viable cell load was 3.93 × 10⁻⁶. 8 CFU / g and 3.82×10 8 The CFU / g values ​​were not significantly different between the two strains. As adsorption time increased, the carrier loading gradually decreased. With further extension of the loading time, a significant decrease in adsorption was observed. To save time and cost, 12 hours was selected as the optimal loading time for *Bacillus lentigines* strains, and 8 hours as the optimal loading time for *Bacillus belyssae* strains. Initially, the loading of both bacteria was relatively low, possibly because the bacteria in the culture medium were in the early stages of growth and reproduction, resulting in a low overall bacterial concentration and insufficient bacterial source for loading.

[0089] Example 4

[0090] Determination of the optimal initial bacterial dose

[0091] According to the method of Example 2, in step 3:

[0092] (1) In the Bacillus spp. K02 loading group, 0.5, 1, 1.5, 2, and 2.5 mL of Bacillus spp. K02 seed culture (OD600 approximately 1.6) were loaded and cultured. The number of viable bacteria loaded was measured to determine the optimal initial bacterial dose.

[0093] (2) In the Bacillus belyss LB002 loading group, 0.5, 1, 1.5, 2, and 2.5 mL of Bacillus belyss LB002 seed culture (OD600 approximately 1.3) were loaded and cultured. The number of viable bacteria loaded was measured to determine the optimal initial bacterial dose.

[0094] The results of the analysis of the bacterial load of calcined oyster shell powder under different inoculum amounts are shown in the figure. Figure 6 .Depend on Figure 6 As can be seen, with increasing initial bacterial agent dosage, the adsorption capacity of oyster shell powder for *Bacillus subtilis* increased. When the initial bacterial agent dosage was greater than 0.5 mL, the viable bacterial load significantly increased. The mean viable bacterial load was 3.07 × 10⁻⁶ when the initial bacterial agent dosage was 1, 1.5, 2, and 2.5 mL. 6 4.33×10 6 3.93×10 6 4.13×10 6 CFU / g, and no significant difference; by Figure 6As shown in b, the carrier loading gradually increased significantly with the increase of the initial addition amount of Bacillus belyceae seed culture. When the initial addition amount of Bacillus belyceae exceeded 1.5 mL, the increase in viable cell loading was not significant. The maximum viable cell loading was observed at an initial addition amount of 2.0 mL, with a mean value of 10.2 × 10⁻⁶. 8 CFU / g. To save time and cost, 1.5 mL was chosen as the optimal amount of inoculum for loading gelatinous Bacillus (carrier), and 2.0 mL was chosen as the optimal amount for loading Bacillus belye from oyster shell powder. Both bacteria were initially loaded at low concentrations, but subsequently increased significantly. This may be because the lower seed culture inoculum resulted in a lower total number of bacteria growing and multiplying, leading to lower overall bacterial activity and concentration.

[0095] Example 5

[0096] Determination of the optimal calcination temperature for oyster shell powder

[0097] According to the method of Example 2, in step 1:

[0098] The calcination temperatures of oyster shell powder were 100℃, 200℃, 300℃, 400℃, 500℃, and 600℃, with a time of 3 hours. Microbial inoculants were prepared according to the method in Example 1, and the number of viable bacteria was measured to determine the optimal calcination temperature for the oyster shell powder.

[0099] Depend on Figure 7 It is evident that there are significant differences in the viable bacterial load on different carriers. Oyster shell powder calcined at different temperatures exhibits different loading performance. With increasing calcination temperature, the overall trend of the viable bacterial load for both types of bacteria is an initial increase followed by no significant change, reaching equilibrium. Figure 7 As the calcination temperature increased, the adsorption capacity of oyster shell powder for *Bacillus subtilis* increased. However, when the calcination temperature exceeded 400℃, the viable bacterial load no longer increased significantly. The average viable bacterial load of oyster shell powder at calcination temperatures of 400℃, 500℃, and 600℃ was 4.4 × 10⁻⁶. 6 4.28×10 6 4.3×10 6 CFU / g, and no significant difference; by Figure 7 As shown in b, the viable bacterial load gradually increases with increasing calcination temperature of oyster shell powder, and this increase is significant. When the calcination temperature of oyster shell powder exceeds 400℃, the increase in viable bacterial load on the carrier becomes insignificant, and the average maximum viable bacterial load at 400℃ is 6.5 × 10⁻⁶. 8 CFU / g. To save time and cost, 400℃ was chosen as the optimal temperature for calcining oyster shells and used in subsequent experiments.

