Construction method of manganese oxidation biological membrane system and application of manganese oxidation biological membrane system in antibiotic removal

Abstract

The invention belongs to the technical field of environmental microbiological engineering, and relates to a construction method of a manganese oxidation biological membrane system and application of the manganese oxidation biological membrane system in antibiotic removal, firstly manganese sand is filled in a filter column, then pseudomonas putida MnB1 bacterial liquid is pumped into the filter column, simulated sewage is continuously pumped into the filter column, incubation is performed at the room temperature at the flow rate of 1 mL * min <-1 > for 7 days, and then the manganese oxidation biological membrane system is obtained. Enabling the pseudomonas putida MnB1 to be tightly adhered to the manganese sand to form a biological membrane, so as to obtain a manganese oxidation biological membrane filter column; the bottom of the manganese oxidation biological membrane filter column is connected with a peristaltic pump; the top of the manganese oxidation biofilm filter column is connected with an oxygen pump and a water outlet pipe. According to the method, the antibiotics can be effectively removed only by using the manganese sand and the pseudomonas putida MnB1, and the removal efficiency is high; the manganese oxidation biological membrane system is simple in structure, easy to operate and low in preparation cost, has long-term effectiveness and low degradation risk, and has practical application value and wide prospects in the aspect of removing trace emerging pollutants.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microbial engineering, and relates to a method for constructing a manganese oxidation biofilm system and an application thereof in removing antibiotics. Background Art

[0002] The overuse of antibiotics (as an emerging pollutant) in aquatic ecosystems not only poses a threat to the health and safety of organisms and damages the ecological environment, but also has the potential to produce drug-resistant pathogens. Therefore, there is an urgent need to control antibiotics in aquatic environments and protect the biological environment. Conventional wastewater treatment cannot remove most antibiotics, resulting in their detection in surface water. Among the many technologies for removing antibiotics, membrane bioreactors combine membrane separation with biological treatment technology. Due to its effective capture function and ability to harbor microorganisms for a long time, it can improve the removal efficiency of antibiotics and other drugs and enable deep purification of wastewater. Manganese sand filter material is a filter material with manganese dioxide as the main component. Due to its strong adsorption capacity and the autocatalytic oxidation of Mn(II) in the presence of manganese oxides, manganese sand is widely used as a biofilter in water plants in China. Manganese oxidizing bacteria oxidize Mn(II) to Mn(III) or Mn(IV), which is generally used to remove manganese or other metal elements. However, there are no reports on the combined use of manganese oxidizing bacteria and manganese sand for the removal of antibiotics. Summary of the invention

[0003] The purpose of the present invention is to solve the technical problem that trace antibiotics in existing sewage are difficult to degrade and remove. The present invention provides a manganese oxidation biofilm system formed by the aggregation of Pseudomonas putida strain MnB1 on the surface of manganese sand, which can effectively remove pollutants such as doxycycline in sewage and realize the purification process of sewage such as medical waste water.

[0004] In order to achieve the above object, the technical scheme adopted by the present invention is: a method for constructing a manganese oxidation biofilm system, wherein the manganese oxidation biofilm system is an upward reactor, wherein manganese sand is first filled in a filter column, and then a Pseudomonas putida MnB1 bacterial solution is pumped into the filter column, and simulated sewage is continuously pumped into the filter column at room temperature at 1 mL min -1 The column was incubated at a flow rate of for 7 days to allow Pseudomonas putida MnB1 to adhere tightly to the manganese sand and form a biofilm, thereby obtaining a manganese oxidation biofilm filter column; a peristaltic pump was connected to the bottom of the manganese oxidation biofilm filter column, and the peristaltic pump was connected to the solution to be filtered; an oxygen pump and a water outlet pipe were connected to the top of the manganese oxidation biofilm filter column, thereby obtaining a manganese oxidation biofilm system.

[0005] The oxygen pump continuously delivers oxygen to ensure an aerobic environment for the microorganisms.

