Immobilized algal-bacterial symbiotic system and application of immobilized algal-bacterial symbiotic system in harmless treatment of spiramycin production wastewater

Abstract

The invention provides an immobilized algal-bacterial symbiotic system and application thereof in harmless treatment of spiramycin production wastewater, and belongs to the technical field of environmental microorganisms. The algal-bacterial symbiotic system comprises bacillus subtilis with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 15560 and microcystis aeruginosa FACHB-315. The immobilized microorganism product prepared from the algal-bacterial symbiotic system and the biological adsorption carrier ball has the following advantages: (1) the floatability of microalgae cells and the negative effects caused by discharge of free cells into natural water can be overcome; (2) the SPI removal rate can reach 98.49%, and the SPI removal capacity is high. Therefore, the method can effectively reduce ecological hazards and the like caused by the SPI entering the environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microorganisms, and in particular relates to an immobilized bacteria-algae symbiotic system, an immobilized microorganism product prepared using the same, and their application in harmless treatment of spiramycin production wastewater. Background Art

[0002] Antibiotics are widely used to treat and prevent various diseases caused by bacterial infections. However, neither humans nor animals can fully absorb antibiotics, and about 70% to 90% of the unmetabolized part will enter the municipal wastewater treatment plants (MWWTPs) through excreta. During the production and application process, a large amount of antibiotics are discharged into the natural environment. It is reported that every ton of antibiotic products produced will produce about 150 to 200 m 3 Industrial wastewater with high concentrations of antibiotics. As municipal wastewater treatment plants cannot completely remove antibiotics from their water, various antibiotics and their metabolites have been found in countries such as India, China, South Korea, Israel, and the United States. They can be detected in sewage, surface water, groundwater, sediments, soil, and sometimes even drinking water. Antibiotics in the environment may cause ecological crises, damage to human health, and other serious problems by disrupting sewage treatment processes, inhibiting the proliferation of beneficial bacteria, changing microbial composition and metabolism, and inducing antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Antibiotic resistance genes are transferred between the environment and human microbiota, posing a serious ecological threat. Spiramycin (SPI) is one of the most common antibiotic contaminants.

[0003] SPI is very stable and hardly undergoes hydrolysis under ambient conditions. Various physical and chemical methods have been used to solve this problem. The commonly used physical removal method, adsorption, requires expensive adsorption materials and does not destroy the antibiotics, but only transfers them. Large amounts of chemical materials, excessive energy consumption, and catalysts (TiO 2 ) and the toxicity of SPI degradation intermediates (such as carcinogenic NDMA) limit the application of chemical removal methods. Microbial methods are cost-effective and environmentally friendly, and are one of the most promising methods for removing antibiotics. According to literature reports, the degradation rate of SPI with an initial concentration of 50 ng / L by Microcystis aeruginosa strains after 7 days of treatment was 12.5% ​​to 32.9%. However, microalgae cells tend to float on the water surface, which limits their application in actual sewage treatment plants. In addition, free bacteria and microalgae may be discharged into natural water bodies and cause potential ecological threats due to their antibiotic resistance genes. Immobilized microorganisms provide an effective option to solve this problem.

[0004] Therefore, the immobilized microorganism method based on the bacteria-algae symbiosis system (MBC) with multiple mechanisms such as biodegradation, biosorption, bioaccumulation and abiotic processes is a cost-effective antibiotic removal method, and this method also has broad application prospects in the removal of antibiotic resistance genes. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an immobilized bacteria-algae symbiotic system and its application in the harmless treatment of spiramycin production wastewater. The bacteria-algae symbiotic system in the present invention includes Bacillus subtilis with a preservation number of CGMCC No. 15560 and Microcystis aeruginosa FACHB-315. The immobilized microbial product made by using the bacteria-algae symbiotic system and biological adsorption carrier balls in the present invention has the following advantages: (1) It can overcome the floating nature of microalgae cells and the adverse consequences of free cells being discharged into natural water bodies; (2) The SPI removal rate can be as high as 98.49%, and the SPI removal ability is strong. Therefore, the method in the present invention can effectively reduce the ecological hazards caused by SPI entering the environment.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] One of the purposes of the present invention is to provide a bacteria-algae symbiotic system, which includes Bacillus subtilis (B. subtilis) and Microcystis aeruginosa (M. aeruginosa) FACHB-315; the preservation number of the Bacillus subtilis is CGMCC No.15560; the cell density ratio of the Bacillus subtilis and the Microcystis aeruginosa is (8-12):1.