[0100] Example 6

[0101] Determination of different cultivation methods

[0102] By comparing static adsorption culture and oscillating adsorption culture, the effect of oscillation conditions on the loading effect was investigated. The culture conditions were the same as in Example 2, and the control group was a static environment. The number of viable bacteria loaded was measured to determine the loading method of oyster shell powder.

[0103] The loading effects of calcined oyster shell powder on two different bacteria under two different loading methods are shown in the figure. Figure 8 The results showed that the viable bacterial load of the shaking adsorption method was greater than that of the static adsorption method. Furthermore, there were significant differences in the viable bacterial load of different carriers in *Bacillus spp.*, and oyster shell powder calcined at different temperatures exhibited different loading performances. With increasing calcination temperature, the overall trend of the viable bacterial load for both bacteria was an initial increase followed by no significant change and eventually reaching equilibrium.

[0104] Example 7

[0105] Comparison before and after bacterial loading

[0106] X-ray diffraction (XRD) analysis was performed on the two bacterial agent samples prepared in Example 2. Figure 9 a) It was found that the mineral composition of oyster shell powder loaded with the two bacteria remained unchanged, with calcite being the dominant mineral peak. The two bacteria did not alter the mineral crystal form of the calcined oyster shell powder. Although the seed cultures of the two bacteria were weakly acidic, this was insufficient to dissolve and destroy the calcium carbonate, thus preserving the original mineral phase of the calcined oyster shell powder. FTIR analysis showed ( Figure 9 b) The two samples were at 1427 cm⁻¹ -1 1083cm -1 875cm -1 713cm -1 Contains CO3 2- Characteristic absorption peaks, of which 1427 cm⁻¹ -1 CO3 2- antisymmetric stretching vibration absorption peak, 879 cm⁻¹ -1 The out-of-plane bending absorption peak is 713 cm⁻¹. -1 This is an in-plane bending absorption peak. Besides the characteristic peaks of calcite, 3382 cm⁻¹ is also present. -1 2883cm -1 2509cm -1 The characteristic peaks at this location are those of -OH, CH, and NH / C=O. K02-COYS has a peak at 1041 cm⁻¹. -1 and 1142cm -1 The presence of an absorption peak at C=O indicates the presence of organic matter.

[0107] To investigate the organic matter content in calcined oyster shell powder after loading with two microorganisms, TG-DTG and DTA were used for analysis. The results are shown below. Figure 10 The mass loss of K02-COYS under conditions of 25–1000℃ mainly occurs in three stages. The first stage of mass loss (1.3 wt%) occurs in the temperature range of 25℃ to 166.5℃, which is due to the evaporation of residual moisture in the sample. The second stage of mass loss occurs in the temperature range of 13.5 wt%, which is between 166.5℃ and 675.7℃. A significant endothermic peak is observed at 190℃ in the DTA curve, which is caused by the combustion of a small amount of organic matter in K02-COYS, further confirming the presence of microorganisms in the calcined oyster shell powder. The third stage of mass loss occurs in the temperature range of 675.7℃ to 759℃, with a significant endothermic peak at 745.3℃ in the DTA curve. This is because, with increasing temperature, CaCO3 in K02-COYS decomposes into CaO, CO2, and CO upon heating. The mass loss of LB002-COYS under the conditions of 25–1000℃ mainly consists of two stages. The first stage of mass loss (4.6 wt%) occurs in the temperature range of 144.4℃ to 218.4℃, which is due to the evaporation of residual moisture and the combustion of organic matter in the sample. This stage is not obvious. The second stage of mass loss (40.2 wt%) occurs in the temperature range of 508.8℃ to 768℃. A significant endothermic peak is observed at 723.8℃ in the DTA curve. This is because as the temperature increases, CaCO3 in LB002-COYS decomposes into CaO, CO2, and CO upon heating.

