Bioremediation method for targeted lysis of mercury methylation bacteria by using specific bacteriophage

Abstract

The invention relates to a bioremediation method for targeted lysis of mercury methylated bacteria by using a specific bacteriophage, which comprises the following steps: separating target bacteria from lake sediments polluted by mercury, screening bacteriophages capable of lysing the bacteria, culturing the bacteriophages and obtaining a high-concentration bacteriophage preparation, and applying the bacteriophage preparation to the sediments. Methylation of mercury is mainly mediated by specific bacteria (such as sulfate reducing bacteria and methanogens), and the bacteria exist in the sediment in a large number. By screening and culturing bacteriophages capable of effectively splitting the bacteria, the number of the bacteriophages can be remarkably reduced, and the generation of methyl mercury is further reduced. Experimental results show that after the bacteriophage is applied, the concentration of methyl mercury in sediment is remarkably reduced and reaches 60% or above, and the method has application potential in lake ecological restoration. The method has the advantages of simple operation, low cost, environmental protection and the like, and is suitable for wide water pollution treatment.

Description

Technical Field

[0001] The present invention relates to a bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages, belonging to the technical field of environmental bioremediation. Background Art

[0002] Mercury is a heavy metal widely present in the environment. Its methylated product, methylmercury, is highly toxic and can accumulate through the food chain, leading to a gradual increase in its concentration in organisms, endangering the ecosystem and human health. The methylation of mercury is mainly mediated by specific bacteria (such as sulfate-reducing bacteria and methanogens), which reproduce rapidly in anoxic environments and are the main sources of methylmercury production.

[0003] Traditional treatment methods include chemical precipitation, biosorption, etc., and certain application technologies already exist. The above traditional methods can remove methylmercury in water to a certain extent, but they also have significant drawbacks. These methods often require high costs, complex operation steps, and long treatment cycles, and may cause secondary pollution or environmental risks in practical applications. In addition, with the increasingly strict environmental regulations and the enhanced awareness of the public about ecological environmental protection, the limitations of traditional treatment methods have become more obvious.

[0004] Therefore, the development of new bioremediation technologies is particularly important. Bioremediation technologies utilize the natural metabolic processes of microorganisms to efficiently and environmentally remove methylmercury in water. By selectively using microorganisms capable of degrading or transforming methylmercury, or by engineering methods to enhance their degradation ability, it is expected to achieve more efficient and low-cost treatment effects. In addition, bioremediation technologies also have good ecological adaptability and can play a role under various environmental conditions, thus providing a sustainable solution for water pollution treatment.

[0005] In summary, for the treatment of methylmercury pollution, there is an urgent need to develop new bioremediation technologies to overcome the deficiencies of traditional methods and protect the ecological environment and human health. Summary of the Invention

[0006] The present invention proposes a bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages. The purpose is to provide a new bioremediation method in view of the defects existing in the prior art, and by selectively infecting and lysing mercury-methylating bacteria with phages, reduce their numbers, thereby reducing the mercury methylation rate.

[0007] The technical solution of the present invention: A bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages, comprising the following steps: Step 1: Isolate harmful bacteria related to mercury methylation from mercury-polluted lake sediments; Step 2: Screen phages that can selectively lyse the harmful bacteria; Step 3: Culture the phages to obtain a high - concentration phage preparation; Step 4: Apply the phage preparation to the mercury - contaminated lake sediment; Step 5: Monitor the change in the concentration of methylmercury in the sediment to evaluate the remediation effect of the phages.

[0008] In the said Step 1, the harmful bacteria are sulfate - reducing bacteria and / or methanogens.

[0009] In the said Step 2, the screening process includes: a) Isolate phages from environmental samples by enrichment culture method; b) Conduct phage infection experiments to evaluate their lysis efficiency against target bacteria; In the said Step 3, the high - concentration phage preparation refers to 600% - 1000% of the original concentration, and the culture conditions of the phages are 37°C and pH 7.0.

