Pseudomonas sp. capable of degrading microcystin, immobilized bacterial agent and application thereof

Abstract

The application discloses a Pseudomonas capable of degrading microcystins, an immobilized bacterial agent and application, and particularly relates to Pseudomonas chengduensis DMC-X1, which is isolated and screened from surface water samples of a cyanobacterial bloom outbreak in Nanyi Reservoir in Nanjing County, Zhangzhou City, Fujian Province. The strain belongs to gamma-proteobacteria, has high degradation capacity for microcystins (MCs), and has a degradation capacity superior to most Pseudomonas reported at present, and can be used for treating water polluted by microcystins. In the application, sodium alginate is used as a carrier, CaCl2 is used as a crosslinking agent, and the Pseudomonas DMC-X1 is embedded to obtain an immobilized bacterial agent, which can degrade microcystins in a short time and can be recycled for multiple times. The immobilized bacterial agent is convenient to store and recycle, and has important application value.

Description

Technical Field

[0001] This invention relates to a Pseudomonas bacterium, specifically a Pseudomonas bacterium capable of degrading microcystin, an immobilized bacterial agent, and its applications. It belongs to the field of water treatment technology. Background Technology

[0002] Harmful algal blooms occur in various aquatic environments, where large amounts of phytoplankton, along with bacteria or protozoa, accumulate and release neurotoxic and hepatotoxic compounds into the water, causing water quality deterioration. When algal bloom pollution is severe, it disrupts the balance of healthy aquatic ecosystems. Furthermore, the algal toxins produced and released by algae are passed down through the food chain to higher trophic levels, seriously threatening human and animal health. Among the algal toxins produced by cyanobacterial blooms, microcystins (MCs) are the most abundant, most frequently detected, and most harmful.

[0003] Microcystins (MCs) are a class of monocyclic heptapeptide compounds with stable chemical properties, which are difficult to rapidly degrade in natural water bodies. The harm of MCs to aquatic environments and human health has become a major environmental issue of global concern. Among the more than 250 MC isomers identified, MC-LR is the most toxic and numerous, and also the most common isomer most closely associated with liver cancer and other diseases in humans and animals. MC-LR is highly toxic due to the presence of a specially structured Adda group. It induces oxidative stress in eukaryotic cells by binding to protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) in the liver. Microcystins can cause deformities and weight loss in plant seedlings, reduce photosynthetic efficiency, and disrupt embryonic development in animals, exhibiting teratogenic effects. They can even accumulate in the human body through drinking water supplies, daily activities, and the food chain, leading to acute gastroenteritis, liver dysfunction, and liver cancer, posing a threat to human health. Therefore, how to effectively remove MC-LR from water and ensure water safety has become a global concern.

[0004] Currently, common toxin treatment methods in drinking water and domestic water treatment can be broadly classified into three categories: physical methods, chemical methods, and biological methods. Compared to physical and chemical methods, which suffer from high costs and secondary pollution, biological methods have attracted widespread attention due to their environmental friendliness, low cost, and high efficiency. Existing research has shown that MC-LR degrading bacteria can achieve low-cost and high-efficiency degradation of MC-LR without producing harmful secondary pollutants, making it the safest and most reliable method for removing MC-LR from natural water bodies. To date, researchers worldwide have discovered indigenous bacteria capable of degrading MC-LR in eutrophic lakes, reservoirs, ponds, river sediments, and sand filters. Most bacteria capable of degrading MC-LR belong to the genus *Sphingomonas* and genus *Sphingopyxis* within the class Alpha-Proteobacteria. There are few reports of γ-Proteobacteria degrading MCs, and studies on immobilizing MC-LR degrading bacteria using encapsulation-crosslinking methods are also scarce. Existing research has shown that after microorganisms are immobilized, a physical or chemical connection is established between the carrier and the microorganisms, providing a survival substrate for the microorganisms, reducing cell loss, and resulting in materials with good stability, pressure resistance, and recyclability, enabling efficient treatment of polluted wastewater. Therefore, screening novel MC-LR degrading bacteria, exploring and expanding the types of MC-LR degrading bacteria, and using immobilization technology to prepare microbial agents can provide a theoretical basis and technical support for the remediation of MC-LR polluted water bodies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Pseudomonas strain that can degrade microcystin, an immobilized bacterial agent, and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] 1. A strain of Pseudomonas chengduensis DMC-X1 that can degrade microcystin is deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 4, 2022. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO.62596.

