Arsenite-tolerant strain as05 and application thereof

Abstract

The application belongs to the technical field of microbiology, and particularly relates to a passivation heavy metal arsenic-like strain As05 and application. The passivation heavy metal arsenic-like strain As05 is taxonomically named Rossellomorea marisflavi As05, and was preserved in the China Center for Type Culture Collection on October 30, 2023, with a preservation number of CCTCC No: M20232082. The application screens an arsenic-resistant strain, identifies the arsenic-resistant strain through morphological observation and 16S rRNA sequence sequencing, and obtains the strain Rossellomorea marisflavi As05 which can passivate heavy metal arsenic, and the strain has strong arsenic resistance, and can provide a strain resource for developing a soil heavy metal pollution microbial remediation technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and in particular relates to a passivation heavy metal arsenic bacterial strain As05 and its application. Background Art

[0002] Arsenic (As) is a highly biotoxic heavy metal, classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Arsenic is widely present in soil and water and can enter the human body through the food chain. Accumulating large amounts of arsenic in the human body can cause birth defects, cancer, and mutations.

[0003] Methods for remediating arsenic-contaminated soil include physical remediation, chemical remediation, and bioremediation. Compared to physical and chemical methods, biological removal has attracted considerable attention due to its advantages such as low cost and environmental friendliness. Research has shown that bioremediation can effectively reduce the bioavailability of heavy metal arsenic, minimizing crop uptake and ensuring agricultural product safety. Bioremediation leverages the arsenic tolerance of natural microorganisms (such as bacteria, algae, and yeast) and their close relationship with arsenic absorption, redox, methylation, compartmentalization, and excretion processes to achieve arsenic transport and transformation within the microorganisms. This can either convert heavy metal arsenic into a non-toxic or low-toxic form or immobilize it within biological cells through adsorption, thereby achieving natural purification. Microorganisms are diverse, grow rapidly, metabolize rapidly, and are highly adaptable to their environments. Therefore, utilizing microbial technologies to manage arsenic contamination in the environment is a current hot topic in environmental pollution control research.

[0004] By studying microorganisms in the soil environment, the inventors screened out a strain with the function of passivating heavy metal arsenic. The strain has strong arsenic resistance and can adsorb and remove arsenic from the solution, providing a strain resource for the development of microbial remediation technology for soil heavy metal arsenic pollution.

[0005] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0006] The purpose of the present invention is to provide a passivation heavy metal arsenic strain As05 and its application.

[0007] The first object of the present invention is to provide a passivation heavy metal arsenic strain As05, whose taxonomic name is Rossellomorea marisflavi As05, which was deposited in the China Center for Type Culture Collection on October 30, 2023, with a preservation number of CCTCC No: M20232082.

[0008] The second object of the present invention is to provide the use of the passivation heavy metal arsenic-like strain As05 in passivating heavy metal arsenic-like environments.

[0009] Compared with the prior art, the present invention has the following beneficial technical effects:

[0010] The present invention screens arsenic-resistant strains and identifies them through morphological observation and 16S rRNA sequence sequencing, obtaining the strain Rossellomorea marisflaviAs05 capable of passivating heavy metal arsenic. The strain has strong arsenic resistance and can adsorb and remove arsenic in the solution, providing a strain resource for the development of microbial remediation technology for soil heavy metal pollution.

[0011] Preservation Information

[0012] The passivating heavy metal arsenic strain As05, with the taxonomic name Rossellomorea marisflavi As05, was deposited in the China Center for Type Culture Collection on October 30, 2023, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC No: M20232082. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the morphological characteristics of the arsenic-resistant strain As05 of the present invention;

[0014] Figure 2 The effect of culture time on the adsorption effect of arsenic-resistant strain As05 of the present invention;

[0015] Figure 3 The effect of the amount of bacterial solution input on the adsorption effect of the arsenic-resistant strain As05 of the present invention;

[0016] Figure 4 This is the effect of pH on the adsorption effect of the arsenic-resistant strain As05 of the present invention. DETAILED DESCRIPTION

[0017] The present invention will be further described in detail below with reference to specific embodiments, which are intended to explain the present invention rather than to limit it.

[0018] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0019] Example 1 :Strain screening and identification

[0020] 1. Soil Sample Collection

[0021] Arsenic-contaminated soil was collected from rice paddies in Quanzhou County, Guangxi on September 25, 2023. A five-point sampling method was used to collect 5-20 cm of surface soil from the rice paddies into 50 mL sterile centrifuge tubes and store them in dry ice boxes for screening of arsenic-resistant strains.

[0022] 2. Culture Medium

[0023] LB solid medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, agar 15 g / L.

[0024] LB broth medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, pH 7.0 ± 0.1 (25°C)

[0025] 3. Strain Isolation and Purification

[0026] Weigh 10.0g of fresh soil sample and place it in a 250mL Erlenmeyer flask containing about 10 sterile glass beads. Add 90mL of phosphate buffer solution (PBS) and shake it at 200r / min at 28°C for 30min. After settling for 15min, discard the supernatant and add 5mL of PBS to suspend the precipitate. Use a sterile pipette to draw 1mL of the above soil suspension into a 10mL centrifuge tube and add 9mL of sterile water to obtain 10 -1 Then draw 10 -1 1 mL of the dilution was added to a test tube containing 9 mL of sterile water, and the 10 -2 , 10 -3 , 10 -4 Dilution solution. Pipette 0.1 mL of each dilution solution onto LB solid medium, invert and culture in a 28°C incubator for 1-3 days. Streak a single colony onto the plate more than 3 times for strain purification and preservation.

[0027] 4. Screening of arsenic-resistant strains

[0028] The purified strains were pressed into 10 -2 and 10 -3 The bacterial solution dilutions were respectively spread on LB solid culture media with final As(V) concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L and 400 mg / L. After colonies grew out, their growth was observed.