[0010] In the said Step 4, the application method of the phage preparation is spraying or mixing, the application concentration is 10 8 to 10 10 pfu / mL, the application frequency is once a week for 4 to 8 weeks.

[0011] During the application process of the phage preparation, the temperature of the sediment is controlled between 15°C and 25°C.

[0012] The storage conditions of the phages are 4°C, avoiding direct sunlight.

[0013] In the said Step 5, the evaluation of the remediation effect is carried out by measuring the concentration of methylmercury in the sediment through gas chromatography - mass spectrometry.

[0014] Advantages of the present invention: The present invention provides a method using phages as bioremediation agents, obtaining, screening, and culturing phages that can effectively infect mercury - methylating bacteria from natural river and lake waters, and then obtaining a high - concentration phage preparation, which can effectively reduce the mercury methylation rate in lake sediments, providing new ideas and technical means for environmental governance. This method has the advantages of simple operation, remarkable effect, environmental friendliness, etc., and is applicable to the treatment of a wide range of water body pollutions. Description of the Drawings

[0015] Figure 1 is a schematic diagram of the specific phage screening process.

[0016] Figure 2 is a schematic diagram of the analysis results of phage lysis characteristics.

[0017] Figure 3 Changes in methylmercury concentration in sediments before and after phage application. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings.

[0019] Compare with Figure 1 , a bioremediation method using specific bacteriophages to target and lyse mercury methylating bacteria, comprising the following implementation steps: 1. Screening and cultivation of bacteriophages: Step 1: Isolation of bacteria associated with mercury methylation from mercury-contaminated lake sediments Steps: (1) Sample processing: Collect samples from mercury-contaminated lake sediments, and take care to avoid external contamination of the samples. Place the sediment sample in a sterile centrifuge tube, add an appropriate amount of sterile physiological saline (such as 0.9% NaCl solution), and gently shake to mix to prepare a dilution solution.

[0020] (2) Inoculation: dilute the diluent in a gradient manner (e.g. 10 -1 Up to 10 -6 ), take samples of each dilution and inoculate them into selective culture medium. Set up at least three parallel experiments for each dilution to ensure the reliability of the results.

[0021] (3) Cultivation: Place the inoculated culture dish in an incubator, set the temperature to 37°C, and culture for 48 hours.

[0022] (4) Observation: Observe the bacterial growth in the culture medium and record the morphology, color, and number of colonies. Pay special attention to colonies with morphological characteristics consistent with sulfate-reducing bacteria, methanogens, etc.

[0023] Step 2: Isolation of phages by enrichment culture Steps: (1) Water sample processing: Collect water samples from rivers and lakes, and try to avoid light and temperature changes to keep the samples fresh. Filter the water samples (using a 0.45 μm filter membrane) to remove large particles.

[0024] (2) Inoculation: The filtered water sample was mixed with the previously isolated sulfate-reducing bacteria and methanogens, and inoculated into a specific culture medium at a ratio of 1:10 (culture medium composition: NaCl 0.5 g / L, MgSO4 0.2 g / L, sodium acetate 2 g / L, HgCl2 0.1 mg / L, pH 6.8-7.2, cultured at 37°C for 7 days).

[0025] (3) Cultivation: Place the inoculated culture medium in an incubator and culture for 48 hours in an anaerobic environment.

[0026] (4)Screening phages: Take the culture medium, centrifuge it (e.g., 8000 rpm, 10 minutes), remove the precipitate, and obtain the supernatant. Use the supernatant for phage infection and lysis experiments, inoculate it into the culture medium containing sulfate-reducing bacteria and methanogens, and observe whether plaque formation occurs.

[0027] Step 3: Conduct phage infection experiments Operating steps: (1)Infection experiment design: Dilute the cultures of sulfate-reducing bacteria, methanogens, etc. to an appropriate concentration (e.g., 10 6 CFU / mL). Add the phage supernatant to the bacterial cultures according to different infection ratios (e.g., 1:10, 1:100, 1:1000), and set up a control group (without adding phages).

[0028] (2)Cultivation: Incubate the infected culture medium in an incubator and observe the growth of the bacteria.

