Two multifunctional alcaligenes strains and their application in cadmium immobilization and fertilizer enhancement in cadmium contaminated soil

Abstract

This invention belongs to the field of biotechnology, specifically relating to the plant growth-promoting properties of two alkali-producing strains, *Rhizobium miltiorrhiza* and *Pseudomonas mossae*, and their applications in soil cadmium fixation and fertilization. This invention provides a multifunctional alkali-producing bacterium, which is any one of the following strains: *Rhizobium miltiorrhiza* (… Rhizobium miluonense WW22, accession number: CGMCC No. 27718; *Pseudomonas molluscum* ( Pseudomonas mohnii WW2, accession number: CGMCC No. 27716. This invention also provides the use of the above-mentioned strain: cadmium fixation and fertilization. The strain of this invention can promote the reduction of cadmium availability in the soil and increase the nutrient content in the soil, providing a technical basis for ensuring the safe production and utilization of crops in cadmium-contaminated farmland, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the plant growth-promoting properties of two alkali-producing strains, Miluo River Rhizobium and Pseudomonas molluscum, and their use in soil cadmium fixation and fertilization. Background Technology

[0002] Soil cadmium pollution poses a serious threat to ecosystems and human health, while microbial remediation technology has received widespread attention due to its environmental friendliness and sustainability. Faced with the severe challenges posed by soil cadmium pollution, traditional physical and chemical remediation methods, although effective to some extent in removing heavy metals from soil (Yang et al., 2018), have significant limitations. Physical remediation technologies, such as topsoil replacement and electroremediation, require substantial human and material resources, and their high economic costs often limit their widespread application. While chemical remediation technologies are faster-acting, the long-term stability of passivating agents is difficult to guarantee, and secondary pollution may occur during treatment due to agent residues or heavy metal transformation, thus exacerbating environmental risks (Jia et al., 2020). More importantly, these traditional methods often cause irreversible damage to the soil ecosystem, leading to negative impacts such as imbalance in soil microbial community structure and reduced enzyme activity, thereby affecting long-term soil productivity and ecological functions. Against this backdrop, the rise of microbial remediation technology provides new ideas and approaches for the treatment of soil heavy metal pollution (Wang et al., 2013). Compared with traditional physicochemical methods, microbial remediation has significant advantages such as being environmentally friendly, low-cost, and easy to operate.

[0003] Studies have found that microbial cell walls and extracellular secretions are important binding sites for the adsorption of heavy metals. This adsorption and chelation is far stronger than electrostatic attraction and exhibits relatively stable characteristics. Microbial extracellular secretions, as key mediators for heavy metal passivation, have been extensively studied for their complex composition and diverse functions. Existing research shows that components such as carboxylic acids, polysaccharides, and proteins in these secretions can interact with heavy metal ions through charged groups such as carboxylate and phosphate groups. Therefore, microorganisms can adsorb and complex soil cadmium using cell wall functional groups and extracellular secretions. Furthermore, the extracellular polysaccharides, organic acids, and amino acids secreted by microorganisms can alter the solubility and speciation of cadmium in the soil by regulating soil pH and ionic strength. In addition, microbial strains with various plant-promoting functions can also interact with soil nutrient cycling, promoting synergistic changes in soil nutrients and enzyme activity while altering cadmium speciation. For example, microorganisms release nitrogen and phosphorus while increasing carbon and nitrogen reserves through the decomposition of organic matter and nitrogen fixation. Although various cadmium-tolerant strains have been discovered, existing research has focused on the specific mechanisms of action of single strains. However, the effects of multifunctional strains on cadmium speciation in soil and their interactions with the soil environment remain unclear. Summary of the Invention

[0004] The problem to be solved by this invention is to provide two multifunctional alkali-producing strains and their application in cadmium fixation and fertilization of cadmium-contaminated soil.

[0005] To solve the above-mentioned technical problems, the present invention provides a multifunctional alkali-producing bacterium, which is any of the following strains:

[0006] Rhizobium miluonense WW22, accession number: CGMCC No. 27718;

[0007] Pseudomonas mohnii WW2, accession number: CGMCC No. 27716.

[0008] The present invention also provides the use of the above-mentioned strain: cadmium fixation and fertilizer enhancement.

