An exogenous bacteriophage inoculation method for enhancing the arsenic methylation capacity of soil microorganisms

Abstract

The application discloses a kind of exogenous bacteriophage inoculation methods for strengthening soil microbial arsenic methylation capacity, comprising the following steps: S1. preparation bacteriophage crude extract;S2. preparation concentrated solution;S3. preparation bacteriophage extract;S4. preparation inoculation soil sample;S5. in the 2, 4, 6th day of culture, bacteriophage extract is added to inoculation soil;S6. after three times inoculation, continue to flood culture 9 days.The application proposes a kind of inoculation method for retaining virulent bacteriophage, not retaining original host bacteria, inoculating after flooding two days, and inoculating three times, so that the proportion of lysogenization of bacteriophage in soil after inoculation is increased by 156%~347%, so that arsenic metabolism genes carried by bacteriophage genome can function, the arsenic methylation capacity of soil microorganism is strengthened, and the arsenic methylation capacity is increased by more than 300%, to efficiently methylate arsenic in soil.

Description

Technical Field

[0001] This invention relates to the technical field of soil microorganisms, and more specifically, to an exogenous phage inoculation method for enhancing the arsenic methylation capacity of soil microorganisms based on mild phage life cycle regulation, which can be used for the remediation of soil arsenic pollution. Background Technology

[0002] Arsenic pollution in soil poses a serious threat to public health. Methylation of arsenic in the soil through microbial metabolic activity, converting it into gaseous methylarsine for natural volatilization, is a promising method for remediating soil arsenic pollution. However, the arsenic methylation rate of soil microorganisms is low, making enhancing this rate crucial for arsenic pollution control. Bacteriophages are viruses that specifically infect bacteria. Temperate phages, in particular, can integrate their own genome's accessory metabolic genes into the host genome, potentially enhancing specific functions of soil microorganisms. However, temperate phages can perform both lysogenic and non-lysogenic cycles after inoculation, leading to poor efficacy of exogenous phage inoculation.

[0003] To enable exogenously inoculated bacteriophages to perform a lysogenic cycle (rather than a lytic cycle) in arsenic-contaminated soil, this invention optimizes the inoculation method based on existing bacteriophage ecology theory. This allows the inoculated exogenous bacteriophages to perform a greater number of lysogenic cycles and integrate their genetic material into soil bacteria, thereby enhancing the host's arsenic methylation capacity through the arsenic methylation genes they carry. Summary of the Invention

[0004] The purpose of this invention is to provide an exogenous bacteriophage inoculation method to enhance the arsenic methylation capacity of soil microorganisms.

[0005] According to one aspect of the present invention, a method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms is provided, comprising the following steps: S1. Collect soil with high arsenic contamination, add sodium acetate solution to flood the soil, induce the release of prephage, centrifuge, take the supernatant and filter it through a membrane to obtain crude phage extract; S2. The crude phage extract was filtered by tangential flow, the phage was concentrated, and after resuspending in sterile buffer, it was concentrated by tangential flow again to remove the arsenic contained in the crude phage extract, and the concentrated solution was obtained. S3. Resuspend the concentrate in sterile buffer and adjust the phage concentration to 10. 7 ~10 10 VLPs / mL were used to obtain phage extract, which was stored at 4°C in the dark for later use. S4. Collect farmland soil with a total arsenic concentration of 50~200mg / kg, air dry, sieve, add deionized water until the soil is completely submerged to form an anaerobic environment, and incubate in a constant temperature water-submerged environment to obtain inoculated soil samples; S5. On the 2nd, 4th and 6th days of constant temperature water immersion culture at 25℃, the phage extract was added to the inoculated soil sample at a ratio of 100 mL / kg soil, and the surface soil was gently stirred to ensure that the phage was in full contact with the soil. S6. After three inoculations, the soil was submerged for another 9 days. The lysogenic ratio of bacteriophages in the soil increased, and exogenous bacteriophages that enhance the arsenic methylation ability of soil microorganisms were present in the soil.

[0006] In some embodiments, the total arsenic content in the highly arsenic-contaminated soil in step S1 is 300~1000 mg / kg, and the concentration of the sodium acetate solution is 10 mmol / L.

[0007] In some implementations, the total arsenic content in the highly arsenic-contaminated soil in step S1 is 674 mg / kg.

[0008] In some embodiments, in step S1, the sodium acetate solution is submerged for 2 days, the centrifugation speed is 8000 r / min, the centrifugation time is 10 min, and the supernatant is filtered sequentially through 0.45 μm and 0.22 μm filter membranes.

[0009] In some embodiments, the crude phage extract in step S2 is concentrated by tangential flow filtration to a volume of 5% of its original volume, and the sterile buffer is sterile SM buffer.

