A strain of oligotrophic monocytosin capable of degrading microcystin, an immobilized bacterial agent and its application

Abstract

The present invention discloses a strain of Stenotrophomonas geniculata that can degrade microcystins, an immobilized bacterial agent, and applications. Specifically, the strain is Stenotrophomonas geniculata DMC-X3, which was isolated and screened from a surface water sample of a cyanobacterial bloom outbreak in Nanyi Reservoir, Nanjing County, Zhangzhou City, Fujian Province. The strain DMC-X3 has good degradation performance for both low and high concentrations of MC-LR, and the degradation rate gradually increases with the increase in the inoculation amount of DMC-X3. At the same time, DMC-X3 can efficiently degrade microcystins under environmental pressures of extreme pH and temperature. The present invention also uses sodium alginate and calcium chloride as immobilization materials to immobilize DMC-X3, and the obtained immobilized bacterial agent can repeatedly and efficiently degrade microcystins.

Description

Technical Field

[0001] The present invention relates to a strain of oligotrophic monocytobacter, in particular to a strain of oligotrophic monocytobacter capable of degrading microcystin, an immobilized bacterial agent and applications thereof, belonging to the technical field of water treatment. Background Art

[0002] With the gradual improvement of social and economic levels and the continuous expansion of human activities, water eutrophication and global warming are becoming increasingly serious, ultimately leading to frequent outbreaks of harmful algal blooms. Harmful algal bloom pollution produces different types of algal toxins, which are usually released when algae die naturally or when physical pressure causes cell lysis. [2] Among them, microcystins (MCs) are the type of algal toxins with the largest production, the highest detection rate and the most serious harm.

[0003] MCs are a group of cyclic heptapeptide hepatotoxins with a common structure: cyclo-(D-Ala-XD-MeAsp-Z-Adda-D-Glu-Mdha), where X and Z are two variable L-amino acids. Over 250 MC isomers have been identified to date, of which MC-LR is the most toxic and numerous. MC-LR inhibits the activities of eukaryotic protein phosphatase 1 and protein phosphatase 2A, causing oxidative stress in eukaryotic cells and threatening their health. MC-LR accumulates in the human body through drinking water, daily activities, and the food chain, causing diseases in various organs, including the liver, intestines, and kidneys. Therefore, developing an efficient method for removing MC-LR is urgently needed.

[0004] The ring structure of MC-LR makes it particularly stable in the environment and can resist various natural factors, including high temperature, extreme pH, sunlight and non-specific enzymes. Therefore, urban water treatment plants have to take a series of measures to treat MC-LR in water. A series of physical and chemical methods such as activated carbon adsorption, chlorination, and ozonation [9]It has been proposed to remove MC-LR from water bodies. However, the implementation cost of physical methods is high and it is difficult to apply them on a large scale to water source treatment. Although chemical methods can remove MC-LR from water with high efficiency, they are prone to cause secondary pollution of water bodies, threatening drinking water safety. Studies have shown that MC-LR degrading bacteria can achieve low-cost and high-efficiency degradation of MC-LR without producing harmful secondary pollutants. It is the safest and most reliable method to remove MC-LR from natural water bodies. To date, researchers around the world have discovered a variety of indigenous bacteria that can degrade MC-LR from eutrophic lakes, reservoirs, ponds, river sediments and sand filters. Most of the MC-LR degrading bacteria belong to Sphingomonas in the class α-Proteobacteria ( Sphingomonas ) and Sphingomonas spp. ( Sphingopyxis ).

[0005] There are currently no reports on the use of Stenotrophomonas for the degradation of microcystins. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a strain of Oligotrophomonas capable of degrading microcystin, an immobilized bacterial agent and applications.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] 1. A strain of Oligotrophomonas that can degrade microcystin Stenotrophomonas geniculata ) DMC-X3, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on July 4, 2022. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC NO.62597.