[0006] The simulated sewage formula is: 100 mg·L -1Glucose: 2.8 mg·L -1 K 2 HPO 4 ; 0.2mg·L -1 MgSO 4 ·

[0007] 7H 2 O; 0.4mg·L -1 CaCl 2 ; 0.7mg·L -1 NaCl; 60.6mg·L -1 NaNO 3 The average chemical oxygen demand (COD) value of the simulated effluent was 40.13.

[0008] The volume of the Pseudomonas putida MnB1 bacterial solution is 50 ml, and the amount of manganese sand used is 295.362 g, accounting for 60% of the total volume of the filter column.

[0009] The Pseudomonas putida MnB1 bacterial solution is obtained by inoculating Pseudomonas putida MnB1 into a conical flask containing 50 mL of sterile activation culture medium, and aerobically culturing the mixture on a shaker at 30° C. and 150 rpm for 12 hours.

[0010] The Pseudomonas putida MnB1 used in the present invention was purchased from the American Type Culture Collection (ATCC) with a deposit number of ATCC 23483.

[0011] Preferably, the strain oxidizes Mn(II) to Mn(III) or Mn(IV), stimulating manganese cycling.

[0012] Preferably, the strain biologically oxidizes Mn(II) faster to produce manganese oxides.

[0013] Preferably, the manganese sand contains trace mineral manganese required for the growth of many organisms, as well as many other essential elements.

[0014] Preferably, Pseudomonas putida MnB1 gathers and reproduces on the surface of manganese sand which is uneven and has many holes, forming a film-like biological community and a biofilm.

[0015] Preferably, the manganese oxidizing biofilm oxidizes and effectively removes antibiotics in sewage.

[0016] The present invention also provides the use of the manganese oxidation biofilm system in removing antibiotics, using a peristaltic pump at 1 mL·

[0017] min -1The antibiotic-containing wastewater is continuously pumped upward through the filter column at a constant flow rate (i.e., 2.6 rpm); at the same time, the oxygen pump continuously delivers oxygen to ensure an aerobic environment for the microorganisms. The manganese oxidation biofilm system can be used to purify medical wastewater contaminated by antibiotics.

[0018] The antibiotics described in the present invention are doxycycline, moxifloxacin or ofloxacin.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention combines membrane separation with biological treatment technology to construct a manganese oxidation biofilm reactor, and uses the Pseudomonas putida strain MnB1 that can quickly oxidize Mn(II) to produce manganese oxides to form a manganese oxidation biofilm on the surface of manganese sand filter material rich in elements such as manganese and iron, effectively removing emerging pollutants such as doxycycline in sewage, thereby purifying medical wastewater contaminated by antibiotics. The present invention can effectively remove antibiotics using only manganese sand and Pseudomonas putida MnB1, and the removal efficiency is high; the constructed manganese oxidation biofilm system has a simple structure, is easy to operate, has a low preparation cost, has long-term effectiveness and low degradation risk, and has been continuously operated for 206 days, with a 10mg·L -1 The maximum removal efficiency of doxycycline reaches 99.62%, which can be applied on a large scale. The present invention is used for the purification of medical wastewater. Within 30 days of continuous operation, the average removal rate of doxycycline is as high as 97.71%, and the average removal rates of moxifloxacin and ofloxacin are 89.32% and 81.6%, respectively. The present invention uses biodegradation to repair the environment polluted by difficult-to-degrade organic pollutants, and has practical application value and broad prospects in removing trace emerging pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Photos of the manganese sand filter column system and the constructed manganese oxidation biofilm system; a: filter column containing only manganese sand; b: used to remove 0.1 mg·L -1 Manganese oxidation biofilm filtration column for doxycycline; c: used to remove 0.5 mg·L -1 Manganese oxidation biofilm filtration column for doxycycline; d: used to remove 1.0 mg·L -1 Manganese oxidation biofilm filtration column for doxycycline; e: used to remove 10 mg·L -1 Manganese oxidation biofilm filtration column for doxycycline; f: Manganese oxidation biofilm filtration column for purification of medical wastewater; g: Manganese oxidation biofilm system.