[0008] The Bacillus subtilis used in the present invention is a Bacillus subtilis with a high degradation rate for SPI. The strain is isolated from the aerobic pool of the Henan Tianfang Pharmaceutical Wastewater Treatment Plant and has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration. The deposit time is April 8, 2018, the deposit number is CGMCC No. 15560, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101.

[0009] The second object of the present invention is to provide a method for constructing the bacteria-algae symbiotic system, which includes the steps of preparing Bacillus subtilis culture solution and Microcystis aeruginosa culture solution, co-immobilizing the two microorganisms, and co-culturing the co-immobilized microorganisms in spiramycin simulated wastewater.

[0010] Furthermore, the step of preparing the Bacillus subtilis culture solution includes an activation culture step and an expansion culture step of the Bacillus subtilis.

[0011] Furthermore, the step of preparing the culture solution of Microcystis aeruginosa is the step of activating the strain of Microcystis aeruginosa, and the activation step comprises: inoculating the strain into a BG11 liquid culture medium, providing a strength of 178 to 222 μmol / m 2 / s of light and CO with a volume ratio of 4-6% and a flow rate of 0.4-0.6 gas / liquid / minute VVM 2 , cultured at 130-150rpm and 24-26℃ for 2-4 days.

[0012] Furthermore, the spiramycin content in the spiramycin simulated wastewater is 500-550 mg / L, and the co-cultivation time is 4-6 days.

[0013] In the present invention, the mixed culture solution of Bacillus subtilis and Microcystis aeruginosa is inoculated into the spiramycin simulated wastewater for a period of time, so as to adapt the microbial inoculum.

[0014] The third object of the present invention is to provide an immobilized microorganism, which includes the bacteria-algae symbiotic system or the bacteria-algae symbiotic system constructed using the construction method.

[0015] Furthermore, the immobilized microorganisms are immobilized with a bio-absorption carrier ball as the immobilized microorganisms; the initial concentrations of bacteria and algae in the immobilized microorganisms are: Bacillus subtilis (1-5) × 10 8 CFU / g, Microcystis aeruginosa (1~5)×10 7 cells / g.

[0016] The fourth object of the present invention is to provide a method for preparing the immobilized microorganism, which comprises the following steps: firstly, loading activated carbon sponge into polyurethane hollow balls to obtain biological adsorption carrier balls, then placing them into spiramycin simulated wastewater, inoculating 8-12% volume ratio of bacteria-algae symbiotic system, culturing for 45-50 hours, and finally air-drying, storing and standby; wherein the volume ratio of bacteria to algae in the bacteria-algae symbiotic system is (0.8-1.2): (0.8-1.2).

[0017] Furthermore, the culture conditions include: a temperature of 24-26°C, an aeration speed of 0.8-1.2 VVM, and a concentration of 178-222 μmol / m 2 / s of light.

[0018] The fifth object of the present invention is to provide an application of the immobilized bacteria-algae symbiotic system in the harmless treatment of spiramycin wastewater.

