Biological method for removing microcystis aeruginosa in fresh water and inhibiting propagation of ARGs

Abstract

The invention discloses a biological method for removing microcystis aeruginosa in fresh water and inhibiting ARGs propagation, and belongs to the technical field of microorganisms. The method comprises the following steps: 1) firstly, culturing escherichia coli (E. coli DH5alpha) carrying RP4 plasmids overnight to a stable growth period; (2) centrifuging a bacterial liquid obtained by culturing, discarding supernate, and cleaning thalli by using PBS (Phosphate Buffer Solution); (3) resuspending the cleaned bacteria to prepare a bacterial suspension with the concentration of 109CFU / mL; and 4) finally adding the prepared bacterial liquid into a target water body containing the microcystis aeruginosa to achieve the purpose of inhibiting the growth of the microcystis aeruginosa. The propagation of ARGs is inhibited by inhibiting the growth of microcystis aeruginosa in a water body. Escherichia coli used in the technology has the advantages of being convenient to store and transport and the like, and the biotechnology is convenient to operate, low in treatment cost and free of secondary pollution.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a biological method for removing Microcystis aeruginosa in fresh water to inhibit the spread of ARGs. Background Art

[0002] In recent years, emerging contaminants, represented by antibiotic resistance genes (ARGs), have become a global concern. ARGs are widely present in surface water, groundwater, and other environmental sources, posing a global public health concern. Plasmid-mediated horizontal conjugation is a major mode of transmission for ARGs. Research has shown that, in addition to the widely accepted selective pressure from antibiotics, other non-antibiotic environmental factors also influence the long-term persistence and spread of ARGs.

[0003] With the continued development of industry and agriculture and the intensification of urbanization, cyanobacterial blooms are becoming more frequent worldwide, with both their scale and duration increasing. These blooms can exacerbate eutrophication and are prone to clustering and multiplying in slow-flowing water bodies, such as reservoirs and lakes, where nutrients accumulate easily. China has numerous freshwater lakes, with Taihu Lake, Chaohu Lake, and Dianchi Lake all experiencing cyanobacterial blooms, creating a severe eutrophication situation. Blooms not only reduce water visibility, hindering photosynthesis in aquatic plants, but also lower dissolved oxygen (DO) levels, leading to suffocation and death of aquatic animals. They also release large amounts of harmful gases and toxic compounds that are detrimental to ecosystems, posing a threat to both ecological safety and human health. Furthermore, cyanobacteria and the cyanotoxins (MCs) they release can accelerate the spread of ARGs. MCs are particularly heat-resistant and difficult to decompose, and their accumulation in water can be toxic to aquatic organisms.

[0004] With the increasing eutrophication of my country's freshwater lakes, water body management has received widespread attention. The methods currently used for the management of eutrophic water bodies are mainly physical, chemical and physical-chemical methods. However, although traditional physical and chemical methods are quick to take effect, the processing cost is high, the operation is complicated and it is easy to cause secondary pollution, which increases the difficulty of treatment. Based on this, the present invention uses a biological method to inhibit the growth of algae, photosynthetic pigment synthesis and MCs production in water bodies, thereby inhibiting the propagation process of ARGs in water bodies, achieving the purpose of purifying water bodies while controlling the spread of ARGs. Through the introduction of bacterial strains, eutrophication of water bodies can be controlled and secondary pollution of water bodies can be avoided. Summary of the Invention

[0005] In order to solve or partially solve the problems existing in the related art, the present invention provides a biological method for removing Microcystis aeruginosa in freshwater to inhibit the spread of ARGs, comprising the following steps: (1) Escherichia coli carrying the RP4 plasmid ( E. coliThe DH5α bacterial solution was inoculated into a culture medium containing three antibiotics: Amp (ampicillin), Km (kanamycin), and Tec (tetracycline) and cultured until the growth stationary phase.

