Acinetobacter pittii WJ2, bacterial agent and application thereof

Abstract

This invention discloses a strain of Acinetobacter piterum WJ2, its inoculum, and its applications. The Latin name of this strain of Acinetobacter piterum WJ2 is... Acinetobacter pittii WJ2, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20251840, exhibits extremely high antimony tolerance, growing normally under 20 mM trivalent antimony conditions. It synergistically reduces antimony availability through multiple pathways, including cell absorption and adsorption, secretion chelation / complexation, alteration of soil C=O bond vibrational modes, and promotion of the formation of insoluble antimony minerals. This strain also possesses the ability to dissolve insoluble calcium phosphate. Pot experiments showed that inoculating antimony-contaminated soil with WJ2 significantly promoted lettuce growth, increased yield, and reduced antimony content in the aboveground parts and roots of lettuce. The strain provided by this invention has a clearly identified species, diverse antimony-reducing mechanisms, and combines phosphate-solubilizing and growth-promoting functions, making it suitable for the microbial remediation of antimony-contaminated soil and the safe production of crops such as lettuce.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and heavy metal pollution control technology, and in particular to Acinetobacter pituitaria WJ2 and its inoculants and applications. Background Technology

[0002] Antimony (Sb) is a highly toxic metalloid element and has been listed as a priority pollutant by many countries and international organizations. With the intensification of human activities such as antimony mining, metallurgical smelting, and chemical production, large amounts of antimony enter the soil environment, causing deterioration of soil physicochemical properties, reduced enzyme activity, and imbalance of microbial community structure. This not only inhibits normal crop growth and development but also threatens human health through bioaccumulation in the food chain. Therefore, the safe remediation and treatment of antimony-contaminated soil has become a research hotspot in the environmental and agricultural fields.

[0003] Antimony-resistant bacteria ( Antimony-resistant Bacteria Antimony-containing bacteria (ACBs) are a class of microorganisms that can still grow and reproduce normally under high antimony stress, and reduce antimony toxicity, alter antimony forms, and mitigate antimony toxicity to plants through specific metabolic pathways. These bacteria are widely distributed in antimony-contaminated mining areas, soils, water bodies, and crop rhizosphere environments. They can adapt to antimony stress through multiple mechanisms such as redox reactions, adsorption and fixation, extracellular isolation, and efflux pump detoxification. They also possess growth-promoting properties such as phosphorus solubilization, potassium solubilization, iron carrier production, and secretion of plant hormones (auxin IAA, gibberellin, cytokinin), making them valuable for both pollution remediation and crop yield enhancement.

[0004] Existing studies have screened multiple antimony-tolerant bacterial strains from antimony-contaminated environments. Current technologies also disclose strains that can reduce heavy metal content in vegetables and promote plant growth. For example, Chinese patent CN111718870A discloses a functional bacterial strain and its application that can reduce heavy metal content in vegetables and improve vegetable quality. This strain includes *Bacillus subtilis* W7 and *Bacillus amyloliquefaciens* W25, both of which have multiple functions, including reducing cadmium (Cd) content in lettuce, increasing vitamin C and soluble protein content in lettuce, and promoting soil microbial proliferation. However, these strains primarily target heavy metals such as cadmium and lead, and do not address antimony tolerance and removal.

[0005] Although antimony-tolerant bacteria have shown great promise in the bioremediation of antimony-contaminated soils, highly efficient antimony-tolerant functional strains remain relatively scarce, especially those capable of tolerating high concentrations of antimony (4–40 mM) and possessing both growth-promoting and antimony-reducing functions. The adaptability, colonization ability, and field stability of these strains need improvement, and standardized screening systems and efficient identification techniques are still incomplete, limiting their large-scale application in actual production. Therefore, there is an urgent need to develop novel strains with higher antimony tolerance and stronger functional integration to provide high-quality inoculants for the remediation of antimony-contaminated soils and safe agricultural production. Summary of the Invention

[0006] The main objective of this invention is to provide Acinetobacter pituitaria WJ2, its inoculant, and its application, aiming to solve the problems that existing microbial strains used for heavy metal pollution remediation generally lack efficient remediation capabilities against antimony, a specific metal class, and that existing antimony-resistant strains suffer from unclear species identification, a single antimony-reducing mechanism, growth-promoting functions limited to IAA / side carriers without phosphorus-solubilizing ability, and a narrow range of applicable crops.

[0007] To achieve the above objectives, the present invention provides Acinetobacter piterum WJ2, wherein the Latin name of Acinetobacter piterum WJ2 is... Acinetobacter pittii WJ2 is deposited at the China Center for Type Culture Collection (CCTCCNO: M20251840) on August 18, 2025.

[0008] Optionally, the 16S rDNA sequence of Acinetobacter pituitaria WJ2 clusters in the same branch as Acinetobacter pituitaria, and its physiological and biochemical characteristics are catalase positive, methyl red negative, starch hydrolysis negative, gelatin hydrolysis negative, nitrate reduction negative, citrate utilization negative, urease reaction negative, sucrose fermentation reaction negative, glucose fermentation reaction negative, and indole reaction negative.

[0009] In addition, to achieve the above objectives, the present invention also provides a microbial agent comprising Acinetobacter pietrogenus WJ2 as described in claim 1 or 2.