[0019] The beneficial effects of the present invention are:

[0020] The present invention: (1) can overcome the adverse consequences caused by the floating nature of microalgae cells and the discharge of free cells into natural water bodies; (2) can achieve an SPI removal rate of up to 98.49%, and has a strong SPI removal capacity. Therefore, the method of the present invention can effectively reduce the ecological harm caused by SPI entering the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a picture of the raw materials used in the preparation of the biosorbent carrier in the present invention, wherein: a polyurethane hollow sphere, b activated carbon sponge;

[0022] Figure 2 is the DO immobilized microorganism concentration change curve in Example 3 of the present invention;

[0023] Figure 3 : are the growth curves of Microcystis aeruginosa (a) and Bacillus subtilis (b) in Example 3 of the present invention;

[0024] Figure 4 is the concentration variation curve of chlorophyll a of Microcystis aeruginosa in Example 3 of the present invention;

[0025] Figure 5 This is the kinetic curve of SPI removal by the BMC system in Example 4 of the present invention;

[0026] Figure 6 The content change of the degradation intermediate detected by HPLC-(+)ESI-MS in Example 5 of the present invention;

[0027] Figure 7 This is a schematic diagram of the SPI degradation pathway proposed in Example 5 of the present invention;

[0028] Figure 8 The abundance changes of RAs of ARGs during the LG biological treatment process in Example 6 of the present invention;

[0029] Fig. 9 These are the results of the effects of SPM on the luminescence of Vibrio fischeri before and after pretreatment in Example 7 of the present invention. DETAILED DESCRIPTION

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. The products, reagents, instruments, equipment, etc. used in the following examples can all be obtained commercially, and the methods used are consistent with the conventional methods unless otherwise specified.

[0031] The SPI standard matrix used in the present invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. BG11 liquid and agar culture medium were purchased from Shandong Top Bioengineering Co., Ltd. Vibrio fischeri water quality biological toxicity detection kit was purchased from Zhejiang Tsinghua Yangtze River Delta Research Institute. Polyurethane hollow spheres (Φ20 mm, mesh surface) and activated carbon sponge ( Figure 1 ) was purchased from Henan Huayu Water Purification Material Factory, Zhengzhou, China. The biosorption carrier spheres were prepared by filling hollow spheres with activated carbon sponge.

[0032] The culture medium formulas used in the present invention are as follows:

[0033] The broth medium formula is: 10g / L peptone, 1g / L beef extract, 5g / L glucose and 5g / L NaCl, pH value is 7.40±0.1; it needs to be autoclaved at 121℃ for 20min.

[0034] The artificial sewage simulated the SPI production sewage of Tianfang Pharmaceutical Co., Ltd. in Zhumadian City, Henan Province. The simulated sewage contained 525.0 mg / L SPI, 2335 mg / L glucose, 47.6 mg / L NH 4 Cl, 32.5mg / L KH 2 PO 4 , 20mg / L MgSO 4· 7H 2 O, 2.5 mg / L CaCl 2 , 0.55mg / L FeSO 4 7H 2 O, pH 8.0. The characteristics of the artificial sewage are as follows (mg / L): COD 3500 ± 305, NH 4 + -N 16.02±0.51,PO 4 3- -P 7.41±0.58, pH 4.5, DO 2.32±0.25.

[0035] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0036] Example 1 Analysis of SPI and its degradation products

[0037] 1. SPI concentration analysis

[0038] The culture broth samples for HPLC analysis of SPI concentration were centrifuged at 8000 rpm for 10 min and then filtered with a 0.45 μm filter membrane. HPLC separation was performed at 30°C using a TC-C18 separation column (Agilent, 4.6×250 mm, 5 μm). The mobile phase flowed at a flow rate of 1 mL / min and consisted of a mixture of acetonitrile and 0.05 mol / L ammonium acetate in a volume ratio of 60:40. The fraction at 232 nm was continuously monitored. The SPI concentration was linearly related to the peak area of ​​the HPLC spectrum in the range of 0 to 10 mg / L, and the linear regression equation was Y=33264X+954574 (R2=0.9965).

[0039] 2. Analysis of SPI degradation products

[0040] The degradation pathway of SPI by BMC system was studied by HPLC / ESI-QqQMS (composed of Agilent 1260HPLC and AB Sciex Q-Trap 5500MS). SPI and its biodegradation products were separated using Kinetex RP C18 column (2.6 μm, 50 mm×2.1 mm).

[0041] Example 2 Preparation of co-immobilized microorganisms

[0042] The Bacillus subtilis slant strain was inoculated into the broth culture medium, and after activation culture at 200 rpm and 37°C for 24 hours, the activated culture solution was inoculated into the broth culture at a volume ratio of 10%, and expanded culture was carried out at 200 rpm and 37°C for 24 hours.