[0009] An improvement to the use of the strain of the present invention: it has plant growth-promoting function.

[0010] As a further improvement to the use of the strain of the present invention: the plant growth-promoting functions include siderophore production, nitrogen fixation, phosphorus solubilization and indoleacetic acid production, which can effectively promote rice growth and nitrogen absorption.

[0011] As a further improvement to the use of the strain of the present invention: increasing the environmental pH (by secreting extracellular metabolites to increase the environmental pH, said environment including soil environment).

[0012] As a further improvement to the use of the strain of the present invention: adsorbing free cadmium, thereby reducing the effectiveness of cadmium.

[0013] As a further improvement to the use of the strain of the present invention: it increases the content of carbon, nitrogen and phosphorus nutrients in the soil, improves enzyme activity, and further promotes the growth of rice and nitrogen absorption, reduces the accumulation of cadmium in rice grains, and realizes the safe production and utilization of crops.

[0014] The strain of this invention was isolated from polluted farmland soil surrounding a waste dismantling site. This strain has the following uses: cadmium-tolerant growth-promoting function, cadmium fixation and fertilization in the soil, secretion of alkaline substances to reduce the availability of cadmium in solution, and growth-promoting functions such as iron carrier production, nitrogen fixation, phosphorus solubilization, and indoleacetic acid production. When added to the soil, it increases the pH within a certain period of time (it can produce alkaline extracellular secretions to increase the pH of the culture medium), increases soil surface functional groups and reduces the availability of cadmium in the soil, increases soil nutrients, alters the activity of enzymes related to the carbon, nitrogen, and phosphorus cycles, and improves fertility, thereby promoting the growth and nutrient absorption of rice and reducing the accumulation of cadmium in the grains.

[0015] In summary, this invention has screened two multifunctional growth-promoting bacteria, Rhizobium miluonense WW22 and Pseudomonas mohnii WW2, which have the ability to adsorb cadmium. These bacteria possess alkali-producing and cadmium-adsorbing functions, which can promote the reduction of cadmium availability in the soil and increase the nutrient content in the soil. This provides a technical basis for ensuring the safe production and utilization of crops in cadmium-contaminated farmland and has broad application prospects. Attached Figure Description

[0016] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0017] Figure 1 Rhizobium miluonense WW22 (magnified 300,000 times);

[0018] Figure 2 Pseudomonas mohnii WW2 (magnified 300,000 times);

[0019] Figure 3 Phylogenetic tree of strains based on 16S rDNA;

[0020] Figure 4 The siderophore production capacity and nitrogenase activity of the strain;

[0021] Figure 5 The strain's ability to solubilize phosphorus and produce indoleacetic acid;

[0022] Figure 6 The strain's cadmium adsorption capacity and pH changes in MSA medium;

[0023] Figure 7 The effects of the strain on soil cadmium speciation and pH;

[0024] Figure 8 The effects of the strain on total organic carbon and soluble organic carbon in the soil;

[0025] Figure 9 The effects of the strain on total nitrogen, soluble organic nitrogen, ammonium nitrogen and nitrate nitrogen in soil;

[0026] Figure 10 The effect of the strain on available phosphorus in the soil;

[0027] Figure 11 The effects of the strain on the activity of carbon, nitrogen, and phosphorus-related enzymes in soil;

[0028] Figure 12 The effects of the strain on rice plant height, chlorophyll, nitrogen uptake, and cadmium content in brown rice were investigated. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0030] Example 1: Screening and Identification of Tolerant Strains

[0031] 1. Materials and Methods

[0032] 1.1 Culture medium and reagents

[0033] LB medium: Dissolve 5 g yeast extract, 10 g tryptone, and 10 g NaCl in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to 7.0, and sterilize at 121℃ for 15 min.

[0034] MSA liquid culture medium: Dissolve 20 g sucrose, 2 g asparagine, 1 g K2HPO4, and 0.5 g MgSO4·7H2O in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to 7.0, and sterilize at 115℃ for 15 min.

[0035] MSA solid culture medium: Dissolve 20 g sucrose, 2 g asparagine, 1 g K2HPO4, 0.5 g MgSO4·7H2O and 18 g agar in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to 7.0, sterilize at 115℃ for 15 min, and obtain the MSA solid detection culture medium after the temperature drops to room temperature.

