A bacteria-algae symbiotic system repairing preparation, a preparation method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a bacteria-algae symbiotic system repair preparation, a preparation method and application thereof. In particular, the bacteria-algae symbiotic system repair preparation comprises acid-resistant microalgae and acid-resistant bacteria, the acid-resistant microalgae is Chlamydomonas SAG 2486, and the acid-resistant bacteria comprises Serratia liquefaciens AN41 and Serratia JG-4-2. The bacteria-algae symbiotic system repair preparation disclosed by the application can naturally improve soil through biological metabolism, and avoids the risks of soil compaction, ion imbalance and secondary salinization caused by chemical acid-adjusting agents.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a fungal-algae symbiotic system repair agent, its preparation method, and its application. Background Technology

[0002] The black soil region of Northeast my country is an important grain production base. However, in recent years, affected by factors such as acid deposition, monoculture farming, and high-intensity fertilization, typical black soil areas in this region (such as the northern Songnen Plain) have shown significant soil acidification, with soil pH values ​​dropping to the range of 4.0–5.5. Soil acidification has triggered a series of ecological problems, including soil structure deterioration, soil fertility decline, reduced microbial diversity, and decreased availability of nutrients such as phosphorus and potassium, seriously restricting sustainable agricultural production and food security in the region.

[0003] To address soil acidification, current technologies commonly employ the application of chemical acidifiers (such as lime). While this method can raise soil pH in the short term, it has drawbacks such as causing soil ion imbalance, inducing secondary salinization, and disturbing the soil micro-ecosystem, thus failing to achieve eco-friendly long-term remediation. On the other hand, bioremediation technologies using single-function microbial agents suffer from low survival rates and poor environmental adaptability under strong acid stress, resulting in unstable remediation effects that are difficult to maintain in the long term. Summary of the Invention

[0004] This invention aims to provide a fungal-algae symbiotic system remediation agent, its preparation method, and its application. This fungal-algae symbiotic system remediation agent naturally improves the soil through biological metabolism, avoiding the risks of soil compaction, ion imbalance, and secondary salinization caused by chemical acidifiers.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A fungus-algae symbiotic system remediation preparation, the remediation preparation comprising acid-resistant microalgae and acid-resistant bacteria, wherein the acid-resistant microalgae is Chlamydomonas (… Chlamydomonas sp. SAG 2486, the acid-resistant bacteria include Serratia liquefaction (Serratia liquefaction). Serratia liquefaciens AN41 and Serratia ( Serratia sp. )JG-4-2.

[0007] Preferably, the Chlamydomonas genus ( Chlamydomonas sp. SAG 2486 was deposited on May 12, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO:M20251014.

[0008] Preferably, the liquefied Serratia ( Serratia liquefaciensAN41 was deposited on May 12, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, with accession number CCTCCNO:M 20251013.

[0009] Preferably, the *Serratia* genus ( Serratia sp. JG-4-2 was deposited on May 12, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO:M20251012.

[0010] Preferably, the repair preparation contains acid-resistant microalgae and acid-resistant bacteria in a cell ratio of 2:1 to 3:2.

[0011] This invention also provides a method for preparing a repair agent for a fungal-algae symbiotic system as described above, comprising the following steps:

[0012] S1. Isolate and screen acid-resistant microalgae and acid-resistant bacteria from acidified soil;

[0013] S2. The acid-resistant bacteria and acid-resistant microalgae selected in S1 were co-cultured in LB:BG11 combined medium at pH 5.0 to obtain a symbiotic solution.

[0014] S3. The symbiotic solution obtained in S2 is mixed with sodium alginate and bentonite, and then added dropwise to CaCl2 solution for cross-linking and molding. After freeze-drying, the repair agent is obtained.

[0015] Preferably, in S2, the co-cultivation conditions are: temperature 25℃, light intensity 2000-3000 Lux, light-dark cycle 16 / 8h, shaker speed 130r / min, cultivation time 48h, and system OD... 600 It reaches 0.8-1.2.

[0016] This invention also provides the application of the described microbial-algae symbiotic remediation formulation in the remediation of acidic soils.

[0017] Preferably, the remediation agent is used to increase soil pH, activate available phosphorus, available potassium and ammonium nitrogen, increase soil organic matter content, and enhance soil urease and catalase activity.

[0018] Preferably, the application rate of the remediation agent is 1-2 g / kg soil, and the remediation period is 30-45 days.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] This invention discloses a microbial-algae symbiotic remediation formulation, its preparation method, and its application. Acid-tolerant microalgae and acid-tolerant bacteria were screened and a synergistic symbiotic system was constructed. The system was then immobilized on a sodium alginate-bentonite composite carrier, successfully preparing a remediation formulation that maintains high activity and structural stability under strong acid and high-temperature stress. This microbial-algae symbiotic system achieves synergistic remediation through acid reduction, fertilization, and growth promotion. The system couples the functions of microalgae photosynthesis (oxygen release and carbon fixation) and bacterial metabolism (alkali production), not only rapidly and persistently increasing soil pH but also efficiently activating fixed nutrients such as phosphorus and potassium in the soil and significantly enhancing the activity of key soil enzymes such as urease and catalase. Simultaneously, this formulation naturally improves the soil through biological metabolism, avoiding the risks of soil compaction, ion imbalance, and secondary salinization caused by chemical acidifiers. It is an environmentally friendly and ecologically harmonious green remediation technology. In addition, this remediation agent is a uniform gel bead dry formulation with the advantages of high strength, good slow release, long shelf life and easy application, which makes it easy to promote and apply on a large scale to acidified farmland, reclaimed land and acid rain affected areas, providing a new technical path with significant comprehensive benefits for solving the current soil acidification problem.

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 This is a streak diagram of Chlamydomonas SAG 2486 on a solid culture medium.