[0009] 2. Application of the above-mentioned oligotrophic bacteria in the treatment of water contaminated by microcystin.

[0010] 3. An immobilized bacterial agent is obtained by using sodium alginate as a carrier and calcium chloride as a cross-linking agent to embed the aforementioned oligotrophomonas.

[0011] 4. The preparation method of the above-mentioned immobilized bacterial agent comprises the following specific steps:

[0012] (1) First, the oligotrophic monocytogenes DMC-X3 was inoculated into MSM inorganic salt liquid culture medium, with microcystin-LR (MC-LR) as the only carbon and nitrogen source, cultured on a shaking table, centrifuged to obtain a precipitate, and then resuspended in MSM inorganic salt liquid culture medium to obtain a bacterial suspension;

[0013] (2) Then, thoroughly mix the 2% sodium alginate aqueous solution and the bacterial suspension of equal volume to obtain a mixed solution. Then, drop the mixed solution dropwise into a 2% calcium chloride aqueous solution and immobilize and cross-link at 4°C for 4 to 6 hours to obtain a small spherical immobilized bacterial agent. Wash with sterile water and air-dry at room temperature.

[0014] 5. Application of the above-mentioned immobilized bacterial agent in the treatment of water contaminated by microcystin.

[0015] 6. Application of the above-mentioned immobilized bacterial agent in the treatment of cyanobacteria blooms.

[0016] Beneficial effects of the present invention:

[0017] The present invention isolates and screens a microcystin-degrading bacterium from a surface water sample of a cyanobacterial bloom in Nanyi Reservoir, Nanjing County, Zhangzhou City, Fujian Province. Stenotrophomonas geniculata DMC-X3, deposited with the Guangdong Provincial Center for Microbial Culture Collection under the accession number GDMCC No. 62597, exhibits excellent degradation performance against both low and high concentrations of MC-LR, with the degradation rate increasing with increasing DMC-X3 inoculum. Furthermore, DMC-X3 is highly effective in degrading microcystins under extreme pH and temperature conditions, with the optimal temperature and pH for microcystin degradation being 30°C and pH 7, respectively.

[0018] The present invention also uses sodium alginate and calcium chloride as immobilization materials to immobilize DMC-X3. The resulting immobilized bacterial agent is capable of repeated and efficient degradation of microcystins. The DMC-X3 immobilized bacterial agent exhibits excellent biological activity and chemical stability, along with advantages such as a simple preparation process, low cost, easy storage, and large-scale field application. It is expected to be used for large-scale industrial remediation of microcystin contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The growth curve of MC-LR degrading bacteria DMC-X3 and its degradation curve for MC-LR.

[0020] Figure 2 The phylogenetic tree and morphological diagram of strain DMC-X3, where (A) is the phylogenetic tree of strain DMC-X3 based on 16SrRNA gene sequence. From (A), we can see that strain DMC-X3 belongs to Stenotrophomonas Genus, named Stenotrophomonas geniculataDMC-X3; (B) shows the colony morphology of the MC-LR-degrading strain DMC-X3 on a plate, and (C) shows a scanning electron micrograph of the MC-LR-degrading strain DMC-X3. As shown in (B) and (C), the colonies of strain DMC-X3 are yellow, opaque, and have smooth edges. The cells are rod-shaped, approximately 1.0-2.0 μm long and 0.20-0.30 μm in diameter.

[0021] Figure 3 The degradation rates of MC-LR by strain DMC-X3 at different concentrations are shown in Table 1. As the initial bacterial solution concentration increases, the degradation efficiency of MC-LR by strain DMC-X3 increases.

[0022] Figure 4 The degradation rates of strain DMC-X3 on MC-LR at different concentrations are shown in Table 1. Strain DMC-X3 can efficiently degrade MC-LR at both low and high concentrations.

[0023] Figure 5 The effect of different pH values ​​on the degradation of MC-LR by strain DMC-X3. The optimal pH value for MC-LR degradation by DMC-X3 is 7.