[0108] Figure 11 SEM images in b show that the pores on the surface of K02-COYS are filled. Further analysis revealed a mixture of broken oyster shell powder and bacterial strains within the pore structure of K02-COYS. A filamentous, adhesive structure was also observed between the oyster shells and bacteria. Figure 11 a). Figure 11 SEM images of LB002-COYS in d and e revealed the surface morphology of the strain attached to oyster shell powder, and were consistent with... Figure 11 Similar to b, bacteria and irregular oyster shell fragments exist on the surface and in the porous structure of oyster shells. Due to electrostatic adsorption, bacteria can enter the pores or attach to the surface of the oyster shell. This relatively loose layered structure and pores ensure sufficient contact between bacterial cells and the carrier, providing conditions for bacterial loading and fixation. Furthermore, due to… Figure 11 c and Figure 11The EDS energy dispersive spectroscopy analysis results show that K02-COYS contains Ca (40.7%), O (52.6%), C (4.1%) and N (2.5%), while LB002-COYS contains Ca (49.6%), O (43.9%), C (3.8%) and N (2.4%), indicating that the main component of these two bacterial agents is still calcium carbonate.

[0109] Example 8

[0110] Soil remediation application trial

[0111] 1. Experimental Design of Suaeda salsa Potted Plants

[0112] In the pot experiment, each pot contained the same number of healthy Suaeda salsa seeds, and 800g of soil substrate (coastal saline-alkali soil from Lianyungang New City, Lianyungang City) was added to each pot. The exogenous additives (oyster shell powder or microbial inoculant) accounted for 1.5% of the total soil mass, and the mass ratio of the mixed inoculant addition group K02-COYS to LB002-COYS (prepared in Example 2) was 1:1. The experiment set up 6 treatment groups, with 6 pots replicated in each group. The original soil was set as the CK group as a blank control. The planting of Suaeda salsa and the setting of inoculant are shown in Table 2.

[0113] Table 2 Different treatment groups

[0114]

[0115] 2. Measurement of physiological indicators of Suaeda salsa growth

[0116] Suaeda salsa plants were collected after 90 days of growth for testing. Figure 12 a represents the aboveground plant height and underground root length of Suaeda salsa in each treatment group. The results showed that the Suaeda salsa in the treatment groups with K02-COYS and LB002-COYS microbial agents (TB, TC, TD groups) grew better than the control group (TE group) without microbial agents and oyster shell powder (OYS) and the group with only oyster shell powder (TA group). Statistical results showed that the average height of the aboveground parts of *Suaeda salsa* in the TB group was 19.88 cm, in the TC group it was 26.65 cm, and in the TD group it was 23.11 cm. The aboveground plant height of each experimental group was significantly increased by 31.36% (TB), 76.10% (TC), and 52.71% (TD) compared to the TE group (p<0.05). The average length of the underground parts of *Suaeda salsa* in the TB group was 8.65 cm, in the TC group it was 8.02 cm, and in the TD group it was 7.93 cm. The root length of each group was increased by 78.90%, 65.93%, and 64.14% compared to the TE group, and there was a significant difference between the two groups (p<0.05). Figure 12b shows the results of the total biomass determination of *Suaeda salsa*. As can be seen from the figure, the dry weights of *Suaeda salsa* in the TA, TB, TC, and TD groups were 0.47 g, 0.5 g, 0.84 g, and 0.57 g, respectively, while the dry weight of the TE group was 0.20 g. The dry weights of the groups treated with the microbial agent increased by 1.46 times, 3.13 times, and 1.82 times compared to the TE group, respectively, and the differences between the groups were statistically significant (p < 0.05). This indicates that the application of microbial agents can promote the accumulation of *Suaeda salsa* biomass. In summary, K02-COYS and LB002-COYS can effectively promote the accumulation of *Suaeda salsa* biomass, meaning that K02-COYS and LB002-COYS have a significant growth-promoting effect on *Suaeda salsa*.

[0117] Plant chlorophyll uses light energy to synthesize carbohydrates from CO2 and water in the air, releasing oxygen. This biological process is called photosynthesis, an essential metabolic activity for plant life. The chlorophyll content of Suaeda salsa leaves in different treatment groups (…) Figure 13 The results showed significant differences in chlorophyll content among the groups of Suaeda salsa leaves. The order of chlorophyll a content from highest to lowest was: TB group (1.53 mg / g), TA group (1.42 mg / g), TD group (1.21 mg / g), TC group (1.04 mg / g), and TE group (0.86 mg / g), with significant differences between TA and TB groups and TC and TD groups. The order of chlorophyll b content from highest to lowest was: TB group (0.68 mg / g), TA group (0.65 mg / g), TD group (0.54 mg / g), TC group (0.44 mg / g), and TE group (0.34 mg / g), with significant differences between TA and TB groups and TD and TE groups. The order of carotenoid content from highest to lowest was: TB group (0.32 mg / g), TD group (0.29 mg / g), TA group (0.24 mg / g), TC group (0.21 mg / g), and TE group (0.16 mg / g). Significant differences were found between the TB group and the TA, TC, and TE groups. The order of total chlorophyll content from highest to lowest was: TB group (2.21 mg / g), TA group (2.07 mg / g), TD group (1.74 mg / g), TC group (1.48 mg / g), and TE group (1.21 mg / g). Significant differences were found between the TA and TB groups and the TD and TE groups.