[0043] Microcystis aeruginosa (M. aeruginosa) FACHB-315, which has the ability to remove SPI in preliminary tests, was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. The freeze-dried tube strain of the algae was inoculated into BG11 liquid culture medium and cultured at 140 rpm and 25°C for 3 days for activation. During the activation process, the intensity of (200±22) μmol / m 2 / s light and 5% (v / v) CO 2 (Flow rate is 0.5 gas / liquid / minute VVM).

[0044] The culture broth of Bacillus subtilis and Microcystis aeruginosa was inoculated into the simulated SPWW at a ratio of 10% (volume ratio) and cultured for 5 days under the above conditions to allow the microbial inoculum to adapt. 100 biosorption carrier balls were placed in a 20L simulated SPWW inoculated with 5% (v / v) bacteria, 5% (v / v) microalgae, or 5% (v / v) bacteria + 5% (v / v) microalgae (to ensure bacterial-algal symbiosis), ensuring that the ratio of carrier to wastewater was 1:23.9. The 25°C, aeration (1VVM wind speed) and light (200±22) μmol / m2 / s) for 48 h, then air-dried and stored at 4°C for later use. The initial cell concentrations in the carriers were: 3.03×10 8 CFU / g of Bacillus subtilis, and 3.08×10 7 cells / g of Microcystis aeruginosa, and the immobilized bacteria-algae co-culture system contained 3.03×10 8 cells / g of Bacillus subtilis and 3.08×10 7 cells / g of Microcystis aeruginosa. Blank carrier spheres were used as controls.

[0045] Example 3 Comparison of the growth of immobilized microorganisms

[0046] The experiments were conducted under the following four conditions: (1) wastewater containing blank spheres as control; (2) wastewater containing immobilized cells of Bacillus subtilis; (3) wastewater containing immobilized cells of Microcystis aeruginosa; and (4) wastewater containing co-immobilized cells of two microorganisms. The carrier: wastewater ratio was 1:30 (v / v). All experiments were conducted at 25°C with lighting and aeration throughout the process. The pH and DO of the wastewater were monitored regularly every 6 h for up to 48 h. Figure 2 ).

[0047] The results showed that the immobilized microbial strains Microcystis aeruginosa and Bacillus subtilis showed similar growth trends ( Figure 3 ). The growth started in the lag phase, followed by the exponential growth phase, and then maintained a nearly constant growth rate until the end of the experiment. The growth rates of the cells of both microorganisms in the co-immobilized BMC system were faster than those of the single-immobilized control test. After 48 hours of cultivation, the final cell concentrations of Microcystis aeruginosa and Bacillus subtilis in the co-immobilized algae symbiotic system reached 8.84×10 8 cells / mL and 9.45×10 9 CFU / mL. Figure 3 It can be seen that in artificial sewage containing 525 mg / L SPI, the immobilized Microcystis aeruginosa, immobilized Bacillus subtilis and co-immobilized BMC systems grew well. Chlorophyll a concentration change curve ( Figure 4 ) is similar to the concentration change curve of Microcystis aeruginosa. After 48 hours of culture, the chlorophyll a concentration of the co-immobilized treatment (3.86 pg / cell) was slightly higher than that of the immobilized Microcystis aeruginosa (3.32 pg / cell), which is consistent with the growth curve of the immobilized Microcystis aeruginosa ( Figure 3 a).