[0022] Figure 2The experimental results of the removal of doxycycline by manganese oxides produced by Pseudomonas MnB1 through manganese oxidation; a: the removal rate of doxycycline by manganese oxides extracted from MnB1 at different culture times; b: the removal rate of doxycycline by manganese oxides extracted from different volumes of culture medium after culturing MnB1 strain for 13 hours; c: the removal rate of doxycycline by manganese oxides after different treatment times.

[0023] Figure 3 is the removal efficiency of manganese oxidation biofilm filtration column for different concentrations of doxycycline in water, C 0 and C t represent the initial doxycycline concentration and the doxycycline concentration at time t, respectively.

[0024] Figure 4 Species analysis of microbial communities; a: Species analysis of microbial communities in pure manganese sand filter column (control group) and manganese oxidizing biofilm filter column system treated with different concentrations of doxycycline (red represents Pseudomonas, of which Pseudomonas putida MnB1 belongs to this genus); b: Heat map of microbial species in the biofilm system.

[0025] Figure 5 Manganese oxidation biofilm system for medical wastewater purification; a: Removal efficiency of doxycycline, ofloxacin and moxifloxacin by manganese oxidation biofilm system; b: Analysis of microbial species and relative abundance after medical wastewater treatment.

[0026] Figure 6 Schematic diagram of the structure of the constructed manganese oxidation biofilm system. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0028] Example 1: Study on the culture of Pseudomonas putida MnB1 and its optimal functional conditions

[0029] The Pseudomonas putida MnB1 used in this example was purchased from the American Type Culture Collection (ATCC) with the accession number ATCC 23483. First, Pseudomonas putida MnB1 was activated in an activation medium (30°C, 150 rpm) for 12 hours, and then inoculated into a fresh growth medium (G / M medium) at a volume ratio of 1%; in order to simulate the environment in which Pseudomonas putida MnB1 oxidizes manganese, 1 mg·L -1 MnCl 2 . And used in the following screening study of the optimal conditions for removing doxycycline.

[0030] The activation medium formula is: deionized water (DIW); 3g·L-1 Beef powder; 10g·L -1 Casein amino acids; 5g·L -1 NaCl.

[0031] The growth medium (G / M medium) formula is: deionized water (DIW); 0.5 g·L -1 Yeast extract; 0.5 g·L -1 Casein amino acids; 1g·L -1 Glucose: 2.38 g·L -1 HEPEs buffer solution (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid); 0.222 g·L -1 CaCl 2 ; 0.8118 g·L -1 MgSO 4 7H 2 O; 0.001g·L -1 FeCl 3 6H 2 O; 1mg·L -1 Trace element solution (6.4 mg·L -1 CuSO 4 ; 44mg·L -1 ZnSO 4 7H 2 O; 20mg·L -1 CoCl 2 6H 2 O; 13mg·L - 1 Na 2 MoO 4 ·2H 2 O).