[0010] In addition, to achieve the above objectives, the present invention also provides a method for reducing the antimony content in an antimony-polluted environment, wherein the aforementioned Acinetobacter piezoides WJ2 or the aforementioned microbial agent is applied to the antimony-polluted environment, and the antimony content is reduced through the synergistic action of at least two mechanisms of action, the mechanisms of action including the absorption and adsorption of antimony by the bacteria, the chelation / complexation of antimony by the bacteria secretions, the alteration of the vibration mode of C=O bonds in the soil, and the promotion of the formation of insoluble antimony minerals.

[0011] In addition, to achieve the above objectives, the present invention provides a method for simultaneously reducing the effective antimony content in antimony-contaminated soil, dissolving insoluble inorganic phosphorus, and promoting plant growth under antimony stress, wherein the aforementioned Acinetobacter pietrogen WJ2 or the aforementioned microbial agent is applied to the antimony-contaminated soil.

[0012] Alternatively, the plant may be lettuce.

[0013] Optionally, the sparingly soluble inorganic phosphorus is calcium phosphate.

[0014] Furthermore, in order to achieve the above objectives, the present invention provides the application of the aforementioned Acinetobacter pietrogenus WJ2 or the microbial agent of claim 3 in the preparation of formulations for reducing the antimony content in the aboveground parts and / or roots of lettuce under antimony stress and for promoting the yield increase of lettuce under antimony stress.

[0015] Furthermore, in order to achieve the above objectives, the present invention provides the application of the aforementioned Acinetobacter pietrogenus WJ2 or the microbial agent of claim 3 in the preparation of a formulation for reducing the available antimony content in soil, wherein the reduction of available antimony content in soil is achieved by altering the vibrational mode of soil C=O bonds and / or promoting the formation of insoluble antimony minerals.

[0016] Furthermore, to achieve the above objectives, the present invention provides a method for culturing *Acinetobacter pituitaria* WJ2, wherein *Acinetobacter pituitaria* WJ2 is inoculated into LB liquid medium and cultured with shaking at 28–30°C and pH 7.0–7.2 until the logarithmic growth phase or stationary phase. Preferably, *Acinetobacter pituitaria* WJ2 is inoculated into LB liquid medium at a volume fraction of 1%–5% and cultured at 28–30°C and 150–200 rpm. -1 Incubate with shaking at pH 7.0–7.2 for 12–16 h.

[0017] Beneficial effects.

[0018] (1) This invention discloses for the first time the application of Acinetobacter piezoides WJ2, a specific species, in the remediation of antimony pollution. Strain WJ2 can grow normally and effectively remove antimony in a culture medium containing 20 mM trivalent antimony; after 7 days of liquid culture, the antimony content in the supernatant decreased by 62.3%. In soil containing 500 mg / kg antimony, inoculation with WJ2 can reduce the available antimony content in the soil by 30%–55%.

[0019] (2) Synergistic effect of multiple antimony reduction mechanisms, antimony fixation and stabilization: This invention reveals the effectiveness of WJ2 in synergistically reducing antimony through four pathways: cell absorption and adsorption, secretion chelation / complexation, alteration of soil C=O bond vibration mode, and promotion of the formation of insoluble antimony minerals. Among them, "alteration of soil C=O bond vibration mode" and "promotion of the formation of insoluble antimony minerals" are antimony reduction mechanisms that have never been disclosed in the prior art. FTIR analysis confirmed that after inoculation with WJ2, the C=O bond (1710 cm⁻¹) in the soil was reduced. -1 The vibration mode (nearby) changed significantly, with weakened peak intensity and shifted peak position, indicating that functional groups such as alcohols, aldehydes, ketones, and carboxylic acids in the strain secretions chelated / complexed with antimony, promoting the transformation of antimony from the exchangeable state to the more stable iron-manganese oxide bound state and residual state, reducing the bioavailability of antimony and the risk of environmental migration.

[0020] (3) It also has the function of phosphorus solubilization and improves the supply of phosphorus in the soil: strain WJ2 has the ability to dissolve insoluble calcium phosphate, which can convert phosphorus in the soil that is difficult for plants to absorb into a usable form. The phosphorus solubilization function not only provides phosphorus nutrition for plant growth, but also reduces the amount of chemical phosphate fertilizer applied, thereby reducing agricultural production costs and environmental burden.

[0021] (4) Significantly promotes lettuce growth and reduces antimony accumulation: Pot experiments confirmed that after inoculating lettuce with WJ2 in antimony-contaminated soil at concentrations of 500 mg / kg and 1000 mg / kg, the fresh weight of lettuce increased by 32.6% and 41.3%, respectively, and the dry weight increased by 28.1% and 36.7%, respectively; the antimony content in the aboveground parts of lettuce decreased by 47.8% and 52.3%, respectively, and the antimony content in the roots decreased by 38.5% and 44.1%, respectively. This indicates that WJ2 can increase yield and income while ensuring the safe production of lettuce, and has important agricultural application value.

[0022] (5) The strain WJ2 of this invention is derived from the natural soil environment and is a natural microorganism that is safe and harmless to the environment. It can be used alone or compounded with other agricultural microorganisms to form a compound microbial agent. It is suitable for various scenarios such as in-situ remediation of antimony-contaminated farmland soil, safe production of leafy vegetables such as lettuce, and activation of soil phosphorus, and has good industrialization prospects. Attached Figure Description

[0023] Figure 1 This is the colony morphology of Acinetobacter piezoides WJ2 of the present invention.