[0118] Soluble sugars in plants not only provide energy and metabolic intermediates for plant growth and development, but also serve as important regulators of plant growth, development, and gene expression. The soluble sugar content of *Suaeda salsa* in different treatment groups (…) Figure 14The results showed that the soluble sugar content of each treatment group was in the following order: TD group (9.80%) > TC group (8.89%) > TB group (6.33%) > TA group (3.31%) > TE group (1.91%). This result indicates that the soluble sugar content of Suaeda salsa plants in the TB, TC, and TD groups treated with the added microbial agent was significantly higher than that in the TA and TE groups.

[0119] When plant organs age or are damaged under adverse conditions, cell membrane lipid peroxidation often occurs. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of damage suffered by the plant under adverse conditions. Figure 15 The results showed that the TA (4.56 μmol / kg), TC (3.9 μmol / kg), and TE (4.01 μmol / kg) groups had lower MDA contents, while the TB (5.97 μmol / kg) and TD (5.38 μmol / kg) groups had relatively higher MDA contents. Significant differences were found between the TC and TE groups and the TB and TD groups. These results indicate that, compared to the two treatment groups treated with the fungal agent alone, the treatment group treated with the mixed fungal agent had the lowest malondialdehyde content, resulting in less stress damage to the plants and promoting the growth and development of *Suaeda salsa*.

[0120] Under stress conditions such as drought and salinity, the proline content in plants increases significantly. The proline content in plants can reflect their stress resistance to some extent. Figure 16 The results showed that the free proline content in the TC and TD groups was significantly lower than that in other treatment groups, indicating that the addition of the microbial agent affected the free proline content of *Suaeda salsa*. The order of free proline content from highest to lowest was TB (292.41 μg / g) > TA (248.84 μg / g) > TE (231.29 μg / g) > TC (185.35 μg / g) ≈ TD (184.94 μg / g). These results indicate that the effects of applying LB002-COYS alone and in combination with K02-COYS have similar effects, both reducing the free proline content in plants. Proline, as a component of plant proteins, is widely distributed in plants. Under adverse conditions such as drought and salinity, plants accumulate a significant amount of proline, and its content reflects the plant's stress resistance. Therefore, it can be inferred that the TD and TC groups of *Suaeda salsa* experienced lower levels of salt stress and had a better growth environment than the other treatment groups.

