Carbonate mineralizing bacteria, composite material and application thereof in repairing heavy metal contaminated soil

Abstract

This invention relates to the field of microbiology and heavy metal immobilization technology, specifically to carbonate mineralizing bacteria, composite materials, and their application in the remediation of heavy metal-contaminated soil. The carbonate mineralizing bacteria is *Bacillus lamellaris* (…). Rummeliibacillus pycnus ANY-2, with accession number CGMCC No. 34595, is a composite material made by loading ANY-2 onto iron-modified biochar. The ANY-2 of this invention exhibits excellent cadmium and arsenic removal and passivation effects, and its ability to increase soil organic matter is significantly stronger than that of the existing ANY-1. The composite material of this invention, under the synergistic effect of iron-modified biochar and ANY-2, demonstrates superior remediation performance compared to treatments using ANY-2 and iron-modified biochar alone. This invention breaks through the bottleneck of traditional "emphasis on remediation and neglect of fertilization," providing a replicable and scalable new approach for the safe utilization and soil fertility restoration of Cd-As contaminated farmland.

Description

Technical Field

[0001] This invention relates to the field of microbial and solidified heavy metal technology, specifically to carbonate mineralizing bacteria, composite materials, and their application in the remediation of heavy metal contaminated soil. Background Technology

[0002] Arsenic (As) and cadmium (Cd) co-contamination leads to a significant decrease in soil enzyme activity, microbial abundance, and biomass carbon and nitrogen, as well as a reduction in soil organic matter and available nutrients (N, P, K). This, in turn, disrupts soil structure and ecological balance, affecting crop growth and quality, and even jeopardizing human health through the food chain. Furthermore, highly reactive forms of Cd and As in the soil (such as acid-extractable forms) are easily absorbed by plants, causing heavy metal accumulation in agricultural products such as rice, threatening food security. Therefore, the remediation of Cd-As co-contaminated soil is not only a bottom-line project to ensure food security but also a strategic necessity for ecological security and public health. Thus, there is an urgent need to develop environmentally friendly remediation products.

[0003] Some microbial agents (such as compound agents of Bacillus and photosynthetic bacteria) can adsorb and fix heavy metals such as Cd and As, reducing their bioavailability, reducing the use of chemical fertilizers and pesticides, and reducing the risk of soil pollution. The inventors previously screened and obtained a strain of Bacillus lamellaris (… Rummeliibacillus sp. ANY-1, disclosed in Chinese Patent Application No. 202411519914.9, has good cadmium and arsenic removal and passivation effects. However, the bacterium has limited effect on increasing soil organic matter content. Therefore, the present invention aims to find a new strain that can both help increase soil organic matter content and have good cadmium / arsenic pollution remediation effect. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a carbonate mineralizing bacterium, a composite material, and its application in the remediation of heavy metal contaminated soil.

[0005] One objective of this invention is to provide a carbonate mineralizing bacterium for increasing soil organic matter content, wherein the carbonate mineralizing bacterium is *Bacillus lamellaris* (…). Rummeliibacillus pycnus ANY-2, the Bacillus lamellaris ANY-2, was deposited on May 19, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34595.

[0006] In preliminary research, the inventors discovered Bacillus lamellaris ( Rummeliibacillus pycnus ANY-1 exhibits excellent passivation ability for cadmium / arsenic, but its effect on improving soil organic matter is extremely low. Figure 8The current situation remains trapped in the dilemma of "emphasizing remediation while neglecting fertilization." To address this, this invention conducted targeted rescreening of strains within the same genus, obtaining a new strain—Bacillus lamellaris ANY-2—belonging to a different species within the same genus as ANY-1. While maintaining comparable cadmium / arsenic remediation efficiency to ANY-1, ANY-2 significantly promotes soil organic matter accumulation, thus overcoming the technical bottleneck of traditional functional bacteria that "only remediate but do not fertilize," achieving simultaneous improvement in pollution remediation and soil fertility enhancement.

[0007] Secondly, the present invention provides a bacterial agent containing the aforementioned carbonate mineralizing bacteria.

[0008] Furthermore, the bacterial agent is OD 600 The bacterial suspension of the carbonate mineralizing bacteria is 1 to 1.8.