[0010] In some embodiments, the phage concentration in the phage extract in step S3 is 5 × 10⁻⁶. 9 VLPs / mL.

[0011] In some implementations, the total arsenic content of the farmland soil collected in step S4 is 145 mg / kg, the mesh size of the sieve is 2 mm, and the culture conditions are water immersion culture in a constant temperature incubator at 25℃ for 2 days.

[0012] In some implementations, in step S5, the instantaneous virus / bacteria ratio in the culture system is 1 at each inoculation.

[0013] In some implementations, the surface soil in step S5 refers to soil at a depth of 0-2 cm from the surface.

[0014] According to another aspect of the present invention, an exogenous phage inoculation method for enhancing the arsenic methylation capacity of soil microorganisms is provided, which is applied to increasing the proportion of phage lysogens in soil, enhancing the arsenic methylation capacity of soil microorganisms, and remediating soil arsenic pollution.

[0015] The beneficial effects of this invention: This invention addresses the gap in the optimization of phage inoculation methods in arsenic-contaminated soil by proposing an optimized inoculation method based on the principle of phage-host cooperation. This method retains virulent phages, does not retain the original host bacteria, inoculates two days after flooding, and performs in three inoculations. It requires no additional equipment and increases the phage lysogenization rate in the soil by 156% to 347% after inoculation. It also enables the arsenic metabolism genes carried by the phage genome to function, enhances the arsenic methylation capacity of soil microorganisms, and increases the arsenic methylation capacity by more than 300%, thereby efficiently methylating arsenic in the soil. Attached Figure Description

[0016] Figure 1 This is a graph showing the change in the content of phosphate-extractable inorganic arsenic in the soil in Example 2 of the present invention.

[0017] Figure 2 This is a graph showing the relative abundance changes of induced prophages in the soil in Example 3 of the present invention.

[0018] Figure 3 This is a diagram showing the types and concentrations of volatile methylarsine after 15 days of soil incubation in Example 4 of the present invention.

[0019] Figure 4 This is a correlation analysis diagram of the abundance of arsM methyltransferase gene (arsM) and the density of prophage in soil in Example 5 of the present invention. Detailed Implementation

[0020] The present invention is further described in detail through specific implementation examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available.

[0021] Example 1

[0022] A method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms includes the following steps: S1. Collect 50 g of high arsenic contaminated soil with a total arsenic content of 300~1000 mg / kg, preferably high arsenic contaminated soil with a total arsenic content of 674 mg / kg, add 150 mL of sodium acetate solution with a concentration of 10 mmol / L and submerge for 2 days to quickly achieve reducing conditions to induce the release of prophages. After centrifugation at 8000 r / min for 10 min, take the supernatant and filter it through 0.45 μm and 0.22 μm filter membranes in sequence to obtain crude phage extract; S2. The crude phage extract was filtered by tangential flow to concentrate the phage contained therein to 5% of the original volume. After resuspending in sterile SM buffer, the extract was concentrated by tangential flow again to reduce the volume to 5% in order to remove the arsenic contained in the crude phage extract and obtain the concentrate. S3. Resuspend the above concentrated solution with sterile SM buffer and adjust the phage concentration to 5 × 10⁻⁶. 9 VLPs / mL were used to obtain phage extract, which was stored at 4°C in the dark for later use. S4. Collect farmland soil with a total arsenic concentration of 145 mg / kg, air dry, pass through a 2 mm sieve, weigh 100 g of the sieved soil and place it in a container, add 150 mL of deionized water until the soil sample is completely submerged to form an anaerobic environment, place the container in a 25℃ constant temperature incubator for incubation, and keep the soil in the container submerged to obtain the inoculated soil sample. S5. On the 2nd, 4th and 6th days of constant temperature water immersion culture at 25℃, 10 mL of phage extract was added to the inoculated soil sample, and the surface soil at a depth of 0~2 cm was gently stirred to ensure that the phage was in full contact with the soil. After each inoculation, the instantaneous virus / bacteria ratio in the container was 1. S6. After three inoculations, the soil was continuously submerged at 25°C for 9 days. The phage lysogenic ratio in the soil was (0.17±0.04) VLP / copy of 16S rRNA gene (VLP is the number of virus-like particles counted by fluorescence microscopy). The arsenic methylation capacity of soil microorganisms was increased by 300%, and exogenous phages that enhance the arsenic methylation capacity of soil microorganisms were present in the soil.

[0023] Example 2

[0024] Following steps S1-S4 of the inoculation method in Example 1, inoculated soil samples were obtained. Sampling of the inoculated soil samples was initiated on the second day of constant temperature water immersion cultivation at 25℃, and repeated every three days for a total of five samplings. After each sampling, the final phosphoric acid concentration was adjusted to 0.1% using a 10% phosphoric acid solution, and extraction was performed at 60℃ for 1 hour. After filtration and purification, the changes in the content of phosphoric acid-extractable inorganic arsenic in the soil were determined using HPLC-ICP-MS. Specific results are shown below. Figure 1 .

