Compound microorganism, microbial inoculant and application thereof in improvement of cadmium-polluted acidified soil

Abstract

This invention provides a composite microorganism, a microbial agent, and its application in the remediation of cadmium-contaminated acidified soil, belonging to the field of environmental microbiology technology. This invention provides a composite microorganism, including Bacillus (…). Bacillus sp H1 and Trichomonas vaginalis ( .) Comamonas sp The Bacillus H1 strain has the preservation number CGMCC NO.29207, and the Trichomonas vaginalis strain Y1 has the preservation number CGMCC NO.29208. The composite microorganisms obtained in this invention have a good removal effect on cadmium (Cd(II)) in the environment, and can improve soil pH while degrading it. The strains have a wide applicable temperature and pH range, and high versatility. The composite microorganisms of this invention show good application prospects in soils with varying degrees of acidification.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and in particular relates to a compound microorganism, a microbial agent, and its application in the improvement of cadmium-contaminated acidified soil. Background Technology

[0002] Soil acidification and heavy metal pollution are prominent soil environmental problems. The main factors contributing to soil acidification are human activities, including atmospheric acid deposition, the excessive use of nitrogen fertilizers in agriculture, and factors affecting normal crop growth and development. Soil acidification leads to decreased soil fertility, damage to soil structure, reduction of beneficial microorganisms in the soil, and alterations in the speciation and transformation balance of heavy metals in the soil, thus affecting the availability of heavy metals and severely impacting crop growth. Soil acidification often overlaps with background heavy metal pollution, exacerbating the harm caused by heavy metal pollution in arable land and posing a serious threat to the quality and safety of agricultural products and human health in the region. Cadmium (Cd) is a major heavy metal pollutant in the agricultural environment. Cadmium (CdII) seriously threatens ecological security and agricultural product quality due to its toxicity, non-degradability, and bioaccumulation. Therefore, improving soil acidification and reducing the content of available cadmium in the soil under the dual influence of severe heavy metal pollution and soil acidification is of significant scientific importance.

[0003] Currently, common remediation measures for heavy metal pollution include physical, chemical, and biological remediation. Among these, biological remediation is more environmentally friendly and poses no risk of secondary pollution compared to traditional physical and chemical methods. Biological remediation primarily utilizes the metabolic processes of microorganisms such as bacteria, fungi, and actinomycetes to convert toxic pollutants into non-toxic substances or transfer pollutants from the soil medium to the organisms. Simultaneously, the introduction of different functional strains can alter the soil microecological structure, effectively improving soil pH and nutrient availability. However, even bacteria specifically screened from environmental media have shown limited effectiveness in removing cadmium and remediating acidic soils. Current research mainly focuses on the single role of microorganisms in remediating acidic soils or degrading heavy metal cadmium; few microbial agents can simultaneously improve soil acidification while efficiently removing cadmium pollution. Summary of the Invention

[0004] The purpose of this invention is to provide a composite microorganism, a microbial agent, and its application in the improvement of cadmium-contaminated acidified soil. The composite microorganism of this invention can not only efficiently remove cadmium pollution, but also improve soil acidification.

[0005] This invention provides a composite microorganism, including Bacillus subtilis deposited at CGMCC on December 1, 2023. Bacillus sp H1 and Trichomonas vaginalis deposited at CGMCC on December 1, 2023 (.) Comamonas spY1; the preservation number of Bacillus H1 is CGMCC NO.29207; the preservation number of Trichomonas vaginalis Y1 is CGMCC NO.29208.

[0006] The present invention also provides a compound microbial agent, comprising the compound microorganisms described in the above-described scheme.

[0007] Preferably, the compound microbial agent comprises a bacterial suspension of Bacillus H1 and a bacterial suspension of Trichomonas vaginalis Y1; the OD values ​​of the bacterial suspensions of Bacillus H1 and Trichomonas vaginalis Y1 are... 600 The ratio of the values ​​is 1:1.

[0008] The present invention also provides the application of the composite microorganisms or composite bacterial agents described above in the remediation of cadmium-contaminated soil or cadmium-contaminated water; the soil includes acidic soil or neutral soil; the water includes acidic water or neutral water.