[0024] Figure 2 This is a scanning electron microscope (SEM) image of Acinetobacter piezoides WJ2 of the present invention.

[0025] Figure 3 A phylogenetic tree constructed based on the 16S rDNA sequence.

[0026] Figure 4 The effect of different culture temperatures on strain WJ2.

[0027] Figure 5 The effect of different pH values ​​on strain WJ2.

[0028] Figure 6 Microscopic images showing the growth of strain WJ2 at antimony contents of 5 mM and 20 mM.

[0029] Figure 7 The results show the phosphate solubilization of strain WJ2 on PKO solid medium.

[0030] Figure 8 Figure 1 shows the growth results of lettuce under different antimony levels / strain WJ2 treatments.

[0031] Figure 9A comparison chart of the fresh and dried weights of lettuce with different antimony contents.

[0032] Figure 10 To investigate the effect of inoculation with strain WJ2 on the antimony content in the aboveground parts and roots of lettuce.

[0033] Figure 11 To investigate the effect of inoculating strain WJ2 on the available antimony content in soil.

[0034] Figure 12 The effect of inoculating strain WJ2 on antimony speciation in soil.

[0035] Figure 13 XRD patterns of soil crystal structures inoculated with strain WJ2 at different antimony concentrations are shown. Figure 13 A is the XRD structure diagram of soil crystals with WJ2 added under Sb0 conditions (0 mg / kg exogenous antimony addition); 13B is the XRD structure diagram of soil crystals with WJ2 added under Sb0.2 conditions (200 mg / kg exogenous antimony addition); 13C is the XRD structure diagram of soil crystals with WJ2 added under Sb0.5 conditions (500 mg / kg exogenous antimony addition); 13D is the XRD structure diagram of soil crystals with WJ2 added under Sb1 conditions (1000 mg / kg exogenous antimony addition); 13E is the XRD structure diagram of soil crystals with WJ2 added to the rhizosphere soil of lettuce under Sb1 conditions (1000 mg / kg exogenous antimony addition).

[0036] Figure 14 Infrared spectra of soil functional groups inoculated with strain WJ2 at different antimony concentrations were obtained. Specifically, 14F represents the infrared spectrum of soil functional groups inoculated with WJ2 under Sb0 conditions (0 mg / kg exogenous antimony); 14G represents the infrared spectrum of soil functional groups inoculated with WJ2 under Sb0.2 conditions (200 mg / kg exogenous antimony); 14H represents the infrared spectrum of soil functional groups inoculated with WJ2 under Sb0.5 conditions (500 mg / kg exogenous antimony); 14I represents the infrared spectrum of soil functional groups inoculated with WJ2 under Sb1 conditions (1000 mg / kg exogenous antimony); and 14J represents the infrared spectrum of soil functional groups in lettuce rhizosphere soil inoculated with WJ2 under Sb1 conditions (1000 mg / kg exogenous antimony).

[0037] Figure 15 The effect of inoculating strain WJ2 on the antimony content of liquid culture medium.

[0038] Figure 16 The antimony content in strain WJ2.

[0039] Figure 17 The effect of fermentation broth of strain WJ2 on the antimony content of liquid culture medium. Detailed Implementation

[0040] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0041] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0042] 1. Isolation and identification of Acinetobacter piezophyllum commune WJ2.

[0043] 1.1 Strains were isolated.

[0044] Take 10g of fresh rhizosphere soil from *Broomcorn millet*, add 90mL of sterile water, and shake at 28℃ and 180r / min for 30min to obtain a soil suspension, which is 10g of the solution. -1 Soil suspension. Let stand until a supernatant appears. Use a sterile pipette to draw 1 mL into a 9 mL sterile water test tube, mix well, and the resulting solution is 10 mL. -2 Diluent: Dilute the soil suspension with sterile water at a ratio of 10:10. -2 Up to 10 -7 After dilution at different dilution ratios, 0.1 mL of each dilution was spread onto LB (broth) solid medium containing 5 mM trivalent antimony (prepared with potassium antimony tartrate) and incubated at 28°C for 24 h.

[0045] After 24 hours, colonies were picked from the plates, and the inoculation and culture steps were repeated on plates containing 10 mM trivalent antimony. After 24 hours, colonies were picked and inoculated onto plates with higher concentrations of trivalent antimony for multiple screenings of antimony-resistant bacteria. The grown bacteria were then inoculated onto plates with even higher concentrations, and the antimony-resistant bacteria were purified and preserved. The antimony-resistant strain obtained was numbered WJ2.

[0046] 1.2 Morphological identification.

[0047] After culturing the WJ2 strain on LB (broth) medium for 24 hours, the macroscopic morphology is as follows: Figure 1 As shown, the strain grows well on the surface of the culture medium, forming uniform, continuous bacterial moss or distinct single colonies. The colonies are grayish-white or pale yellow, which helps to visually distinguish this strain from other bacteria that may be present in the environment. Furthermore, strain WJ2 is Gram-negative.

[0048] The strain was also imaged using a scanning electron microscope (SEM), and the results are as follows: Figure 2 As shown, the microorganisms in this image are distinctly short rod-shaped or oval, with some cells arranged in pairs or short chains, featuring neat edges and smooth surfaces. This visually demonstrates the actual size of the strain (typically around 1-2 μm).

[0049] The morphological and macroscopic characteristics of the above-mentioned strains are consistent with the general morphology of Acinetobacter spp.