[0023] Figure 2 This is a streak diagram of two acid-resistant bacteria on a solid culture medium. Figure 2 In this context, A represents Serratia liquefiedis AN41. Figure 2 B in the text refers to Serratia spp. JG-4-2;

[0024] Figure 3 This is a flowchart of the compounding and synthesis process of the bacteria-algae symbiotic repair system in Example 2;

[0025] Figure 4 The image shown is a scanning electron microscope (SEM) image of the bacterial-algae symbiotic remediation system in Example 2. Figure 4 In the image, A represents the scanning electron microscope image taken on days 0-2. Figure 4 B in the image represents a scanning electron microscope image taken at 2-4 days. Figure 4 C in the image represents a scanning electron microscope image taken at 4-6 days. Figure 4 D in the image represents a scanning electron microscope image taken at 6-8 days. Figure 4 E in the image represents a scanning electron microscope image taken at 8-10 days. Figure 4 F in the image represents a scanning electron microscope image taken at 10-12 days. Figure 4 G in the image represents a scanning electron microscope image taken at 12-14 days. Figure 4 H in the image represents a scanning electron microscope image taken at 14-16 days. Figure 4 In the image, I represents a scanning electron microscope image taken at 16-18 days. Figure 4 J in the image represents a scanning electron microscope image taken at 18-20 days.

[0026] Figure 5 The Fourier transform infrared (FTIR) spectrum of the bacterial-algae symbiotic remediation system in Example 2 is shown below.

[0027] Figure 6 Figure 1 shows the environmental stability experiment of the bacterial-algae symbiotic remediation system.

[0028] Figure 7 The results show the synergistic acid-reducing efficacy of the bacterial-algae symbiotic remediation system under microcosmic conditions, among which, Figure 7 In this context, A represents the nitrate nitrogen content. Figure 7 In this context, B represents the ammonium nitrogen content.

[0029] Figure 8 The results show the synergistic acid-reducing efficacy of the bacterial-algae symbiotic remediation system under microcosmic conditions, among which, Figure 8 In this context, A represents the content of readily available potassium. Figure 8 In this context, B represents the available phosphorus content.

[0030] Figure 9 The effect of pH value on carbon increase and acid reduction in acidic soil based on the bacterial-algae symbiotic system;

[0031] Figure 10 The graph shows the changes in organic matter content in acidic soils based on the carbon-increasing and acid-reducing efficiency of the bacteria-algae symbiotic system.

[0032] Figure 11 Changes in catalase activity of soil microorganisms in soil remediation based on a bacteria-algae symbiotic system;

[0033] Figure 12 Changes in soil microbial acid phosphatase activity during remediation based on a microbial-algae symbiotic system;

[0034] Figure 13 This study investigated changes in soil microbial urease activity during remediation based on a microbial-algae symbiotic system. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0037] BG11 liquid culture medium: commercially available, prepare according to instructions;

[0038] LB medium (200mL formula): 2g tryptone, 1g yeast extract, 2g sodium chloride, distilled water to a final volume of 200mL, pH adjusted to 5.0 by HCl, sterilized at 121℃ for 20min;

[0039] PBS (pH 7.4): NaCl 8.0g, potassium dihydrogen phosphate 0.2g, Na₂HPO₄ 2.9g of 12H2O and 0.2g of potassium chloride were added and brought to a final volume of 1000mL. The mixture was then sterilized at 112kPa for 20min.

[0040] S-LB (200mL): Same as LB, but adjust the volume to 200mL with soil extract, pH 5.0; pH gradient selection: adjust the above culture medium to pH 5.0, 5.5, 6.0, 6.5 as needed.

[0041] Note: Plates and solutions should be autoclaved routinely, and ultraviolet sterilization for 30 minutes may be necessary.

[0042] The potassium-solubilizing bacterium Paenibacillus mucilaginosus K1 was deposited on November 20, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO:M 20242542.

[0043] The phosphate-solubilizing bacterium Enterobacter sp. P1 was deposited on November 20, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO:M 20242544.

[0044] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0045] Example 1

[0046] Screening and molecular identification of acid-resistant algae

[0047] Acidified black soil suspension was spread on pH 5.0 LB plates. Single colonies with obvious morphological differences were picked and subjected to gradient subculturing to pH 3.5. The gradient points were: pH 5.0→4.5→4.0→3.5, with 3 subculturings per step.

[0048] Enrichment of Chlamydomonas SAG 2486 algal strain: single algal colonies were picked from BG11 plates, transferred to pH 5.0 BG11 liquid, and cultured at 25℃, 2500 Lux, 16 / 8h light / dark for 7-10 days;

[0049] Acid resistance evaluation: Under different pH and time conditions, individuals with excellent proliferation rate and survival rate were screened.

[0050] Molecular identification: Genomic DNA was extracted from the above-mentioned selected strains, and their 16S rRNA gene sequences were amplified. After sequencing, the sequences were compared with the NCBI database, and the strains were identified as Chlamydomonas SAG 2486, Serratia liquefaction AN41, and Serratia JG-4-2.

[0051] Genomic DNA was extracted from the selected algal strains, and their 16S rDNA / ITS sequences were amplified, identifying them as Chlamydomonas SAG2486. All identified bacteria and algae are deposited at the China Center for Type Culture Collection (CCTCC).

[0052] Example 2

[0053] Preparation of fungal-algae symbiotic repair agents

[0054] Preparation of bacterial culture: The identified acid-resistant core strains *Serratia liquefaction* AN41 and *Serratia* JG-4-2 were inoculated into LB liquid medium at pH 5.0 and cultured with shaking at 28°C and 130°C until the logarithmic growth phase (OD200). 600 0.8).

[0055] Microbial community optimization: The above bacterial solutions were mixed in different ratios (1:1, 1:2, 2:1) and transferred to fresh 200 mL LB medium (pH 5.0) at an inoculation rate of 2%. The mixture was then cultured at 28 °C and 130 r / min for 48 h to obtain the compound bacterial solution.

[0056] Algal solution preparation: Chlamydomonas SAG 2486 was inoculated into BG11 liquid medium at pH 5.0 and cultured for 7 days at 25℃, 2500 Lux, and 16 / 8h light / dark conditions to obtain the algal solution.