[0006] (2) Centrifuge the bacterial solution obtained in step (1) that has grown to the stable phase, discard the supernatant, wash the bacteria and resuspend them, and adjust the bacterial solution concentration to the target concentration.

[0007] (3) Add the bacterial solution with the concentration adjusted in step (2) into the water containing Microcystis aeruginosa.

[0008] Preferably, the culture medium used in step (1) is LB culture medium.

[0009] Preferably, the culture conditions in step (1) are: temperature of 25-37°C, culture speed of 180-200 rpm, and culture in the dark.

[0010] More preferably, the culture conditions in step (1) are: temperature of 37° C., and culture speed of 200 rpm.

[0011] Preferably, the concentration of the bacterial solution adjusted in step (2) is 10 8 ~10 9 CFU / mL.

[0012] More preferably, the corresponding concentration in step (2) is 10 9 CFU / mL.

[0013] Preferably, PBS buffer (phosphate buffered saline) is used for washing and resuspending the bacteria in step (2).

[0014] Preferably, the growth conditions of the bacterial solution and Microcystis aeruginosa in step (3) are: temperature of 25±1°C, culture speed of 120-150 rpm, light intensity of 2000-3500 Lux, and light-dark cycle ratio of 12h:12h.

[0015] More preferably, the growth conditions of the bacterial liquid and Microcystis aeruginosa in step (3) are: a culture speed of 150 rpm and a light intensity of 3000 Lux.

[0016] Preferably, the volume ratio of the bacterial liquid to the water in step (3) is 10:210 to 23:210.

[0017] More preferably, the volume ratio of the bacterial liquid to the water in step (3) is 23:210.

[0018] Preferably, in step (3), the algae density of Microcystis aeruginosa reaches 10 6 ~10 7 cells / mL.

[0019] More preferably, in step (3), the density of Microcystis aeruginosa reaches 10 6 cells / mL.

[0020] The technical solution provided by the present invention can have the following beneficial effects: Escherichia coli carrying the RP4 plasmid ( E. coli The biological method involving DH5α) can efficiently purify Microcystis aeruginosa, significantly reducing its density, chlorophyll a, carotenoids and microcystin toxins, and simultaneously achieving freshwater algal bloom control and inhibiting the evolution and spread of ARGs. The technology is easy to operate, has low processing costs and no secondary pollution. In addition, the Escherichia coli used in this technology has the advantage of being easy to store and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For the cells carrying the RP4 plasmid E. coli DH5α removal effect on Microcystis aeruginosa.

[0022] Figure 2 For the cells carrying the RP4 plasmid E. coli DH5α removal effect on chlorophyll a.

[0023] Figure 3 For the cells carrying the RP4 plasmid E. coli DH5α's effect on carotenoid removal.

[0024] Figure 4 For the cells carrying the RP4 plasmid E. coli Comparison chart of DH5α's improvement on water chromaticity.

[0025] Figure 5 For the cells carrying the RP4 plasmid E. coli Comparison of the inhibition of DH5α on the MCs content of Microcystis aeruginosa.

[0026] Figure 6 For the cells carrying the RP4 plasmid E. coli Comparative chart of the effects of DH5α on the morphology and quantity of Microcystis aeruginosa.

[0027] Figure 7 For the cells carrying the RP4 plasmid E. coli Graph showing changes in bacterial count of DH5α.

[0028] Figure 8 For the cells carrying the RP4 plasmid E. coli Comparison of the effects of Microcystis aeruginosa on the horizontal transfer of ARGs under DH5α exposure. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0030] In LB medium, the working concentration of Amp is controlled at 25ug / ml to 50ug / ml, and the working concentrations of Km and Tec are controlled at 10ug / ml to 50ug / ml.