[0036] The cadmium-containing MSA liquid medium is prepared by adding sterilized and cooled MSA medium to a filtered and sterilized cadmium solution (cadmium concentration of 1000 mg / L). -1 The aqueous solution is filtered through a membrane with a 0.45 μm filter diameter to achieve sterilization, resulting in a final concentration of 10 mg / L. -1 .

[0037] The preparation method of the blue solid detection culture medium is the same as that in 202410369553.8, that is, specifically:

[0038] CAS medium: per 100 mL contains 1 mL of 20% sucrose solution, 3 mL of 10% acid-hydrolyzed casein, and 1 mmol / L -1 100 μL of CaCl2, 1 mmol / L -1 2 mL MgSO4·7H2O, 1.8 g agar, and the remainder deionized water. The percentages mentioned above are by mass.

[0039] When the CAS medium is at approximately 60°C and not yet fully solidified, slowly add 5 mL each of phosphate buffer (pH 6.5) and CAS staining solution. After the temperature drops to room temperature, a blue solid detection medium is obtained.

[0040] 1.2 Selection of Tolerant Strains

[0041] 1.2.1 Source of strain

[0042] Soil samples were collected from cadmium-contaminated farmland in Taizhou, Zhejiang Province (soil samples from cadmium-contaminated farmland surrounding a waste electrical appliance dismantling site in Taizhou, Zhejiang Province).

[0043] 1.2.2 Isolation, purification, and screening of resistant strains

[0044] Take 2 g of each cadmium-contaminated farmland soil sample and, under aseptic conditions, add it to a 50 mL Erlenmeyer flask containing 18 mL of sterile water. Shake at 180 r / min for 1 hour at 28°C. After serial dilution in sterile water, place 100 μL of the diluted soil suspension on a blue solid detector and incubate at 28°C for 7 days. Select single colonies with a large orange halo and repeatedly perform streak plating and purification until single colonies are obtained. Inoculate each single colony into LB liquid medium and incubate with shaking until turbid. Take 900 μL of the bacterial suspension and add it at a 1:1 ratio to a storage tube containing 50% glycerol, then store at -80°C.

[0045] The selected strains were named WW22 and WW2, respectively.

[0046] 1.2.3 Strain Identification

[0047] The strains were identified using 16S rDNA sequencing. Total DNA from strains WW22 and WW2 were used as templates for PCR amplification of the 16S rDNA gene using universal primers. The amplified fragments were recovered and sequenced to determine their size. The sequencing results were then compared for homology with sequences in NCBI using MEGA software.

[0048] Strain WW22 showed high homology with *Rhizobium miluonense*, with a homology of approximately 87%; strain WW2 showed high homology with *Pseudomonas mohnii*, with a homology of approximately 70%. Figure 3 ).

[0049] 1.2.4 Morphological characteristics and physiological and biochemical properties of the two strains were observed and determined.

[0050] Two different strains were inoculated into MSA liquid medium. After 72 h, the strains were pretreated and their morphological characteristics were observed by scanning electron microscopy. The purified strains were subjected to Gram staining during the logarithmic growth phase.

[0051] Strain WW22 is a bacillus with a rough surface, does not produce spores, lacks flagella, and is Gram-negative. The cells are approximately 0.8–1.5 μm long and 0.2–0.4 μm wide, exhibiting a densely aggregated state with abundant cross-links between cells and a surface covered with filamentous material (such as...). Figure 1 Strain WW2 is a bacillus with a rough surface, no flagella, and is Gram-negative. The cell size is approximately (1.2–1.8) μm long and (0.2–0.3) μm wide, with obvious flocculation and precipitate on the cell surface (e.g., ...). Figure 2 On MSA solid medium, both strains appeared as regular round dots with smooth surfaces, raised centers, and moist textures; however, WW22 appeared pale white, while WW2 appeared pale pink.