[0057] Construction of the symbiotic system: The compound bacterial solution and algal solution were mixed at bacterial:algal cell ratios of 1:1, 1:2, 2:1, 2:3, and 3:2, and inoculated into LB:BG11 (1:1, v / v) combined medium (pH 5.0). The mixture was co-cultured for 48 hours at 25℃, 2500 Lux, 16 / 8h light / dark cycle, and 130 rpm to determine the optimal bacterial:algal ratio as 3:2.

[0058] The bacterial-algae symbiotic solution was mixed with an equal volume of a mixed carrier solution containing 2% (w / v) sodium alginate and 1% (w / v) bentonite, and then dropped into a 3% (w / v) CaCl2 solution under magnetic stirring at a uniform rate to form uniform gel beads. The gel beads were rinsed with sterile water and then freeze-dried in a freeze dryer at -40℃ for 24 hours to obtain an immobilized bacterial-algae symbiotic repair preparation with an average particle size of about 3 mm, which was stored at 4℃ in the dark.

[0059] The repair formulations prepared in the above examples were characterized and their stability was tested.

[0060] The acid-tolerant microalgae *Chlamydomonas* SAG 2486, which had been acclimatized to liquid culture, was streaked onto BG11 solid medium (pH 5.0) and cultured for 7 days at 25°C, 2000 Lux light intensity, and a 16-hour light / 8-hour dark cycle. The results are as follows: Figure 1 As shown.

[0061] Depend on Figure 1 As can be seen, the algal cells formed a continuous and dense green algal band along the doodled trajectory. The colonies were uniform in color, with clear edges, and no obvious fading or necrotic areas, indicating that the algal strain could maintain good growth vitality even in a weakly acidic environment. Combined with the results of proliferation experiments at different pH gradients (pH 3.5–7.0), the algal strain showed stable growth and rapid recovery at pH 5.0, exhibiting significant acid resistance, and can be considered a preferred algal strain for subsequent bacterial-algal symbiotic remediation preparations.

[0062] The acid-resistant strain of *Serratia liquefaction* AN41, initially screened from acidified black soil, was inoculated into LB solid medium at pH 5.0 and incubated upside down in a 28°C incubator for 48 hours. The strain was purified by three consecutive passages using the streak plating method, designated as CYH01, CYH02, and CYH03 plates, to assess its purity and morphological stability.

[0063] The selected acid-resistant *Serratia* strain JG-4-2 was inoculated into LB solid medium at pH 5.0 and incubated at 28°C for 48 h. To verify its morphological stability and inoculation reproducibility, six parallel plates were prepared, all inoculated using a triangular streak method and cultured under identical conditions. The results are as follows: Figure 2 As shown.

[0064] Results of Serratia liquefaction AN41 Figure 2 As shown in A in the diagram.

[0065] Depend on Figure 2 As shown in A, in terms of colony quantity and distribution, the colonies on each plate grew relatively concentratedly, with evenly distributed striped areas and no obvious contaminating bacteria, indicating good separation. In terms of colony morphology, the surfaces were smooth, the color uniform, and the edges regular, exhibiting typical characteristics of Serratia marcescens colonies. The high morphological consistency among the three plates indicates a stable genetic background of the strain. Judging from purity, no heterogeneous colonies were observed visually, and all three plates showed the same morphology, which can be considered a single pure culture.

[0066] The results of Serratia spp. JG-4-2 are as follows: Figure 2 As shown in B in the diagram.

[0067] Depend on Figure 2As shown in section B, in terms of colony quantity and growth performance, all six plates exhibited abundant striped growth, with uniform colony coverage, indicating good growth and strong viability. Regarding colony morphology, the shape, color, and edge characteristics remained consistent across all plates, with no significant variation observed. In terms of purity and stability, the plates showed good reproducibility and no interference from other microorganisms, indicating that the strain maintains high stability even after continuous subculturing.

[0068] In summary, both groups of strains showed good culture purity, and no heterologous colonies were observed. The colony morphology of *Serratia liquefaction* AN41 was consistent across different plates, indicating stable strain quality; *Serratia* JG-4-2 showed high consistency across six replicate plates, demonstrating even greater stability. Based on these results, both strains meet the quality requirements for subsequent experiments.

[0069] Two core acid-fast bacteria, *Serratia liquefaction* AN41 and *Serratia* JG-4-2, were selected and inoculated into LB liquid medium at pH 5.0 at volume ratios of 1:1, 1:2, and 2:1, respectively, and cultured at 28℃ and 130 rpm for 48 h. The soluble protein content and ammonia nitrogen (NH4+) in the supernatant were then compared. + A comprehensive evaluation radar chart was constructed using five indicators: total nitrogen (TN) concentration, extracellular polysaccharide (EPS) content, and soluble phosphorus concentration (characterizing phosphorus solubilization efficiency). The results are as follows: Figure 3 .

[0070] Depend on Figure 3 It can be seen that the 2:1 treatment showed significantly higher levels of protein, polysaccharide, ammonia nitrogen, and total nitrogen accumulation compared to the 1:1 and 1:2 treatments. The system also exhibited the highest soluble phosphorus content and the best overall score, indicating that the bacterial community exhibited more vigorous metabolism and optimal phosphorus solubilization efficiency at this ratio. Therefore, the optimal ratio of *Serratia liquefiedis* AN41 to *Serratia japonica* JG-4-2 (2:1) was determined to be the optimal ratio for acid-tolerant bacterial communities.

[0071] Optimization of the compound ratio of bacteria-algae symbiotic system

[0072] Based on the determined optimal bacterial ratio (Serratia liquefaction AN41 : Serratia spp. JG-4-2 = 2 : 1), a mixed liquid with the optimal ratio of potassium-solubilizing bacteria and two acid-tolerant bacteria was selected and cultured at volume ratios of 1:1, 1:2, and 2:1. The results showed significantly higher levels of protein, polysaccharide, ammonia nitrogen, and total nitrogen accumulation compared to the 1:1 and 1:2 treatments, indicating that the optimal ratio of potassium-solubilizing bacteria to the mixed bacterial solution was 2:1. The results are as follows: Figure 3 Based on this, a mixed bacterial solution with a ratio of 2:1 (phosphate-solubilizing bacteria and mixed bacterial solution:potassium-solubilizing bacteria) was selected and cultured again. The same indicators were measured, and the optimal ratio was found to be 2:1, resulting in the most effective compound bacterial solution.