[0008] 2. Application of the above-mentioned Pseudomonas bacteria in the treatment of microcystin-contaminated water.

[0009] 3. An immobilized bacterial agent, which is obtained by encapsulating the aforementioned Pseudomonas bacteria using sodium alginate as a carrier and calcium chloride as a cross-linking agent.

[0010] 4. The specific steps of the above-mentioned method for preparing an immobilized bacterial agent are as follows:

[0011] (1) First, the Pseudomonas DMC-X1 was inoculated into MSM inorganic salt liquid medium, with microcystin-LR (MC-LR) as the only carbon and nitrogen source, cultured on a shaker, centrifuged to collect the precipitate, and then resuspended in MSM inorganic salt liquid medium to obtain a bacterial suspension.

[0012] (2) Then mix the 2% sodium alginate aqueous solution and an equal volume of bacterial suspension thoroughly to obtain a mixture. Then add the mixture dropwise to the 2% calcium chloride aqueous solution and immobilize and crosslink at 4°C for 4-6 hours to obtain small spherical immobilized bacterial agent. Wash with sterile water and air dry at room temperature.

[0013] 5. The application of the above-mentioned immobilized bacterial agent in the treatment of microcystin-contaminated water.

[0014] 6. Application of the above-mentioned immobilized bacterial agent in the treatment of cyanobacterial blooms.

[0015] The beneficial effects of this invention are:

[0016] This invention isolates and screens a microcystin-degrading bacterium, *Pseudomonas chengduensis* DMC-X1, from surface water samples of a cyanobacterial bloom outbreak at Nanyi Reservoir in Nanjing County, Zhangzhou City, Fujian Province. This strain belongs to the class Gamma-Proteobacteria and exhibits high degradation capacity for microcystins (MCs), surpassing that of most previously reported Pseudomonas strains. It can be used for the treatment of microcystin-contaminated water.

[0017] This invention also uses sodium alginate as a carrier and CaCl2 as a cross-linking agent to encapsulate Pseudomonas DMC-X1, obtaining an immobilized bacterial agent that can degrade microcystin in a short time and can be recycled multiple times. The immobilized bacterial agent exhibits rapid, stable, and recyclable degradation of microcystin, maintaining stable efficacy within a certain pH and temperature range (25–40°C). It can save on the cost of treating toxin-contaminated water and efficiently degrade MC-LR in aquatic ecosystems, showing great application potential. It is expected to be used for large-scale degradation of microcystin during cyanobacterial blooms, thus controlling water pollution. This immobilized bacterial agent is easy to store and recycle, possessing significant application value. Attached Figure Description

[0018] Figure 1 The growth curve and degradation curve of MC-LR by the MC-LR degrading bacterium DMC-X1 are shown.

[0019] Figure 2The figures show the phylogenetic tree and morphological diagram of strain DMC-X1. (A) is the phylogenetic tree of strain DMC-X1 constructed based on the 16S rRNA gene sequence. As shown in the figure, strain DMC-X1 belongs to the genus Pseudomonas and is named Pseudomonas chengduensis DMC-X1. (B) and (C) are morphological diagrams of MC-LR degrading bacteria DMC-X1. (B) shows the morphology of plate colonies, and (C) is a scanning electron microscope image of the strain. As shown in the figure, the colonies of strain DMC-X1 are flesh-colored, opaque, and have rough edges; the cells are rod-shaped, 2.8-3.5 μm in size, and about 0.25-0.40 μm in diameter.

[0020] Figure 3 The figure shows the degradation rate of MC-LR by DMC-X1 bacteria at different concentrations. As can be seen from the figure, the degradation rate of MC-LR by DMC-X1 gradually increases with the increase of bacterial concentration.