[0121] 3. Results of soil properties and soil mineral composition analysis

[0122] Soil pH has a significant impact on plant growth and soil fertility. Excessively acidic or alkaline soils can impair nutrient absorption by plants, thus affecting their growth and development. In this study, the soil pH values ​​of the treatment groups with different microbial agents were all lower than those of the control group. Figure 17The soil pH of group CK was 8.35. The soil pH values ​​of groups TA (oyster shell powder), TB (K02-COYS), and TD (LB002-COYS) were 8.27, 8.18, and 8.14, respectively. The soil pH value of group TC (mixed microbial inoculant) was the lowest at 8.07. The results indicate that the mixed microbial inoculant treatment had the most significant effect, followed by the inoculant-only treatments, while the effect of adding only oyster shell powder was the worst. EC value indicates the total concentration of soluble salts; a higher soil EC value indicates a greater concentration of soluble salt ions in the soil. Figure 17 The results showed that the conductivity of the TA, TB, TC, and TD groups with added soil conditioners was lower than that of the CK group, with the CK group having a conductivity of 1617.67 μS / cm. The TE group, which only had Suaeda salsa, showed no significant decrease in conductivity. The TD treatment group had the lowest conductivity (783.67 μS / cm), followed by the TB group (980 μS / cm), TC group (985.33 μS / cm), and TA group (1124 μS / cm). The following are the chemical analysis results of the soil from different treatment groups in the pot experiment. As shown in Table 3, compared with the initial CK group, the total carbon (TC) content of each group increased. There was no significant difference between each treatment group and the CK group (0.83 mg / g), but a significant difference existed between the TB group and the TA group. The total sulfur (TS) content of each group changed relative to the CK group (0.06 mg / g), with the largest increase in the TB group (0.076 mg / g) and the largest decrease in the TA group (0.04 mg / g). Water-soluble salts in soil are an important property of saline-alkali soils and a limiting factor for crop growth. The water-soluble cations (Na+) in soil samples from each treatment group... + K + Mg 2+ Ca 2+ ) and anions (Cl) - , The results of the determination are shown in Table 3. The results show that, compared with the CK group, the concentrations of all cations in the treatment groups generally decreased. Among them, the sodium ion content (81.89 mg / kg) and potassium ion content (232.36 mg / kg) decreased the most in the TB group, the magnesium ion content (21.90 mg / kg) decreased the most in the TD group, and the calcium ion content (991.55 mg / kg) decreased the most in the TA group. The chloride ion content generally decreased, with the chloride ion content decreasing in the following order: TE (2.49 mg / kg) > CK (2.45 mg / kg) > TA (2.22 mg / kg) > TB (1.92 mg / kg) ≈ TC (1.92 mg / kg) > TD (1.53 mg / kg). The TD group showed the most significant reduction in chloride ion content. The sulfate ion content of each group, from highest to lowest, was TB (3.18 mg / kg) > TA (2.99 mg / kg) ≈ TD (2.99 mg / kg) > TC (2.59 mg / kg) > TE (2.25 mg / kg) > CK (2.4 mg / kg). The bicarbonate ion content of each group, from highest to lowest, was TB (0.39 mg / kg) > TC (0.34 mg / kg) ≈ TD (0.34 mg / kg) > TA (0.32 mg / kg) > TE (0.31 mg / kg) > CK (0.29 mg / kg), with the TB group showing a significantly higher bicarbonate content.

[0123]

[0124] Figure 18 Figure a shows the differences in soil moisture content among different treatment groups. The soil moisture content of the CK group was 11.56%, while the soil moisture contents of the TA, TB, TC, TD, and TE groups were 11.45%, 12.64%, 11.93%, 13.70%, and 13.45%, respectively, which were -0.09%, 1.09%, 0.38%, 2.15%, and 1.90% higher than that of the CK group, respectively. However, only the TD group showed a significant difference from the CK group (p<0.05). Figure 18 b represents the available phosphorus and potassium contents in the soil of different treatment groups. The available potassium contents of each group were as follows: TA group (48.36 mg / kg), TB group (47.93 mg / kg), TC group (44.71 mg / kg), TD group (44.74 mg / kg), TE group (49.23 mg / kg), and CK group (47.58 mg / kg). Among them, the TC group and TD group were significantly lower than the CK group, decreasing by 6.02% and 5.97%, respectively. The analysis results of the available phosphorus contents in the soil of different treatment groups showed that ( Figure 18(b) The available phosphorus content in the TC group (89.3 mg / kg) was significantly higher than that in other treatment groups, and significantly higher than that in the CK group (58.3 mg / kg) by 53.17%. The available phosphorus contents in the soil of the TA, TB, TD, and TE groups were 53.7 mg / kg, 59 mg / kg, 55.3 mg / kg, and 59.2 mg / kg, respectively.

[0125] Soil organic matter is the foundation of soil fertility and one of the main indicators of soil fertility level. Figure 19 The soil organic matter content was measured in different treatment groups. The results showed that compared with the control group (1.84%), the soil organic matter content in all other groups increased to some extent, with the highest values ​​for each treatment group being TA (3.49%), TB (3.87%), TC (3.95%), TD (3.83%), and TE (3.06%). There were no significant differences among the TB, TC, and TD groups treated with microbial agents (p>0.05). However, compared with the control group, the application of K02-COYS and LB002-COYS significantly increased the soil organic matter content, with increases of 110.33%, 114.67%, and 108.15% in the three treatment groups, respectively. The TA group, which only added oyster shell powder, also showed a significant increase compared to the CK group (89.67%), but there was no significant difference compared to the TB, TC, and TD groups, which added microbial agents. Meanwhile, the TE group, which only planted plants, also showed a significant increase compared to the CK group (66.30%), but there was no significant difference compared to the TA group.