[0029] (3)Record data: Measure the optical density (OD600) value of the bacteria every 1 hour, record the growth curve of the bacteria, and evaluate the lysis efficiency of the phages on the bacteria. Determine the infection effect of the phages by comparing the OD600 values of the control group and the experimental group.

[0030] (4)Select highly efficient phages: Screen out phages with a lysis efficiency > 90% at pH 6.5 - 7.5 and temperature 25 - 35°C. Select the phages with the best performance according to the lysis efficiency for subsequent cultivation and amplification to obtain sufficient phage preparations for subsequent bioremediation experiments.

[0031] (II)Evaluate the stability of phages and their activity under different environmental conditions: Operating steps: (1)Phage stability test: Divide the phage supernatant into multiple sterile centrifuge tubes, add buffer solutions with different pH values (pH 5.0 - 8.0) to each tube to prepare phage solutions with the same concentration. Let it stand at room temperature for 1 hour, and then let it stand in a 37°C incubator for 1 hour. Take samples (e.g., 100 μL from each tube), inoculate them into the culture medium of sulfate-reducing bacteria and methanogens, and conduct infection experiments.

[0032] (2)Temperature stability test: Divide the phage supernatant into multiple sterile centrifuge tubes, and store them at different temperatures (e.g., 4°C, 25°C, 37°C, 45°C) for 24 hours. Take samples (e.g., 100 μL from each tube), inoculate them into the culture medium of mercury-methylating bacteria, and conduct infection experiments.

[0033] (3) Record data: Observe and record the bacterial growth in each experimental group, measure the OD600 value, and evaluate the activity of phages at different pH values and temperatures.

[0034] (4) Data analysis: Compare the bacterial growth under different pH values and temperatures, and plot the growth curves. Evaluate the stability and activity of phages by calculating the lysis efficiency (such as the change in OD600 value relative to the control group), and then determine the optimal pH and temperature ranges of phages to guide subsequent application research.

[0035] (III) Bioremediation experiment: Step 1: Apply phage preparation to mercury-contaminated lake sediments Operating procedures: (1) Sediment collection: Collect sediment samples from mercury-contaminated lakes, taking care to avoid external contamination of the samples. Place the samples in sterile containers and bring them back to the laboratory as soon as possible.

[0036] (2) Sediment treatment: Homogenize the sediment samples under sterile conditions, divide them into multiple small portions, and prepare for application.

[0037] (3) Apply phage preparation: According to the experimental design, select the application concentration (e.g., 10 8 、10 9 and 10 10 pfu / mL), and dilute the phage preparation to the required concentration. Uniformly apply the diluted phage preparation to the sediments to ensure that each sample comes into contact with the phages.

[0038] (4) Record initial data: Record the pH value of the sediments and other relevant environmental parameters (such as temperature, humidity, etc.) before application.

[0039] Step 2: Apply once a week for 4 to 8 weeks Operating procedures: (1) Application plan: Develop an application plan to apply the phage preparation once a week for 4 to 8 weeks. According to the experimental design, select an appropriate application concentration.

[0040] (2) Application process: Prepare the phage preparation according to the method in Step 1. Uniformly apply the phage preparation to the sediments every week to ensure that the application amount is the same as before.

[0041] (3) Monitoring and recording: After each application, record the environmental parameters such as the pH value and temperature of the sediments to ensure the consistency of the experimental conditions.

[0042] Step 3: Regularly monitor the change in the concentration of methylmercury in the sediments Operating procedures: (1)Sample collection: Samples were taken from the sediment weekly (or at intervals according to the experimental design) after the application of the phage preparation to ensure the representativeness of the samples at each time point. Sterile sampling tubes were used to collect sediment samples, minimizing the exposure time of the samples.

[0043] (2)Sample treatment: The collected sediment samples were appropriately treated (such as drying, grinding) for subsequent analysis. According to the requirements of GC-MS, the samples were extracted and concentrated for analysis.