[0031] Example 1 A biological method for removing Microcystis aeruginosa in freshwater to inhibit the horizontal spread of ARGs, specifically comprising the following steps: (1) Add 1 mL of RP4 plasmid E. coli The DH5α bacterial liquid was inoculated into 100 mL of liquid LB medium containing three antibiotics: Amp, Km, and Tec. The culture conditions were set as follows: 37°C, 200 rpm, and cultured in the dark for 12 h.

[0032] (2) Take 40 mL of the cultured bacterial solution and centrifuge it at 4°C and 6000 rpm for 5 min. Discard the supernatant and wash the bacteria 2-3 times with PBS. Adjust the concentration of the bacterial solution to 10 9 CFU / mL (OD 600 ≈2.0).

[0033] (3) Use a 3L conical flask to culture Microcystis aeruginosa to the logarithmic phase (algae density reaches 10 6 cells / mL), 10 mL of dark green Microcystis aeruginosa algae solution cultured to the logarithmic phase was inoculated into 200 mL of BG11 culture medium and 23 mL of the bacterial solution prepared in step (2) was added, and the culture was placed in a shaker at a temperature of 25°C, a light intensity of 3000 Lux, a light-dark cycle ratio of 12h:12h, and a rotation speed of 150 rpm. During the culture period, the algae density was tested at intervals of 24h starting from the logarithmic phase (day 6); chlorophyll a and carotenoids were tested at intervals of 48h; and microcystin was tested at intervals of 48h starting from day 7. The algae solution and algae-bacteria mixture on days 8, 10, 12, and 14 were used for conjugation and transfer experiments.

[0034] Example 2 A biological method for removing Microcystis aeruginosa in freshwater to inhibit the horizontal spread of ARGs, specifically comprising the following steps: (1) Add 1 mL of RP4 plasmid E. coli The DH5α bacterial liquid was inoculated into 100 mL of liquid LB medium containing three antibiotics: Amp, Km, and Tec. The culture conditions were set as follows: 37°C, 180 rpm, and cultured in the dark for 12 h.

[0035] (2) Take 40 mL of the cultured bacterial solution and centrifuge it at 6000 rpm for 5 min at 4°C. Discard the supernatant and wash the bacteria 2-3 times with PBS. Adjust the concentration of the bacterial solution to 10 9 CFU / mL (OD 600 ≈2.0).

[0036] (3) Use a 3L conical flask to culture Microcystis aeruginosa to the logarithmic phase (algae density reaches 10 6 cells / mL), 10 mL of dark green Microcystis aeruginosa cultured to the logarithmic phase was inoculated into 200 mL of BG11 culture medium and 23 mL of the bacterial solution prepared in step (2) was added, and the culture was placed in a shaker at a temperature of 25°C, a light intensity of 2000 Lux, a light-dark cycle ratio of 12h:12h, and a rotation speed of 150 rpm. During the culture period, the algae density was tested at intervals of 24h starting from the logarithmic phase (day 6); chlorophyll a and carotenoids were tested at intervals of 48h; and microcystin toxins were tested at intervals of 48h starting from the 7th day.

[0037] Example 3 A biological method for removing Microcystis aeruginosa in freshwater to inhibit the horizontal spread of ARGs, specifically comprising the following steps: (1) Add 1 mL of RP4 plasmid E. coli The DH5α bacterial liquid was inoculated into 100 mL of liquid LB medium containing three antibiotics: Amp, Km, and Tec. The culture conditions were set as follows: 37°C, 200 rpm, and cultured in the dark for 12 h.

[0038] (2) Take 40 mL of the cultured bacterial solution and centrifuge it at 6000 rpm for 5 min at 4°C. Discard the supernatant and wash the bacteria 2-3 times with PBS. Adjust the concentration of the bacterial solution to 10 8 CFU / mL (OD 600 ≈2.0).