[0126] Figure 20 Mineral analysis results of the root zone soil of *Suaeda salsa* grown under different growing conditions were presented. XRD analysis revealed that the main mineral component in the saline-alkali soil was quartz (ICDD PDF NO. 85-0794), which is the matrix mineral in the original culture system. The mineral phases found in each group are as follows: Mineral phases in CK soil: Quartz (SiO2), Halloysite (Al2[Si2O5](OH)4), Roscoelite (Al2Si3O4) 10 (OH)2}, Gismondine (calcium hydrate zeolite CaAl2Si2O4·4H2O), Muscovite {mica, KAl2[Si3AlO] 10Mineral phases in TA soils: Quartz (SiO2), Albite (NaAlSi3O8); Mineral phases in TB soils: Quartz (SiO2), Halloysite (Al2[Si2O5](OH)4); Mineral phases in TC soils: Quartz (SiO2), Gismondine (CaAl2Si2O4·4H2O), Halloysite (Al2[Si2O5](OH)4), Muscovite (KAl2[Si3AlO4](OH)4). 10 Mineral phases in TD soil: Quartz (SiO2), Albite (NaAlSi3O8); Mineral phases in TE soil: Quartz (SiO2), Gismondine (CaAl2Si2O4·4H2O), Calcite (CaCO3), Halloysite (Al2[Si2O5](OH)4), Muscovite (KAl2[Si3AlO3]). 10 ](OH,F)2}.

[0127] Different soil amendments have a significant impact on soil carbonate content, and the changes in soil carbonate content are as follows: Figure 21 As shown, the soil carbonate content in the TB group (2.05%) ≈ TE group (2.04%) > TD group (1.83%) > TA group (1.63%) > TC group (1.34%) > CK group (1.14%). Except for the TC group with added microbial agents, the soil carbonate content in all other treatment groups was significantly higher than that in the CK group. The TC group was slightly higher than the CK group, but the difference was not significant. There were no significant differences among the TB, TD, and TE groups.

[0128] 4. Correlation analysis among environmental factors

[0129] Spearman correlation analysis was performed on the growth, physiological indicators, soil physicochemical properties, and nutrient indicators of Suaeda salsa under different treatments. Figure 22A) is a heatmap showing the correlation between soil physicochemical indicators. The results indicate that soil pH was significantly positively correlated with potassium ion content and significantly negatively correlated with soil organic matter and total carbon content (p < 0.05). Soil electrical conductivity was significantly positively correlated with soil salinity, chloride ion, sodium ion, and potassium ion concentrations, and significantly negatively correlated with soil total carbon content (p < 0.05). Soil salinity was significantly positively correlated with soil chloride ion, sodium ion, and potassium ion concentrations, and significantly negatively correlated with soil organic matter content (p < 0.05). Organic matter is a crucial component of soil nutrients. This study shows a significant positive correlation between soil organic matter content and soil total carbon content, possibly because organic matter is primarily carbon-based. It was also significantly negatively correlated with potassium and sodium ion concentrations (p < 0.05). Soil total carbon was significantly negatively correlated with soil chloride ion content (p < 0.05). Soil sodium ions showed a significant negative correlation with sulfate and bicarbonate ions, soil potassium ions showed a significant positive correlation with calcium and magnesium ions, and soil magnesium ions showed a significant positive correlation with calcium ions (p < 0.05).

[0130] Figure 22 b is a heatmap showing the correlation between Suaeda salsa biomass and soil indicators. The plant height and fresh weight of Suaeda salsa were significantly negatively correlated with soil pH (p < 0.05), while fresh weight was significantly positively correlated with soil available phosphorus and calcium ion content (p < 0.05). Root length was significantly negatively correlated with soil salinity and soluble potassium ion content (p < 0.05), and significantly positively correlated with soil organic matter content (p < 0.05). Regarding physiological indicators of Suaeda salsa, chlorophyll content was significantly negatively correlated with soil sulfate ions (p < 0.05), soluble sugar content was significantly positively correlated with soil organic matter content, and significantly negatively correlated with soil electrical conductivity, salinity, and chloride ion content (p < 0.05).