[0044] (3)GC-MS analysis: The extracted samples were analyzed using gas chromatography-mass spectrometry (GC-MS) to determine the concentration of methylmercury in the sediment. Quantification was performed according to the standard curve, and the methylmercury concentration data at each time point were recorded.

[0045] (4)Data recording and analysis: The weekly methylmercury concentration data were recorded in a table, and a concentration change curve was plotted to evaluate the effect of the phage preparation on the removal of methylmercury in the sediment of the contaminated lake.

[0046] Example 1: Specific phage screening and verification (1)Sediments were collected from the mercury-polluted area of Lake Taihu, and Desulfovibrio sp. DS-1 was isolated. (2)Isolation of phage ΦDS-1: The host bacteria were cultured to the logarithmic phase, and the sediment filtrate was added. Plaques were obtained by the double-layer plate method. (3)Host range test: ΦDS-1 only lysed DS-1 and strains of the same genus and was inactive against Methano-12, a methanogen. (4)Lysis efficiency: As shown in the appendix, at pH 7.0 and 30 °C, the lysis rates of different phage concentrations (such as 10 Figure 2 6 7 8 9 9 pfu / mL) on DS-1 and strains of the same genus within 24 hours were shown, and a phage concentration of 10 9 pfu / mL could achieve a lysis rate of >80%.

[0047] pfu / mL of the phage could achieve a lysis rate of >80%.

[0047] Example 2: Sediment remediation effect (1)The preparation (10 9 PFU / mL) was added to the sediment containing 20 ng / g of methylmercury. (2)As shown in the appendix, after 5 weeks, the methylmercury concentration decreased to 3.7 ng / g (a decrease of more than 80%), while that of the control group (untreated) was 18.6 ng / g. Figure 3 As shown in the appendix, after 5 weeks, the methylmercury concentration decreased to 3.7 ng / g (a decrease of more than 80%), while that of the control group (untreated) was 18.6 ng / g. (3)The restoration effect of multiple sediment applications was found. The concentration reduction of methylmercury in the sediment can exceed 60%, indicating the application potential of this method in lake ecological restoration.

Claims

1. A bioremediation method for specifically targeting and lysing mercury-methylating bacteria by using specific phages, characterized in that It includes the following steps: Step 1: Isolate harmful bacteria related to mercury methylation from mercury - polluted lake sediments; Step 2: Screen phages that can selectively lyse the harmful bacteria; Step 3: Culture the phages to obtain a high - concentration phage preparation; Step 4: Apply the phage preparation to the mercury - polluted lake sediments; Step 5: Monitor the change in the concentration of methylmercury in the sediments to evaluate the remediation effect of the phages.

2. The bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages according to claim 1, characterized in that In the said Step 1, the harmful bacteria are sulfate - reducing bacteria and / or methanogens.

3. The bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages according to claim 1, characterized in that In the said Step 2, the screening process includes: a) Isolate phages from environmental samples by enrichment culture method; b) Conduct phage infection experiments to evaluate their lysis efficiency against target bacteria.

4. The bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages according to claim 1, characterized in that In the said Step 3, the high - concentration phage preparation refers to 600% - 1000% of the original concentration, and the culture conditions of the phages are 37°C and pH 7.

0.

5. The bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages according to claim 1, characterized in that In the step 4, the bacteriophage preparation is applied by spraying or mixing, and the application concentration is 10 8 to 10 10 pfu / mL, and the application frequency is once a week for 4 to 8 weeks.

6. The bioremediation method for specifically targeting and lysing mercury-methylating bacteria by using specific phages according to claim 1 or 5, characterized in that During the application process of the phage preparation, the temperature of the sediments is controlled between 15°C and 25°C.

7. The bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages according to claim 1, characterized in that The storage conditions of the phages are 4°C, avoiding direct sunlight.

8. The bioremediation method for specifically targeting and lysing mercury-methylating bacteria using specific phages according to claim 1, characterized in that In the said Step 5, the evaluation of the remediation effect is to evaluate by measuring the concentration of methylmercury in the sediments through gas chromatography - mass spectrometry.

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