[0032] The MnB1 culture solution (containing manganese oxide precipitate) was extracted at different incubation times (0-18 h) to evaluate its ability to remove 1 mg·L -1 The most effective extraction time was determined by centrifuging the extracted MnB1 culture solution at 10,000 rpm for 5 min to obtain manganese oxide precipitate, which was then mixed with a 1 mg·L -1 The doxycycline aqueous solution was mixed and shaken in a vertical shaking incubator for 0.5 hours. The manganese oxide precipitate and the treated doxycycline solution were separated by a 0.22 μm filter membrane. The concentration of the remaining doxycycline in the eluate after removal was detected by HPLC, and the removal rate was calculated; the results are shown in Figure 2 As shown in a. Figure 2a It can be seen that after 13 hours of incubation with MnB1, the extracted manganese oxide showed the highest doxycycline removal efficiency (66.6%). Subsequently, under the optimal incubation time of 13 h, the optimal extraction volume (8-48 mL) of the MnB1 culture medium of Pseudomonas putida was determined. Specifically, different volumes of MnB1 growth medium were aspirated, centrifuged to obtain manganese oxide, and the obtained manganese oxide precipitate was added to a 1 mg·L -1 After 0.5 hours of oscillation reaction in a doxycycline aqueous solution, the solution was separated by a 0.22 μm filter membrane, and the concentration of doxycycline was detected by HPLC, and the removal rate was calculated. Figure 2 As shown in b. Figure 2 b It can be seen that when the volume of MnB1 medium reached 24 mL, the removal rate reached 89.01%, and then the removal effect almost reached a stable state. Under the optimal extraction volume of 24 mL and incubation time of 13 h, the optimal removal time was determined by increasing the removal time from 0 to 6 h. The results are shown in Figure 2 c. The results showed that as the treatment time of manganese oxide increased, the removal efficiency of doxycycline increased and stabilized at about 2 hours.

[0033] From the above experimental results, it can be seen that Pseudomonas putida MnB1 can produce manganese oxides under the action of manganese, and manganese oxides have the effect of removing antibiotics.

[0034] Example 2: Construction and use of manganese oxidation biofilm system

[0035] A filtration column system with biofilm formed by Pseudomonas putida MnB1 on manganese sand was designed for continuous removal of antibiotics from water.

[0036] (1) Pseudomonas putida MnB1 was inoculated into a conical flask containing 50 mL of sterile activated medium and cultured aerobically at 150 rpm on a shaker at 30°C for 12 h;

[0037] (2) An acrylic column with a height of 20 cm and an inner diameter of 5 cm was used as a filter column, and 60 v / v% (i.e., 295.36 g) of manganese sand (equivalent to a column bed height of 12 cm) was filled in the filter column. 50 mL of the cultured Pseudomonas putida MnB1 solution was stirred at 20 mL min -1 The simulated sewage was pumped into the filter column through a peristaltic pump (LabN1) at a flow rate of 1 mL min-1, and then the simulated sewage was continuously pumped into the adsorption column at room temperature. -1 The column was incubated at a flow rate of 100 for 7 days to allow Pseudomonas putida MnB1 to adhere tightly to the manganese sand and form a biofilm, which is a manganese oxidation biofilm filter column. During this period, oxygen was continuously supplied by a continuous aeration system. Both the bacterial solution and sewage were pumped in from the bottom of the column upward;

[0038] The simulated sewage formula is: 100 mg·L -1 Glucose: 2.8 mg·L -1 K 2 HPO 4 ; 0.2mg·L -1 MgSO 4 ·

[0039] 7H 2 O; 0.4mg·L -1 CaCl 2 ; 0.7mg·L -1 NaCl; 60.6mg·L -1 NaNO 3 The average chemical oxygen demand (COD) value of the simulated effluent was 40.13.

[0040] (3) Constructing a manganese oxidation biofilm system: The bottom of the manganese oxidation biofilm filter column is connected to a peristaltic pump, which is connected to the solution to be filtered; the top of the manganese oxidation biofilm filter column is connected to an oxygen pump and a water outlet pipe, which is the manganese oxidation biofilm system. The method of use is: use a peristaltic pump at 1 mL min -1 The doxycycline solution or medical wastewater is continuously pumped upward at a constant flow rate to pass through the manganese oxidation biofilm filter column; at the same time, the oxygen pump continuously delivers oxygen to ensure an aerobic environment for the microorganisms. The manganese oxidation biofilm system is operated at room temperature (about 20°C). The effluent is collected once a day. The pH value of the effluent sample is almost the same as that of the influent solution (pH7.0±0.2), indicating that there is no significant change in pH value in the entire filter column system and no acid-base change is involved.

[0041] The collected effluent samples were filtered through a 0.45 μm filter membrane, and then the concentration of doxycycline in the treated water samples and the concentration of other antibiotics in medical wastewater were analyzed using a high-performance liquid chromatography (HPLC) system (LC-20A, Shimadzu, Japan) to determine the removal of doxycycline and other antibiotics.

