Alkali-producing cadmium-tolerant acinetobacter capable of improving pH of soil and application thereof

Abstract

The invention discloses alkali-producing cadmium-tolerant acinetobacter capable of increasing the pH value of soil and application of the alkali-producing cadmium-tolerant acinetobacter, and belongs to the technical field of microorganisms, the alkali-producing cadmium-tolerant acinetobacter is preserved in the China General Microbiological Culture Collection Center (CGMCC), the number of the alkali-producing cadmium-tolerant acinetobacter is CGMCC No.33619, and the alkali-producing cadmium-tolerant acinetobacter belongs to proteobacteria and pseudomonas. The strain can increase the environmental pH value from 6.0 to 8.0 through metabolism, and stably grows in an environment with the cadmium (Cd < 2 + >) concentration as high as 20 ppm. The strain can be applied to the fields of acid soil improvement, cadmium pollution remediation, industrial wastewater treatment, fermentation engineering and the like. The method has the advantages of being environmentally friendly, efficient, stable, applicable to multiple scenes and the like, and a novel microbial resource is provided for heavy metal pollution treatment and soil ecological restoration.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly relates to an alkali-producing and cadmium-tolerant Acinetobacter that can increase soil pH and its application. Background Art

[0002] In the fields of agriculture and environmental engineering, the problem of soil acidification seriously affects the stability of farmland ecosystems and the growth of crops. Existing alkalization techniques mainly rely on chemical methods such as lime application, but they have disadvantages such as soil compaction, acid return, and poor persistence.

[0003] In recent years, microbial remediation technology has received extensive attention due to its environmental protection and sustainable characteristics. Compared with physical and chemical methods, bioremediation uses the metabolism of microorganisms for remediation, which can achieve harmlessness, no secondary pollution, and low treatment costs. It is an important method for treating environmental pollution and has advantages such as economy, effectiveness, sustainability, and environmental friendliness. Some microorganisms can regulate the environmental pH through metabolic actions. However, there are few known highly efficient alkali-producing strains, and their applications still have limitations. The treatment of cadmium pollution in acidic soil has become an important task in current soil pollution remediation and safe crop production in China. Developing microbial resources with both cadmium tolerance and pH regulation ability is of great significance for the remediation of polluted soil. Summary of the Invention

[0004] To solve the above problems, the present invention provides an alkali-producing and cadmium-tolerant Acinetobacter that can increase soil pH and its application. This bacterium can increase the environmental pH value through metabolic actions during growth, and at the same time has strong cadmium tolerance. The maximum cadmium tolerance concentration under solution culture conditions is 20 ppm. The strain of the present invention can increase the pH of the solution environment from 6 to 8 under suitable conditions and maintain good growth performance in cadmium-polluted soil environments, showing strong cadmium tolerance.

[0005] An alkali-producing and cadmium-tolerant Acinetobacter that can increase soil pH, the strain belongs to Proteobacteria, Pseudomonadales, and is classified and named as: Acinetobacter sp. It is preserved in the General Microbiological Center of the China Center for Type Culture Collection, with the address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is February 24, 2025, and the preservation number is: CGMCC No. 33619.

[0006] In the above technical solution, the strain raises the environmental pH value to 7.5 - 8.0 within 48 hours when cultured in a medium with a pH of 5.0 - 6.0, and the maximum cadmium tolerance concentration is 20 ppm.

[0007] A screening method for an alkali-producing and cadmium-tolerant Acinetobacter that can increase soil pH, comprising the following steps:

[0008] (a)Primary screening: Mix the cadmium - contaminated acidic soil sample with sterile water, shake and disperse, and take the supernatant.

[0009] (b)Isolation and purification: Obtain a soil dilution suspension by gradient dilution method, spread it on LB solid medium containing 17 ppm (about 0.15 mM) cadmium, screen for cadmium - tolerant single colonies and purify them.

[0010] (c)Molecular identification: Extract the DNA of the strain, amplify the 16S rRNA gene and sequence it, and determine the taxonomic status through Blast comparison in the NCBI database.

[0011] Application of an alkaligenic cadmium - tolerant Acinetobacter that can increase soil pH in the remediation of cadmium - contaminated acidic soil. By inoculating the above - mentioned strain into cadmium - contaminated soil, the soil pH value is increased by 0.3 - 0.5 units, and the available cadmium content is reduced by ≥30%.

[0012] In the above technical solution, the application includes at least one of neutralizing acidified water bodies, adjusting the pH value of industrial wastewater, and optimizing the pH of the fermentation system.