[0009] Thirdly, the present invention provides a composite material, which is formed by loading the carbonate mineralizing bacteria onto iron-modified biochar.

[0010] Fourthly, the present invention provides a method for preparing the composite material, comprising the following steps:

[0011] S1. Using soybean straw biochar as raw material, KOH and Fe(NO3)3 were used for modification to obtain α-FeOOH modified biochar;

[0012] S2. Add α-FeOOH modified biochar, the bacterial suspension of the carbonate mineralizing bacteria and urea to the culture medium. The precipitate obtained after cultivation is the composite material.

[0013] Further, the specific steps of S1 are as follows: soybean straw biochar is dispersed in a KOH solution with a concentration of 4~6 mol / L and activated for 1~2 h, then Fe(NO3)3 solution with a concentration of 0.8~1.2 mol / L is added, and the mixture is stirred for 1~2 h to produce a red precipitate. The mixture is then allowed to stand at 65~70℃ for 50~60 h, and then allowed to settle naturally for 20~24 h. The resulting precipitate is dried to obtain the α-FeOOH modified biochar.

[0014] Furthermore, the mass-to-volume ratio of the soybean straw biochar, KOH solution, and Fe(NO3)3 solution is 2-3 g: 100 mL: 100 mL.

[0015] Furthermore, the soybean straw biochar is obtained by cutting, washing, and drying soybean straw, and then pyrolyzing it at 380-400℃ for 2-3 hours in a nitrogen atmosphere.

[0016] Fifthly, the present invention provides the application of the carbonate mineralizing bacteria, the bacterial agent, or the composite material in the remediation of heavy metal contaminated soil and the improvement of soil physicochemical properties.

[0017] Furthermore, the improvement of soil physicochemical properties includes increasing soil organic matter content and pH value, and the heavy metal is cadmium and / or arsenic.

[0018] The present invention has the following beneficial effects:

[0019] The carbonate mineralizing bacteria of this invention exhibit good mineralization ability. Under the same conditions, compared with ANY-1, their ability to remove arsenic and cadmium is comparable. However, ANY-2's ability to increase soil organic matter is significantly stronger than ANY-1. The carbonate mineralizing bacteria of this invention show good removal effects under both single and combined arsenic and cadmium pollution conditions. This invention further demonstrates the passivation effect of the aforementioned carbonate mineralizing bacteria on combined arsenic and cadmium pollution in farmland soil. Attached Figure Description

[0020] Figure 1 This is a Gram staining image of the strain.

[0021] Figure 2 Scanning electron microscope images of iron-modified biochar loaded with ANY-1(a) and ANY-2(b).

[0022] Figure 3 To show the removal efficiency of different treatments for Cd(II) and As(III) combined pollution, (a) Cd(II), (b) As(III).

[0023] Figure 4 Scanning electron microscope and energy dispersive spectroscopy (EDS) images of mineralized products from different treatments: (a) ANY-1, (b) ANY-2, (c) GBC+ANY-1, (d) GBC+ANY-2.

[0024] Figure 5 XRD spectra of mineralized products: (a) mineralized product of As, (b) mineralized product of Cd.

[0025] Figure 6 The effects of different treatments on the available Cd and As content in soil, (a) Cd, (b) As.

[0026] Figure 7 The effect of the same treatment on soil pH.

[0027] Figure 8 The organic matter content of soil under different treatments. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0029] In the following examples, all liquid-liquid percentages refer to volume percentages.

[0030] In the following examples, Cd(II) refers to cadmium chloride and As(III) refers to sodium arsenite.

[0031] The culture medium composition involved in the following examples:

[0032] Enrichment medium (g / L): 20g glucose, 2g sodium acetate, 2g potassium dihydrogen phosphate, 5g sodium chloride, 20g urea, and the remainder is distilled water.

[0033] Screening medium (g / L): 3g beef extract, 5g peptone, 5g sodium chloride, 20g urea, 10mL 0.2% phenol red solution, and the remainder is distilled water.

[0034] NBU medium (g / L): 3g beef extract, 5g peptone, 5g sodium chloride, 20g urea, and the remainder is distilled water.

[0035] Autoclave at 121℃ for 25 minutes. Add urea, calcium chloride, glucose and phenol red solution after filtering through a 0.22μm filter membrane. If solid culture medium is required, add 2% agar powder.