[0042] Test conditions for doxycycline: isocratic elution analysis of doxycycline in samples. Liquid chromatograph (LC-20A, Shimadzu, Japan), chromatographic column: ODS-3C18, 5 μm, 4.6×150 mm. The detection wavelength is 280 nm. The detection mobile phase is MeOH: 0.01 M oxalic acid (55:45, v:v), and the flow rate is 1 mL min -1 The injection volume was 15 μL. The column temperature was 35°C. The peak time was 2.1 min.

[0043] After sampling, moxifloxacin and ofloxacin need to be concentrated to near dryness by nitrogen purging at 40°C, and then re-dissolved to 1 mL using HPLC mobile phase, vortexed for 1 min, transferred to the injection bottle and tested on the machine. Detection method of moxifloxacin and ofloxacin: Samples collected from the outlet of the manganese oxidation biofilm system are analyzed by isocratic elution by high performance liquid chromatography. Chromatographic column: ODS-3C18, 5μm, 4.6×150mm. The detection wavelength is set to 290nm. The mobile phase used for analysis is methanol: 0.05% trifluoroacetic acid (TFA) (38:62, v / v), with a flow rate of 1.1mL·min -1 The injection volume was 50 μL. The column chamber temperature was 45°C. Standard solution (100 μg ml each) -1 ) was prepared with methanol, stored in a brown reagent bottle at -20°C, and diluted with the mobile phase in a gradient manner.

[0044] (2) Effect of removing doxycycline from wastewater by manganese oxidation biofilm system

[0045] The five manganese oxidation biofilm systems were used to continuously filter and treat simulated sewage containing different concentrations of doxycycline according to the above method; a filter column system consisting of manganese sand alone was used as a control to treat 1.0 mg·L -1 Doxycycline; Figure 1 The actual photographic images of the constructed manganese oxidation biofilm system are provided. All the filter column systems were operated continuously for 206 days. The removal rate of doxycycline at different times during the 206 days was tested, and the results are as follows: Figure 3 shown.

[0046] from Figure 3 It can be seen that within 206 days, the average removal rate of doxycycline in the control group was 21.54%. -1 , 0.5mg·L -1 , 1.0mg·L -1 and 10mg·L -1 The average removal efficiency of doxycycline at different concentrations was 82.36%, 84.16%, 91.35% and 93.25%, respectively. Correspondingly, the maximum removal efficiency reached 94.58%, 95.12%, 97.43% and 99.62%. These data indicate that the manganese oxidizing biofilm system exhibits a high efficiency in removing doxycycline. Given its long-term effectiveness and low degradation risk, the biosystem has greater application potential in water pollutant purification.

[0047] The 16S rRNA gene sequencing was used to analyze the microbial species composition of the pure manganese sand filter column system (as a control group) and the microbial species composition of the manganese oxidizing biofilm system before and after treating simulated sewage containing doxycycline. Figure 4 As shown. Figure 4 It can be seen that no Pseudomonas bacteria were detected in the control group; however, the relative abundance of Pseudomonas in the manganese oxidizing biofilm obtained after the Pseudomonas putida MnB1 of the Pseudomonas genus was pumped into the manganese sand column system and cultured for 7 days (i.e. before doxycycline treatment) reached 44.92%. When the doxycycline concentration increased, the relative abundance of Pseudomonas gradually decreased, while the microbial diversity increased significantly ( Figure 4 ), which may contribute to the stability of the microbial community and promote more effective doxycycline removal.