[0013] A microbial inoculant containing an alkaligenic cadmium - tolerant Acinetobacter that can increase soil pH. The inoculant is composed of the above - mentioned strain and a carrier, and the carrier is selected from at least one of humic acid, bentonite or biochar.

[0014] A method for remediating cadmium - contaminated soil, comprising the following steps:

[0015] (a)Inoculate the Acinetobacter into cadmium - contaminated soil, and the inoculation amount is 10 6 -10 8 CFU / g soil;

[0016] (b)Cultivate at 25 - 35 °C for 10 - 15 days to increase the soil pH value to 5.7 - 6.0 and reduce the available cadmium content to ≤3.0 ppb.

[0017] A culture medium for the above - mentioned screening method. The culture medium is YP5.5 medium, and its composition is: yeast extract 0.25 g / L, peptone 0.25 g / L, CaCl2·2H2O 0.01 g / L, MgSO4·7H2O 0.5 g / L, pH 5.5.

[0018] In the above technical solution, the 16S rRNA gene sequence of the strain contains the nucleotide sequence shown in SEQ ID NO:1.

[0019] Application of an alkaligenic and cadmium-tolerant Acinetobacter strain capable of increasing soil pH, which reduces the bioavailability of cadmium by adjusting the soil pH value, resulting in a 30% - 35% reduction in the content of available cadmium in cadmium-contaminated soil.

[0020] Beneficial effects:

[0021] 1. High alkalization ability: This strain can increase the pH value from 6 to 8 within a short time (such as within 48 hours), with remarkable effects.

[0022] 2. Environmentally friendly: Compared with the traditional method of chemically adjusting pH, this method is more environmentally friendly and has no secondary pollution.

[0023] 3. Wide application: It can be used in multiple fields such as agriculture, environmental remediation, and industry, with strong applicability.

[0024] 4. Good stability: This strain can stably survive under different environmental conditions and maintain its alkalization ability.

[0025] 5. Strong cadmium tolerance: It can stably grow in cadmium-contaminated environments and effectively reduce the plant availability of cadmium, reducing the harm of heavy metals to crops. Description of the drawings

[0026] Figure 1 : Colony morphology of Acinetobacter in the examples.

[0027] Figure 2 : Phylogenetic tree diagram of Acinetobacter strains in the examples and other strains of the same genus in the database.

[0028] Figure 3 : Acinetobacter strains in the examples have extremely strong alkalization ability in the YP5.5 medium.

[0029] Figure 4 : Acinetobacter strains in the examples have cadmium tolerance.

[0030] Figure 5 : Acinetobacter in the examples significantly increases the pH value of the soil.

[0031] Figure 6 : Acinetobacter in the examples significantly reduces the content of available cadmium in the soil. Detailed implementation manners

[0032] The present invention will be described in detail below with reference to the drawings and specific examples. However, the following examples are only for explaining the present invention, and the protection scope of the present invention should include all the contents of the claims. Moreover, through the description of the following examples, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0033] Example 1: Screening and Identification of Alkaligenic Cadmium-Tolerant Acinetobacter

[0034] 1. Primary screening: Accurately weigh 5 g of cadmium-polluted acidic soil into a 250-ml conical flask containing 100 ml of sterile water, shake it on a shaker at 30 °C and 200 rpm for 48 h to completely disperse the soil. After standing for 5 min, transfer it into a laminar flow hood, and use a sterile pipette to take 1 ml of the soil supernatant into a 1.5-ml sterile centrifuge tube.

[0035] 2. Isolation and purification: Obtain a gradient dilution suspension of the enriched soil by the gradient dilution method, and spread it on an LB solid medium supplemented with 10 mM cadmium. Carry out isolation culture under aerobic conditions, and streak and purify the grown single colonies to obtain multiple purified strains, as Figure 1 shown;

[0036] 3. Molecular identification: Extract the DNA of the strains isolated in step 2 above, select representative strains for PCR amplification and sequencing of the full-length fragment of the 16S rRNA gene. The obtained sequence is shown as SEQ ID NO:1 in the sequence listing. Using the NCBI database for Blast alignment, the homology with Acinetobacter is ≥99%. The phylogenetic tree ( Figure 2 ) shows its independent branch and is deposited in CGMCC with the accession number 33619.

[0037] Example 2: Alkalinity Production Ability Detection

[0038] 1. Medium preparation

[0039] YP5.5 medium (pH 5.5): Yeast extract 0.25 g, peptone 0.25 g, calcium chloride dihydrate 0.01 g, magnesium sulfate heptahydrate 0.5 g, dd water 1000 ml, pH 5.5.