[0036] Example 1: Screening and identification of carbonate mineralizing bacteria.

[0037] 1. Materials and Methods

[0038] (1) Screening

[0039] 1g of soil sample collected from farmland in Guichi District, Chizhou City, Anhui Province was inoculated into 100mL enrichment medium (100mL / 250mL Erlenmeyer flask) and incubated at 30℃ with shaking at 160r / min for 24h. 1mL of the supernatant suspension was then added to a centrifuge tube containing 9mL of sterile water and mixed thoroughly by pipetting (to obtain 10...). -1 (Diluent), serially diluted to 10 using the same steps. -7 Take 10 respectively -5 10 -6 10 -7 Three concentrations of dilution were spread at 0.2 mL onto the selection medium and incubated upside down at 30°C for 72 h. Strains with distinctly different colony characteristics and whose surrounding medium turned red were selected for streak isolation. This step was repeated at least three times for purification until pure single bacteria were obtained.

[0040] (2) Identification

[0041] Morphological identification: Single bacteria were inoculated into NBU medium and cultured at 30°C for 24 hours before observing colony morphology.

[0042] Gram staining: Take a clean glass slide, place a drop of sterile distilled water in the center of the slide, use a sterile inoculation loop to pick up a small amount of bacterial inoculum and spread it on the slide. Break up the colonies, mix them thoroughly with sterile water, and spread them into a uniform thin layer. After air-drying, quickly pass the slide over a flame three times to fix the bacteria on the slide. Add a drop of crystal violet staining solution and stain for 1-2 minutes, then rinse with water. Add a drop of iodine solution and stain for 1-2 minutes, then rinse with water. Next, add 95% ethanol and stain for 20-30 seconds, then rinse with water. Finally, add safranin and stain for 2 minutes, then rinse with water and air-dry. After air-drying, the slide preparation is complete and it is ready for microscopic examination.

[0043] 16S rDNA sequence identification: PCR amplification was performed using universal primers for bacterial 16S DNA.

[0044] The reaction system consisted of: 5 µL 10×Ex Taq Buffer, 5 µL dNTP Mixture, 2 µL upstream primer, 2 µL downstream primer, 5 µL template DNA, 1 µL Ex Taq DNA polymerase, and 30 µL sterile ddH2O. The PCR program was: 94℃ for 5 min; 94℃ for 30 s; 55℃ for 30 s; 72℃ for 90 s, 35 cycles; 72℃ for 10 min; and storage at 4℃. After the PCR reaction, all the products were spotted onto a 1.2% large-well agarose gel and electrophoresed at a constant voltage of 100 V for 45 min. The electrophoresis results were observed using a UV gel imaging system, and the products were purified.

[0045] The purified samples were sequenced. A phylogenetic tree was constructed based on the 16S rRNA genes of the finally determined strains to determine their position in the taxonomic system. The specific steps for constructing the phylogenetic tree were as follows: Homology alignment analysis of the 16S rRNA sequences obtained from sequencing was performed using the BLAST function on the National Center for Biotechnology Information (NCBI) website. The corresponding gene sequences of type strains closely related to the determined gene sequences were selected, and these sequences were used as analysis objects. The Clustal W function of MEGA7 was used to align the target gene sequence with related sequences. Finally, the phylogenetic tree was constructed using the Neighbor-joining method with MEGA7 software.

[0046] 2. Results

[0047] After culturing the strain on NBU solid medium, smooth, well-defined, opaque colonies were observed. Under a light microscope, the bacterial cells appeared as short rods, and Gram staining results showed... Figure 1 The results showed that the strain was Gram-positive. The strain screened in this invention is similar to strain 22879. RummeliibacilluspycnusThe strain showed high homology. It was ultimately identified as *Bacillus lamellaris*. Rummeliibacillus pycnus ), named ANY-2.