[0048] Example 4 Performance and mechanism of removing spiramycin from sewage by bacterial-algal symbiotic system

[0049] The experiment was carried out for 6 days under the same conditions as Example 3. In addition to the light group (Light group, LG), a dark group (Dark group, DG by wrapping the flask with aluminum foil) was set up to study the removal of SPI in the absence of photosynthesis. In order to study the role of environmental microorganisms and abiotic factors in the removal of SPI, sewage control (EC, sterile non-inoculated SPWWTPE) and pure water control (WC, sterile non-inoculated ultrapure water) experiments were carried out under the same conditions as LG. All experiments were repeated three times. During the experiment, carrier samples were taken daily to monitor the changes in ARGs. The pH and DO of the sewage system were also monitored every day. In addition, each experimental sewage was sampled every day to monitor SPI and its degradation intermediates (DIs). The biosorption, bioaccumulation and biodegradation of SPI were evaluated by analyzing the SPI concentration at the end of the biological treatment. The carrier balls collected on the last day were crushed into 1 mL 3 About 1000 μg of particles were mixed with 9 times the volume of sterile water, ultrasonically treated (40kHz, 2.2Kw) for 30 minutes at 25°C, and filtered with sterile filtered water. Repeat the above carrier pretreatment operation 3 times. The obtained filtrates were combined and centrifuged at 5000rpm for 20 minutes. Then, the supernatant was subjected to low-temperature distribution extraction to obtain the extract product used to determine the concentration of SPI adsorbed on the cell surface. The cell pellet was suspended in 5mL of dichloromethane-methanol (1:2v / v), ultrasonically treated for 30 minutes, and then centrifuged at 4000rpm for 10 minutes. The SPI concentration in the supernatant is the concentration of bioaccumulated SPI.

[0050] The biodegradation rate (Bd) of SPI was calculated by formula (1):

[0051] Bd(%)=(At-Ar-Ad-Ap-Ac)×100 / At(1)

[0052] At and Ar are the initial and final SPI concentrations in the biological treatment system, respectively. Ad is the concentration of SPI adsorbed by cells, Ap is the amount of SPI removed by photodegradation (result of WC), and Ac is the concentration of SPI bioaccumulated in cells.

[0053] The SPI degradation rate constant of the BMC system is calculated by formula (2):

[0054] lnCt=-kt+lnC0(2)

[0055] C0 and Ct are the SPI concentrations at time 0 and time t, respectively, and k is the degradation rate constant (d -1 ), t is the degradation duration.

[0056] The results showed that 92.7% and 83.6% of SPI were degraded in the LG and DG experiments, respectively, after 7 days of biological treatment (Table 1). The trends of SPI removal in LG and DG were similar ( Figure 5 ). The SPI degradation curve decreased rapidly in the first 24 h and then decreased at a smaller slope in the following 5 days. The potential reasons for this trend may be that in the first stage, sufficient SPI-degrading enzymes could rapidly degrade SPI, and subsequently, the related enzymes were inhibited by the degradation intermediates. Another reason is that SPI was rapidly absorbed by bacterial cells and then slowly degraded. After 144 h of biological treatment, the SPI removal rates of LG and DG reached 98.49% and 84.05%, respectively. The kinetic removal rate constant (k) of SPI in LG was 0.15 / d, which was higher than that in DG (0.128). The half-life values of SPI in LG and DG were 3.43 d and 4.09 d, respectively, indicating that the half-life of SPI in LG was shorter than that in DG (Table 1). This result shows that the photodegradation caused by autotrophic microalgae in the BMC system significantly promoted the degradation of SPI.

[0057] Table 1 Mass balance of SPI removal in the BMC system (%)

[0058]

[0059] <MQL = below the method quantification limit

[0060] Example 5 SPI biodegradation pathway in the BMC system

[0061] The mass-to-charge ratio (m / z), formula, and proposed chemical structures of SPI and its degradation intermediates (DIs) are listed in Table 2.

[0062] Table 2 Chemical formulas, molecular weights, mass-to-charge ratios, and chemical structures of SPI and its degradation intermediates.

[0063]

[0064] During the whole biodegradation process, the peak area changes of 6 DIs are as Figure 6 shown. With the progress of SPI degradation, the peak areas of detected DIs showed a trend of first increasing and then slowly decreasing, except for DI384. And DI384 continuously increased during the degradation process. In addition, the peak intensity of DI830 was the highest but decreased significantly later. Based on the identified DIs, two SPI degradation pathways were proposed: the glycosidic bond hydrolysis pathway and the demethylation pathway ( Figure 7 ).