[0073] A compound bacterial culture (containing two core acid-tolerant bacteria, one potassium-solubilizing bacterium, and one phosphate-solubilizing bacterium) was mixed with acid-tolerant microalgae inoculum at different bacterial-to-algae cell ratios of 1:1, 1:2, 2:1, 2:3, and 3:2. This mixture was then inoculated into a combined LB:BG11 medium (1:1 v / v) at pH 5.0 and co-cultured for 48 hours at 25°C, 2500 Lux, 16 hours light / 8 hours dark, and 130 rpm. The soluble protein, ammonia nitrogen, total nitrogen, extracellular polysaccharide content, and phosphate-solubilizing efficiency were simultaneously measured, and a comprehensive score was calculated. The results are shown below. Figure 3 As shown.

[0074] Depend on Figure 3 It was found that the bacteria:algae ratio of 3:2 showed the highest overall performance across the five indicators, with a significant increase in protein and polysaccharide accumulation, improved ammonia and total nitrogen conversion efficiency, and the highest soluble phosphorus concentration. This indicates that the nutrient exchange and metabolic synergy between bacteria and algae were most significant at this ratio. Based on the comprehensive evaluation results, the bacteria:algae ratio of 3:2 was determined to be the optimal compound ratio for the bacteria-algae symbiotic remediation preparation.

[0075] Structural characterization: The internal structure of the immobilized bacteria-algae symbiotic remediation agent was observed using scanning electron microscopy (SEM). The results are as follows: Figure 4 .

[0076] Depend on Figure 4 It can be seen that bacteria are attached to the surface of algal cells and form a porous chimeric network; the immobilized beads have uniform pore size, which is conducive to material transport and enzymatic reactions.

[0077] Fourier transform infrared spectroscopy (FTIR) analysis results are as follows Figure 5 As shown.

[0078] Depend on Figure 5 It can be seen that at 3420cm -1 A broad peak of OH stretching vibration appears at 1650 cm⁻¹. -1 The presence of a C=O stretching vibration peak indicates that the bacterial / algal cells and the carrier are tightly bound together by forces such as hydrogen bonds.

[0079] Acid-tolerant bacterial consortium (Serratia liquefaction AN41:Serratia spp. JG-4-2 = 2:1) and acid-tolerant microalgae Chlamydomonas sp. SAG 2486, obtained from previous screening, were mixed at the optimal bacterial:algae cell ratio of 3:2 and inoculated into a combined medium of LB:BG11 = 1:1 (v / v) and pH 5.0. The medium was co-cultured for 48 h at 25℃, 2500 Lux, 16 h light / 8 h dark, and 130 r / min. Separate bacterial consortium, separate microalgae, and blank control groups were also included. Dissolved oxygen (DO), carbon dioxide release, soluble organic carbon (DOC), and inorganic nitrogen (NH4+) were measured. + -N, NO3- The content of N, available phosphorus and micronutrients was analyzed to determine the material exchange relationship between bacteria and algae.

[0080] like Figure 6 As shown, under light conditions, microalgae fix CO2 and release O2 through photosynthesis, and secrete soluble organic carbon and some extracellular polysaccharides, providing carbon and energy sources for bacterial growth. Bacteria utilize the algal organic carbon and consume O2, releasing CO2 during the decomposition of organic matter, while simultaneously mineralizing organic nitrogen, phosphorus, and some trace elements into NH4. + PO4 3- The bacteria are recycled back to the microalgae in their usable forms. Compared with treatments involving bacteria or algae alone, the dissolved oxygen level in the co-culture system was significantly increased, the turnover of soluble organic carbon was accelerated, and the concentrations of inorganic nitrogen and available phosphorus were significantly increased, indicating that a feedback loop of "algal oxygen supply and carbon source - bacterial mineralization and nutrient regeneration" was formed between bacteria and algae. This synergistic mechanism is beneficial for maintaining the high activity state of the system, providing the material and energy basis for subsequent acidity buffering and nutrient activation in acidified soils.

[0081] Stability test:

[0082] Acid stability: The immobilized bacteria-algae symbiotic remediation preparation was placed in a buffer solution at pH 3.5 and treated with shaking at 25°C for 72 hours. The stability was assessed by viable bacteria count and algal cell chlorophyll fluorescence activity (Fv). Fm) detection was used to calculate the survival rate. 80%, the results are shown in Table 1.

[0083] Table 1 Results of acid stability test

[0084]

[0085] Table 1 shows that, under the dual testing conditions of acid stability and potassium solubilization efficiency, both strains maintained high activity across different pH ranges (4.5-6.5) and exhibited significant differences. *Serratia liquefaction* AN41 showed highly consistent data across replicates at pH 4.5-6.5, with minimal overall variation, demonstrating stable potassium solubilization efficiency. Its activity did not decrease significantly with increasing pH, indicating good acid tolerance and functional stability, maintaining a relatively constant metabolic level in weakly acidic to neutral environments. *Serratia* JG-4-2 showed significantly higher potassium solubilization efficiency than *Serratia liquefaction* AN41 at all pH levels, peaking at pH 5.0-5.5, demonstrating strong acid adaptability and enhanced functional response. Although efficiency decreased slightly at pH 6.0-6.5, it remained at a high level, indicating good activity over a wide acidity range.

[0086] In summary, both strains were able to maintain high functional performance under acidic conditions, consistent with their survival rate. The 80% acid stability evaluation standard is used. Serratia liquefaction strain AN41 exhibits strong stability and low fluctuations, making it suitable for applications requiring high acid environment stability. JG-4-2 generally outperforms Serratia liquefaction strain AN41, particularly under weakly acidic conditions of pH 5.0-5.5, making it suitable for applications requiring high potassium solubilization capacity. Both strains demonstrate good acid stability and application potential, but differ in potassium solubilization efficiency and pH adaptability, allowing for targeted selection based on the specific application environment.

[0087] Thermal stability: After treatment at 60℃ for 1 hour, its activity was tested, and the activity retention rate was measured. 70%, the results are shown in Table 2.