[0050] 1.3 Determination of physiological and biochemical characteristics.

[0051] The strain was identified using routine physiological and biochemical methods. The specific assay methods are as follows.

[0052] 1.3.1 Determination of catalase Take 100 mL of 30% hydrogen peroxide solution, dissolve it in 900 mL of distilled water, mix well, and dispense into 5 mL sterile test tubes for later use. Pick a fresh bacterial culture and inoculate it into 3% hydrogen peroxide solution, observing whether bubbles are produced. The production of bubbles indicates a positive reaction, while the absence of bubbles indicates a negative reaction.

[0053] 1.3.2 Methyl red test (MR test) MR medium: 5.0 g dipotassium hydrogen phosphate (K₂HPO₄), 5.0 g glucose, 7.0 g peptone, 1000 mL distilled water, pH 6.7-7.1, autoclaved at 121 °C for 20 min. Aliquot into 5 mL sterile test tubes for later use.

[0054] Methyl red indicator: Dissolve 10 mg of methyl red in 30 mL of 95% ethanol, then add 20 mL of distilled water. Pick fresh bacterial culture, inoculate it into a test tube, and incubate at the appropriate temperature for 24-48 hours. After incubation, add 2-3 drops of methyl red reagent, mix well, and observe the results. If a red color appears, the result is positive; if a yellow color appears, the result is negative.

[0055] 1.3.3 Starch hydrolysis test Soluble starch culture medium: 10g soluble starch, 1g dipotassium hydrogen phosphate (K₂HPO₄), 1g magnesium sulfate (MgSO₄), 2g calcium carbonate (CaCO₃), 2g ammonium sulfate ((NH₄)₂SO₄), 1g sodium chloride (NaCl), 0.001g zinc sulfate (ZnSO₄), 0.001g manganese chloride (MnCl₂), 0.001g ferrous sulfate (FeSO₄), 1000mL distilled water, pH 7.0-7.4, autoclaved at 121℃ for 20min. Dispense the sterilized liquid culture medium into 5mL sterile test tubes. After 24h of culture, transfer 50-100μL of the bacterial suspension to soluble starch test tubes and incubate at 36℃ for 48-96h. After incubation, add Lugol's iodine solution and observe the color change; no blue color indicates a positive result, and a blue color indicates a negative result.

[0056] 1.3.4 Gelatin Hydrolysis Test Culture medium: 10g peptone, 120g gelatin, 3g beef extract, 5g sodium chloride (NaCl), 1000mL distilled water, pH 7.2-7.4. Sterilize at 121℃ for 20min. Dispense the sterilized gelatin culture medium into 5mL sterile test tubes and hold them vertically until the gelatin solidifies. Before use, liquefy the gelatin in a 30-40℃ incubator until it reaches a semi-frozen liquid state. Place the tubes upright on a test tube rack and inoculate 50-100μL of a bacterial suspension cultured for 24 hours into each gelatin tube. Seal the tubes with film and include two blank controls. Incubate at 36℃. Since the liquefaction time of the gelatin varies with bacteria, observations were performed at 1, 2, 3, 7, 10, 14, and 30 days. Before observation, refrigerate the gelatin tubes at 2-8℃ for 10-30min and observe whether the gelatin has liquefied. If liquefied, record as positive; otherwise, record as negative.

[0057] 1.3.5 Nitrate Reduction Test Nitrate broth (nitrate medium): 5g peptone, 1.0g KNO3, 1000mL distilled water, pH 7.0~7.5, autoclave at 121℃ for 20min, and dispense the nitrate medium into 5mL sterile test tubes for later use.

[0058] Solution A (p-toluenesulfonic acid solution): Dissolve 0.8g p-aminobenzenesulfonic acid in 100mL of 5mol / L acetic acid. Solution B: Dissolve 5g α-naphthol in 100mL of 5mol / L acetic acid. After 24 hours of culture, transfer 50-100μL of the bacterial culture to nitrate broth and incubate at approximately 36℃ for 1-5 days. Use two test tubes of nitrate broth as blank controls. After incubation, add 2-3 drops each of solution A and solution B, mix well, and immediately observe the color change. If it turns red, it is a positive reaction; if it does not change color, add a small amount of zinc powder. If it still does not change color, it is also a positive reaction; if it turns red, it is a negative reaction.

[0059] 1.3.6 Utilization of Citrate Simon's Citrate Medium: Sodium chloride (NaCl) 5.0 g, dipotassium hydrogen phosphate (K₂HPO₄) 1.0 g, magnesium sulfate (MgSO₄) 0.5 g, potassium dihydrogen phosphate (KH₂PO₄) 1.0 g, sodium citrate (C₆H₅Na₃O₇) 1.0 g, agar 14.0 g, bromothymol blue (C 27 H 28 0.08 g of O5SBr2, 1000 mL of distilled water, pH 6.7-6.9, autoclave at 121℃ for 20 min. Dispense into 5 mL sterile test tubes, tilt and solidify to form slant agar. Pick fresh colonies, streak them onto the slant, and incubate at the appropriate temperature for 24-96 h. Observe the color of the culture medium; blue indicates a positive result, and green indicates a negative result.