[0131] In this embodiment, a pot experiment was set up using coastal saline-alkali soil as the test soil and Suaeda salsa as the test plant to study the effects of two microbial agents, K02-COYS and LB002-COYS, prepared from waste oyster shells and different application methods on the improvement of saline-alkali soil quality and the growth promotion effect on Suaeda salsa. The results showed that compared with the control group, the treatment group with biological agents significantly increased the content of available phosphorus and organic matter in the soil, significantly reduced the content of soluble salts, significantly improved soil nutrients, significantly improved the growth of Suaeda salsa, improved the quality of saline-alkali soil, and provided a new method for solving the pollution and reuse of waste shellfish resources. (1) In terms of plant growth promotion, the original saline-alkali soil had no significant growth promotion effect on Suaeda salsa. However, in the treatment group with added microbial agents, the physiological indicators of Suaeda salsa were significantly improved. Among them, the TC treatment group had the best growth promotion effect and the best effect on improving stress resistance, the TB treatment group showed a significant improvement in photosynthesis of Suaeda salsa, and the TD treatment group had the best effect on the accumulation of sugar in Suaeda salsa. The results of various plant measurements showed that the application of oyster shell powder could promote the growth and development of Suaeda salsa, but the effect was not significant. However, the application of microbial agents, whether applied alone or in combination, could significantly promote the growth of Suaeda salsa and improve its stress resistance. The results showed that the combined application of microbial agents had the best effect on the growth and development of Suaeda salsa. (2) In terms of soil salinity reduction, the addition of microbial agents could significantly reduce the pH and EC of saline-alkali soil, and the reduction of salt reduced the salt stress on plants. The comparative analysis of the experimental results of each group showed that the TC group treatment performed the best, with the pH and EC decreasing by 0.34 and 632.33 μS / cm, respectively. (3) In terms of soil nutrient and mineral changes, compared with each group, the TC treatment group had the best soil improvement effect, with the soil moisture content and organic matter increasing by 13% and 115.34%, respectively, and the available phosphorus content increasing by 53.06%. This indicates that the combined application of microbial agents and plant treatment had a better effect.

Claims

1. A microbial inoculant based on oyster shell powder load, characterized by, The oyster shell powder-based microbial agent takes calcined oyster shell powder as a carrier, and the carrier is loaded with bacillus.

2. The oyster shell powder-based microbial inoculant according to claim 1, characterized by, The bacillus is Pasteuria sp. K02 or Bacillus velezensis LB002.

3. A method for preparing a microbial inoculant based on oyster shell powder as described in claim 1, characterized in that, The method comprises the following steps: (1) washing and drying oyster shells, crushing the dried oyster shells, and calcining the crushed oyster shell powder to obtain an oyster shell powder carrier; (2) taking the prepared oyster shell powder carrier, placing it in a culture solution, sterilizing it, adding bacillus seed liquid, and culturing and loading the bacillus, then naturally settling the culture, removing the supernatant, centrifuging and washing to collect the precipitate, and obtaining an oyster shell powder-loaded microbial agent.

4. The production method according to claim 3, characterized by, In step (1), the oyster shells are scrubbed and soaked in water overnight to remove salt, biomass and other surface residues, rinsed with deionized water, dried, and then ground with a grinder, sieved, and stored for use.

5. The preparation method according to claim 3, characterized in that, In step (1), the calcination temperature is 100-600°C, and the calcination time is 3-4h.

6. The preparation method according to claim 3, characterized in that, In step (1), the culture medium is a nitrogen-containing culture medium or an LB culture medium.

7. The preparation method according to claim 3, characterized in that, The mass-volume ratio of the oyster shell powder carrier, the bacillus spore seed liquid and the culture solution in step (2) is 1 g: 1-2 mL: 15-25 mL, and the OD 600 of the bacillus spore seed liquid is 1-2.

8. The preparation method according to claim 3, characterized in that, In step (2), the culturing and loading time is preferably 8-12h, the culturing temperature is 30-37°C, and the rotation speed is 150-200rpm.

9. Use of the oyster shell powder-loaded microbial agent of claim 1 in the remediation of coastal saline-alkali soil and the promotion of the growth of salt-tolerant plant Suaeda glauca.

10. Use according to claim 9, characterized in that, The oyster shell powder-loaded microbial agent is combined with salt-tolerant plant Suaeda glauca for use in the remediation of coastal silty saline-alkali soil.

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