[0021] Figure 4 The figure shows the degradation rate of MC-LR by strain DMC-X1 at different concentrations. As can be seen from the figure, strain DMC-X1 can not only degrade low concentrations of MC-LR in a short time, but also rapidly degrade high concentrations of MC-LR.

[0022] Figure 5 The effect of different pH values ​​on the degradation of MC-LR by strain DMC-X1 is shown in the figure. As can be seen from the figure, the optimal pH value for MC-LR degradation by DMC-X1 is 7-8.

[0023] Figure 6 The effect of different temperatures on the degradation of MC-LR by strain DMC-X1 is shown in the figure. As can be seen from the figure, the optimal MC-LR degradation temperature of DMC-X1 is 25-40℃.

[0024] Figure 7 The effect of immobilized bacterial agents on the degradation of MC-LR is shown in the figure. (A) Immobilized bacterial agent pellets without air drying treatment, (B) Immobilized bacterial agent pellets after air drying at room temperature, and (C) Effect of different numbers of immobilized bacterial agent pellets on the degradation of MC-LR. As can be seen from the figure, the more pellets added, the better the degradation effect on MC-LR. The degradation effect tends to stabilize at 48h, and the degradation effect of all treatment groups reaches more than 90%.

[0025] Figure 8 The results of the test on the recycling of the immobilized bacterial agent are shown in the figure. As can be seen from the figure, the microspheres still have good MC-LR degradation ability after three uses, indicating that the immobilized bacterial agent microspheres can be reused.

[0026] Preservation Information

[0027] Classification and nomenclature: Pseudomonas

[0028] Latin scientific name: Pseudomonas chengduensis

[0029] Name of depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC)

[0030] Address of the repository: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0031] Deposit date: July 4, 2022 Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0033] I. Isolation, Screening and Identification of Strains

[0034] (1) Surface water samples from the Nanyi Reservoir in Nanjing County, Zhangzhou City, Fujian Province, which had experienced an outbreak of cyanobacterial blooms, were taken and serially diluted 10 times with MSM inorganic salt liquid medium (MgSO4·7H2O 180mg / L, CaCl2 20mg / L, Na2MoO4·2H2O 2.5mg / L, ZnSO4·7H2O 8mg / L, FeCl3·6H2O 0.25mg / L, KH2PO4 300mg / L, NaHPO4·12H2O 868mg / L liquid medium without agar powder). 10mL of the diluted supernatant was inoculated into 90mL of MSM inorganic salt liquid medium, with 200μg / L MC-LR as the sole carbon and nitrogen source, and cultured at 30℃ and 150rpm with shaking. The concentration of MC-LR was detected using a microcystin detection kit (Beacon, Cat. #20-0068). After MC-LR was completely degraded, 100 μL of culture medium was serially diluted, and after shaking and mixing, 200 μL was spread on MSM inorganic salt solid medium (MSM inorganic salt liquid medium with 15 g / L agar powder added) with a final MC-LR concentration of 200 μg / L. The medium was then incubated at 30°C in the dark for 10 days.

[0035] (2) Based on the differences in colony morphology, size, color, etc., different colonies were picked and inoculated into 20 mL of MSM inorganic salt liquid medium containing 200 μg / L MC-LR (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), and placed in a shaker (30℃, 150 rpm) and cultured in the dark until the medium became turbid.

[0036] (3) Different cultures were streaked again on MSM inorganic salt solid plates containing 200 μg / L MC-LR and incubated at 30°C in the dark for 3 days, and single colonies appeared.

[0037] (4) Pick a single colony and repeat steps (2) and (3) multiple times until a pure culture of a single colony is obtained;

[0038] (5) The obtained single colonies were inoculated into 20 mL of MSM liquid medium containing 200 μg / L MC-LR and placed in a shaker at 30℃ and shaken at 150 rpm for 8 days. The MSM liquid medium containing 200 μg / L MC-LR without colonies was used as a blank control. The culture medium was taken every day, and the absorbance of the culture medium was measured at 600 nm to plot the bacterial growth curve. At the same time, the concentration of MC-LR in the culture medium was detected by the microcystin detection kit, and the MC degradation curve of the bacteria was plotted. Three replicates were set up for each group.