[0088] Table 2 Results of thermal stability test

[0089]

[0090] As shown in Table 2, based on the activity test results after treatment at 60℃ for 1 hour, both strains maintained a high activity level, and their physiological functions did not show significant decline, demonstrating good heat resistance. Detailed analysis is as follows:

[0091] Serratia liquefaction strain AN41 exhibits high overall stability. Under different testing temperatures ranging from 15 to 40°C, its functional performance remained within the range of 5.80-6.50, with minimal fluctuations and good consistency among replicates. Furthermore, it showed no significant signs of thermal inhibition; even after heat treatment at 60°C, its potassium-solubilizing efficiency remained close to the untreated level, indicating minimal heat damage to the strain's cell structure and metabolic function, demonstrating good heat stability.

[0092] Serratia japonica JG-4-2 exhibited significantly higher potassium-solubilizing efficiency than Serratia liquefaction strain AN41. Across all tested temperatures (15-40℃), the efficiency of this strain remained within the range of 47.63-59.33, consistently higher than that of Serratia liquefaction strain AN41, indicating a stronger metabolic capacity. Furthermore, its function remained high even after heat treatment; after treatment at 60℃ for 1 hour, its efficiency still maintained a high value, demonstrating strong heat resistance and functional recovery ability.

[0093] In summary, both strains met the requirements for activity retention rate. The 70% thermal stability evaluation standard indicates its potential for use in high-temperature environments or processes requiring heat treatment. Liquefied Serratia AN41 exhibits higher stability at different temperatures, but its absolute efficacy is relatively low. Serratia JG-4-2 maintains a high potassium solubility level after heat treatment, making it suitable for applications seeking highly efficient functional expression.

[0094] Microcosmic Synergistic Acid-Reducing Efficacy Verification:

[0095] Experimental Design: Natural black soil samples were collected from farmland in Northeast China (initial pH ≈ 5.8) and sieved through a 2mm sieve. Two groups were set up: ① blank control group (no preparation added); ② preparation group (soil pH was adjusted to simulate acidified black soil, and the remediation preparation prepared in Example 2 was added at 1.5 g / kg of soil dry weight). Each group had 3 replicates. The soil moisture content was adjusted to 65% of field capacity, and the soil was incubated at a constant temperature of 25℃ for 30 days.

[0096] Soil samples were taken and tested for pH, available phosphorus, available potassium, ammonium nitrogen, and nitrate nitrogen. Samples were taken on day 0 and day 30 of the culture.

[0097] pH: The pH was determined by potentiometric method, and the results are shown in Tables 3-5.

[0098] Table 3. pH Measurement Results of Serratia liquefaction AN41

[0099]

[0100] From the overall growth trend of the bacterial community, the bacterial cell count showed a steady upward trend with increasing culture time under all pH conditions, indicating that *Serratia liquefaction* AN41 has a stable growth capacity under weakly acidic to neutral conditions. From 0 hours to 24 hours, its growth index increased from approximately 0.15-0.18 to approximately 2.32-2.39, an increase of approximately 14-15 times, indicating that the strain has good proliferation ability.

[0101] Growth differences under different pH conditions: pH 4.5 is a low acid condition, with a low initial absorbance value (0.1533 at 0h), which reaches 2.328 at 24h. Compared with other pH conditions, the final value at pH 4.5 is the lowest, indicating that the strain can still grow in a slightly acidic environment, but the proliferation rate is slightly inhibited. pH 5.0 is a weak acid condition, with the growth curve generally higher than that at pH 4.5, reaching 2.3317 at 24h, which is significantly better than that at pH 4.5. The weak acid condition is more suitable for the growth of the strain. pH 5.5 is the optimal growth range, and the growth curve remains at a high level. The 24-hour absorbance was 2.3789, one of the higher values ​​among all pH conditions, indicating that pH 5.5 is close to the optimal acidity range for this strain, which is conducive to its rapid proliferation. At pH 6.0, a slightly acidic-neutral environment, the growth performance was similar to that at pH 5.5, reaching 2.377 at 24 hours, the second highest among the five groups. At pH 6.5, a near-neutral environment, the growth rate was the fastest over time, especially prominent in the 8-24 hour range, with a 24-hour absorbance of 2.3932, the highest among the five pH groups. This indicates that the strain exhibits the most vigorous metabolic activity and proliferation capacity in a weakly acidic to near-neutral environment. In summary, the optimal range is pH 5.5-6.5, with rapid cell proliferation and high final growth. Growth is inhibited at pH 4.5-5.0, but it is still sustainable, indicating a certain degree of acid tolerance. The time-series curves under all pH conditions showed high consistency and good repeatability, reflecting stable internal metabolic regulation of the strain. Serratia liquefaction strain AN41 can grow normally within a pH range of 4.5-6.5, exhibiting good acid tolerance and proliferation ability. The optimal proliferation range is pH 5.5-6.5, which can be used as the recommended pH condition for its environmental applications, providing a basis for subsequent bacterial agent preparation and application scenario design.

[0102] Table 4. pH Measurement Results of Serratia spp. JG4-2

[0103]

[0104] The results showed that the strain had significant proliferation ability under weakly acidic to neutral conditions, and its overall growth level was higher than that of Serratia liquefiedii AN41.

[0105] From the overall growth trend, the growth curves under all pH conditions showed obvious time dependence, rising from about 0.12-0.18 at 0 hours to about 2.27-2.35 at 24 hours, with a growth rate of 13-18 times, indicating that the strain is active and has vigorous metabolism.