[0052] 1.3 Determination of the functions of WW22 and WW2 strains

[0053] 1.3.1 Determination of the plant growth-promoting function of the strain

[0054] I. Iron production capacity:

[0055] Preparation of modified sucrose-asparagine (MSA) liquid culture medium: Dissolve 20 g sucrose, 2 g asparagine, 1 g K2HPO4, and 0.5 g MgSO4·7H2O in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to 7.2~7.4, sterilize at 115℃ for 15 min, and set aside for use.

[0056] 0.2 mL of bacterial culture stored in glycerol at -80℃ was inoculated (transferred) into 20 mL of sterile LB liquid medium. After pre-culturing in a constant temperature air bath with shaking (28℃, 180 r / min) for 48 hours, the culture was transferred to a sterile centrifuge tube and centrifuged at low temperature (4℃, 6000 g, 5 min) to collect the bacterial cells. The cells were washed three times with MSA liquid medium and resuspended. The OD of the bacterial culture was adjusted. 600 = 1, take 200 μL of the obtained bacterial suspension and add it to a solution with a Cd concentration of 10 mg / L. -1 After adding 20 mL of MSA culture medium, place it in a constant temperature shaker (180 rmin). -1 Incubate at 28℃ for 72 h at 4000 r / min -1After centrifugation for 10 min, the sample was filtered through a 0.22 μm filter to obtain a sterile supernatant, from which the content of siderophores secreted by bacteria was determined. The sterile supernatant was mixed with CAS staining solution at a 1:1 ratio, allowed to stand for 2 h, and the OD was measured using a UV spectrophotometer. 630 The value obtained is A. Using a sterile culture medium that has not been cultured with microorganisms as a control, the value obtained is Ar. The formula for calculating the relative content of siderophores (SU) is: SU = 1-A / Ar.

[0057] II. Nitrogenase activity:

[0058] Preparation of Ashby's nitrogen-free medium: Dissolve 10.0 g mannitol, 5.0 g CaCO3, 0.2 g KH2PO4, 0.2 g MgSO4·7H2O, 0.2 g NaCl, and 0.1 g CaSO4·2H2O in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to 7.2-7.4, sterilize at 121℃ for 15 min, and set aside for use.

[0059] After activating the strain according to the aforementioned steps, the bacterial culture was centrifuged (6000 g, 5 min), washed three times with MSA liquid medium, resuspended, and adjusted to OD. 600 = 1, 200 μL of the obtained bacterial suspension was inoculated into 20 mL of Ashby nitrogen-free liquid medium and cultured at a constant temperature (28℃) for 72 h. The nitrogenase content was determined according to the Kote kit (Wuhan Bio-Vivan Biotechnology Co., Ltd., China).

[0060] III. Phosphorus solubility:

[0061] Preparation of Monkina organic phosphorus culture medium: Dissolve 10.0 g glucose, 5.0 g CaCO3, 0.5 g (NH4)2SO4, 0.4 g yeast extract, 0.3 g NaCl, 0.3 g KCl, 0.2 g egg yolk lecithin, 0.03 g FeSO4, 0.03 g MnSO4, and 15 g agar in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to about 7.0, sterilize at 121℃ for 15 min, and set aside for use.

[0062] Preparation of Monkina Inorganic Phosphorus Medium: Take 10.0 g glucose, 10.0 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, and 15 g agar. Adjust the pH to about 7.0, dissolve in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to about 7.0, and sterilize at 121℃ for 15 min before use.

[0063] After activating and culturing the strain according to the aforementioned steps, take 1 μL of bacterial suspension (OD) 600 = 1) Inoculate the strain into organic and inorganic phosphorus solid culture medium of Monkina and incubate at 28℃ for 5-7 days. Evaluate the phosphorus solubility of the strain based on the phosphorus solubility zone.

[0064] IV. Ability to produce indoleacetic acid:

[0065] Preparation of Kings medium: Dissolve 20.0 g tryptone, 1.5 g K2HPO4, 1.5 g MgSO4, and 0.2 g tryptophan in an appropriate amount of deionized water, bring the volume to 1000 mL, adjust the pH to about 7.2, and sterilize at 121℃ for 30 min before use.

[0066] Preparation of Salkowski reagent colorimetric solution: Take 50 mL of 35% HClO4 and 1 mL of 0.5 mol / L... -1 It is obtained by mixing FeCl3.