[0009] Preferably, the cadmium pollution includes cadmium (Cd(II)) pollution.

[0010] This invention also provides a method for remediating cadmium-contaminated acidified soil, comprising the following steps: The composite microorganisms or composite bacterial agents described in the above scheme are applied to cadmium-contaminated acidified soil for improvement; or, the composite microorganisms or composite bacterial agents described in the above scheme are mixed with cadmium-contaminated acidified soil for improvement.

[0011] Preferably, the concentration of cadmium (Cd(II)) in the cadmium-contaminated acidified soil is 3-7 mg / kg.

[0012] Preferably, the amount of the compound microorganisms or compound bacterial agent used, calculated based on the total effective viable count of Bacillus H1 and Trichomonas vaginalis Y1, is 10. 5 cfu / g soil.

[0013] Preferably, the improved ambient temperature is 25~45℃.

[0014] Preferably, the pH value of cadmium-contaminated acidified soil is ≥6.

[0015] This invention provides a composite microorganism, including Bacillus subtilis deposited at CGMCC on December 1, 2023. Bacillus sp H1 and Trichomonas vaginalis deposited at CGMCC on December 1, 2023 (.) Comamonas spThe Bacillus H1 strain has the preservation number CGMCC NO.29207, and the Trichomonas vaginalis strain Y1 has the preservation number CGMCC NO.29208. The composite microorganisms obtained in this invention have a good removal effect on cadmium (Cd(II)) in the environment, and can improve soil pH while degrading it. The strains have a wide applicable temperature and pH range, and high versatility. The composite microorganisms of this invention show good application prospects in soils with varying degrees of acidification.

[0016] Biological Preservation Instructions Bacillus ( Bacillus sp H1 was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29207.

[0017] Trichomonas vaginalis ( Comamonas sp Y1 was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29208. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a technical roadmap for screening and validating the strains of the present invention; Figure 2 Mapping of soil samples; Figure 3 The results of the NCBI nucleotide database comparison for strain H1; Figure 4 The results of the NCBI nucleotide database comparison for strain Y1; Figure 5 The images show the morphological diagram and SEM electron microscope image of the strain of the present invention; wherein, H represents Bacillus H1 and Y represents Trichomonas vaginalis Y1. Figure 6 The present invention describes the removal capacity of cadmium (Cd(II)) under different temperature / pH conditions in laboratory settings for single bacteria / complex microbial communities; wherein H represents Bacillus H1, Y represents Trichomonas vaginalis Y1, and HY represents the complex microorganism HY; Figure 7The present invention describes the removal capacity of the composite microbial community of cadmium (Cd(II)) in soil from different regions; wherein, H represents Bacillus H1, Y represents Trichomonas vaginalis Y1, and HY represents the composite microorganism HY; Figure 8 The percentage change in pH varies in different regions; where H represents Bacillus H1, Y represents Trichomonas vaginalis Y1, and HY represents the complex microbial HY. Detailed Implementation

[0020] This invention provides a composite microorganism, including Bacillus subtilis deposited at CGMCC on December 1, 2023. Bacillus sp H1 and Trichomonas vaginalis deposited at CGMCC on December 1, 2023 (.) Comamonas sp Y1; the preservation number of Bacillus H1 is CGMCC NO.29207; the preservation number of Trichomonas vaginalis Y1 is CGMCC NO.29208.

[0021] The composite microorganism HY in this invention has a highly efficient removal effect on cadmium (Cd(II)) pollution in soil under different temperature (-5-45℃) and different pH (3-7) conditions. It has good application prospects in the removal of heavy metal cadmium (Cd(II)) pollution in acidified soil or water bodies, and is of great significance to soil quality, human health and ecological environment improvement.

[0022] The Bacillus H1 and Trichomonas vaginalis Y1 in the composite microorganisms of this invention can efficiently remove cadmium pollution from soil under individual or combined culture conditions, while improving soil pH.

[0023] In this invention, the Bacillus H1 and Trichomonas vaginalis Y1 are microbial strains isolated and screened from soil in the black soil region of Northeast China, capable of efficiently removing cadmium (Cd(II)) from the environment. Based on classical microbiological morphological classification and bioinformatics classification, the strains were named H1 and Y2, respectively belonging to the Bacillus (…). Bacillus sp.) and Trichomonas vaginalis (sp.) Comamonas sp.).