[0039] (6) By Figure 1 It was found that a strain DMC-X1 with good MC-LR degradation ability was isolated. This strain grew slowly in MSM liquid medium containing MC-LR, reaching the stationary phase on day 8. At this time, OD 600 =0.23. On the other hand, strain DMC-X1 rapidly degraded MC-LR, completely degrading 200 μg / L of MC-LR within 24 h.

[0040] (7) The genome of strain DMC-X1 was extracted and 16S rRNA gene amplified. The PCR product was sent to Xiamen Platinum Biotech for sequencing. After primer removal, the sequence was uploaded to the Korean EzBioCloud website (https: / / www.ezbiocloud.net / ) for comparison and analysis. A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 7.0 software. The results are shown in […]. Figure 2 (A) Through comparative identification, strain DMC-X1 showed a 100% similarity to *Pseudomonas chengduensis*. Therefore, strain DMC-X1 belongs to the genus *Pseudomonas* and is named *Pseudomonas chengduensis* DMC-X1. Its 16S rRNA sequence was uploaded to the NCBI database, with GenBank accession number ON920515. Morphological characteristics of DMC-X1 are shown in [reference needed]. Figure 2 (B) and (C).

[0041] II. Study on the Degradation Characteristics of Strains DMC-X1

[0042] 1. Degradation of MC-LR by DMC-X1 at different cell densities

[0043] Strains DMC-X1 were inoculated into LB liquid medium and cultured in a constant temperature shaker at 30℃ and 150rpm in the dark for 36h. After centrifugation at 6000rpm for 6min, the bacterial pellet was collected, washed three times with MSM inorganic salt liquid medium, and then an appropriate amount of MSM inorganic salt medium was added to prepare a bacterial suspension of a certain concentration for later use.

[0044] The DMC-X1 bacterial suspension was prepared at 1.1 × 10⁻⁶. 5 5.5×10 5 1.1×10 6 5.5×10 6 1.1×10 7 The CFU / mL dose was inoculated into MSM inorganic salt liquid medium containing 200 μg / L MC-LR, with 3 replicates per group. The medium was incubated at 30℃ and 150 rpm in the dark with shaking. Samples were taken every 4 h, and the concentration of the toxin was detected using a microcystin detection kit. The degradation rate of MC-LR was calculated according to Formula 1.

[0045] Degradation rate (%) = (C0 - C) t ) / C t ×100%(Formula 1)

[0046] Where C0 is the concentration of MC-LR in the bacterial culture medium at 0h (200 μg / L); C t The concentration of MC-LR in the bacterial culture medium at the corresponding sampling time.

[0047] Depend on Figure 3 It can be seen that the degradation effect of DMC-X1 on MC-LR exhibits a concentration gradient effect; the degradation effect increases with increasing bacterial concentration. At 12 h, 1.1 × 10⁻⁶... 7 The degradation rate of MC-LR by DMC-X1 in the CFU / mL inoculation dose group was 76.56%, which is 1.1 × 10⁻⁶. 5 The CFU / mL inoculation dose was 1.59 times that of the control group, but at 24 h, there was no significant difference in the degradation rate of MC-LR among all concentrations of DMC-X1, all reaching over 95%.

[0048] DMC-X1's ability to rapidly degrade MC-LR within 24 hours is significantly superior to other strains reported to date (Table 1).

[0049] Table 1. Comparison of the degradation capacity of DMC-X1 with that of previously reported Pseudomonas species.

[0050]

[0051]

[0052] The literature sources of each strain in Table 1 are as follows:

[0053] 1. S. Takenaka, Watanabe, M. F. Microcystin LR degradation by Pseudomonas aeruginosa alkaline protease. Chemosphere, 1997, 34(4): 749-757. http: / / doi.org / 10.1016 / s0045-6535(97)00002-7.