[0060] 1.3.7 Urease reaction Urease agar medium: 1.0g peptone, 5.0g sodium chloride (NaCl), 1.0g glucose, 2.0g potassium dihydrogen phosphate (KH2PO4), 0.012g phenol red, 15.0g agar, 1000mL distilled water, pH 6.9~7.1, autoclaved at 121℃ for 20min. Dispense into 5mL sterile test tubes, tilt and allow to solidify into slant agar. Pick fresh inoculum, streak it onto the slant, and puncture the bottom of the medium. Seal the tubes with sealing film. Set up two blank controls and incubate at 36℃ for 18~24h. After incubation, observe the color of the medium; a change to rose red indicates a positive result, while no color change or a change to yellow indicates a negative result.

[0061] 1.3.8 Utilization of sucrose Sucrose fermentation medium: 10.0g peptone, 10.0g sucrose, 30.0g sodium chloride (NaCl), 3.0g beef extract, 0.04g bromocresol purple, 1000mL distilled water, pH 6.8-7.2, autoclaved at 121℃ for 20min. Dispense into 5mL sterile test tubes for later use. Inoculate fresh inoculum into the test tubes and incubate at the appropriate temperature for 18-24h. Observe the color of the medium in the test tubes; yellow indicates a positive result, while blue or blue-green indicates a negative result.

[0062] 1.3.9 Glucose utilization Glucose fermentation medium: Sodium chloride (NaCl) 30.0g, peptone 10.0g, beef extract 3.0g, bromocresol purple 0.04g, glucose 10.0g, distilled water 1000mL, pH 6.8~7.2, autoclaved at 121℃ for 20min. Dispense into 5mL sterile test tubes for later use. Inoculate fresh inoculum into the test tubes and incubate at room temperature for 18~24h. Observe the color of the medium in the test tubes; yellow indicates a positive result, and blue or blue-green indicates a negative result.

[0063] 1.3.10 Peptone water reaction (indole reaction) Tryptone aqueous medium: 10.0g tryptone, 5.0g sodium chloride (NaCl), 1000mL distilled water, pH 7.2-7.4, autoclaved at 121℃ for 20min. Dispense the sterilized liquid medium into 5mL test tubes for later use. Kovacs' indole matrix reagent: Dissolve 5.0g p-dimethylaminobenzaldehyde in 75mL pentanol, then slowly add 25mL concentrated hydrochloric acid. Inoculate an appropriate amount of bacterial suspension into the above medium and incubate at 36℃ for 18-24h. After incubation, add 2-3 drops of Kovacs' indole matrix reagent to the test tube, shake well, and observe the results within a few seconds. A rose-red ring indicates a positive reaction; no color change (or a yellow ring) indicates a negative reaction.

[0064] Table 1 shows the physiological and biochemical characteristics of strain WJ2 as follows: catalase positive, methyl red (MR) negative, starch hydrolysis negative, gelatin hydrolysis positive, nitrate reduction positive, citrate utilization negative, urease reaction negative, sucrose fermentation reaction positive, glucose fermentation reaction positive, and indole reaction negative. Note: "+" indicates positive; "-" indicates negative.

[0065] As shown in Table 1, the final physiological and biochemical characteristics obtained are: catalase positive, methyl red (MR) negative, starch hydrolysis negative, gelatin hydrolysis negative, nitrate reduction negative, citrate utilization negative, urease reaction negative, sucrose fermentation reaction negative, glucose fermentation reaction negative, and indole reaction negative.

[0066] 1.4 16S rDNA sequence analysis Genomic DNA was extracted from the WJ2 strain isolated using the above method, and PCR amplification was performed using universal bacterial primers 27F and 1492R to obtain a 16S rDNA fragment of approximately 1400 bp. This fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained 16S rDNA sequence was BLAST aligned in the NCBI database, and a phylogenetic tree was constructed using MEGA software. Figure 3 As shown, the 16S rDNA sequence of WJ2 is similar to that of Acinetobacter piezophyllum (…). Acinetobacter pittii Clustered in the same branch, with a similarity ≥99%. Based on morphological and physiological-biochemical characteristics, it was identified as Acinetobacter pitera (…). Acinetobacter pittii WJ2).

[0067] The 16S rDNA sequence of WJ2 is shown in SEQ ID NO.1, specifically as follows:

[0068] The universal primer 27F sequence is shown in SEQ ID NO.2, specifically: 5'-AGAGTTTGATCCTGGCTCAG-3'; The sequence of universal primer 1492R is shown in SEQ ID NO.3, specifically: 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0069] 1.5 Preservation of strains This strain was deposited at the China Center for Type Culture Collection (CCTCC) on August 18, 2025, with accession number CCTCC NO: M20251840, and the deposit address is No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0070] 2. Growth characteristics of strain WJ2.

[0071] 2.1 Effect of temperature on growth Activated WJ2 cells were inoculated into LB liquid medium and cultured at 25℃, 27℃, 29℃, 31℃, 33℃, and 35℃, with four replicates for each treatment. The cells were shaken at 180 rpm for 48 h, and OD values ​​were measured. 600 Value. Result as follows Figure 4 As shown, strain WJ2 grows well in the range of 29~35℃, with the optimal growth temperature being 33℃.

[0072] 2.2 Effect of pH on growth The initial pH of LB liquid medium was adjusted to 4, 5, 6, 7, 8, 9, and 10, respectively. After inoculation with WJ2, the medium was cultured at 28℃ with shaking at 180 r / min for 48 h, and the OD was measured. 600 Value. Result as follows Figure 5 As shown, strain WJ2 grows best in the pH range of 6 to 7, with the optimal pH being 7.