[0048] (3) Application of manganese oxide biofilm filtration column system to remove doxycycline and other antibiotics from medical wastewater

[0049] The manganese oxidation biofilm system of the present invention was used to treat doxycycline in the medical wastewater of Qingdao University Affiliated Hospital, and the concentration of doxycycline (DOX) was determined by HPLC. The results are as follows Figure 5 As shown in a. The results show that the manganese oxidation biofilm system exhibits a very efficient and long-lasting removal ability for doxycycline in medical wastewater. Within 30 days, the average removal rate of doxycycline was as high as 97.71%, and the maximum and minimum removal rates were 98.49% and 96.91%, respectively. In addition, the manganese oxidation biofilm system of the present invention has the ability to remove other typical high-concentration antibiotics in medical wastewater (such as moxifloxacin MOX and ofloxacin OFLO). Figure 5 As shown in a, the removal efficiency of the system for the antibiotics moxifloxacin and ofloxacin was relatively high, with the average removal rates reaching 89.32% and 81.6% respectively within 30 days, indicating the application value of the manganese oxidation biofilm system in medical wastewater purification.

[0050] Figure 5 b shows the composition of microbial species in the biofilm of the manganese oxidation biofilm system used to remove medical wastewater. Figure 4 ), the microbial species in the manganese-oxidizing biofilm after actual medical wastewater treatment were also richer, and the relative abundance of Pseudomonas, including Pseudomonas putida MnB1, was also higher, accounting for 15.89% of all microorganisms. The above studies show that the system has a considerable degree of universality in effectively removing antibiotics from water bodies and has a wide range of application potential in treating emerging pollutants.

Claims

1. A method for constructing a manganese oxidation biofilm system, characterized in that: The manganese oxidation biofilm system is an upward reactor, in which manganese sand is first filled into the filter column, and then the Pseudomonas putida MnB1 bacterial solution is pumped into the filter column, and simulated sewage is continuously pumped into the filter column at room temperature at 1 mL min -1 The column was incubated at a flow rate of for 7 days to allow Pseudomonas putida MnB1 to adhere tightly to the manganese sand and form a biofilm, thereby obtaining a manganese oxidation biofilm filter column; a peristaltic pump was connected to the bottom of the manganese oxidation biofilm filter column; and an oxygen pump and a water outlet pipe were connected to the top of the manganese oxidation biofilm filter column.

2. The method for constructing a manganese oxidation biofilm system according to claim 1, characterized in that: The oxygen pump continuously delivers oxygen to ensure an aerobic environment for the microorganisms.

3. The method for constructing a manganese oxidation biofilm system according to claim 1, characterized in that: The simulated sewage formula is: 100 mg·L -1 Glucose: 2.8 mg·L -1 K2HPO4; 0.2mg·L -1 MgSO4·7H2O; 0.4mg·L -1 CaCl2; 0.7mg·L -1 NaCl; 60.6mg·L -1 NaNO3.

4. The method for constructing a manganese oxidation biofilm system according to claim 1, characterized in that: The volume of the Pseudomonas putida MnB1 bacterial solution is 50 ml, and the amount of manganese sand used is 295.362 g, accounting for 60% of the total volume of the filter column.

5. The method for constructing a manganese oxidation biofilm system according to claim 1, characterized in that: The Pseudomonas putida MnB1 bacterial solution is obtained by inoculating Pseudomonas putida MnB1 into a conical flask containing 50 mL of sterile activation culture medium, and aerobically culturing the mixture on a shaker at 30° C. and 150 rpm for 12 hours.

6. The manganese oxidation biofilm system obtained by the construction method according to any one of claims 1 to 5.

7. Use of the manganese oxide biofilm system according to claim 6 in removing antibiotics, characterized in that: Can be used to purify wastewater contaminated by antibiotics.

8. The use of the manganese oxide biofilm system in removing antibiotics according to claim 7, characterized in that: A peristaltic pump was used at 1 mL min -1 The antibiotic-containing wastewater is continuously pumped upward through the filter column at a constant flow rate; at the same time, the oxygen pump continuously delivers oxygen to ensure an aerobic environment for the microorganisms.

9. The use of the manganese oxide biofilm system in removing antibiotics according to claim 7, characterized in that: The antibiotic is doxycycline, moxifloxacin or ofloxacin.

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