[0024] Figure 6 The effect of different temperatures on the degradation of MC-LR by strain DMC-X3. The optimal MC-LR degradation temperature of DMC-X3 is 30℃.

[0025] Figure 7 The results of DMC-X3 immobilized microbial pellets and their effect on MC-LR degradation are shown in Figures (A) and (B). (A) shows the immobilized microbial pellets before air drying, and (B) shows the immobilized microbial pellets air-dried at 30°C. (C) shows the effect of different numbers of immobilized microbial pellets on MC-LR degradation. The degradation rate of MC-LR by the microbial pellets was only slightly lower than that of free bacteria at the same dosage.

[0026] Figure 8 The results of the recycling test of the immobilized bacterial agent are shown in Figure 2. After three uses, the pellets still have good MC-LR degradation ability, indicating that the bacterial agent has good recycling ability.

[0027] Preservation Information

[0028] Classification name: Stenotrophomonas

[0029] Latin name: Stenotrophomonas geniculata

[0030] Name of depository institution: Guangdong Provincial Microbiological Culture Collection Center (GDMCC)

[0031] Address of the preservation unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0032] Date of deposit: July 4, 2022 DETAILED DESCRIPTION

[0033] 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 contents.

[0034] 1. Isolation, screening and identification of strains

[0035] (1) Surface water samples from the Nanyi Reservoir in Nanjing County, Zhangzhou City, Fujian Province, where a cyanobacterial bloom occurred, were obtained and serially diluted 10-fold (by volume) with MSM inorganic salt liquid medium (MgSO4∙7H2O 180 mg / L, CaCl2 20 mg / L, Na2MoO4∙2H2O 2.5 mg / L, ZnSO4∙7H2O 8 mg / L, FeCl3∙6H2O 0.25 mg / L, KH2PO4 300 mg / L, NaHPO4∙12H2O 868 mg / L). Ten mL of the diluted supernatant was inoculated into 90 mL of MSM inorganic salt liquid medium, and 200 μg / L MC-LR was used as the sole carbon and nitrogen source. The culture was shaken at 30°C and 150 rpm. The concentration of MC-LR was detected using a microcystin detection kit (Beacon, Cat. #20-0068). When MC-LR was completely degraded, 100 μL of the culture medium was diluted serially. After oscillation and mixing, 200 μL was spread on MSM inorganic salt solid medium (MSM inorganic salt liquid medium supplemented with 15 g / L agar powder) with a final MC-LR concentration of 200 μg / L. The culture was then incubated in the dark at 30°C for 10 days.

[0036] (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 (MC-LR final concentration was 200 μg / L), placed in a shaker (30°C, 150 rpm), and cultured in the dark until the medium became turbid;

[0037] (3) Re-streak the culture onto LB solid plates (yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar powder 15 g / L) and incubate in the dark at 30°C for 2-3 days;

[0038] (4) Repeat step (3) multiple times until a pure culture is obtained;

[0039] (5) The purified single colony was inoculated into 20 mL of MSM liquid culture medium with a final MC-LR concentration of 200 μg / L, and cultured in a shaker at 30°C and 150 rpm for 8 days. MSM-MC-LR without single bacteria inoculation was used as a blank control. The culture medium was taken every other day and its absorbance at a wavelength of 600 nm was measured. At the same time, the concentration of MC-LR was detected using a microcystin detection kit. The concentration of MC-LR and OD were respectively determined. 600 Growth curves and degradation curves were drawn, with three parallel sets for each group.

[0040] (6) By Figure 1 It can be seen that a strain DMC-X3 with good MC-LR degradation ability was isolated. The strain grew slowly in the MSM liquid medium containing MC-LR and reached the growth stability phase on the 8th day. At this time, OD 600 =0.28. On the other hand, the degradation rate of MC-LR increased rapidly with the growth of strain DMC-X3, reaching 90.33% within 24 h, and 200 μg / L MC-LR was completely degraded within 48 h.