[0048] Example 2: Preparation, characterization, and effect verification of composite materials

[0049] 1. Test Methods

[0050] (1) Preparation of composite materials

[0051] Preparation of iron-modified biochar: Using soybean straw biochar as raw material, KOH and Fe(NO3)3 were used for modification to obtain α-FeOOH modified biochar, denoted as GBC; the specific steps are as follows:

[0052] Biochar preparation: Soybean straw was used as raw material, collected from Lianyungang City, Jiangsu Province, and biochar was obtained through pyrolysis. Before preparation, the soybean straw was cut into small pieces (3–5 cm long), washed with deionized water, and dried in an oven at 60°C until a constant weight was obtained. The dried biochar was then ground through a 0.15 mm sieve and pyrolyzed with nitrogen at a flow rate of 0.5 L / min, with the temperature increased at a rate of 10°C / min. The biochar was then pyrolyzed in a tube furnace at 400°C for 2 hours. Finally, the biochar was cooled and ground to pass through a 100-mesh nylon sieve, denoted as BC.

[0053] Preparation of modified biochar: 2.8 g of BC was dispersed in 100 mL of 5 mol / L KOH solution and activated for 1 h. Then, 100 mL of 1 mol / L Fe(NO3)3 solution was added to the mixture, and the mixture was stirred with a magnetic stirrer for 1 h to ensure thorough mixing and the formation of a red precipitate. The final volume of the mixture was adjusted to 1 L using ultrapure water, and the mixture was placed in an oven at 70 °C for 60 h. After natural settling for 24 h, the mixture was cooled, centrifuged, the supernatant was removed, and the precipitate was washed with deionized water. The precipitate was then dried in air at 60 °C to constant weight to obtain α-FeOOH modified soybean biochar, denoted as GBC.

[0054] Add 1g of iron-modified biochar (GBC) to 100mL of NB medium (NBU medium omits urea) and sterilize at 121℃ for 25min. Inoculate the sterilized medium with 2% OD. 600 ≈1.5g of ANY-1 and ANY-2 bacterial suspensions and 20g / L sterile urea were added and incubated at 30℃ with shaking at 160r / min for 18h. Afterwards, the mixture was centrifuged at 8000r / min for 10min, and the supernatant was discarded. The precipitate was washed with deionized water and centrifuged, and this process was repeated three times. The precipitate was then filtered and dried at room temperature for storage. The composite materials were labeled GBC+ANY-1 and GBC+ANY-2.

[0055] (2) The removal effect of different treatments on As and Cd combined pollution

[0056] GBC (1 g / L), ANY-1 (2%), ANY-2 (2%), GBC+ANY-1 (1 g / L), and GBC+ANY-2 (1 g / L) were added to 100 mL of NBU medium containing 10 ± 5 mg / L Cd(II) + As(III). For the microbial treatment, 5 g / L CaCl2 was added. The mixtures were incubated at 30 °C with shaking at 160 rpm. Samples were collected at 0, 6, 12, 24, 48, 96, and 144 h. After centrifugation at 8000 rpm for 10 min, the supernatant was filtered through a 0.22 μm filter to determine the heavy metal concentration.

[0057] (3) Sample analysis and characterization of mineralized products

[0058] 2% bacterial suspension was inoculated into NBU + 5 g / L CaCl2 liquid medium containing 1 mg / L Cd(II) and 5 mg / L As(III), respectively. After incubation at 30℃ and 160 r / min for 96 h, the product was filtered out, pretreated, and then subjected to scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction energy dispersive spectroscopy (XRD).

[0059] Take an appropriate amount of mineralized sample into a 50 mL centrifuge tube, centrifuge at 4000 r / min for 10 min, discard the supernatant, fix with 2.5% glutaraldehyde pre-cooled at 4℃ for 8-12 h; then wash and mix with phosphate buffer at pH=7.2, centrifuge again to precipitate the bacteria to the bottom of the centrifuge tube, repeat the operation 3 times, 15 min each time; then dehydrate sequentially with different concentrations of ethanol gradient (30%, 50%, 70%, 80%, 90%, 100%), each concentration for 15 min, after the last centrifugation, discard the supernatant, place the sample in a CO2 critical point desiccator to dry for 2 h, after coating treatment, perform scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis to observe the bacterial structure and analyze the elemental composition of the mineralization products.