[0067] After activating and culturing the strain according to the aforementioned steps, aspirate 200 μL (OD) of the cultured product. 600 = 1) The bacterial suspension was inoculated into 20 mL of Kings liquid medium and incubated at 130 r min. -1 After culturing the bacterial culture in a shaker at 28℃ for 3 days, the supernatant was obtained by centrifugation at 10000g for 10 min at low temperature (4℃). 200 μL of the supernatant was transferred to a white porcelain plate, and 200 μL of Salkowski reagent was added. The plate was then wrapped in aluminum foil and placed in the dark for 30 min. The results were observed. The color of Kings medium served as a control; a pink color indicated that the strain could secrete auxins or indole compounds, with a darker color representing a stronger ability.

[0068] The results showed that strains WW22 and WW2 possess multiple plant growth-promoting functions, such as... Figure 4 As shown, the relative siderophore yields of strains WW22 and WW2 were 63.20% and 80.76%, respectively, and their nitrogenase activities were 231.80 and 104.62 ng / mL, respectively. -1 .like Figure 5 As shown, strains WW22 and WW2 can effectively dissolve both organic and inorganic phosphorus, and can produce indoleacetic acid.

[0069] 1.3.2 Determination of the strain's ability to adsorb cadmium

[0070] Take 0.2 mL of the bacterial culture stored in glycerol at -80℃ and inoculate (transfer) it into 20 mL of sterile MSA liquid medium. After pre-culturing in a constant temperature air bath with shaking (28℃, 180 r / min) for 48 hours, transfer it to a sterile centrifuge tube and centrifuge at low temperature (4℃, 6000 g / min). -1 Collect bacterial cells (5 min), using MSA liquid medium as a blank, and dilute the bacterial solution to OD. 600 = 1, take 1 mL and add it to a solution with a Cd concentration of 10 mg / L. -1 The samples were cultured in 20 mL of MSA liquid medium, with three replicates. Samples were collected after 72 hours and centrifuged (12,000 g min). -1 (5 min) to separate the supernatant and particles. Take 10 mL and filter the supernatant through a 0.22 µm filter membrane. Determine the Cd concentration in the supernatant by ICP-MS. The ICP-MS determination method is in accordance with HJ 776-2015. The pH of the remaining 10 mL was determined by a pH meter.

[0071] Under aerobic conditions, pH 7.0, temperature 28℃, OD 600 1.0, Cadmium concentration 10 mg / L -1 180 r / min -1 Under MSA liquid culture conditions, after 3 days of culture, centrifugation and filtration, the cadmium ion removal rates of strains WW22 and WW2 were 58.84% and 65.83%, respectively. Figure 6 In a culture medium with an initial pH of around 7.0, the pH of the medium with different cadmium concentrations could be increased by 1.73 and 1.74 units, respectively. This indicates that the *Rhizobium WW22* and *Pseudomonas mossii* WW2 isolated and screened in this invention can secrete some alkaline substances to increase the environmental pH and effectively adsorb free cadmium ions, showing potential in the remediation of heavy metal pollution in soil.

[0072] In summary, this invention isolated, purified, and screened two cadmium-tolerant alkali-producing bacteria, belonging to *Rhizobium miluonense* and *Pseudomonas mohnii*, respectively, and named WW22 and WW2. Comprehensive physiological and biochemical properties and 16S rDNA phylogenetic analysis show that both strains possess high siderophore production capacity and nitrogenase activity, can dissolve both organic and inorganic phosphorus, and can produce indoleacetic acid, exhibiting multiple plant growth-promoting functions.

[0073] Strains WW22 and WW2 are effective against 10 mg L -1 The cadmium removal rates were 58.84% and 65.83%, and the pH values ​​were increased by 1.73 and 1.74 units.

[0074] The preservation information for WW22 is as follows:

[0075] Preservation Name: Rhizobium miluonense, Preservation Institution: China General Microbiological Culture Collection Center, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Accession Number: CGMCC No. 27718, Preservation Date: June 27, 2023.