[0040] 2. Alkalinity production experiment

[0041] Inoculate the strain into a medium with pH 5.5, culture it on a shaker at 30 °C and 200 rpm for 48 h, and regularly detect the pH value of the culture solution. The experimental results show that the strain can increase the pH value to 8 within 24 hours, as Figure 3 , and the pH of the control group (uninoculated) is maintained at 5.5 ± 0.1.

[0042] Example 3: Cadmium Tolerance Experiment

[0043] Inoculate the strain into a medium containing different concentrations of cadmium (Cd 2+ ), and detect the growth of the strain. The results are shown as Figure 4, this bacterium can grow well in an environment polluted by cadmium at a relatively high concentration and reduce the solubility of cadmium. The maximum cadmium tolerance concentration can be seen from the figure as 20 ppm.

[0044] Example 4: Cadmium-polluted soil remediation experiment

[0045] Take 100 g of cadmium-polluted soil (0.5 ppm), repeat three times, inoculate 0.5 ml of bacterial solution with OD = 1, and set 100 g of cadmium-polluted soil without added bacterial solution as the control group. After culturing for 15 days, air-dry the soil samples naturally, sieve them through a 10-mesh sieve, measure the soil pH with a pH meter, and measure the change in the content of available cadmium using the ICP-MS measurement method.

[0046] The results show that compared with the non-inoculated control group, the pH value of the control group is 5.3, and the soil pH of the inoculated group is 5.78, which is significantly increased, as Figure 5 shown; the content of available cadmium in the control group is 4.30 ppb, and the content of available cadmium in the inoculated group is 2.98 ppb. The availability of cadmium is significantly reduced and the reduction rate reaches 30.7%, as Figure 6 shown.

[0047] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. An alkaligenic and cadmium-tolerant Acinetobacter that can increase soil pH, characterized by: The strain belongs to the Pseudomonadales of Proteobacteria and is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC No. 33619.

2. The Acinetobacter according to claim 1, characterized in that: The strain can increase the environmental pH value to 7.5 - 8.0 within 48 hours when cultured in a medium with a pH of 5.0 - 6.0, and the maximum tolerance concentration to cadmium is 20 ppm.

3. A screening method for the Acinetobacter as claimed in claim 1 or 2, comprising the following steps: (a) Primary screening: Mix the cadmium - contaminated acidic soil sample with sterile water and shake to disperse, then take the supernatant. (b) Isolation and purification: Obtain a soil dilution suspension by gradient dilution method, spread it on an LB solid medium containing 17 ppm (about 0.15 mM) cadmium, screen for cadmium - tolerant single colonies and purify them. (c) Molecular identification: Extract the DNA of the strain, amplify the 16S rRNA gene and sequence it, and determine the taxonomic status through Blast comparison in the NCBI database.

4. Use of the Acinetobacter according to claim 1 or 2 in the remediation of cadmium-contaminated acidic soil, characterized in that: By inoculating the strain into cadmium - contaminated soil, the soil pH value is increased by 0.3 - 0.5 units, and the available cadmium content is reduced by ≥30%.

5. The application according to claim 4, wherein: The application includes at least one of neutralizing acidified water bodies, adjusting the pH value of industrial wastewater, and optimizing the pH of the fermentation system.

6. A microbial inoculant comprising the Acinetobacter described in claim 1 or 2, characterized in that: The microbial agent is composed of the strain and a carrier, and the carrier is selected from at least one of humic acid, bentonite, or biochar.

7. A method for remediating cadmium - contaminated soil, characterized in that: Comprising the following steps: (a) Inoculate the Acinetobacter described in claim 1 or 2 into cadmium-contaminated soil at an inoculation amount of 10 6 -10 8 CFU / g of soil; (b) Cultivate at 25 - 35 °C for 10 - 15 days to increase the soil pH value to 5.7 - 6.0 and reduce the available cadmium content to ≤3.0 ppb.

8. A culture medium for the screening method according to claim 3, characterized in that: The medium is YP5.5 medium, and its composition is: yeast extract 0.25 g / L, peptone 0.25 g / L, CaCl2·2H2O 0.01 g / L, MgSO4·7H2O 0.5 g / L, pH 5.

5.

9. The Acinetobacter according to claim 1 or 2, characterized in that: The 16S rRNA gene sequence of the strain contains the nucleotide sequence shown in SEQ ID NO:

1.

10. Use of the Acinetobacter according to claim 1 or 2, characterized in that: By adjusting the soil pH value, the bioavailability of cadmium is reduced, and the available cadmium content in cadmium - contaminated soil is reduced by 30% - 35%.

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