[0073] 3. Antimony tolerance of strain WJ2.

[0074] Activated WJ2 cells were inoculated at a rate of 2% (v / v) into LB liquid medium containing different concentrations of trivalent antimony (5 mM, 20 mM), and cultured at 28°C with shaking at 150 rpm for 48 h. Growth was then observed. Results are as follows: Figure 6 As shown, at a concentration of 5 mMSb, WJ2 grew extremely vigorously, with colonies covering the plate; while under the high concentration stress of 20 mMSb, although the background of the culture medium was clear (indicating that antimony has a strong inhibitory effect on most microorganisms), WJ2 was still able to grow well and form distinct pale yellow colonies. These results indicate that WJ2 has extremely strong tolerance to trivalent antimony and can survive in extreme environments with high concentrations of antimony contamination.

[0075] 4. Phosphate-solubilizing ability of strain WJ2.

[0076] raw material: PKO solid culture medium: C6H 12 O6 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, MgSO4 0.3g, MnSO4 0.03g, Fe2SO4·7H2O 0.03g, Ca3(PO4)2 5.0g, yeast extract 0.5g, agar 18g, bring to a final volume of 1L, pH 7.0±0.2. PKO solid medium contains insoluble calcium phosphate (Ca3(PO4)2) as the sole phosphorus source.

[0077] Beef extract peptone liquid culture medium: 3g beef extract, 10g peptone, 5g NaCl, bring to a final volume of 1L, pH 7.0±0.2.

[0078] The bacterial strain was inoculated into 50 mL of beef extract peptone liquid medium and incubated in a 37°C shaker at 180 rpm for 24 h to fully activate the cells. 2.5 µL of the activated bacterial suspension was precisely pipetted onto the surface of a PKO solid medium plate. The inoculated plates were then placed in a 37°C incubator for daily observation of colony growth and phosphate-solubilizing zone formation. After incubation, if… Figure 7 As shown, a distinct phosphate-solubilizing zone is present next to WJ2. This indicates that the strain can dissolve insoluble calcium phosphate by secreting acidic metabolites (such as organic acids) or biological enzymes, releasing soluble phosphorus for its own growth. In other words, WJ2 has a significant inorganic phosphorus-solubilizing ability.

[0079] 5. Effects of strain WJ2 on the growth promotion and antimony reduction of lettuce in antimony-contaminated soil.

[0080] The strain WJ2 obtained above has a significant growth-promoting effect on lettuce in soil contaminated with antimony. This is illustrated below through a pot experiment covering the entire growth period: The lettuce variety tested was Italian lettuce 333, purchased from Zhengdong Flower Market; the soil tested was loamy alluvial soil, and its basic physical and chemical properties are shown in Table 2. Three antimony levels were set up [0 (Sb0), 500 (Sb0.5), 1000 (Sb1) mg / kg] and one inoculum level (WJ2). The tested strain WJ2 was cultured and prepared into a bacterial suspension, at a concentration of 2.7 × 10⁻⁶ mg / kg. 8Five treatments were applied to the soil at an inoculum of CFU / g, with four replicates for each treatment. Each pot was inoculated with 0.2 g / kg pure N, 0.15 g / kg P2O5, and 0.2 g / kg K2O to meet the N, P, and K requirements, respectively. The N, P, and K sources were CO(NH2)2, KH2PO4, and KCl, respectively.

[0081] Air-dried soil was sieved through a 20-mesh sieve and placed into 3L plastic basins, 2.5kg of soil per basin. The above-mentioned fertilizers and experimental treatments were added to the soil and mixed thoroughly. The soil was then watered until completely saturated. Plump lettuce seeds were selected and sown evenly in the basins, with a 2-3mm layer of dry soil covering the seeds. When the lettuce reached about three leaves and a bud, 7 seedlings were thinned per basin. During the lettuce growing period, approximately 300mL of water was added every 3 days. Samples were collected after 40 days of cultivation. The dry and fresh weight of the lettuce and its antimony content were measured.

[0082] from Figure 8 As can be seen, in the uninoculated control groups (labeled Sb0.5 and Sb1), compared with Sb0 (test soil, without additional antimony), lettuce plants showed significant growth inhibition and leaf yellowing with increasing antimony concentration. This indicates that high concentrations of antimony have a significant toxic effect on plants, hindering normal plant development. Comparing the uninoculated and inoculated groups (labeled Sb0.5WJ2 and Sb1WJ2), it can be found that after inoculation with WJ2, even under higher concentrations of antimony stress, lettuce still maintained relatively good growth. This shows that WJ2 can effectively alleviate the toxicity of antimony to plants and exert a significant growth-promoting and protective effect.

[0083] And from Figure 9 As can be seen, in the control group without WJ2 inoculation, the biomass of lettuce initially stabilized and then decreased sharply with increasing exogenous antimony concentration; however, after inoculation with WJ2, the biomass recovered significantly. The reason why high concentrations of antimony (Sb1) caused such a precipitous drop in biomass is mainly because the large-scale absorption of antimony ions by plants interfered with intracellular metabolic processes. After WJ2 inoculation, although the available antimony content in the soil remained high, the accumulation of antimony in the plant body may have been effectively controlled, thus allowing photosynthesis and water metabolism to partially recover. WJ2 not only reduces plant absorption (blocking) by lowering available antimony in the soil, but may also enhance the stress resistance (antimony tolerance) of lettuce by improving the rhizosphere microenvironment or secreting plant hormones, achieving a synergistic effect of "toxicity reduction" and "growth promotion".