[0029] A strain with high arsenic tolerance was selected and named As05. The arsenic tolerance of strain As05 is shown in Table 1 below.

[0030] Table 1 Growth status of strain As05 at different arsenic concentrations

[0031]

[0032] Note: + indicates growth; - indicates no growth

[0033] 5. Morphological observation of arsenic-resistant strain As05

[0034] Arsenic-resistant strain As05 was inoculated into LB solid medium and cultured at 37°C. The colony morphology was shown in Figure 1 .

[0035] Depend on Figure 1 It can be seen that the cells of the arsenic-resistant strain As05 are straight rod-shaped, the colonies are beige, opaque, fried egg-shaped, with irregular edges and a smooth surface.

[0036] 6. Identification of strain 16S rRNA

[0037] The arsenic-resistant strain As05 was sequenced using the 16S Sanger method: 16S rRNA gene amplification was performed using the extracted genomic DNA as a template and bacterial universal primers 27F and 1492R. The primer sequences are shown in Table 2, and the PCR reaction system is shown in Table 3.

[0038] Table 2 16S rRNA primers

[0039]

[0040] Table 3 PCR reaction system

[0041]

[0042] The PCR program was as follows: 95°C, 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 90 s, 25 cycles; 72°C, 10 min.

[0043] The purified PCR product was subjected to 16S rRNA sequencing, and the sequencing result is shown in SEQ ID No. 3. The 16S rRNA gene sequence amplified by the primers was compared with the NCBI database to obtain species information of similar sequences and construct a phylogenetic tree. Using homology alignment to assist in species identification, As05 was determined to be Rossellomorea marisflavi and named Rossellomorea marisflaviAs05.

[0044] Example 2 :Experiment on arsenic removal by strain As05

[0045] 1. Bacterial Liquid Preparation

[0046] The strain As05 was inoculated into LB broth medium and cultured in a shaking incubator at 28°C and 180 rpm until the logarithmic growth phase (about 12 h). The culture was centrifuged at 4°C for 10 min. The precipitate was washed with sterile deionized water, resuspended, and centrifuged three times. Finally, the bacteria were resuspended in 0.9% sodium chloride (sterile saline) solution for later use. After resuspension, the OD 600 =1.

[0047] 2. Effect of incubation time on adsorption effect

[0048] Prepare 50 mL of LB broth with a final As(V) concentration of 9.01 mg / L. After sterilization, add 2.5% bacterial solution and incubate in a biochemical incubator at 28°C for 24 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours. Repeat each treatment three times. After incubation, centrifuge at 12,000 rpm for 5 minutes. The supernatant is filtered through a 0.45 μm filter membrane and the heavy metal solution concentration is determined by ICP-OES. The results are shown in the figure. Figure 2 .

[0049] Depend on Figure 2 It can be seen that the arsenic removal efficiency of the arsenic-resistant strain As05 in the solution was 59.46-78.79% within 24-120 hours; among them, when the culture time was 72 hours, the arsenic removal effect of the arsenic-resistant strain As05 was the best.

[0050] 3. Effect of bacterial solution dosage on adsorption effect

[0051] Prepare 50 mL of LB broth with a final As(V) concentration of 9.01 mg / L. After sterilization, inoculate the culture solution into 50 mL of LB broth at inoculum sizes of 2.5%, 5%, 10%, 15%, and 20%. Incubate in a biochemical incubator at 28°C for 24 hours, then centrifuge at 12,000 rpm for 5 minutes. Filter the supernatant through a 0.45 μm filter membrane and determine the concentration of the heavy metal arsenic solution using ICP-OES. Results are shown in the table. Figure 3 .

[0052] Depend on Figure 3 It can be seen that when the bacterial liquid input is 2.5-15%, the arsenic removal efficiency of the arsenic-resistant strain As05 in the solution is 59.39-63.87%. Among them, when the bacterial liquid input is 15.0%, the arsenic-resistant strain As05 has the best arsenic removal effect.

[0053] 4. Effect of pH value on adsorption effect

[0054] Prepare 50mL of LB broth medium with a final As(V) concentration of 9.01mg / L, and adjust the pH to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 (±0.1) with 0.1mol / LNaOH solution and 0.1mol / L HCl solution, respectively, to obtain 6 As(V) solutions with different pH values, with 3 replicates for each treatment. After sterilization, add 2.5% bacterial solution, culture at 28℃ in a biochemical incubator for 24h, then centrifuge at 12000r / min for 5min, take the supernatant and filter it through a 0.45μm filter membrane, and determine the concentration of heavy metal solution by ICP-OES. The results are shown in Figure 4 .

[0055] Depend on Figure 4 It can be seen that within the pH range of 3.0-8.0, the arsenic removal efficiency of the arsenic-resistant strain As05 in the solution is 52.56-58.18%; among them, when pH=6, the arsenic removal effect of the arsenic-resistant strain As05 is the best.

[0056] Removal efficiency = (initial solution arsenic concentration - treated solution arsenic solubility) / initial solution arsenic concentration × 100%

[0057] In summary, the arsenic-resistant strain As05 of the present invention has strong arsenic resistance and can provide bacterial resources for the development of microbial remediation technology for soil heavy metal arsenic pollution.

[0058] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A heavy metal arsenic passivation strain As05, characterized in that: The taxonomic name of the passivation heavy metal arsenic strain As05 is Rossellomorea marisflavi As05 was deposited in the China Center for Type Culture Collection on October 30, 2023, with the accession number CCTCC No: M20232082.

2. Use of the passivation heavy metal arsenic-like strain As05 according to claim 1 in passivating heavy metal arsenic-like bacteria in an environment.

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