[0060] Take an appropriate amount of the mineralized sample into a 50 mL centrifuge tube, centrifuge at 8000 r / min for 15 min, discard the supernatant, wash 2-3 times, freeze-dry the collected precipitate for 24 h, grind it with an agate mortar and pestle, and then perform Fourier transform infrared spectroscopy (FTIR) analysis with a scanning range of 4000 cm⁻¹. -1 -400cm -1 X-ray diffraction energy dispersive spectroscopy (XRD) analysis, with a scanning angle (2θ) of 10°–90°, a step size of 0.02, and a counting rate of 8°min. -1 .

[0061] (4) The remediation effects of different treatments on As and Cd in soil

[0062] 100g of cadmium-arsenic contaminated farmland soil was weighed into a 250mL beaker, and 1g of GBC, 1%ANY-1, 1%ANY-2, 1g of GBC+ANY-1, and 1g of GBC+ANY-2 were added respectively. For the microbial treatment, 1% urea and 1% CaCl2 were added. The moisture content was maintained at 70% of the field capacity (384.85g / kg). An equal volume of ultrapure water was added to the control group. Three control groups were set up for each treatment. Samples were collected at 1, 7, 14, 21, and 28 days under 25℃ day-night light and shadow conditions to determine the concentrations of available Cd and As in the soil.

[0063] (5) Effects of different treatments on soil conditions

[0064] Soil pH determination: Weigh 10g of air-dried soil through a 2mm pore size into a beaker, add 25mL of ultrapure water (soil-to-liquid ratio of 1:2.5), stir for 1min to fully disperse the soil particles, and then measure the pH after 30min.

[0065] Determination of organic matter in soil: Soil organic content was determined by potassium dichromate-sulfuric acid oxidation method.

[0066] 2. Results

[0067] (1) Characterization of composite materials

[0068] Figure 2 Scanning electron microscopy (SEM) images of iron-modified biochar loaded with ANY-1(a) and ANY-2(b). The images show a large number of microorganisms attached to the modified biochar. The iron-modified biochar has an irregular morphology and deep cracks. This rough surface structure provides ample surface area and attachment sites for the large-scale attachment of microorganisms and facilitates the diffusion of pollutants and metabolites. A large number of short rod-shaped microorganisms were successfully loaded onto the biochar.

[0069] (2) The removal effect of different treatments on As and Cd combined pollution

[0070] This experiment subsequently used iron-modified biochar-supported strains ANY-1 and ANY-2 to preliminarily explore the removal effects of different treatments on As and Cd under combined pollution conditions. Results are shown below. Figure 3When the concentrations of Cd(II) and As(III) were 10 mg / L and 5 mg / L, respectively, the removal efficiencies of GBC+ANY-1 and GBC+ANY-2 treatments for Cd(II) reached 97.24% and 97.92%, respectively, which were superior to the removal efficiencies of microbial treatment alone (81.76% and 77.31%) and GBC treatment alone (82.39%). The removal efficiencies for As(III) reached 77.85% and 72.32%, respectively, which were superior to the removal efficiencies of microbial treatment alone (21.93% and 19.66%) and GBC treatment alone (69.44%).

[0071] All five treatments effectively removed Cd(II), but the composite material showed significantly better removal performance than GBC and microorganisms. For As(III) removal, the composite material's performance was similar to that of modified biochar, but significantly better than the microbial treatment. The immobilization effect of iron-modified biochar effectively alleviated the stress on microbial activity caused by uncontaminated heavy metals. The adsorption of iron-modified biochar synergistically with the mineralization of carbonate mineralizing bacteria, covering a wider range of heavy metal speciations and significantly improving removal efficiency. Through comparison, the composite material in this study demonstrates significant remediation potential, providing a theoretical basis for the remediation of As and Cd co-contaminated soils.

[0072] (3) Characterization of mineralized products

[0073] To investigate the mechanism by which iron-modified biochar-supported microbial composites remove Cd and As, the precipitates generated after microbial and composite material treatment were analyzed. The dried precipitate powder was analyzed by SEM and EDS; the image results are shown below. Figure 4 As shown, the mineralization products mainly exhibit three typical morphologies: irregular network structure, layered superimposed structure, and aggregated particles. These products have loose and porous surfaces, mostly clustering around bacteria, with some even adhering to them. This unique spatial distribution and structural characteristics fully demonstrate that microbial-induced mineralization precipitation is the dominant mechanism for heavy metal fixation. Under heavy metal pollution, the metabolic activity of microorganisms changes to varying degrees. On the one hand, heavy metal stress weakens microbial respiration, reducing microbial activity and leading to physiological cell death or rupture; on the other hand, when heavy metals act on the cell surface, they easily cause changes in the morphology and surface structure of microbial cells. Through analysis of… Figure 4 The comparison revealed that, compared with microbial mineralization treatment, the microbial cells remained intact after composite material treatment, confirming the protective effect of biochar on microorganisms.