[0084] Further, the lettuce samples were separated into above-ground parts and root systems respectively, and the antimony contents in the above-ground parts and root systems were measured respectively. Specifically, 35 days after the growth of lettuce, the lettuce was divided into above-ground parts and root systems with scissors disinfected by 75% alcohol, and the total fresh weight of the above-ground parts and root systems was measured with an electronic balance. After the measurement, the fresh samples were placed in an oven, de-enzymed at 105°C for 15 min, and dried at 80°C, then the total dry weight was measured. For the determination of plant antimony content: the antimony content of the plant was determined with reference to GB5009.137-2016. In short, 0.1-0.5g of plant was weighed, digested with nitric acid-perchloric acid (4:1) mixed acid, and the total Sb concentration of the digested solution was measured by a dual-channel atomic fluorescence spectrometer. The specific results are as Figure 10 shown, the antimony content in the above-ground parts (left graph) shows that under non-inoculated conditions, as the antimony concentration increases, the antimony content in the above-ground parts increases significantly (Sb0<Sb0.5<Sb1). After inoculation with WJ2, the antimony content in the above-ground parts decreases greatly. Especially in the high-concentration group (Sb1WJ2), its content (about 16mg / kg) is significantly lower than that of the non-inoculated high-concentration group (Sb1, about 22mg / kg). It can be seen that inoculation with WJ2 mainly plays a role of "detoxification" (relative to the non-inoculated high-concentration group Sb1 of the same level), rather than the effect of "reversing" or "being lower than" low-concentration pollution. That is: although WJ2 is inoculated, the antimony content in the above-ground parts of lettuce grown in a high-antimony (Sb1) environment is still significantly higher than that of lettuce grown in a low-antimony (Sb0.5) environment.

[0085] Meanwhile, the antimony content in the root system (right graph) shows that: the antimony content in the root system is generally higher than that in the above-ground parts, and the content of the non-inoculated group increases with the increase of antimony concentration. After inoculation with WJ2, the antimony content in the root system also decreases significantly. For example, in the high-concentration group (Sb1WJ2), the antimony content in its root system (about 90mg / kg) is significantly lower than that of the non-inoculated group (Sb1, about 135mg / kg). This indicates that WJ2 can not only prevent antimony from transferring to the above-ground parts of plants, but also effectively reduce the retention and enrichment of antimony in the root system.

[0086] 6. Effects of strain WJ2 on the available antimony content and antimony speciation in soil.

[0087] The strain WJ2 of the present invention has the effect of reducing the available antimony content in soil under medium antimony concentration conditions, which is illustrated below through a soil culture test: Four antimony levels [0 (Sb0), 200 (Sb0.2), 500 (Sb0.5), 1000 (Sb1) mg / kg] and two inoculation levels [non-inoculation (None), WJ2] were set. The test strain WJ2 was cultured to prepare a bacterial suspension, which was prepared at 2.7×10 8 CFU / g inoculation amount was inoculated into the soil, with a total of 16 treatments, 4 replicates for each treatment, and the basic physical and chemical properties of the soil are the same as those in Table 2.

[0088] The experiment used 260mL PVC cups, into which 250g of air-dried soil (passed through a 20-mesh sieve) was added. After uniformly mixing the soil with the experimental treatment, deionized water was added until the total soil mass reached 290g, maintaining the soil moisture content at 60% of field capacity. The cups were sealed with a breathable film and incubated at 25℃ and 50% humidity for 30 days, after which soil samples were collected. After incubation, the soil samples were air-dried and sieved. The available antimony content in the soil was determined using the DTPA extraction method (atomic fluorescence spectrometry). Figure 11 As can be seen, the available antimony content in the soil decreased significantly under all antimony concentration treatments after WJ2 inoculation, with a reduction of 30% to 55%.

[0089] The Tessier sequential extraction method was used to analyze the distribution of antimony speciation in soil (exchangeable, carbonate-bound, iron-manganese oxide-bound, organic matter-bound, and residual forms). Figure 12 It can be seen that after inoculation with WJ2, the proportion of exchangeable antimony forms decreased, while the proportions of iron-manganese oxide bound and residual forms increased. This indicates that WJ2 promoted the transformation of antimony into more stable forms, reducing its bioavailability and environmental migration risk.

[0090] X-ray diffraction (XRD) was used to analyze changes in soil crystal structure. Figure 13 The AE data shows that SbOF and Pb5Sb2S appear at the Sb0.2 and Sb0.5 levels. 11 The characteristic peaks of Sb2MnO6 crystals were observed at the Sb1 level, while at the Sb1 level, the characteristic peaks of Sb2MnO6 crystals appeared after the addition of WJ2. This indicates that under certain Sb concentration conditions, WJ1 can promote the formation of stable crystal phases of Sb with multiple elements (Pb, Mn, Ag, Nb, etc.), thereby reducing the bioavailability of Sb.