[0024] The present invention also provides a compound microbial agent, comprising the compound microorganisms described in the above-described scheme.

[0025] In one embodiment, the compound microbial agent comprises a bacterial suspension of Bacillus H1 and a bacterial suspension of Trichomonas vaginalis Y1; the OD values ​​of the bacterial suspensions of Bacillus H1 and Trichomonas vaginalis Y1 are... 600 The ratio of the values ​​is 1:1.

[0026] The present invention also provides the application of the composite microorganisms or composite bacterial agents described above in the remediation of cadmium-contaminated soil or cadmium-contaminated water; the soil includes acidic soil or neutral soil; the water includes acidic water or neutral water.

[0027] As one implementation, the cadmium pollution includes cadmium (Cd(II)) pollution; the pH value of the cadmium-polluted soil or cadmium-polluted water body is 6.5-7.

[0028] This invention also provides a method for remediating cadmium-contaminated acidified soil, comprising the following steps: The composite microorganisms or composite bacterial agents described in the above scheme are applied to cadmium-contaminated acidified soil for improvement; or... The composite microorganisms or composite bacterial agents described in the above scheme are mixed with cadmium-contaminated acidified soil for improvement.

[0029] In one embodiment, the concentration of cadmium (Cd(II)) in the cadmium-contaminated acidified soil is 3-7 mg / kg.

[0030] As one implementation method, the amount of the compound microorganisms or compound bacterial agent used, calculated based on the total effective viable count of Bacillus H1 and Trichomonas vaginalis Y1, is 10. 5 cfu / g soil.

[0031] As one implementation method, the improved ambient temperature is 25~45°C, and more specifically 35°C.

[0032] As one implementation method, the pH value of cadmium-contaminated acidified soil is ≥6, further 6~7, and even further 6.5~7.

[0033] In one implementation, the improvement takes 14 days.

[0034] The composite microorganisms of this invention can completely remove cadmium pollution from soil within 14 days, improve soil acidification, and greatly promote the degradation rate of cadmium in the system when combined.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a composite microorganism, inoculant, and its application in the remediation of cadmium-contaminated acidified soil, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 Potentially functional bacterial strains were isolated from soil in the black soil region of Northeast China using a conditional screening method.

[0037] For the technical roadmap of strain screening and validation, please refer to [link / reference]. Figure 1 .

[0038] (1) Sample collection: Soil samples were collected in July 2023 from Tieling City (Changtu), Liaoning Province; Changchun City (Kuancheng), Jilin Province; Suihua City (Hailun, Beilin); and Heihe City (Beian). See the soil sampling map below. Figure 2 .

[0039] (2) Sample enrichment: Accurately weigh 100 g of soil sample into a 250 mL sterile Erlenmeyer flask, add 50 mM cadmium (Cd(II)) each, stir gently and incubate at 28℃ for 1 week, and add sterile water to keep the moisture content unchanged.

[0040] (3) Strains were isolated: 10 g of cadmium (Cd(II)) enriched soil sample was accurately weighed into an Erlenmeyer flask containing 90 mL of sterile physiological saline, and shaken in a shaker at 28°C for 1 h. After shaking for 2 h, the sample was removed and allowed to stand. 1 mL of the diluted sample was then gradually diluted to 10 mL of sterile physiological saline. -3 10 -4 10 -5 Spread 0.1 mL of each dilution onto R2A solid medium plates containing 50 mM cadmium (Cd(II)). Spread three plates for each dilution and incubate at 28°C for 5 days. The resulting bacterial strains are resistant to Cd(II). Store the plates at 4°C for later use. The R2A medium formula (1 L) is as follows: 0.5 g yeast extract, 0.5 g soluble starch, 0.5 g peptone, 0.3 g dipotassium hydrogen phosphate, 0.5 g casein amino acids, 0.3 g sodium pyruvate, 0.5 g glucose, and 0.05 g MgSO4∙7H2O. Sterilize at 121°C for 20 min.