[0065] The molecular structure of SPI is composed of a 16-membered lactone ring with a fulvesamine group and a mycamine-mycamine disaccharide group connected by a glucosidic bond. It is speculated that the fulvesamine or mycamine group is hydrolyzed first to produce DI702 or P2DI699, which loses the mycamine or fulvesamine group in the next step and is converted to DI558. Then DI558 loses mycamine and is converted to DI384. On the other hand, DI702 may also lose the disaccharide fragment composed of mycamine and mycamine and be directly converted to DI384. Mycamine, fulvesamine and fulvesamine that fall off from the parent compound may be metabolized by bacteria once they fall off, so they are not detected. Finally, all SPI intermediates are mineralized into carbon dioxide and water, and Bacillus subtilis strains can produce extracellular glycosidases.

[0066] In the demethylation pathway, one methyl group in the dimethylamine (DMA) group attached to the fulvestrant or mycamino group is first cleaved to form two isomeric intermediates DI829-1 or DI829-2, which lose the second methyl group in the other DMA group and are converted to DI815.

[0067] Example 6 SPI resistance gene assessment

[0068] The vectors sampled daily in the LG experiment were pretreated using the method described in Example 5 to obtain cell pellets. DNA was extracted using the DNeasy PowerSoil Kit Its concentration was detected by Ultra-micro UV spectrophotometer (Quawell Q3000, USA). With sterile water as negative control, PCR was performed with the primers listed in Table 3 to detect 15 ARGs, including 8 rRNA methylase genes (ermA, ermB, ermC, ermF, ermG, ermQ, ermT, ermX), 3 efflux genes (msrA, msrD, mefA), 2 esterase genes (ereA, ereB), 2 phosphorylase genes (mphA, mphB) and 3 mobile elements (intI1, Tn916 / 1545, ISCR1).

[0069] Four ARGs (ermB, ermF, ermT, msrD), two mobile elements (intI1, Tn916 / 1545) and 16SrRNA gene were detected by SYBR-Green real-time fluorescence quantitative PCR.

[0070] For primers, please refer to the literature: Liu, M., Ding, R., Zhang, Y., Gao, Y., Tian, ​​Z., Zhang, T., Yang, M., 2014. Abundance and distribution of Macrolide-Lincosamide-Streptograminresistance genes in an anaerobic-aerobic system treating spiramycinproduction wastewater.Water.Res.63,33-41.https: / / doi.org / 10.1016 / j.watres.2014.05.045.

[0071] The standard plasmid concentration (ng / mL) was detected by Nanodrop ND-1000 (Nanodrop, USA) and converted to measure the copy number μL -1 The absolute abundances (gene copies per sample size) of MLS resistance genes and mobile elements are shown in Table 3 .

[0072] Table 3 Changes of 16srRNA, AAs and RAs after 6 days of biological treatment

[0073]

[0074] To reduce the differences caused by bacterial concentration and DNA extraction efficiency, we normalized the copy numbers of MLS resistance genes and mobile elements to the relative abundance of the 16S rRNA gene. During the 6-day biotreatment, the total relative abundance of ARGs ranged between 4.65 and 9.50 copies / 16s rRNA. The mobile factors ranged between 0.96 and 6.76 copies / 16s rRNA. The most highly expressed rRNA methylase gene was ermB, followed by ermF and ermT, and msrD had a lower expression level of RA. intI1 was the main mobile factor.

[0075] Changes of ARGs and RAs of intI1 treated with LG Figure 8As shown. The results showed that during the 6-day biological treatment, the RAs of total GRAs and intI1 increased significantly (2.04-fold and 7.47-fold, respectively), especially on the second day. For individual genes, the efflux gene msrD showed a downward trend throughout the process. In contrast, the RAs of other ARGs increased during the biological treatment. The resistance genes ermB, ermF, and ermT were positively correlated with class 1 integrons and Tn916 / 1545, suggesting that there was horizontal transfer during their proliferation. The increase in ARGs and intI1 suggests that the microorganisms used in this study may pose potential risks to the ecological environment.