[0091] Soil functional group changes were analyzed using Fourier transform infrared spectroscopy (FTIR) (KBr pellet method, scanning range 4000–400 cm⁻¹). -1 ).from Figure 14 As can be seen from FJ: after inoculation with WJ2, the C=O bonds (1710 cm⁻¹) in the soil FTIR spectrum are less visible. -1 The vibrational modes (near the target area) underwent significant changes, manifested as decreased peak intensity and peak position shift. This indicates that functional groups such as alcohols, aldehydes, ketones, carboxylic acids, and their derivatives in the strain's secretions chelated / complexed with antimony, promoting the formation of insoluble antimony minerals and thus reducing antimony availability.

[0092] 7. The mechanism by which strain WJ2 and its fermentation broth reduce the antimony content in liquid culture medium.

[0093] Based on the fact that strain WJ2 and its fermentation broth can reduce the effective antimony content in the solution and adsorb antimony at a concentration of 20 mM MSb, the following shake-flask experiment illustrates this: 20 mM trivalent antimony (prepared from potassium antimony tartrate) was inoculated into LB liquid medium in Erlenmeyer flasks. Antimony-resistant bacteria WJ2, after activation, were inoculated into the above antimony-containing liquid medium at a ratio of 2% (v / v) and continuously cultured in a constant temperature shaking incubator at 28℃ and 150 rpm for 7 days. During the culture period, samples were taken every 24 hours. The bacterial suspension was transferred to 10 mL centrifuge tubes, centrifuged at 8000 rpm, and 1 mL of the supernatant was collected. After appropriate dilution, the solution was filtered through a 0.45 μm filter membrane, and the total antimony concentration in the solution was determined using atomic fluorescence spectrometry within 24 hours. The bacterial cells in the centrifuge tubes were freeze-dried and digested using a microwave digester to determine the antimony content in the bacterial cells. Figure 15 It can be seen that after 7 days of cultivation, the antimony content in the supernatant decreased by 62.3%. Figure 16 The results showed that the antimony content in the bacteria gradually increased over time, indicating that the bacteria removed antimony through absorption and adsorption.

[0094] Furthermore, the bacterial culture obtained from the shake-flask experiment was transferred to a 50 mL centrifuge tube, repeatedly centrifuged to remove the bacterial cells, and the supernatant (i.e., fermentation broth) was added to an Erlenmeyer flask containing 20 mM Sb. The mixture was cultured for 7 days, and the antimony content in the solution was detected using atomic fluorescence spectrometry. Figure 17 It can be seen that the antimony content in the fermentation broth treatment group was reduced by 37.8% compared with the control group without fermentation broth, indicating that the strain secretions have the ability to chelate / complex antimony.

[0095] In summary, WJ2 reduces antimony content through a dual pathway of "bacterial cell absorption and adsorption" and "secretion chelation / complexation".

[0096] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. An Acinetobacter piezophyllum commune strain WJ2, characterized in that, The Latin name of the Acinetobacter piezophyllum WJ2 is... Acinetobacter pittii WJ2 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M20251840 and deposit date of August 18, 2025.

2. The Acinetobacter piezophyllum commune WJ2 as described in claim 1, characterized in that, The 16S rDNA sequence of Acinetobacter pituitaria WJ2 clusters in the same branch as Acinetobacter pituitaria, and its physiological and biochemical characteristics are catalase positive, methyl red negative, starch hydrolysis negative, gelatin hydrolysis negative, nitrate reduction negative, citrate utilization negative, urease reaction negative, sucrose fermentation reaction negative, glucose fermentation reaction negative, and indole reaction negative.

3. A microbial inoculant, characterized in that, It includes Acinetobacter piterum WJ2 as described in claim 1 or 2.

4. A method for reducing the antimony content in an antimony-polluted environment, characterized in that, The Acinetobacter pietrogenus WJ2 as described in claim 1 or 2, or the microbial agent as described in claim 3, is applied to an antimony-contaminated environment, and the antimony content is reduced through the synergistic action of at least two mechanisms of action, including the absorption and adsorption of antimony by the bacteria, the chelation / complexation of antimony by the bacteria's secretions, the alteration of the vibrational mode of C=O bonds in the soil, and the promotion of the formation of insoluble antimony minerals.

5. A method for simultaneously reducing the content of available antimony in antimony-contaminated soil, dissolving insoluble inorganic phosphorus, and promoting plant growth under antimony stress, characterized in that, Apply the Acinetobacter piterum WJ2 as described in claim 1 or 2, or the microbial agent as described in claim 3, to antimony-contaminated soil.

6. The method according to claim 5, characterized in that, The plant in question is lettuce.

7. The method according to claim 5, characterized in that, The insoluble inorganic phosphorus is calcium phosphate.

8. The use of Acinetobacter pietrogenis WJ2 as described in claim 1 or 2, or the microbial agent as described in claim 3, in the preparation of formulations for reducing the antimony content in the aboveground parts and / or roots of lettuce under antimony stress and for promoting increased lettuce yield under antimony stress.

9. The use of Acinetobacter pietrogenis WJ2 as described in claim 1 or 2, or the microbial agent as described in claim 3, in the preparation of a formulation for reducing the available antimony content in soil, characterized in that, The reduction of available antimony content in the soil is achieved by altering the vibrational mode of C=O bonds in the soil and / or promoting the formation of insoluble antimony minerals.

10. A method for culturing Acinetobacter piezophyllum as described in claim 1 or 2, characterized in that, Acinetobacter piezophyllum WJ2 was inoculated into LB liquid medium and cultured with shaking at 28–30°C and pH 7.0–7.2 until the logarithmic growth phase or stationary phase.

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