[0039] (3) Use a 3L conical flask to culture Microcystis aeruginosa to the logarithmic phase (algae density reaches 10 6 cells / mL), 10 mL of dark green Microcystis aeruginosa cultured to the logarithmic phase was inoculated into 90 mL of BG11 culture medium and 10 mL of the bacterial solution prepared in step (2) was added, and the culture was carried out in a shaker at a temperature of 25 ° C, a light intensity of 3000 Lux, a light-dark cycle ratio of 12h:12h, and a rotation speed of 120 rpm. During the culture period, the algae density was tested at intervals of 24h starting from the logarithmic phase (day 6); chlorophyll a and carotenoids were tested at intervals of 48h; and microcystin toxins were tested at intervals of 48h starting from the 7th day.

[0040] Comparative Example 1 As a comparison, the difference between this comparative example 1 and Example 1 is that the cells carrying the RP4 plasmid were cultured. E. E. coli When preparing DH5α bacterial solution, use a culture medium that does not contain the three antibiotics Amp, Km, and Tec. The specific steps are as follows: (1) Add 1 mL of RP4 plasmid coli The DH5α Escherichia coli liquid was inoculated into 100 mL of liquid LB medium without the three antibiotics Amp, Km, and Tec. The culture conditions were set as follows: 37° C., 200 rpm, and cultured in the dark for 12 h.

[0041] (2) Take 40 mL of the cultured bacterial solution and centrifuge at 6000 rpm for 5 min at 4°C. Discard the supernatant and wash the bacteria 2-3 times with PBS to adjust the concentration of the bacterial solution to 10 9 CFU / mL (OD 600 ≈2.0).

[0042] (3) Use a 3L conical flask to culture Microcystis aeruginosa to the logarithmic phase (algae density reaches 10 6 cells / mL), 10 mL of dark green Microcystis aeruginosa cultured to the logarithmic phase was inoculated into 200 mL of BG11 culture medium and 23 mL of the bacterial solution prepared in step (2) was added, and the culture was placed in a shaker at a temperature of 25°C, a light intensity of 3000 Lux, a light-dark cycle ratio of 12h:12h, and a rotation speed of 150 rpm. During the culture period, the algae density was tested at intervals of 24h starting from the logarithmic phase (day 6); chlorophyll a and carotenoids were tested at intervals of 48h; and microcystin toxins were tested at intervals of 48h starting from the 7th day.

[0043] Comparative Example 2 For comparison, the difference between this comparative example 2 and example 1 is that the Escherichia coli adhesive extracellular polymer (AD-EPS) is added to the culture medium in step (3). The specific steps are as follows: (1) Add 1 mL of RP4 plasmid E. coli The DH5α bacterial liquid was inoculated into 100 mL of liquid LB medium containing three antibiotics: Amp, Km, and Tec. The culture conditions were set as follows: 37°C, 200 rpm, and cultured in the dark for 12 h.

[0044] (2) Take 40 mL of the cultured bacterial solution and adjust the volume to 10 using a UV spectrophotometer. 9 CFU / mL (OD 600≈2.0). Centrifuge at 4500 r / min, 4°C for 5 min, discard the supernatant, add 15 mL of PBS to the precipitate, and centrifuge at 4500 r / min, 4°C for 20 min to obtain the supernatant, which is the Escherichia coli adhesive extracellular polymer (AD-EPS).

[0045] (3) Use a 3L conical flask to culture Microcystis aeruginosa to the logarithmic phase (algae density reaches 10 6 cells / mL), 10 mL of dark green Microcystis aeruginosa liquid cultured to the logarithmic phase was inoculated into 200 mL of BG11 culture medium and 23 mL of Escherichia coli adhesive extracellular polymers (AD-EPS) prepared in step (2) was added, and the cells were cultured in a shaker at a temperature of 25 ° C, a light intensity of 3000 Lux, a light-dark cycle ratio of 12 h:12 h, and a rotation speed of 150 rpm. During the culture period, the algae density was tested at intervals of 24 h starting from the logarithmic phase (day 6); chlorophyll a and carotenoids were tested at intervals of 48 h; and microcystin toxins were tested at intervals of 48 h starting from the 7th day.