[0041] (4) Streak plating: Select colonies of different morphologies from the resistant bacteria obtained in step (3) and repeat the streaking process several times to obtain single clones. Use 1 / 10 LB agar plates for streaking. After the bacteria have grown, store them in glycerol tubes at -80°C. The formula for 1 / 10 LB agar is as follows (1L): 1g tryptone, 0.5g yeast extract, 1g sodium chloride. Adjust the pH of the agar to 7.4 with NaOH. Weigh and dissolve the components of the agar according to the proportions and autoclave at 121°C for 20 min. Cultivation on 1 / 10 LB solid medium is the same as on liquid medium, except that 1.5% agar needs to be added.

[0042] (5) Screening for strains with cadmium removal capabilities: The single clones obtained in step (4) were transferred to 1 / 10 LB of 200 mM cadmium Cd(II) and cultured in a shaker at 28°C with shaking at 150 rpm / min. After 72 h, 2 mL of bacterial culture was centrifuged at 12000 rpm for 2 min, the supernatant was collected and filtered, and the filtrate was used to determine the cadmium removal by atomic absorption spectrometry. Strains that could remove more than 50% of cadmium Cd(II) were selected as preliminary screening strains, and strains H1 and Y1 were obtained.

[0043] (6) Classification and identification of strains H1 and Y1: First, 16S rDNA was used for identification. This involved PCR amplification using universal prokaryotic 16S rDNA primers 27F (5'-agagtttgatcmtggctcag-3', SEQ ID NO.1) and 1492R (5'-ggytaccttgttacgactt-3', SEQ ID NO.2) (for specific PCR methods, please refer to a method for rapid extraction of total soil DNA in small quantities disclosed in Chinese patent CN1990863A), followed by sequencing. The results were then compared with the international NCBI GenBank nucleotide database (www.ncbi.nlm.nih.gov), showing 99% nucleotide homology. The comparison results are shown below. Figure 3 and Figure 4 .

[0044]

[0045]

[0046] Second, morphological and growth characteristic identification was performed using transmission electron microscopy. The morphological characteristics are as follows: Both strains H1 and Y1 have yellowish colonies, are long rod-shaped, and can grow well in both standard R2A and LB media. (See...) Figure 5 ) The strains are preserved as follows: Bacillus H1 and Trichomonas Y1 can be cultured on 1 / 10 LB liquid or solid medium at 28°C, and can be stored for a short period at 4°C after culture. For long-term storage, glycerol cryovials or freeze-dried tubes are more suitable for preserving the strains (for specific methods, please refer to: Zhao Bin, He Shaojiang, Microbiology Experiment, First Edition, Science Press, Beijing, 2002: pp. 202-205).

[0047] Example 2 Degradation of heavy metal cadmium by composite microorganism HY under different temperature and pH conditions Selected monoclonal Bacillus H1 and Trichomonas Y1 strains were inoculated into 100 mL of 1 / 10 LB liquid medium and cultured in a shaker at 28°C until OD. 600 The initial OD value was approximately 0.5. This was used as the seed culture and inoculated at a 1% inoculum (or 50% inoculum for each culture under co-culture conditions) into 100 mL of fresh 1 / 10 LB liquid medium (initial OD). 600 <0.01), the initial concentration of cadmium (Cd(II)) was 200 mM. Experimental groups were set up as follows: ①pH=7, different temperature gradients: -5, 0, 10, 15, 25, 35, 45℃; ② Temperature 35℃, different pH values: 3, 4, 5, 6, 7, 8; The sample was placed in a shaker at 28°C and cultured for 48 hours. Samples were taken to determine the cadmium removal efficiency. After centrifugation at 12000 rpm for 2 min, the cadmium (Cd(II)) concentration was determined by atomic absorption spectrometry. The determination method is described in Example 1.