[0106] The growth under different pH conditions is as follows: At pH=4.5, the initial growth value is low (0.1255) and the rate of increase is slow. It reaches 2.324 after 24 hours, which, although the lowest among the five groups, still shows good proliferative capacity. This indicates that stable growth can be maintained under slightly acidic conditions, although the rate is slightly inhibited. At pH=5.0, significant growth begins from 3 hours (0.4714). It reaches 2.3223 after 24 hours, slightly higher than pH 4.5. A weakly acidic environment is more suitable for this strain, significantly improving growth efficiency. At pH=5.5, the growth is stable throughout, with a faster growth rate than at pH 5.0. The highest value reaches 2.3342 after 24 hours, the second highest among the five groups. This indicates that pH 5.5 is the optimal growth range for this strain. At pH=6.0, initial growth is relatively fast (0.1758 at 0h, the highest among the five groups). The value reaches 2.2758 after 24 hours, slightly lower than at pH 5.5 and 6.5. The results indicate that a slightly acidic to neutral environment is conducive to the rapid adaptation of the strain, but the final growth rate is slightly lower than that under optimal pH conditions. At pH 6.5, proliferation is faster at all time points, especially between 1 and 8 hours. The growth rate reaches 2.3506 after 24 hours, the highest among the five groups. This suggests that the strain is most metabolically active under weakly acidic to near-neutral conditions, which represents its optimal growth range.

[0107] In summary, *Serratia* JG4-2 exhibits good tolerance to acidic environments, maintaining stable and rapid proliferation within a pH range of 4.5-6.5. Its optimal growth range is pH 5.5-6.5, where its growth volume and rate are particularly outstanding. Compared to *Serratia liquefied* AN41, *Serratia* JG4-2 demonstrates stronger overall growth and adaptability, exhibiting higher metabolic potential and environmental resilience. *Serratia* JG4-2 displays high activity, high proliferation, and strong adaptability within a weakly acidic to neutral pH range, making it suitable for applications requiring rapid growth or high efficiency in acidic environments. These results can serve as key criteria for its application in microbial preparations, soil amendment, and screening of acid-tolerant functional bacteria.

[0108] Table 5. pH Measurement Results of Chlamydomonas SAG 2486

[0109]

[0110] The results showed that Chlamydomonas SAG 2486 had good adaptability to weakly acidic environments, and its growth rate and final growth amount showed regular differences with pH.

[0111] Under all pH conditions, the absorbance of Chlamydomonas SAG 2486 gradually increased over time, exhibiting a typical growth pattern of lag phase-exponential phase-stationary phase:

[0112] Initial stage (0-8h): Slow growth, in the adaptation period.

[0113] Mid-term (12-56h): Entering the logarithmic growth period, the absorbance value increases rapidly.

[0114] Later stage (56-72h): tends to stabilize, but still maintains a certain growth rate.

[0115] The overall growth rate increased from 0 hours (approximately 0.07) to 72 hours (approximately 0.52-0.73), a magnification of approximately 7-10 times, indicating that the algal species has good metabolic activity.

[0116] The growth differences under different pH conditions are as follows: At pH 4.5, a slightly acidic environment, the initial absorbance was the lowest (0.0715), and the growth rate was slow. It reached 0.6019 after 72 hours, which, although lower than the other group, still maintained continuous growth. This indicates that it can tolerate a slightly acidic environment, but the growth rate is slightly inhibited. At pH 5.0, a slightly acidic environment, the overall growth level was higher than at pH 4.5, especially with significant growth between 12 and 56 hours. It reached 0.6235 after 72 hours, showing stable performance. It thrives under slightly acidic conditions, with enhanced growth capacity. At pH 5.5, the optimal growth condition, the highest data was observed throughout, especially with rapid growth between 24 and 72 hours. It reached 0.7322 after 72 hours, the highest value among the five groups. The data shows that this algal species grows most vigorously at pH 5.5, which is its optimal pH range. At pH 6.0, the environment is slightly acidic to neutral. Growth is stable in the mid-stage (24-56 hours), but the rate is lower than at pH 5.5. The value reaches 0.5165 after 72 hours, one of the lowest values ​​among the five groups. This indicates that the algal species' proliferation ability in a slightly neutral environment is not as significant as under acidic conditions. At pH 6.5, the environment is close to neutral. Growth is relatively rapid in the initial stage (0-12 hours), but the proliferation rate is lower than at pH 5.5 in the mid-to-late stages. The value reaches 0.5758 after 72 hours, which is above average. Normal growth is possible, but this is not the optimal environment.

[0117] Overall, *Chlamydomonas* can tolerate a wide pH range of 4.5-6.5, demonstrating good acid adaptability. The optimal growth range is pH 5.0-5.5, with the most rapid growth and largest final growth volume at pH 5.5. pH 6.0-6.5 somewhat limits algal growth, but activity is still maintained, indicating suitability for slightly acidic environments. Unlike bacteria, *Chlamydomonas* performs better in the more acidic range (5.0-5.5), reflecting the unique acid-tolerant ecological characteristics of algae.

[0118] Therefore, *Chlamydomonas* exhibits the strongest proliferative capacity in weakly acidic environments, particularly at pH 5.5. This growth characteristic provides a basis for pH regulation in bacterial-algal symbiotic systems and can be used to optimize the environmental adaptability of immobilized symbiotic remediation agents.

[0119] Available phosphorus: 0.5 mol / L NaHCO3 extraction - molybdenum antimony colorimetric assay, 880 nm colorimetric assay.

[0120] Available potassium: 1 mol / L ammonium acetate extraction - atomic absorption.

[0121] Ammonium nitrogen: Extracted with 2 mol / L KCl, then phenol-sodium hypochlorite was used for color development at 630 nm.

[0122] Nitrate nitrogen: 0.01 mol / L CaCl2 extraction-ion chromatography.

[0123] The specific groups are as follows:

[0124] CK-CK (blank control group): contains only collected soil samples (black soil from farmland in Northeast China, pH=5.8), without pH adjustment or the addition of any microorganisms / exogenous substances.

[0125] CK (acidification control): pH was adjusted to 5.0 without the addition of any microorganisms or exogenous substances.

[0126] Z (Single Algae): Adjust pH to 5.0+ and add only microalgae (Chlamydomonas SAG 2486).

[0127] J (Compound bacterial solution): Adjust pH to 5.0+ and add bacterial solution (liquefied Serratia AN41: Serratia JG-4-2: potassium-solubilizing bacteria: phosphate-solubilizing bacteria = 8:4:2:1).

[0128] BA (Bacterial-Algal Symbiotic): Adjust pH to 5.0+ and add bacterial-algal symbiotic (the above screening ratio of bacteria:algal = 3:2).