[0054] 2. L. Eleuterio, Batista, J. R. Biodegradation studies and sequencing of microcystin-LR degrading bacteria isolated from a drinking water biofilter and a fresh water lake. Toxicon, 2010, 55(8): 1434-1442. http: / / doi.org / 10.1016 / j.toxicon.2010.02.020.

[0055] 3. Y. H. Kang, et al. Pseudomonas aeruginosa UCBPP-PA14 a useful bacterium capable of lysing Microcystis aeruginosa cells and degrading microcystins. Journal of Applied Phycology, 2012, 24(6): 1517-1525. http: / / doi.org / 10.1007 / s10811-012-9812-6.

[0056] 4. H. Li, Pan, G. Enhanced and continued degradation of microcystins using microorganisms obtained through natural media. Journal of Microbiological Methods, 2014, 96: 73-80. http: / / doi.org / 10.1016 / j.mimet.2013.11.005.

[0057] 5.GAFLemes, et al. Biodegradation of D-Leu(1)microcystin-LR by abacterium isolated from sediment of Patos Lagoon estuary, Brazil. Journal of Venomous Animals and Toxins Including Tropical Diseases, 2015, 21. http: / / doi.org / 10.1186 / s40409-015-0001-3.

[0058] 2. Degradation of MC-LR by DMC-X1 at different concentrations

[0059] DMC-X1 bacterial suspensions were inoculated into MSM inorganic salt liquid medium containing 200, 400, 800, and 1000 μg / L MC-LR, respectively, so that the bacterial cell concentration in each concentration group was 2.4 × 10⁻⁶. 6 CFU / mL (OD) 600 =0.1), with 3 replicates per group, incubated at 30℃ and 150 rpm in the dark with shaking. Samples were taken every 4 hours, and the concentration of microcystin was detected using a microcystin detection kit. The degradation rate of MC-LR was calculated according to Formula 1. The results showed that DMC-X1 had good MC-LR degradation performance: strain DMC-X1 could completely degrade 200 μg / L MC-LR within 24 hours, and within 48 hours, it could degrade 400, 800, and 1000 μg / L MC-LR by 95.15%, 95.33%, and 94.87%, respectively. This indicates that DMC-X1 has a high efficiency in degrading high concentrations of MC-LR and can be an excellent strain for treating toxin-contaminated water.

[0060] 3. Effects of pH and temperature on the degradation of MC-LR by DMC-X1

[0061] (1) The pH values ​​of the MSM inorganic salt liquid medium were adjusted to 5, 6, 7, 8, and 9 using HCl and NaOH, respectively. The DMC-X1 bacterial suspension was inoculated into the MSM inorganic salt liquid medium (containing 200 μg / L MC-LR) at the above different pH values, resulting in an initial concentration of 2.4 × 10⁻⁶. 6 CFU / mL (OD) 600 =0.1), with 3 replicates per group, incubated at 30℃ and 150 rpm in the dark with shaking. Samples were taken every 4 hours, and the concentration of microcystin was detected using a microcystin detection kit. The MC-LR degradation rate was calculated according to Formula 1. The effect of the initial pH of the culture medium on the MC-LR degradation rate of DMC-X1 is as follows: Figure 5As shown, when the initial pH of the culture medium was 7 and 8, strain DMC-X1 could completely degrade 200 μg / L MC-LR in 24 h. When the initial pH of the culture medium was 6, the degradation ability of strain DMC-X1 on MC-LR weakened, and the degradation rate was only 68.85% in 24 h. At a higher pH (pH=9), the degradation ability of DMC-X1 on MC-LR also decreased significantly, and the degradation rate was 82.37% in 24 h. This indicates that the optimal pH for strain DMC-X1 to degrade MC-LR is 7-8.