[0076] The preservation information for WW2 is as follows:

[0077] Preservation Name: Pseudomonas mohnii, Preservation Institution: China General Microbiological Culture Collection Center, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Accession Number: CGMCC No. 27716, Preservation Date: June 27, 2023

[0078] Example 2: Verification of the strain reducing cadmium availability and increasing nutrients in soil

[0079] 1. Materials and Methods

[0080] 1.1 Experimental Setup

[0081] Cadmium-contaminated farmland soil from Taizhou, Zhejiang Province was selected as the test soil (cadmium concentration in the soil was 1.87 mg / kg). -1 Soil samples were collected and air-dried naturally. After removing mixed plant debris and mineral fragments manually, they were passed through a 10-mesh nylon sieve before use in subsequent experiments. To restore the activity of native microorganisms in the experimental soil, the soil was placed at room temperature for 24 hours for activation before the experiments. Then, 100 g (dry weight) of the soil was accurately weighed and placed into 250 mL Erlenmeyer flasks for subsequent experiments. Three treatments were set up: no inoculation, inoculation with Rhizobium sp. WW22, and Pseudomonas sp. WW2, with three replicates for each treatment. The Erlenmeyer flasks were inoculated with the strains according to the treatment settings: the OD values ​​of the activated strains WW22 and WW2 were... 600 Adjust to approximately 1.5 and 2.0 respectively (using MSA liquid culture medium), at approximately 10 6 CFU g -1 Inoculation was performed at a dry weight of approximately [amount missing], with an equal volume of sterile water added to the control (CK) treatment. Each sample was thoroughly mixed for 2 minutes and then placed in a 28°C incubator in the dark for static incubation. During incubation, the samples were weighed periodically and water was replenished to maintain maximum field capacity (27%). Samples were taken for analysis on day 21 of incubation.

[0082] 1.2 Determination and treatment of soil properties

[0083] After a portion of the sample was air-dried, the soil pH, DOC, DON, TC, TN, TS, AP, and NH4 were determined according to the following steps. + -N、 Different forms of cadmium were classified. The activities of soil β-glucosidase (S-β-GC), soil cellulase (S-CL), soil urease (SUE), soil arylamidase (S-AAS), soil N-acetyl-β-D-glucosidase (S-NAG), and soil alkaline phosphatase (S-ALP) were determined according to the instructions of the enzyme kit (Beijing Box Sangon Biotech Co., Ltd., China).

[0084] Soil cadmium speciation: Using the BCR three-step extraction method proposed by the European Standards Institute, Cd was separated into four forms: exchangeable, reducible, oxidizable, and residual. The extract was filtered through a 0.45 μm filter membrane and then analyzed by ICP-MS.

[0085] Soil pH: Take 4g of air-dried soil sample, add 10ml of deionized water, shake for 30min, let stand for 30min, and measure its pH with a soil pH meter.

[0086] Total organic carbon (TOC) in soil: After removing inorganic carbon with dilute hydrochloric acid, weigh approximately 15 mg of a 100-mesh soil sample, wrap it in tin foil, and then measure it using a total organic carbon analyzer based on the dry burning method.

[0087] Total nitrogen (TN) in soil: Weigh approximately 15 mg of a 100-mesh soil sample, wrap it in tin foil, and measure it using an elemental analyzer.

[0088] Soil soluble organic carbon (DOC) and soluble organic nitrogen (DON): Take 5g of air-dried soil sample, add 25 mL of deionized water (soil-to-water ratio 1:5), shake for 1 h, and then spray at 4000 r / min. -1 Centrifuge for 10 min, filter using a 0.45 μm filter membrane, collect the supernatant, and measure it using a TOC / TN analyzer.

[0089] Soil ammonium nitrogen (NH4) + -N) and nitrate nitrogen ( ): Take 5 g of fresh soil and add it to 25 mL of 1 mol / L KCl solution. Shake at a constant temperature for 1 h (25℃, 250 r / min).-1 Centrifuge the soil suspension (4000 r min). -1 (20 min), take the supernatant and filter it with a 0.45 μm aqueous phase filter membrane (or filter paper) to obtain soil leachate, which is then measured using a continuous flow colorimetric analyzer.