[0041] (7) The morphological characteristics of MC-LR degrading bacteria DMC-X3 are shown in Figure 2 The genome of strain DMC-X3 was extracted and the 16S rRNA gene was amplified. The PCR product was sent to Xiamen Borui Biotechnology for sequencing. After removing the primers, the sequence was uploaded to the EzBioCloud website in South Korea (https: / / www.ezbiocloud.net / ) for comparison analysis. The neighbor-joining method of MEGA7.0 software was used to construct the phylogenetic tree. The results are shown in Figure 2 (A). After comparison and identification, strain DMC-X3 and Stenotrophomonas geniculata The similarity rate is as high as 100%, so the strain DMC-X3 belongs to the genus Stenotrophomonas and is named Stenotrophomonas geniculata DMC-X3, whose 16S rRNA sequence was uploaded to the NCBI database, has the GenBank accession number ON999189.

[0042] 2. Study on the degradation characteristics of strain DMC-X3

[0043] 1. Degradation of MC-LR by DMC-X3 at different concentrations

[0044] The strain DMC-X3 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 30°C and 150 rpm in the dark for 36 hours. The bacterial precipitate was collected after high-speed centrifugation at 6000 rpm for 6 minutes, 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.

[0045] DMC-X3 bacterial suspension was prepared at 3×10 6 , 6 × 10 6 , 1.2×10 7 , 2.4×10 7 , 4.8×10 7 The concentration of CFU / mL was inoculated into MSM inorganic salt liquid culture medium containing 200 μg / L MC-LR. Three biological replicates were set for each treatment group. The culture was shaken at 30°C and 150 rpm in the dark. Samples were taken every 4 h, and the toxin concentration was detected using a microcystin detection kit. The MC-LR degradation rate was calculated according to Formula 1.

[0046] Degradation rate (%) = (C0-C t ) / C t ×100% (Formula 1)

[0047] Where C0 is the concentration of MC-LR at 0 h; C t is the concentration of MC-LR corresponding to the treatment time.

[0048] Depend on Figure 3 It can be seen that there is a concentration gradient effect in the degradation effect of DMC-X3 on MC-LR. As the concentration of bacterial solution increases, the degradation effect increases. 6 , 6 × 10 6 , 1.2×10 7 , 2.4×10 7 , 4.8×10 7 The degradation rates of DMC-X3 to MC-LR in the CFU / mL inoculation dose groups were 60.39%, 74.95%, 88.55%, 90.31% and 94.17%, respectively. However, at 48 h, except for 3×10 6 The degradation rate of DMC-X3 to MC-LR in the CFU / mL inoculation dose group was 79.13%, and the degradation rates of other inoculation dose groups were all above 90%.

[0049] The total bacterial count in water is often used as an important indicator for assessing the degree of water pollution. When using MC-LR degrading bacteria for large-scale water purification, excessively high strain inoculation levels can cause certain pollution to the surrounding water. Compared with other oligotrophic monocytogenes (Table 1), the DMC-X3 used in this invention can still efficiently degrade MC-LR at low inoculation levels, showing certain advantages.

[0050] Table 1 Comparison of degradation ability of DMC-X3 and other oligotrophic monocytogenes

[0051]

[0052] 100% of the rest of the world:

[0053] 1. Yang , F , Zhou , YL , Yin , LH , et al. Microcystin-degradingactivity of an indigenous bacterial strain Stenotrophomonas acidaminiphilaMC-LTH2 isolated from Lake Taihu. Plos One, 2014, 9(1):e8 http: / / doi.org / 10.1371 / journal.pone.0086216.

[0054] 2. Idroos, ES, De Silva, B., Manage, PM Biodegradation ofmicrocystin analogues by Stenotrophomonas maltophilia isolated from BeiraLake Sri Lanka. Journal of the National Science Foundation of Sri Lanka,2017, 45(2):91-99. http: / / doi.org / 10.4038 / jnsfsr.v45i2.8175.