[0062] (2) Inoculate the DMC-X1 bacterial suspension into MSM inorganic salt liquid medium containing 200 μg / L MC-LR to achieve an initial concentration of 2.4 × 10⁻⁶. 6 CFU / mL (OD) 600 =0.1), and were incubated at 20, 25, 30, 35, and 40℃ respectively, with three biological replicates for each temperature group. The cultures were incubated at 150 rpm with shaking in the dark, and samples were taken every 4 hours. The concentration of the microcystin was detected using a microcystin detection kit, and the MC-LR degradation rate was calculated according to Formula 1. The results are as follows: Figure 6 As shown, the optimal temperature for DMC-X1 to degrade MC-LR is 30-35℃. Within this temperature range, the strain can completely degrade 200 μg / L of MC-LR within 24 hours. When the temperature is increased to 40℃, the strain degrades only 82.02% of MC-LR after 24 hours. When the temperature decreases (25℃ and 20℃), the strain's ability to degrade MC-LR also weakens, with degradation rates of 77.16% and 59.44% after 24 hours, respectively.

[0063] III. Preparation of Immobilized Bacterial Agent and Detection of its MC-LR Degradation Ability

[0064] (1) Preparation of bacterial suspension: The strain DMC-X1 was inoculated into 20 mL of MSM inorganic salt liquid medium, with 200 μg / L MC-LR as the sole carbon and nitrogen source, and placed in a shaker (30℃, 150 rpm) in the dark until the strain reached the stationary phase. 10 mL of the stationary phase bacterial suspension was centrifuged at 6000 rpm for 6 min, the supernatant was discarded, and the suspension was resuspended in MSM inorganic salt liquid medium to obtain the DMC-X1 bacterial suspension.

[0065] (2) Preparation of sodium alginate (SA) solution: Weigh 2g of sodium alginate, dissolve it in 98mL of distilled water, heat and stir with a magnetic stirrer until dissolved, and obtain a sodium alginate solution with a mass fraction of 2%. After high temperature and high pressure sterilization, it is ready for use.

[0066] (3) Preparation of CaCl2 solution: Weigh 2g of CaCl2 solid, dissolve it in 98mL of distilled water, and filter it through a 0.22μm filter membrane to remove bacteria, and obtain a CaCl2 solution with a mass fraction of 2%.

[0067] (4) Preparation of immobilized bacterial agent: 2 mL of 2% sodium alginate solution and an equal volume of DMC-X1 bacterial suspension were thoroughly mixed. Then, using a sterile syringe, the sodium alginate-DMC-X1 mixture was dripped dropwise from a height into a beaker containing 50 mL of 2% CaCl2 cross-linking agent solution (each bacterial agent pellet was approximately 1.2 × 10⁻⁶ oz.). 5 DMC-X1 cells (CFU / mL) were immobilized and cross-linked at 4°C for 6 hours. Blank control pellets (containing no DMC-X1 cells) were prepared by mixing 2% sodium alginate solution with an equal volume of MSM inorganic salt liquid medium.

[0068] (5) After immobilization and cross-linking, remove the beaker from the 4°C refrigerator and wash the pellets repeatedly with sterile water;

[0069] (6) Place the prepared microbial agent pellets and blank pellets in a room-temperature air-drying environment and store them for later use;

[0070] (7) The prepared microbial pellets of 2, 4, 6, 8 and 20 were added to MSM inorganic salt medium containing 200 μg / L MC-LR, respectively, so that the amount of bacteria added to each treatment was 2.4 × 10⁻⁶. 5 4.8×10 5 7.2×10 5 9.6×10 5 2.4×10 6 The samples were collected at CFU / mL and incubated at 30°C in the dark. Samples were taken every 4 hours, and the toxin concentration was detected using a microcystin detection kit. The degradation rate of MC-LR was calculated according to Formula 1. A treatment with an equal amount of free bacteria served as a positive control, and a treatment with an equal amount of blank microspheres served as a negative control. All treatments were performed in triplicate.

[0071] (8) Figure 7As shown in Table 2, within 24 hours, the degradation rates of MC-LR in treatment groups with 2, 4, and 6 immobilized microbial pellets were 70.45%, 77.61%, and 86.56%, respectively, while the corresponding degradation rates of free bacteria were 92.05%, 93.22%, and 95.12%. This indicates that immobilized microbial pellets of different numbers exhibit efficient degradation capabilities for MC-LR, with degradation rates only slightly lower than those of free bacteria at the same dosage. Furthermore, the more immobilized microbial pellets used, the better the degradation effect on MC-LR; 20 pellets achieved a degradation rate of over 90% for MC-LR within 24 hours. With prolonged treatment time, after 48 hours, the degradation rate of MC-LR by immobilized microbial pellets was no different from that of free bacteria, both reaching over 90%. This demonstrates that immobilized microbial pellets have a highly efficient degradation capability for MC-LR, which is beneficial for the preservation and field application of the degrading bacteria DMC-X1.