[0090] Available phosphorus (AP) in soil: According to the national standard (HJ 704-2014), take 2.50 g of air-dried soil and place it in a centrifuge tube. Add 50 mL of extraction solvent and shake at a constant temperature for 30 min (25℃, 180 r / min). -1 Immediately filter with phosphorus-free filter paper, measure 10 mL of supernatant, add water to about 20 mL, add 1 drop of indicator, then add sulfuric acid solution dropwise until the solution is colorless, add 0.75 mL of ascorbic acid, mix well, add 5 mL of molybdate solution after 30 seconds, dilute with water to 50 mL, mix well, and then perform color development and measurement using an ELISA reader.

[0091] Soil enzyme activity: Carbon, nitrogen, and phosphorus-related enzyme activities were determined according to the instructions of the enzyme kit (Beijing Box Sangon Biotech Co., Ltd., China). All soil enzyme activity results were expressed in U / d. -1 g -1 Expressed in units, specifically defined as: the amount of product (e.g., urease to generate NH4) produced by each gram of dry soil (based on oven-dried soil mass) within 24 hours (1 day) through enzymatic reactions catalyzing the substrate. + The enzyme activity units (U) corresponding to the amount of -N, the amount of phosphatase and the amount of phenol produced, etc., are calculated based on the standard curve and reaction system parameters of the corresponding assay method.

[0092] 2 Results

[0093] The results are as follows Figure 7 As shown, after the two strains were inoculated into the soil, they could reduce the proportion of exchangeable cadmium in the soil by 2.63% and 2.40%, increase the proportion of residual cadmium by 3.73% and 4.98%, and increase the soil pH by 0.17 and 0.32 units, respectively. Figure 8-9 This indicates that some available nutrients in the soil have been significantly increased, with total organic carbon in the soil increasing by 1.30 g / kg and 0.90 g / kg, respectively. -1 The levels of soluble organic carbon increased by 6.45 and 66.49 mg kg, respectively. -1 The levels of soluble organic nitrogen increased by 25.67 and 3.79 mg / kg, respectively. -1 Ammonium nitrogen increased by 6.86 and 11.84 mg / kg, respectively. -1 Available phosphorus increased by 3.34 and 3.21 mg / kg, respectively. -1 Strain WW22 can also increase total nitrogen in the soil by 0.11 g / kg.-1 Nitrate nitrogen 2.12 mg kg -1 ( Figure 10 Furthermore, the activity of carbon, nitrogen, and phosphorus-related enzymes in the soil was activated, with WW22 increasing soil cellulase activity by 3.49 U / d. -1 g -1 Soil β-glucosidase activity 2.05 U / d -1 g -1 Soil urease activity 37.64 U d -1 g -1 Soil N-acetyl-β-D-glucosidase activity was 0.81 Ud. -1 g -1 Soil arylsulfatase activity 1.68 U / d -1 g -1 Soil alkaline phosphatase activity was 0.58 U / d. -1 g -1 WW2 increased soil cellulase activity by 12.50 U / d. -1 g -1 Soil urease activity 25.85 U / d -1 g -1 Soil N-acetyl-β-D-glucosidase activity was 4.31 U / d. -1 g -1 Soil arylsulfatase activity was 0.81 U / d. -1 g -1 Soil alkaline phosphatase activity was 0.82 U / d. -1 g -1 ( Figure 11 ).

[0094] Example 3: The strain promotes rice growth and nitrogen absorption in cadmium-contaminated soil and reduces cadmium accumulation in brown rice.