[0055] 3. Krishnan, A., Zhang, YQ, Mou, XZ Isolation and characterization of microcystin-degrading bacteria from Lake Erie. Bulletinof Environmental Contamination and Toxicology, 2018, 101(5):617-623. http: / / doi.org / 10.1007 / s00128-018-2468-4.

[0056] 2. DMC-X3 is a free MC-LR mount

[0057] The DMC-X3 bacterial suspension was inoculated into MSM inorganic salt liquid medium containing 200, 400, 800, and 1000 μg / L MC-LR, respectively. The inoculation dose of the strain was 1.7×10 7 CFU / mL (OD 600 =0.1), with three biological replicates in each group, cultured at 30°C, 150 rpm in the dark, and sampled every 4 h. The concentration of the toxin was detected using a microcystin detection kit, and the MC-LR degradation rate was calculated according to Formula 1. The results showed that ( Figure 4 ), the degradation effect of DMC-X3 was affected by different concentrations of MC-LR: strain DMC-X3 could degrade 200, 400, 800, and 1000 μg / L MC-LR by 99.96%, 91.80%, 91.37%, and 90.66%, respectively, within 48 h, indicating that DMC-X3 has good MC-LR degradation performance and great potential in the treatment of polluted water.

[0058] 3. Effects of pH and temperature on the degradation of MC-LR by DMC-X3

[0059] HCl and NaOH were used to adjust the pH of the MSM inorganic salt liquid medium to 5, 6, 7, 8, and 9, respectively. The DMC-X3 bacterial suspension was inoculated into the MSM inorganic salt liquid medium containing 200 μg / L MC-LR at different pH values, so that the initial concentration was 1.7×10 7 CFU / mL (OD 600 =0.1), with three replicates per group, cultured at 30°C, 150 rpm, and protected from light. Samples were taken every 4 h, and the toxin concentration was detected using a microcystin detection kit. The MC-LR degradation rate was calculated according to Formula 1. The efficiency of DMC-X3 in degrading MC-LR under different pH conditions is shown in Figure 1. Figure 5 As shown in the figure, at pH 7, the MC-LR degradation rates were 90.33% and 99.96% after 24 and 48 h, respectively. At pH 8, the MC-LR degradation rates were 86.20% and 98.35% after 24 and 48 h, respectively, which were not significantly different from the degradation rates at pH 7. This indicates that the optimal pH for MC-LR degradation by DMC-X3 is between 7 and 8. At pH 9, the MC-LR degradation rates decreased to 70.40% and 92.94% after 24 and 48 h, respectively. Furthermore, pH 5 and 6 were also not conducive to the degradation of MC-LR by DMC-X3, with MC-LR degradation rates as low as 71.25% and 82.96% after 48 h, respectively.

[0060] (1) The DMC-X3 bacterial suspension was inoculated into the MSM inorganic salt liquid medium containing 200 μg / L MC-LR to an initial concentration of 1.7×10 7 CFU / mL (OD 600 =0.1), and cultured at 20, 25, 30, 35, and 40°C, with three biological replicates for each temperature group. Cultures were shaken at 150 rpm in the dark, and samples were taken every 4 h. The concentration of the toxin was detected using a microcystin detection kit, and the MC-LR degradation rate was calculated according to Formula 1. The results are shown in Figure 1. Figure 6 As shown, the optimal temperature for DMC-X3 to degrade MC-LR is 30°C. At this temperature, DMC-X3 degraded 90.33% of MC-LR within 24 hours. At 35°C and 40°C, DMC-X3 achieved 88.15% and 84.26% degradation of MC-LR within 24 hours, respectively. However, at 20°C and 25°C, the degradation rates dropped to 63.23% and 76.91% within 24 hours. However, at 48 hours, with the exception of the 20°C treatment, which achieved a MC-LR degradation rate of 81.21%, the degradation rates of the 25, 30, 35, and 40°C treatments all exceeded 90%, reaching 90.36%, 99.96%, 98.42%, and 96.75%, respectively.