[0129] M (Maltose group): Adjust pH to 5.0 + bacterial-algal symbiosis (bacteria:algal = 3:2) + maltose.

[0130] P (proline group): pH adjusted to 5.0 + bacterial-algal symbiosis (bacteria:algal = 3:2) + proline, where the amount of each substance added was 5% of the culture medium volume. Results are as follows. Figures 7-8 As shown.

[0131] Nitrate nitrogen and ammonium nitrogen are the most important and direct forms of inorganic nitrogen that can be absorbed and utilized by plants in the soil. If ammonium nitrogen and nitrate nitrogen increase significantly over time, it indicates that the microorganisms have recovered or been enhanced, the nitrogen cycle is becoming more active, and the soil ecological function has been improved.

[0132] Depend on Figure 7As shown in A, the soil nitrate nitrogen content under each treatment generally showed a gradual upward trend with incubation time, but there were significant differences between the different treatments. CK-CK (blank control): The content remained at a low level throughout the process, with a relatively slow increase, indicating that nitrification was somewhat limited under conditions without pH adjustment, addition of microorganisms, or exogenous substances. CK (pH adjustment only): The content was slightly higher than CK-CK overall, but still lower than the biological treatment group, indicating that while adjusting acidity alone can improve environmental conditions, the improvement is limited. J and Z (single-bacterial / single-algae treatment): The nitrate nitrogen content increased significantly from the initial stage of incubation and continued to rise over time, indicating that the intervention of a single microorganism can promote the nitrogen conversion process. BA (bacterial-algae symbiotic system): The nitrate nitrogen content at each stage was generally higher than that of groups J and Z, and the difference further widened in the middle and late stages, indicating that the interaction between bacteria and algae is conducive to the continuous nitrification process. M and P (symbiotic system involving exogenous carbon sources): The content reached the highest level based on BA, with group P slightly better than group M, indicating that exogenous organic matter can further improve nitrogen conversion efficiency.

[0133] Depend on Figure 7 As shown in B, the ammonium nitrogen content in all treatments also gradually increased over time, but the overall level and treatment differences were slightly smaller than those for nitrate nitrogen. CK-CK: remained at the lowest level throughout, with only a slight increase, indicating that nitrogen mineralization and conversion capacity is weak under natural conditions. CK: slightly higher than CK-CK, but still significantly lower than the biological treatment group, suggesting that simply adjusting acidity has limited effect on ammonium nitrogen accumulation. J and Z: both significantly higher than the two control groups, and increased significantly with culture time. BA: the overall level was higher than the single-bacterial or single-algae groups, and the advantage was more obvious in the middle and late stages. M and P: remained at the highest level among all treatments, with group P slightly higher than group M.

[0134] Figure 8 The measurements focus on available potassium and available phosphorus, which are the main forms that crops can directly absorb and utilize. The results can be used to evaluate soil nutrient availability and fertility levels, and to reflect whether nutrient passivation in acidic soils has been alleviated.

[0135] from Figure 8 As shown in A, the content of this indicator under each treatment generally increased with the culture time, and the differences between different treatments were relatively clear: CK-CK remained at a low level with limited growth, showing only slow accumulation over time. The overall level of CK was slightly higher than CK-CK, but still significantly lower than each biological treatment group, indicating that simply adjusting acidity could only bring limited improvement. J and Z were significantly higher than the two control groups in the early stage and continued to increase during the culture process, indicating that the introduction of microorganisms could promote the accumulation of this indicator. BA remained at a high level, and its advantage became more apparent in the middle and late stages, exceeding that of single bacterial or algal treatments. M and P were at the highest levels among all treatments, with group P slightly higher than group M, and the upward trend was still quite obvious in the later stages, indicating that the promoting effect of exogenous organic matter combined with the bacterial-algal symbiotic system was more significant.

[0136] Figure 8 The B index also showed a rapid initial increase, followed by a slowdown and plateau, but the overall level differed slightly from the treatment. In summary, while adjusting pH can partially alleviate acid stress, its effect on restoring the biological activity and function of acidic soils is limited.

[0137] Soil remediation efficacy verification

[0138] Experimental design: A pot experiment was conducted using acidified black soil. The remediation agent prepared in Example 2 was applied (at a rate of 2 g / kg soil). The incubation period was 45 days.

[0139] Indicator detection method: After the cultivation is completed, soil samples are collected for testing.

[0140] Soil pH and organic matter: pH was determined by potentiometric method, and organic matter was determined by potassium dichromate oxidation-external heating method.

[0141] Soil enzyme activity: Soil urease, acid phosphatase, and catalase activities were measured using commercial kits. The results are as follows: Figures 9-13 .

[0142] Figure 9 The graph shows the pH measurement results. Soil pH is a fundamental constraint indicator spanning the chemical environment, nutrient availability, and microbial activity, playing a leading and interpretive role in acidic soil remediation research; therefore, continuous monitoring is conducted. Figure 9 It can be seen that CK-CK consistently maintained the highest initial pH and remained stable. The pH of CK was stabilized at a low level and recovered extremely slowly, indicating that the system lacked active recovery momentum after simple acidification. In contrast, the pH values ​​of the exogenous addition group and the bacterial-algae symbiotic treatment group recovered to a greater extent, indicating that bacterial-algae symbiosis is superior to single bacterial or algae treatment, and the exogenous treatment group also has a significant enhancing effect on pH recovery.

[0143] Soil organic matter is one of the core indicators of soil fertility, directly reflecting the strength of overall soil fertility. It is also a major explorer and energy source for soil microorganisms, and in the process of acidic soil remediation, it is a key indicator reflecting the long-term cumulative effects. Figure 10 It can be seen that the increase in CK-CK was the smallest, showing only a slow, natural accumulation. CK accumulation was slightly higher than CK-CK, but the promoting effect was weak. The organic matter content in each treatment group generally showed a slow upward trend with the culture time. However, the organic matter content in the exogenous treatment group and the bacterial-algal symbiotic system treatment group showed a greater increase in the later stage, indicating that the effect of simply adjusting pH is limited, and that the introduction of the bacterial-algal symbiotic system can promote organic matter accumulation to a certain extent.