[0025] Depend on Figure 1 It can be seen that starting from the second day of flooding, the concentration of trivalent arsenic rapidly increased, thus forming a mild stress of As(III) on soil microorganisms.

[0026] Example 3

[0027] This embodiment sets up three treatments: Treatment 1 (optimized low V / B group): Following the inoculation method of Example 1, after 2 days of submersion in step S4, bacteriophages were inoculated three times, with an inoculation amount of 100 mL / kg each time. The instantaneous virus / bacteria ratio (V / B) in the container after each inoculation was 1. Treatment 2 (optimized high V / B group): Following steps S1-S4 of Example 1, bacteriophages were inoculated once after 2 days of submersion in the container, with an inoculation amount of 300 mL / kg. The instantaneous virus / bacteria ratio (V / B) in the container after inoculation was 3. Treatment 3 (unoptimized control group): Following steps S1-S4 of Example 1, bacteriophages were directly inoculated during submersion, with an inoculation amount of 300 mL / kg. The first sampling was conducted for each treatment two days after submersion, with subsequent sampling intervals of 3 days, for a total of 5 samplings. After sampling, free bacteriophages were removed, and the soil and bacterial sediment was resuspended. Mitomycin C was then used for induction to obtain induced bacteriophages. The induced bacteriophages were then counted using a fluorescence microscope. Detailed results are shown below. Figure 2 Note: The virus in the virus / bacteria ratio (V / B) refers to the bacteriophages added to the soil at the time of inoculation.

[0028] Depend on Figure 2 Dynamic monitoring showed that after the first inoculation on the second day of flooding (the induction point of As(III) mild stress), the relative abundance of prophages in the soil showed a steady upward trend. Mild phages in an integrated state after the lysogenic cycle were considered prophages. After 15 days of flooding culture, the lysogenic ratio (calculated as the ratio of induced phages to 16S rRNA copy number) in treatment group 1 (optimized low V / B group) was 180% of that in treatment group 2 (optimized low V / B group), and significantly increased by 260% compared to treatment group 3 (unoptimized control group). This result strongly demonstrates that multiple inoculations not only effectively overcome the physical adsorption loss of virus particles by the complex soil matrix, but also precisely match the recovery kinetics of soil microorganisms under flooding conditions, maximizing the induction of the transformation of native arsenic-resistant bacteria into lysogenic bacteria, and achieving efficient colonization of phages in the in-situ soil environment.

[0029] Example 4

[0030] In this embodiment, a treatment group and a control group were set up. The treatment group followed the inoculation method of Example 1. After the first inoculation, the soil was submerged and cultured for 15 days. Then the soil was drained and the soil moisture was controlled at 30% of the maximum field capacity. The soil was then transferred to a reactor that could collect methylarsine for incubation. Volatile methylarsine volatilized in the air was collected daily using an air pump. The arsine species oxides adsorbed on silica beads soaked in silver nitrate were analyzed using HPLC-ICP-MS to detect the types and concentrations of captured volatile methylarsine.

[0031] Control group: 50 g of highly arsenic-contaminated soil with a total arsenic content of 674 mg / kg was collected and eluted with SM buffer to remove free phage particles and exclude virulent phages. The soil was then submerged in 150 mL of 10 mmol / L sodium acetate solution for 2 days to rapidly establish reduction conditions and induce phage release. After centrifugation at 8000 r / min for 10 min, the supernatant was filtered through a 0.45 μm filter membrane and then subjected to steps S2-S3 of Example 1 to obtain an extract free of virulent phages. Farmland soil samples with a total arsenic concentration of 145 mg / kg were collected, air-dried, and passed through a 2 mm sieve. 100 g of the sieved soil (dry weight) was placed in a container, and 30 mL of the extract (without virulent bacteriophages) was evenly sprayed onto the soil surface. The top 0-2 cm of soil was gently stirred to ensure thorough contact between the sprayed bacteriophages and the soil, completing the inoculation. The soil was then submerged at 25°C for 15 days, followed by drainage. The soil moisture was controlled at 30% of maximum field capacity, and the soil was transferred to a reactor capable of collecting methylarsenic for further incubation. Volatile methylarsenic was collected daily using an air pump, along with arsenic species oxides adsorbed onto silica beads soaked in silver nitrate. The types and concentrations of captured volatile methylarsenic were detected using HPLC-ICP-MS. Specific results are detailed below. Figure 3 .