[0095] 1. Materials and Methods

[0096] 1.1 Experimental Setup

[0097] The aforementioned cadmium-contaminated farmland soil from Taizhou, Zhejiang Province, was selected as the test soil (cadmium concentration in the soil was 1.87 mg / kg). -1The donor strains were WW22 and WW2, and the donor rice was Xiushui 14. Three treatments were set up: no inoculation and inoculation with either Rhizobium sp. WW22 or Pseudomonas sp. WW2. Each treatment had three replicates, for a total of nine pots, with 4.5 kg (dry weight) of soil per pot. Rice seeds were disinfected and germinated according to standard procedures. Three seedlings were transplanted into each pot, and after one week of stable growth, two seedlings with good and similar growth were retained for further cultivation. The OD values ​​of the activated strains WW22 and WW2 were... 600 Adjust to approximately 1.5 and 2.0 respectively (using MSA liquid culture medium), at approximately 10 6 CFU g -1 Approximately 100 mg (dry weight) of the inoculum was applied to the roots of the rice plants. An equal volume of sterile water was added to the control (CK) treatment. Inoculation was repeated every 14 days (post-inoculation, the bacterial concentration in the soil was 10-1). 6 CFUg -1 For the CK treatment, an equal volume of sterile water was added, and the plants were regularly watered to maintain a water level of 3-5 cm. Plant samples were collected at maturity (110 days after transplanting, after 6 inoculations). One day before harvest sampling, the chlorophyll content of leaves (leaves with fully swollen tops) was measured at 10:00 AM using a SPAD-502PLUS chlorophyll meter, and plant height was recorded simultaneously. Rice plants were divided into roots, stems, leaves, and panicles, and rinsed several times in deionized water. The samples were then blanched at 105℃ for 2 hours and dried to constant weight at 75℃. Each part of the plant was weighed and pulverized, and the nitrogen content of each part was determined using an elemental analyzer. The cadmium content of a portion of the brown rice from the panicles was determined using microwave digestion.

[0098] 2 Results

[0099] like Figure 12 As shown, the addition of the strains to the rice rhizosphere increased the rice biomass and aboveground nitrogen content, indicating that the two multifunctional growth-promoting strains promoted rice growth to some extent in cadmium-stressed soil. Strain WW22 increased rice plant height by 10.93%, increased chlorophyll content by 12.52%, increased whole-plant nitrogen content by 13.29%, and reduced the cadmium concentration in brown rice by 69.24%, from 0.63 mg / kg. -1 Reduced to 0.19 mg / kg -1 The strain WW2 increased rice plant height by 13.04%, chlorophyll content by 16.29%, and overall nitrogen content by 6.87%. It also reduced cadmium concentration in brown rice by 62.78%, to a mere 0.19 mg / kg. -1 Both strains resulted in cadmium concentrations in brown rice that were lower than the standard requirement (0.2 mg / kg) for brown rice in the National Food Safety Standard for Maximum Levels of Contaminants in Food (GB2762).-1 ).

[0100] It is evident that the *Rhizobium miluonense* WW22 and *Pseudomonas mohnii* WW2 discovered in this invention are multifunctional alkali-producing bacteria capable of efficiently removing cadmium. These strains can efficiently adsorb free cadmium ions and increase the environmental pH. After inoculation into the soil, they can still reduce exchangeable cadmium in the soil and increase soil pH. Furthermore, they can improve the content of available carbon, nitrogen, and phosphorus in the soil and their related enzyme activities to varying degrees, improving the soil environment and promoting rice growth and nutrient absorption. Simultaneously, they effectively reduce cadmium accumulation in brown rice. This discovery not only provides a novel microbial resource for the remediation of heavy metal-contaminated farmland, possessing functions such as efficient cadmium fixation, alkali production and pH adjustment, soil fertility enhancement, and enzyme activity improvement, but also provides theoretical support for the remediation technology of multifunctional plant growth-promoting bacteria for the safe production and utilization of rice in cadmium-contaminated environments.

[0101] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A multifunctional alkali-producing bacterium, characterized in that... It is any of the following strains: Miluo River Rhizobium ( Rhizobium miluonense WW22, Collection Number: CGMCC No. 27718; Pseudomonas molluscum ( Pseudomonas mohnii )WW2, Collection Number: CGMCC No.27716.

2. The use of the strain as described in claim 1, characterized in that: Cadmium fixation and fattening.

3. The use of the strain according to claim 2, characterized in that: It has plant growth-promoting function.

4. The use of the strain according to claim 3, characterized in that: The plant growth-promoting functions include siderophore production, nitrogen fixation, phosphorus solubilization, and indoleacetic acid production.

5. The use of the strain according to claim 4, characterized in that: Increase the pH level of the environment.

6. The use of the strain according to any one of claims 2 to 5, characterized in that: It adsorbs free cadmium, thereby reducing the bioavailability of cadmium.

7. The use of the strain according to any one of claims 2 to 6, characterized in that: It increases the content of carbon, nitrogen, and phosphorus nutrients in the soil, promotes rice growth and nitrogen absorption, reduces cadmium accumulation in grains, and enables its application in the remediation of heavy metal pollution.

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