[0061] 3. Preparation of immobilized bacterial agent and detection of its MC-LR degradation ability

[0062] (1) Preparation of bacterial suspension: Stenotrophomonas geniculata DMC-X3 was inoculated into 20 mL of MSM inorganic salt liquid medium with 200 μg / L MC-LR as the sole carbon and nitrogen source. The culture was incubated in a shaker (30°C, 150 rpm) in the dark until the strain reached the stationary phase. A 10 mL suspension of the stationary phase culture was centrifuged at 6000 rpm for 6 minutes, the supernatant discarded, and the suspension resuspended in MSM inorganic salt liquid medium to obtain a DMC-X3 bacterial suspension.

[0063] (2) Preparation of sodium alginate (SA) solution: Weigh 2 g of sodium alginate and dissolve it in 98 mL of distilled water. Heat and stir with a magnetic stirrer until dissolved to obtain a 2% sodium alginate solution. Sterilize at high temperature and high pressure for later use.

[0064] (3) Preparation of CaCl2 solution: Weigh 2 g of CaCl2 solid, dissolve it in 98 mL of distilled water, and filter sterilize it using a 0.22 μm filter membrane to obtain a 2% CaCl2 solution;

[0065] (4) Preparation of immobilized bacterial agent: Mix 2 mL of 2% sodium alginate solution with an equal amount of DMC-X3 bacterial suspension, mix thoroughly to obtain SA-DMC-X3 mixed solution, and set aside. Use a sterile syringe to draw the SA-DMC-X3 mixed solution from a high place drop by drop into a beaker containing 50 mL of cross-linking agent 2% CaCl2 solution (each bacterial pellet can embed 5.0×10 7 DMC-X3 bacterial cells) were placed in a 4°C refrigerator for immobilization and crosslinking for 6 h. A blank pellet containing no bacteria, DMC-X1, was prepared as a control by mixing a 2% sodium alginate solution with an equal volume of MSM inorganic salt liquid medium in the same manner.

[0066] (5) After immobilization and cross-linking, remove the beaker from the 4°C refrigerator and wash the microbial pellets several times with sterile water;

[0067] (6) Air-dry the pellets at 25-30°C and store at room temperature for later use.

[0068] (7) 2, 4, 6, 8, and 20 DMC-X3 pellets were added to 20 mL of MSM inorganic salt medium containing 200 μg / L MC-LR, and the corresponding free bacterial counts were 2.5×10 6 , 5.0×10 6 , 7.5×10 6 、10.0×10 6 , 2.5×10 7 CFU / mL, with three biological replicates in each group. Equal amounts of free bacteria and blank pellets were used as controls. The cells were cultured statically at 30°C in the dark. Samples were taken every 4 h. The concentration of the toxin was detected using a microcystin detection kit, and the MC-LR degradation rate was calculated according to Formula 1.

[0069] (8) If Figure 7As shown in Table 2, within 24 h, the degradation rates of MC-LR in the treatment groups with 2, 4, 6, 8 and 20 DMC-X3 microbial pellets were 53.10%, 64.84%, 71.51%, 78.42% and 87.92%, respectively, while the degradation rates of the corresponding free bacteria were 56.61%, 69.31%, 76.12%, 82.13% and 90.31%, respectively. This shows that different amounts of immobilized microbial pellets have high efficiency in degrading MC-LR, and their degradation rates are only slightly weaker than those of free bacteria at the same dose. In addition, the more immobilized microbial pellets are added, the better the degradation effect on MC-LR. As treatment time increased, after 48 hours, the degradation rates of the treatments containing 6, 8, and 20 DMC-X3 pellets showed no significant difference from those of the free bacteria, all exceeding 90% (90.01%, 91.96%, and 96.29%, respectively). This suggests that the DMC-X3 immobilized pellets are highly efficient in degrading MC-LR, which is beneficial for the preservation and field application of the degrading bacteria DMC-X3.