[0048] Depend on Figure 6It can be seen that at different temperatures, the removal capacity of a single strain for cadmium ranged from 8.97% to 57.77%, while the removal capacity of the composite microbial HY increased to 27.47% to 93.77%. The removal efficiency of the composite microbial HY was significantly higher than that of the single strain across all temperature ranges. Under different pH conditions, the removal capacity of a single strain for cadmium ranged from 4.77% to 48.93%, while the removal capacity of the composite microbial HY increased to 29% to 91.77%. The removal efficiency of the composite microbial HY was significantly higher than that of the single strain across all pH ranges. The composite microbial HY significantly promoted cadmium removal, achieving almost 100% removal of cadmium in the system at the optimal temperature of 35℃ and pH 7.0. Furthermore, its applicable temperature and pH range was far greater than that of the strains / composite microbial HY previously observed.

[0049] Example 3 Cadmium removal and pH changes in soil by compound microorganism HY Initial cadmium content and soil pH were determined using in-situ black soil samples. Cadmium (Cd(II)) (mg / kg): Chang Figure 3 0.79, Kuancheng 4.51, Beilin 3.94, Hailun 5.17, Bei'an 5.97; pH value: Chang Figure 6 0.5, Kuancheng 7, Beilin 6.5, Hailun 6.8, Bei'an 6.5.

[0050] Add compound microbial HY at a rate of 10% of the total biomass of the compound microbial HY. 5 The soil sample was thoroughly mixed with a CFU / g soil meter, and after standing at room temperature for 14 days, samples were taken to test the cadmium content and pH of the soil. Figure 7 and Figure 8 As can be seen, the composite microorganism HY in this invention can remove cadmium in soil at a rate of 81.47% to 90.91%, which is 1 to 3.5 times higher than that of a single strain. At the same time, by measuring the soil pH value after 14 days, it was found that the composite microorganism HY can increase the soil pH value in a short period of time, with a pH value change of 2.86% to 12.31%, which is 1 to 10 times that of a single strain, showing significant effect.

[0051] In summary, the composite microorganism HY obtained by this invention has a good removal effect on cadmium (Cd(II)) in the environment, and can improve soil pH while degrading it. The strain has a wide applicable temperature and pH range and high versatility. It shows that the composite microorganism HY of this invention has good application prospects in soils with different degrees of acidification.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite microorganism, characterized in that, Bacillus (S.) deposited at CGMCC on December 1, 2023 Bacillus sp. H1 and Trichomonas vaginalis deposited at CGMCC on December 1, 2023 ( Comamonas sp. Composed of Y1; The preservation number of the Bacillus H1 is CGMCC NO.29207; The preservation number of the *Trichomonas vaginalis* Y1 is CGMCC NO.29208.

2. A compound microbial agent, characterized in that, Includes the composite microorganisms described in claim 1.

3. The compound microbial agent according to claim 2, characterized in that, The compound bacterial agent includes a bacterial suspension of Bacillus H1 and a bacterial suspension of Trichomonas vaginalis Y1; the ratio of the OD600 values ​​of the bacterial suspension of Bacillus H1 and the bacterial suspension of Trichomonas vaginalis Y1 is 1:

1.

4. The application of the composite microorganisms of claim 1 or the composite bacterial agent of claim 2 or 3 in the remediation of cadmium-contaminated soil; wherein the soil includes acidic soil or neutral soil.

5. The application according to claim 4, characterized in that, The cadmium pollution includes cadmium (Cd(II)) pollution.

6. A method for remediating cadmium-contaminated acidified soil, characterized in that, Includes the following steps: The composite microorganisms described in claim 1 or the composite bacterial agent described in claim 2 or 3 are applied to cadmium-contaminated acidified soil for improvement. or, The composite microorganisms described in claim 1 or the composite bacterial agent described in claim 2 or 3 are mixed with cadmium-contaminated acidified soil for improvement.

7. The method according to claim 6, characterized in that, The concentration of cadmium (Cd(II)) in the acidified soil contaminated with cadmium was 3-7 mg / kg.

8. The method according to claim 6, characterized in that, The dosage of the compound microorganisms or compound bacterial agent, calculated based on the total effective viable count of Bacillus H1 and Trichomonas vaginalis Y1, is 10. 5 cfu / g soil.

9. The method according to claim 6, characterized in that, The improved ambient temperature is 25~45℃.

10. The method according to claim 6, characterized in that, The pH value of cadmium-contaminated acidified soil is ≥6.

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