[0072] Table 2. Comparison of MC-LR degradation rates

[0073]

[0074] IV. Testing the Recyclability of Immobilized Microbial Agents

[0075] (1) First use of the immobilized bacterial agent: Eight immobilized bacterial agent microspheres were added to MSM inorganic salt culture medium containing 200 μg / L MC-LR and cultured statically at 30℃ in the dark. Samples were taken every 4 hours, and the concentration of microcystin was detected using a microcystin detection kit. The MC-LR degradation rate was calculated according to Formula 1. A control group was prepared by adding the same number of blank microspheres. All treatments were performed in triplicate. Figure 8 As shown in Figure A, when the immobilized microspheres were used for the first time, the degradation rate of MC-LR was 89.13% after 24 hours and 98.74% after 48 hours.

[0076] (2) Second use of the immobilized bacterial agent: Remove the immobilized bacterial agent pellets and blank pellets used in step (1) from the culture medium, wash them three times with sterile water, and then air-dry them at room temperature (25-30℃). After air-drying, store the immobilized pellets at room temperature for 3 days before conducting a second MC-LR degradation experiment. Refer to step (1) above for specific steps. Figure 8 As shown in Figure B, when the immobilized microspheres were reused for the second time, the degradation rate of MC-LR was 87.83% after 24 hours and 97.49% after 48 hours.

[0077] (3) Third use of immobilized bacterial agent: Remove the immobilized bacterial agent pellets and blank pellets used in step (2) from the culture medium, wash them three times with sterile water, and then air-dry them at 25-30℃. After air-drying, store the immobilized pellets at room temperature for 5 days, and then conduct the third MC-LR degradation experiment. For specific procedures, refer to step (1) above. Figure 8 As shown in Figure C, when the immobilized bacterial agent microspheres were reused for the third time, their degradation ability against MC-LR decreased significantly after 24 hours, with a degradation rate of only 57.61%. However, after 48 hours, the immobilized bacterial agent microspheres still maintained a high degradation ability against MC-LR, with a degradation rate of 91.68%.

[0078] In summary, the immobilized bacterial pellets formed by coating MC-degrading bacteria DMC-X1 with sodium alginate still maintain a high degradation capacity for MC after being reused three times, which is beneficial for the future preservation and reuse of the bacterial agent.

[0079] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A strain of Pseudomonas that can degrade microcystin ( Pseudomonas chengduensis DMC-X1, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, on July 4, 2022, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO.62596.

2. The application of the Pseudomonas bacillus of claim 1 in the treatment of microcystin-LR contaminated water.

3. An immobilized bacterial agent, characterized in that, It is obtained by encapsulating the Pseudomonas bacteria described in claim 1 using sodium alginate as a carrier and calcium chloride as a cross-linking agent.

4. The method for preparing an immobilized bacterial agent according to claim 3, characterized in that, The specific steps are as follows: (1) First, the Pseudomonas DMC-X1 was inoculated into MSM inorganic salt liquid medium, with microcystin-LR as the only carbon and nitrogen source, cultured on a shaker, centrifuged to collect the precipitate, and then resuspended in MSM inorganic salt liquid medium to obtain a bacterial suspension. (2) Then mix the 2% sodium alginate aqueous solution and an equal volume of bacterial suspension thoroughly to obtain a mixture. Then add the mixture dropwise to the 2% calcium chloride aqueous solution and immobilize and crosslink at 4°C for 4-6 hours to obtain small spherical immobilized bacterial agent. Wash with sterile water and air dry at room temperature.

5. The application of the immobilized bacterial agent according to claim 3 in the treatment of microcystin-LR contaminated water.

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