[0046] Effect Example 1 Test samples: Materials prepared in Examples 1-3 and Comparative Examples 1-2 Carrying RP4 plasmid E. coli The effects of DH5α on the removal of Microcystis aeruginosa are as follows: (1) Shake the Microcystis aeruginosa thoroughly and take 30 μL of the mixture onto a cell counting plate.

[0047] (2) Place the cell counting plate on the cell counter to count the algae cells. Calculate the algae density based on the data obtained. Measure each sample three times. The calculation formula is: (3) Compare the experimental group with the control group and calculate E. coli The inhibition rate of Microcystis aeruginosa is shown in E. coli DH5α , the calculation formula is: like Figure 1 As shown, compared with Comparative Example 1 and Comparative Example 2, the addition of the RP4 plasmid Figure 1 DH5α bacterial solution has obvious purification effect. In the experiment, it was found that the bacteria carrying RP4 plasmid E. coli DH5α has a significant inhibitory effect on the growth of Microcystis aeruginosa, with an inhibition rate of more than 90%. E. coli DH5α has a significant effect in purifying Microcystis aeruginosa in water bodies and improving algal blooms in freshwater bodies.

[0048] The prepared sample carrying the RP4 plasmid E. coli DH5α bacterial solution, in which E. coli is cultured without the addition of Amp, Km, and Tec antibiotics, has an inhibition rate of about 85% ( E. coli The purification effect of Example 1 is better in terms of the inhibition rate on algae. This may be because the comparative example 1 is used to culture the microorganisms carrying the RP4 plasmid. Figure 1 The lack of Amp, Km, and Tec antibiotics in DH5α resulted in the E. coli DH5α was not screened for bacteria. There may be other microorganisms in the culture medium that compete for nutrient resources, resulting in insufficient nutrition for E. coli. E. coli The morphology of DH5α is smaller and less active than that of E. coli in Example 1. Therefore, its ability to inhibit algae is not as good as that of Example 1.

[0049] The prepared sample of Comparative Example 2 carrying the RP4 plasmid E. coli The supernatant of DH5α is the extracellular polymer of E. coli adhesion. E. coli As shown in Figure 2, the water purification effect is poor, with an inhibition rate of only about 5% to 15%. This is because only the RP4 plasmid was added. Figure 1 Although the adhesive extracellular polymers of DH5α have a certain inhibitory effect on algae, they do not have the effect of carrying the RP4 plasmid. E. coli The addition of DH5α did not have a significant anti-algae effect and the water purification effect was also poor.

[0050] In addition, there is no significant difference in the above-mentioned operation examples 1 to 3, and the inhibition rate of algae can reach more than 90%. Therefore, in actual experiments, the parameters can be appropriately adjusted according to the actual situation.

[0051] Effect Example 2 Test samples: Materials prepared in Examples 1-3 and Comparative Examples 1-2 Carrying RP4 plasmid E. coli The effects of DH5α on the removal of chlorophyll a and carotenoids from Microcystis aeruginosa are as follows: (1) Shake the cultured Microcystis aeruginosa thoroughly, take 10 mL of algae solution and 10 mL of algae-bacteria mixture into a centrifuge tube, centrifuge at 10000 r / min for 10 min in a 10°C cooling centrifuge, discard the supernatant, and collect the deposited algae cells.

[0052] (2) Resuspend the sediment in 10 mL of 99.9% chromatography-grade methanol and incubate in a water bath at 60°C in the dark for 24 h.

[0053] (3) After the water bath, centrifuge at 6000 r / min for 10 min and collect the supernatant.

[0054] (4) Take 200 μL of supernatant and place it in a 96-well plate. Make three parallel samples for each sample.