[0074] EDS results showed that the precipitate induced by carbonate mineralizing bacteria contained Ca, Cd, and As, further indicating that As and Cd had been adsorbed and immobilized by the loaded material.

[0075] To further determine the composition of the mineralization products of carbonate-mineralizing bacteria, XRD phase analysis was performed on the precipitates generated after treatment with microorganisms and composite materials. Figure 5 As shown in the figure, the results indicate the presence of characteristic peaks for calcite (PDF#72-1937) and CdCO3 (PDF#42-1342) in the mineralization products. This is due to the production of carbonate ions (CO3-) by carbonate-mineralizing bacteria from the decomposition of urea. 2- ), then CO3 2- With free Ca 2+ Cd 2+ The formation of stable metal precipitates with sharp characteristic diffraction peaks indicates good crystallinity. Furthermore, the mineralization products of As mainly exist as co-precipitates of Ca and As (CaO4) (PDF#89-1365). Combined EDS analysis confirmed that microorganisms remove Cd and As from the solution by generating CdCO3 and co-precipitated arsenic-cadmium minerals through mineralization.

[0076] (4) The remediation effects of different treatments on As and Cd in soil

[0077] Heavy metals in farmland soil mainly enter the food chain through crops, posing a serious threat to human health. There is a significant correlation between the available content of heavy metals in soil and the content of heavy metals in crops. Their content directly affects the migration and transformation of heavy metals in soil and the absorption and utilization of heavy metals by organisms. Figure 6 The contents of available As and Cd in the soil at days 1, 7, 14, 21, and 28 of bioremediation were compared between the blank control group (CK), iron-modified biochar (GBC), microbial treatment groups (ANY-1, ANY-2), and iron-modified biochar-loaded microbial groups (GBC+ANY-1, GBC+ANY-2). Figure 6 (a) This indicates that the bioavailable Cd content in each treatment group decreased from 0.085 mg / kg on day 1 to 0.028, 0.017, 0.019, 0.012, and 0.012 mg / kg, respectively, compared to the control (CK). The highest immobilization efficiency reached 84.38%. Figure 6 (b) The results showed that all treatments promoted the conversion of available As to stable forms in the soil during As fixation. The treatments showed the best results at day 14 of the remediation experiment, at which point the largest reductions in available As content were observed under microbial treatment conditions: ANY-1: 22.20% and ANY-2: 23.61%, respectively. The As fixation effect of each treatment gradually weakened with increasing remediation time, possibly due to the NH4 produced from urea decomposition. +Raising soil pH weakens the anion fixation effect of bacterial mineralization, but the immobilization effect of the composite material is still better than that of single-strain treatment. Analysis of the differences in effects between different treatments clearly shows that both groups of iron-modified biochar-supported microorganisms showed better fixation effects on available Cd and As than single-strain and GBC treatments. The GBC in the loading material adsorbs heavy metals, reducing their toxicity to microorganisms, and improves bacterial metabolism and urease activity, thereby enhancing the remediation effect.

[0078] (5) Effects of different treatments on soil conditions

[0079] 1) Effects on soil pH

[0080] Soil pH is an important physicochemical property of soil and a key indicator reflecting soil characteristics. Compared with the unremediated control (CK) soil, the pH values ​​of all treatments increased significantly. Figure 7 After 28 days, the final pH values ​​were: 6.46 (GBC), 7.28 (ANY-1), 7.27 (ANY-2), 7.32 (GBC+ANY-1), and 7.29 (GBC+ANY-2). The pH values ​​of each treatment group were consistently higher than the control group during the remediation process. Furthermore, the pH values ​​of the microbial treatment groups (ANY-1, ANY-2) and the composite material treatment groups (GBC+ANY-1, GBC+ANY-2) rapidly increased in the first 7 days, indicating that the strain rapidly secreted urease in the first 7 days, utilizing the complex nitrogen-containing compounds in the soil to produce NH4+. + and OH - This increases the soil pH value.