[0144] Excessive hydrogen peroxide accumulation inhibits microbial growth. Catalase is a functional indicator reflecting soil "biological vitality." Increased catalase activity indicates a stronger oxidative stress buffering capacity in the soil and is also an important indicator of soil ecological health and remediation effectiveness. Under all treatments, soil catalase activity gradually increased with incubation time, indicating that soil microbial activity gradually strengthened as incubation progressed. Figure 11 Data trends indicate that the control (CK) remained at the lowest level with limited increases. Single-bacterial (J) and single-algae (Z) treatments showed significant increases in activity from the initial incubation period, followed by continued increases, indicating that microbial input effectively activated soil redox-related enzymes. The enzyme activity of the symbiotic microbiome (BA) treatment was generally higher than that of the single-microbial treatment, with the advantage further expanding in the later stages. The M and P groups, with the addition of exogenous organic matter, exhibited the highest catalase activity, maintaining their leading position throughout the entire incubation period, with the P group slightly higher than the M group. This suggests that the enhancement of catalase activity in acidic soils primarily depends on microbial input and the establishment of a symbiotic system, while exogenous organic matter can further amplify its promoting effect.

[0145] Acid phosphatase is an important hydrolytic enzyme in soil and a key link in soil phosphorus cycling and the supply of available phosphorus to crops. Measuring its activity can, on the one hand, evaluate the soil's organic phosphorus mineralization capacity and the potential supply level of available phosphorus, reflecting the soil fertility status; on the other hand, it can serve as a sensitive indicator of the intensity of microbial and root metabolism, used to determine whether phosphorus passivation in acidic soils has been alleviated and whether soil ecological functions have been activated under different remediation and bioregulation measures, and can be corroborated with nutrient indicators such as available phosphorus content.

[0146] from Figure 12 As can be seen, the overall acid phosphatase activity under each treatment showed an increasing trend with culture time, but significant differences existed between different treatments: CK-CK remained at a low level throughout, increasing only slowly; CK, although slightly higher than CK-CK, fluctuated significantly and showed limited increase, indicating that simply adjusting pH had a weak effect on improving phosphorus hydrolysis. After the addition of biological treatment, enzyme activity significantly increased, with single bacteria (J) and single algae (Z) gradually widening the gap with the control group after 10 days. The activity of the bacterial-algal symbiotic (BA) was significantly higher than that of J and Z from the mid-term, indicating that bacterial-algal interaction had a synergistic promoting effect on organophosphate mineralization; the M and P groups, with the addition of exogenous organic matter, showed the highest acid phosphatase activity, rapidly increasing and reaching a high plateau in the 10–20 day stage, followed by a slight increase thereafter. The P group was slightly lower than or close to the M group but remained in the same high-value tier overall. This indicates that in acidic soils, constructing a bacterial-algal symbiotic system combined with the addition of exogenous organic matter is most beneficial for improving acid phosphatase activity, thereby enhancing soil phosphorus supply function.

[0147] Urease is an important nitrogen metabolism enzyme in soil. Measuring urease activity is mainly used to: evaluate soil nitrogen transformation and supply capacity; characterize microbial activity and nitrogen cycle intensity (higher activity usually indicates more vigorous microbial metabolism); and determine whether remediation and improvement measures have activated nitrogen cycle function, especially in acidic soils. If urease recovery is significant, it indicates that nitrogen utilization potential has been simultaneously enhanced.

[0148] Depend on Figure 13 It was found that the soil urease activity under each treatment generally showed a gradual upward trend with the incubation time, but there were significant differences between the different treatments: the CK-CK group remained at a low level throughout the entire process, increasing only slowly; although the CK group was slightly higher than the CK-CK group, the overall increase was limited. The single-bacterial treatment (J) and the single-algae treatment (Z) were significantly higher than the two controls after 10 days, and continued to increase over time, indicating that microbial input could significantly promote the activity of nitrogen transformation-related enzymes. The bacterial-algal symbiotic treatment (BA) was higher than the single-microorganism treatment at all stages, and the advantage gradually expanded in the middle and late stages. The M and P groups with the superimposed exogenous organic matter had the highest urease activity, entering the high value range in the 10-20 day stage, and continuing to increase slightly in the later stage. Among them, the P group was slightly higher than the M group, indicating that the bacterial-algal symbiotic system and exogenous organic matter had the most significant activation effect on nitrogen cycle function.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A fungal-algae symbiotic system repair preparation, characterized in that, The remediation agent includes acid-resistant microalgae and acid-resistant bacteria. The acid-resistant microalgae is Chlamydomonas sp. SAG 2486, and the acid-resistant bacteria include Serratia liquefaciens AN41 and Serratia sp. JG-4-2. The Chlamydomonas species SAG 2486 was deposited at the China Center for Type Culture Collection (CCTCC) on May 12, 2025, at Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO:M 20251014. The liquefied Serratia AN41 was deposited at the China Center for Type Culture Collection (CCTCC) on May 12, 2025, at Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO:M 20251013. The *Serratia* genus JG-4-2 was deposited on May 12, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO:M 20251012. The acid-resistant bacteria also include potassium-solubilizing bacteria (Paenibacillus mucilaginosus) K1 and phosphate-solubilizing bacteria (Enterobacter sp.) P1; The potassium-solubilizing bacterium K1 was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2024, at Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO:M 20242542. The phosphate-solubilizing bacterium P1 was deposited on November 20, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO:M 20242544.

2. The bacterial-algae symbiotic system repair preparation according to claim 1, characterized in that, The repair preparation contains acid-resistant microalgae and acid-resistant bacteria in a cell ratio of 2:1 to 3:

2.

3. The application of the bacterial-algae symbiotic remediation system preparation as described in claim 1 in the remediation of acidic soil.

4. The application according to claim 3, characterized in that, The remediation agent is used to increase soil pH, activate available phosphorus, available potassium and ammonium nitrogen, increase soil organic matter content, and enhance soil urease and catalase activity.

5. The application according to claim 3, characterized in that, The remediation agent is applied at a rate of 1-2 g / kg of soil, with a remediation period of 30-45 days.

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