[0055] (5) The absorbance response values ​​of photosynthetic pigments at different absorbances were measured using a visible spectrophotometer. The concentrations of different chlorophyll components were calculated as follows: Chlorophyll a=16.72A 665 -9.16A 652 Carotenoids = (1000A 470 -1.63×chlorophyll a) / 221 Where A 665 、A 652 、A 470 and A 750 The absorbances of the photosynthetic pigment extract at 665 nm, 652 nm, 470 nm, and 750 nm are shown in Table 1. Note that the absorbances at 470 nm, 652 nm, and 665 nm are corrected for turbidity by subtracting the absorbance at 750 nm.

[0056] (6) Calculate the inhibition rate based on the absorbance response value. The calculation formula is as follows: Chlorophyll a inhibition rate (%) = [(chlorophyll a - chlorophyll a1) / chlorophyll a] * 100 Carotenoid inhibition rate (%) = [(carotenoid - carotenoid 1) / carotenoid] * 100 Wherein, chlorophyll a and carotenoids are the contents of photosynthetic pigments in the algal sap containing only Microcystis aeruginosa, and chlorophyll a1 and carotenoids 1 are the contents of photosynthetic pigments in the algae-bacteria symbiosis.

[0057] The experiment started on the 6th day and ended on the 14th day, and the chlorophyll a and carotenoid contents in the water were tested every two days.

[0058] like E. coli and Figure 2 As shown, compared with Comparative Example 1 and Comparative Example 2, the addition of the RP4 plasmid Figure 3 After the DH5α bacterial solution was added, the chlorophyll a and carotenoids were significantly reduced, with the maximum degradation of chlorophyll reaching 98% and carotenoids reaching 95%. E. coli DH5α has a significant effect on purifying Microcystis aeruginosa in water, and the color of the water is also effectively improved. E. coli a.

[0059] The prepared sample carrying the RP4 plasmid Figure 4 DH5α bacterial solution has a degradation rate of up to 85% for chlorophyll a and 90% for carotenoids. E. coli andFigure 2 As shown, the chromaticity in the water body has also been significantly improved ( Figure 3 b). However, compared with the degradation level of Example 1, the degradation ability of Comparative Example 1 is slightly inferior.

[0060] The prepared sample of Comparative Example 2 carrying the RP4 plasmid Figure 4 The supernatant of DH5α is E. coli AD-EPS. Its degradation effect on chlorophyll a and carotenoids in water is poor, less than 10% ( E. coli and Figure 2 ), and its ability to improve the chromaticity of water bodies is also poor and still appears light green ( Figure 3 c).

[0061] According to the above operating examples 1 to 3, there is no significant difference in the inhibition rate of photosynthetic pigments, and all have high inhibitory effects. Therefore, in actual experiments, the parameters can be appropriately adjusted according to actual conditions.

[0062] Effect Example 3 Test samples: Materials prepared in Examples 1-3 and Comparative Examples 1-2 Carrying RP4 plasmid Figure 4 The effects of DH5α on the MCs content of Microcystis aeruginosa are as follows: (1) Using the microcystin test kit, add 40 μL of sample diluent and 10 μL of 7-day cultured Microcystis aeruginosa algae solution and algae-bacteria mixture (the sample is diluted 5 times) into the sample well to be tested. Make three parallel samples for each sample.

[0063] (2) Seal the plate with sealing film and place in a 37°C warm bath for 30 minutes.

[0064] (3) After the warm bath, discard the liquid and spin dry. Add detergent and let it stand for 30 seconds before discarding it. Repeat 5 times and pat dry.

[0065] (4) Add 50 μL of enzyme-labeled reagent to each well and repeat steps (2) and (3).

[0066] (5) After washing, add 50 μL of color developer A and 50 μL of color developer B to each well, shake gently to mix, and incubate at 37°C in the dark for 10 minutes.

[0067] (6) After incubation, add 50 μL of stop solution to each well and the sample will turn from blue to yellow.