[0081] Increased soil pH significantly affects the speciation and immobilization efficiency of heavy metals. More adsorption sites are created on soil organic-inorganic colloids and clay particles, making it easier to form organometallic complexes, metal hydroxides, and carbonates. Simultaneously, increased soil pH may also promote the formation of iron and manganese oxides, providing additional binding sites for heavy metals in the soil. Therefore, increased soil pH significantly reduces the bioavailability of heavy metals through multiple synergistic pathways, ultimately reducing the accumulation of heavy metals in plants.

[0082] 2) Impact on soil organic matter

[0083] Soil organic matter, as a core component of the soil ecosystem, generally originates from plant residues, microbial metabolism, and animal decomposition products in the soil environment. In terms of nutrient supply, it is the main energy source for microorganisms, promotes plant root development, and accelerates nutrient cycling. Regarding soil improvement, it significantly improves soil structure, enhances soil fertility, and strengthens buffering capacity. In terms of interactions, it promotes N and P activation and regulates K and S forms. Within a certain range, soil organic matter content is positively correlated with overall soil fertility.

[0084] Figure 8 The figure shows the total organic matter content in the soil 28 days after different treatments. As can be seen from the figure, there was no significant difference in the total organic matter content between the ANY-1 treatment group and the control group. The soil organic matter content under the ANY-2, GBC+ANY-1, and GBC+ANY-2 treatments was significantly higher than that under the control group. The total organic matter content of the composite material (GBC+ANY-1, GBC+ANY-2) treatment group increased the most, by 8% and 10% respectively compared with the control. This may be due to the loose and porous structure of iron-modified biochar, which can improve the physical and chemical properties of the soil, thereby improving the living environment of soil organisms and promoting the increase of soil organic matter content.

[0085] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0086] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. The application of a carbonate-mineralizing bacterium in increasing soil organic matter content, characterized in that, The carbonate mineralizing bacteria is Bacillus lamellaris (B. lamellaris). Rummeliibacillus pycnus ANY-2, the Bacillus lamellaris ANY-2, was deposited on May 19, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34595.

2. An inoculum containing the carbonate mineralizing bacteria described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is OD 600 The bacterial suspension of the carbonate mineralizing bacteria is 1 to 1.

8.

4. A composite material, characterized in that, The composite material is made by supporting the carbonate mineralizing bacteria of claim 1 with α-FeOOH modified biochar, and the preparation of the composite material includes the following steps: S1. Using soybean straw biochar as raw material, KOH and Fe(NO3)3 were used for modification to obtain α-FeOOH modified biochar; S2. Add α-FeOOH modified biochar, the bacterial suspension of the carbonate mineralizing bacteria and urea to the culture medium. The precipitate obtained after cultivation is the composite material.

5. The composite material according to claim 4, characterized in that, The specific steps of S1 are as follows: soybean straw biochar is dispersed in a KOH solution with a concentration of 4~6 mol / L and activated for 1~2 h, then Fe(NO3)3 solution with a concentration of 0.8~1.2 mol / L is added and stirred for 1~2 h to produce a red precipitate. The mixture is then allowed to stand at 65~70℃ for 50~60 h, and then allowed to settle naturally for 20~24 h. The precipitate is then dried to obtain the α-FeOOH modified biochar.

6. The composite material according to claim 5, characterized in that, The mass-to-volume ratio of soybean straw biochar, KOH solution, and Fe(NO3)3 solution is 2-3 g: 100 mL: 100 mL.

7. The composite material according to claim 6, characterized in that, The soybean straw biochar is obtained by cutting soybean straw into shorter pieces, washing and drying it, and then pyrolyzing it at 380~400℃ for 2~3 hours in a nitrogen atmosphere.

8. The application of the microbial agent of claim 2 or the composite material of claim 4 in the remediation of heavy metal contaminated soil and the improvement of soil physicochemical properties, characterized in that, The improvement of soil physicochemical properties includes increasing soil organic matter content and pH value, and the heavy metal is cadmium and / or arsenic.

Images