[0076] Example 7 Acute toxicity assessment of spiramycin degradation products

[0077] Using Promega -Multi Jr (TextS3) was used to measure the bioluminescence inhibition of Vibrio fischeri to study the acute toxicity of SPI biotreatment products. The specific operation was carried out according to the instructions. L0 and Lt are the luminescence intensity of the wastewater samples before and after treatment, respectively. Lt / L0 can be used to evaluate acute toxicity. The results showed that the change trend of the acute toxicity of SPI degradation products was opposite to the change trend of SPI concentration, showing a logarithmic curve ( Fig. 9 In addition, the luminescence intensity values ​​of the sewage sample collected at 144 h were similar to those of deionized water (Table 4), indicating that the degradation products of SPI did not have antibacterial properties.

[0078] Table 4 Luminescence intensity of SPM samples treated with BMC system and deionized water

[0079]

[0080] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A bacteria-algae symbiotic system, characterized in that: The bacteria-algae symbiotic system comprises Bacillus subtilis (B. subtilis) and Microcystis aeruginosa (M. aeruginosa) FACHB-315; the preservation number of the Bacillus subtilis is CGMCC No. 15560; the cell density ratio of the Bacillus subtilis and the Microcystis aeruginosa is (8-12):

1.

2. The method for constructing the bacteria-algae symbiotic system according to claim 1, characterized in that: The construction method comprises the steps of preparing a culture solution of Bacillus subtilis and a culture solution of Microcystis aeruginosa, a co-immobilization step of the two microorganisms and a co-culturing step of the co-immobilized microorganisms in spiramycin simulated wastewater.

3. The construction method according to claim 2, characterized in that: The preparation steps of the Bacillus subtilis culture solution include an activation culture step and an expansion culture step of the Bacillus subtilis.

4. The construction method according to claim 3, characterized in that: The preparation step of the culture solution of Microcystis aeruginosa is the activation step of the Microcystis aeruginosa strain, and the activation step includes: inoculating the strain into a BG11 liquid culture medium, providing a strength of 178-222 μmol / m 2 / s of light and a volume ratio of 4-6% and a flow rate of 0.4-0.6 gas / liquid / minute VVM of CO2 are cultured at 130-150rpm and 24-26℃ for 2-4 days.

5. The construction method according to claim 4, characterized in that: The spiramycin content in the spiramycin simulated wastewater is 500-550 mg / L, and the co-cultivation time is 4-6 days.

6. An immobilized microorganism, characterized in that: It includes the bacteria-algae symbiotic system according to claim 1 or the bacteria-algae symbiotic system constructed using the construction method according to any one of claims 2 to 5.

7. The immobilized microorganism according to claim 6, characterized in that The immobilized microorganisms are immobilized with biological adsorption carrier balls as the immobilized materials. The initial concentrations of bacteria and algae in the immobilized microorganisms are: Bacillus subtilis (1-5) × 10 8 CFU / g, Microcystis aeruginosa (1~5)×10 7 cells / g.

8. The method for preparing the immobilized microorganism according to claim 7, characterized in that: The preparation method comprises the following steps: firstly, loading activated carbon sponge into polyurethane hollow balls to obtain biological adsorption carrier balls, then placing the balls into spiramycin simulated wastewater, inoculating 8-12% by volume of a bacteria-algae symbiotic system, culturing for 45-50 hours, and finally air-drying, storing, and using for later use; wherein the volume ratio of bacteria to algae in the bacteria-algae symbiotic system is (0.8-1.2): (0.8-1.2).

9. The preparation method according to claim 8, characterized in that: The culture conditions include: a temperature of 24-26°C, an aeration wind speed of 0.8-1.2 VVM, and a pressure of 178-222 μmol / m 2 / s of light.

10. Use of the bacteria-algae symbiotic system of claim 1, the method for constructing the bacteria-algae symbiotic system of any one of claims 2 to 5, the immobilized microorganism of any one of claims 6 to 7, or the method for preparing the immobilized microorganism of any one of claims 8 to 9 in the harmless treatment of spiramycin wastewater.

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