[0068] (7) Measure the absorbance (OD value) of each well in turn at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0069] (8) Calculate the MCs concentration of the sample based on the standard curve. The calculation formula is as follows: MCs content (μg / L) = (376.1*OD450 -11.7)*N N——dilution multiple.

[0070] The experiment started on the 7th day and ended on the 15th day, and the MCs content in the water was tested every two days.

[0071] like E. coli As shown, compared with Comparative Example 1 and Comparative Example 2, the addition of the RP4 plasmid Figure 5 The MCs content was significantly reduced after the DH5α bacterial solution was added. E. coli DH5α has a significant effect in reducing microcystin toxins in water and improving the water environment.

[0072] The prepared sample carrying the RP4 plasmid E. coli DH5α bacterial solution also has a good inhibitory effect on MCs ( E. coli ), but compared with the inhibition level of Example 1, the degradation ability of Comparative Example 1 is slightly inferior.

[0073] The prepared sample of Comparative Example 2 carrying the RP4 plasmid Figure 5 The supernatant of DH5α is E. coli AD-EPS, which has a poor inhibitory effect on MCs in water ( E. coli ).

[0074] According to the above operating examples 1 to 3, the accumulation of MCs in the water body was significantly reduced and the water environment was improved, and the effects were significantly better than those of comparative examples 1 and 2. Therefore, in actual experiments, the parameters can be appropriately adjusted according to actual conditions.

[0075] Effect Example 4 Test sample: Material prepared in Example 1 Take 30 μL of each of the logarithmic phase Microcystis aeruginosa and algae-bacteria symbiotic system and place them on a cell counting plate. Then place them on a cell counter to observe the number of cells carrying the RP4 plasmid. Figure 5 Effects of DH5α on the number of Microcystis aeruginosa, such as E. coli As shown. Figure 6 The content of Microcystis aeruginosa in the logarithmic phase under DH5α was significantly reduced, proving that the RP4 plasmid E. coli DH5α has the ability to remove Microcystis aeruginosa in water bodies and improve the algal bloom phenomenon in water bodies.

[0076] Effect Example 5 Test sample: Material prepared in Example 1 Take 30 μL of each liquid of the logarithmic phase algae-bacteria symbiotic system and place it on a cell counting plate. Then, place it on a cell counter to observe the cells carrying the RP4 plasmid. E. coli The activity status and quantity of DH5α, such asE. coli As shown. In the experiment, E. coli grew rapidly in the early logarithmic phase, robbing Microcystis aeruginosa of its nutrient source, and even significantly inhibited the growth of Microcystis aeruginosa by using Microcystis aeruginosa as a nutrient source. From the 11th day of the logarithmic phase, the bacteria began to die gradually, the number decreased significantly, and the activity became slow. By the 13th day, almost no bacterial activity was observed. Figure 7 DH5α has a significant inhibitory effect on Microcystis aeruginosa, improving water quality and achieving purification. E. coli DH5α will also die naturally and will not cause secondary pollution to the water body.

[0077] The phenomena presented in Examples 2 and 3 are highly similar to those in Example 1. Therefore, in actual experiments, the parameters can be appropriately adjusted according to the actual situation.

[0078] Effect Example 6 Test sample: Material prepared in Example 1 Microcystis aeruginosa carrying the RP4 plasmid E. coli The effects of DH5α exposure on the spread of antibiotic resistance genes (ARGs) were investigated in the following steps: (1) Set up three groups, A, B, and C, each containing 4.95 mL of RP4 plasmid. E. coli DH5α bacterial solution (concentration of 10 8 CFU / mL) and 4.95 mL without RP4 plasmid E. coli HB101 bacterial solution (concentration of 10 8 CFU / mL), group A was blank, and 100 μL PBS was added; group B was the Microcystis aeruginosa cultured on the 8th, 10th, 12th and 14th days, and the addition amount was 100 μL; group C was the Microcystis aeruginosa carrying the RP4 plasmid obtained in Example 1 on the 8th, 10th, 12th and 14th days. E. E. coli Add 100 μL of the mixed solution of DH5α bacterial solution and algae solution.