[0032] like Figure 3 As shown, the significant enhancement of soil arsenic speciation and arsenic methylation capacity was demonstrated by comprehensive analysis of soil arsenic speciation using HPLC-ICP-MS. The results proved that the mild As(III) stress driven by the treatment group successfully activated the metabolic defense mechanism of lysogens. In terms of yield, the production of methylarsenic compounds (mainly DMA and MMA) in the soil of the treatment group showed an explosive increase, which was 385% higher than that of the control group.

[0033] Example 5

[0034] Following the inoculation method of Example 1, samples were collected every three days for 15 days after the first inoculation (considered day 0). Changes in the relative abundance of prophages and the abundance of the arsM gene in the microbiome were detected, and the correlation between the two was analyzed. The relative abundance of prophages (VLP) was counted using fluorescence microscopy, and the abundance of the arsM gene in the microbiome was quantified using qPCR. Specific results can be found in [link to specific results]. Figure 4 .

[0035] Depend on Figure 4qPCR quantitative analysis revealed a highly significant positive correlation between the abundance of the arsenic methyltransferase gene (arsM) in soil and the density of prephages (p < 0.01). Mechanistic evidence indicates that the inoculation method of this invention does not simply utilize phages as vectors for horizontal gene transfer, but rather induces significant activation or enhancement of the host bacteria's original arsenic detoxification metabolic pathways during lysogenic processes through precise environmental stress induction. This strategy, based on a combination of environmental sensing and phased enhancement, increases soil arsenic methylation capacity by over 300% without the need for additional equipment, achieving a qualitative leap in soil arsenic methylation remediation efficiency.

[0036] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms, characterized in that, Includes the following steps: S1. Collect soil with high arsenic contamination, add sodium acetate solution to flood the soil, induce the release of prephage, centrifuge, take the supernatant and filter it through a membrane to obtain crude phage extract; S2. The crude phage extract is filtered by tangential flow to concentrate the phage. After resuspending in sterile buffer, it is concentrated by tangential flow again to remove the arsenic contained in the crude phage extract, and a concentrated solution is obtained. S3 resuspends the concentrate in sterile buffer and adjusts the phage concentration to 10. 7 ~10 10 VLPs / mL were used to obtain phage extract, which was stored at 4°C in the dark for later use. S4. Collect farmland soil with a total arsenic concentration of 50~200mg / kg, air dry, sieve, add deionized water until the soil is completely submerged to form an anaerobic environment, and incubate in a constant temperature water-submerged environment to obtain inoculated soil samples; S5. On the 2nd, 4th and 6th days of constant temperature water immersion culture at 25℃, the bacteriophage extract was added to the inoculated soil sample at a ratio of 100 mL / kg soil, and the surface soil was gently stirred to ensure that the bacteriophage was in full contact with the soil. S6. After three inoculations, the soil was submerged for another 9 days. The lysogenic ratio of bacteriophages in the soil increased, and exogenous bacteriophages that enhance the arsenic methylation ability of soil microorganisms were present in the soil.

2. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 1, characterized in that, In step S1, the total arsenic content in the highly arsenic-contaminated soil is 300~1000 mg / kg, and the concentration of the sodium acetate solution is 10 mmol / L.

3. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 1, characterized in that, The total arsenic content in the highly arsenic-contaminated soil in step S1 is 674 mg / kg.

4. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 1, characterized in that, In step S1, the sodium acetate solution is submerged for 2 days, the centrifugation speed is 8000 r / min, and the centrifugation time is 10 min. The supernatant is filtered sequentially through 0.45 μm and 0.22 μm filter membranes.

5. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 1, characterized in that, In step S2, the crude phage extract is concentrated by tangential flow filtration to a volume of 5% of its original volume, and the sterile buffer is a sterile SM buffer.

6. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 1, characterized in that, In step S3, the phage concentration in the phage extract is 5 × 10⁻⁶. 9 VLPs / mL.

7. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 6, characterized in that, The total arsenic content of the farmland soil collected in step S4 was 145 mg / kg, the sieve aperture was 2 mm, and the culture conditions were water immersion culture in a 25℃ constant temperature incubator for 2 days.

8. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 1, characterized in that, In step S5, the instantaneous virus / bacteria ratio in the culture system is 1 at each inoculation.

9. The method for inoculating exogenous bacteriophages to enhance the arsenic methylation capacity of soil microorganisms according to claim 8, characterized in that, In step S5, the surface soil refers to the soil at a depth of 0-2 cm from the surface.

10. The exogenous phage inoculation method for enhancing the arsenic methylation capacity of soil microorganisms according to any one of claims 1-9 is applied to increasing the proportion of phage lysogens in soil, enhancing the arsenic methylation capacity of soil microorganisms, and remediating soil arsenic pollution.

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