[0070] Table 2. Comparison of MC-LR degradation rates

[0071]

[0072] 4. Detection of the recycling ability of immobilized bacterial agents

[0073] (1) First use of immobilized bacterial agent: Take 20 prepared immobilized bacterial agent pellets (each bacterial agent pellet contains 5.0×10 7 DMC-X3 bacterial cells), the pellets were added to 20 mL MSM inorganic salt medium containing 200 μg / L MC-LR, and the same number of blank pellets were added as the control group. All treatments were set up with 3 biological replicates, and the cells were cultured statically at 30°C in the dark. Samples were taken every 4 hours, and the toxin concentration was detected using a microcystin detection kit, and the MC-LR degradation rate was calculated according to Formula 1. Figure 8 As shown in (A), when the immobilized beads were used for the first time, 87.76% of MC-LR could be degraded within 24 hours, and 96.29% of MC-LR could be degraded within 48 hours.

[0074] (2) Second use of the immobilized bacterial agent: The immobilized bacterial agent pellets and blank pellets used in step (1) were removed from the culture medium, washed three times with sterile water, and then air-dried at room temperature (25-30°C). The air-dried immobilized pellets were stored at room temperature for 3 days before the second MC-LR degradation experiment. The specific steps refer to the above step (1). Figure 8As shown in (B), when the immobilized beads were reused for the second time, the degradation rate of MC-LR was 66.93% in 24 h and 93.12% in 48 h.

[0075] (3) The third use of the immobilized bacterial agent: The immobilized bacterial agent pellets and blank pellets used in step (2) were removed from the culture medium, washed three times with sterile water, and then air-dried at room temperature (25-30°C). The air-dried immobilized pellets were stored at room temperature for 5 days before the second MC-LR degradation experiment. The specific steps are as follows (1). Figure 8 As shown in (C), the degradation ability of the immobilized microspheres on MC-LR was significantly reduced after the third reuse, with the degradation rate being only 55.35% after 24 h. However, after 48 h, the immobilized microbial agent microspheres still maintained a high degradation ability on MC-LR, with a degradation rate of 83.06%.

[0076] In summary, the immobilized bacterial agent formed by encapsulating the MC-LR degrading bacteria DMC-X3 with sodium alginate still maintained a good MC-LR degradation ability after being reused three times, indicating that the bacterial agent is expected to be used for large-scale sewage treatment.

[0077] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A strain of Oligotrophomonas DMC-X3 capable of degrading microcystin, characterized by: The classification of the Stenotrophomonas DMC-X3 is named Stenotrophomonas geniculata , the deposit number is GDMCC NO.62597.

2. Use of the oligotrophomonas DMC-X3 according to claim 1 in the treatment of water contaminated by microcystin-LR.

3. An immobilized bacterial agent, characterized in that: The invention is obtained by using sodium alginate as a carrier and calcium chloride as a cross-linking agent to embed the oligotrophomonas DMC-X3 described in claim 1.

4. The method for preparing the immobilized bacterial agent according to claim 3, characterized in that: The specific steps are as follows: (1) First, the oligotrophic monocytogenes DMC-X3 was inoculated into MSM inorganic salt liquid culture medium, with microcystin-LR as the only carbon and nitrogen source, cultured on a shaking table, centrifuged to obtain a precipitate, and then resuspended in MSM inorganic salt liquid culture medium to obtain a bacterial suspension; (2) Then, thoroughly mix the 2% sodium alginate aqueous solution and the bacterial suspension of equal volume to obtain a mixed solution. Then, drop the mixed solution dropwise into a 2% calcium chloride aqueous solution and immobilize and cross-link at 4°C for 4 to 6 hours to obtain a small spherical immobilized bacterial agent. Wash with sterile water and air-dry at room temperature.

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

Images