[0079] (5) The different treatment groups were placed in a shaker at 25°C and 200 rpm in the dark for 4 hours to perform the conjugation reaction.

[0080] (6) Dilute the conjugated bacterial solution with PBS, take 100 μL of the 50-fold bacterial solution and apply it on the surface of the plate containing Amp R , Km R 、Tec R Str R On solid LB of four resistance strains, dilute 10 4 times the bacterial solution was applied on a surface containing only Str R A resistant solid LB.

[0081] (7) Place the coated plate upside down in a 37°C incubator in the dark for 48 hours, then count the colonies and calculate the conjugation frequency. The specific conjugation frequency calculation formula is as follows: Engagement frequency calculation: f – junction transfer frequency; N c ——Four-resistance board (Amp R , Km R 、Tec R Str R ) multiplied by the dilution factor (CFU / mL); N R ——Monoclonal antibody board (Str R ) multiplied by the dilution factor (CFU / mL).

[0082] The mixture of bacterial liquid and algae liquid cultured on the 8th, 10th, 12th and 14th days of Example 1 was subjected to the above conjugation transfer experiment. The results are as follows: coli As shown, under the exposure of only Microcystis aeruginosa (Group B), the frequency of ARGs conjugation transfer was significantly increased compared with the natural conjugation system (Blank Group A), up to 3.00 times the conjugation frequency of Group A, proving that Microcystis aeruginosa can promote the conjugation transfer of ARGs and pose a health risk to the environment. Figure 8 The conjugation frequency of Microcystis aeruginosa treated with DH5α was not significantly different from that of group A. However, compared with group B, the conjugation frequency of group C was significantly reduced, especially on the 12th day, with an inhibition rate of up to 59.18% (P < 0.001). E. E. coli coli DH5α intervention can effectively reduce the environmental health risk of ARGs conjugation and transmission caused by Microcystis aeruginosa.

[0083] While various embodiments of the present invention have been described above, the foregoing description is intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A biological method for removing Microcystis aeruginosa from freshwater to inhibit the spread of ARGs, characterized by: The following steps are involved: (1) Inoculate the Escherichia coli carrying the RP4 plasmid into a culture medium containing three antibiotics: ampicillin, kanamycin, and tetracycline, and culture until the growth reaches the stable phase; (2) Centrifuge the bacterial solution obtained in step (1) that has grown to the stationary phase, discard the supernatant, wash the bacteria, resuspend the bacteria, and adjust the bacterial solution concentration to the target concentration; (3) The bacterial solution with the concentration adjusted in step (2) is added to the water containing Microcystis aeruginosa according to a specific volume ratio.

2. The biological method for removing Microcystis aeruginosa from freshwater to inhibit the spread of ARGs according to claim 1, characterized in that: The culture medium used in step (1) is LB culture medium.

3. The biological method for removing Microcystis aeruginosa from freshwater to inhibit the spread of ARGs according to claim 1, characterized in that: The culture conditions in step (1) are: temperature of 25-37°C, culture speed of 180-200 rpm, and culture in darkness.

4. The biological method for removing Microcystis aeruginosa from freshwater to inhibit the spread of ARGs according to claim 1, characterized in that: The target concentration in step (2) is 10 8 ~10 9 CFU / mL.

5. The biological method for removing Microcystis aeruginosa from freshwater to inhibit the spread of ARGs according to claim 1, characterized in that: In step (2), PBS buffer is used to wash and resuspend the bacteria.

6. The biological method for removing Microcystis aeruginosa from freshwater to inhibit the spread of ARGs according to claim 1, characterized in that: The volume ratio of bacterial liquid to water in step (3) is 10:210~23:210.

Images