Application of a filamentous microalgae in removing enrofloxacin from water

Abstract

The application discloses application of a filamentous microalgae in removal of enrofloxacin in water bodies. The application is obtained by the inventors of the application who find that Nostoc sp.SCAU-13 has the effect of removing enrofloxacin in water bodies. The algae strain can grow in water bodies containing enrofloxacin and can efficiently remove enrofloxacin, which indicates that the algae has the application potential in removing antibiotics in sewage. The application not only provides a biological treatment method of enrofloxacin, but also is easy to collect and separate from water bodies, so that subsequent treatment of the water bodies is simple.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment of pollutants, and specifically relates to the application of a filamentous microalga in the removal of enrofloxacin from water. Background Technology

[0002] Enrofloxacin (ENR), also known as enrofloxacin, is a quinolone antibiotic commonly used in livestock and aquaculture. It is characterized by its broad-spectrum antibacterial activity, strong antibacterial activity, high lipophilicity, strong accumulation, persistence, high biotoxicity, and difficulty in degradation. Surveys have revealed varying levels of quinolone antibiotics in rivers, sewage treatment plants, and waterworks both domestically and internationally, with concentrations reaching μg / L and even mg / L. Studies show that enrofloxacin is difficult to degrade and accumulates in fat; large-scale use can lead to long-term accumulation in animals and transmission to humans through the food chain. Its entry into the environment can easily lead to the development of antibiotic-resistant bacteria and resistance genes, thus posing a potential threat to human health and environmental safety. The most effective and commonly used methods for treating antibiotic contamination are chemical and physical methods, but these carry the risk of secondary pollution and are costly. For low-concentration antibiotic contamination, environmentally friendly and inexpensive biological treatment methods have greater application potential. Studies have shown that many microorganisms have the ability to enrich, remove, or degrade antibiotics, making them a crucial biological treatment method. Recent research also indicates that microalgae show promising applications in treating pollutants such as antibiotics in water bodies. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of a filamentous microalga in the removal of enrofloxacin from water.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] The application of a filamentous microalga in removing enrofloxacin from water is based on the inventor's discovery that a filamentous microalga can remove enrofloxacin from water, and that the growth rate of the filamentous microalga is not affected by enrofloxacin.

[0006] The name of the filamentous microalgae is Nostoc sp.SCAU-13.

[0007] The application of the above-mentioned filamentous microalgae in the removal of enrofloxacin from water includes the following steps: adding filamentous microalgae strains or filamentous microalgae solutions to enrofloxacin-contaminated water for cultivation to remove enrofloxacin.

[0008] The filamentous microalgae culture solution is a filamentous microalgae culture solution that has been cultured to the logarithmic growth phase or the stationary phase; preferably, it is obtained by the following steps: inoculating the filamentous microalgae seed into the culture medium and culturing it to the logarithmic growth phase or the stationary phase.

[0009] The culture is preferably carried out at 25-30℃, light intensity of 8000-10000 lux, and light-dark time of 10-14h:10-14h; more preferably, it is carried out at 28±0.5℃, light intensity of 9000±1000 lux, and light-dark time of 12h:12h.

[0010] The culture medium is preferably BG110 liquid culture medium.

[0011] The composition of BG110 liquid culture medium is as follows: K2HPO4 4. 3H₂O 0.04g / L, MgSO₄ 4. 7H2O 0.075g / L, CaCl 2. 2H2O 0.036g / L, citric acid 0.006g / L, ferric ammonium citrate 0.006g / L, EDTA 0.001g / L, Na2CO3 0.02g / L, trace element A5 1mL.

[0012] The composition of trace element A5 is as follows: H3BO3 2.860 g / L, NaMoO 4. 2H₂O 0.021g / L, ZnSO₄ . 7H2O 0.222g / L, CuSO4 . 5H₂O 0.079 g / L, MnCl₂ . 4H₂O 1.810 g / L, NiSO₄ . 6H2O 0.479g / L.

[0013] The amount of filamentous microalgae species or microalgae solution added is based on the OD of algal cells in the water. 680 Calculated as 0.1 to 0.2; more preferably based on the OD of algal strains in the water. 680 Calculated as 0.2.

[0014] The present invention has the following advantages and effects compared with the prior art:

[0015] This invention discovers that Nostoc sp. SCAU-13 can efficiently remove enrofloxacin, thus demonstrating its potential application in eliminating antibiotics from wastewater. This not only provides a biological treatment method for enrofloxacin, but also makes Nostoc sp. SCAU-13 easy to collect, readily separable from water bodies, and simple to treat subsequently. Attached Figure Description

[0016] Figure 1 This is a growth curve of three algae in BG110 medium containing 200 μg / L enrofloxacin.

[0017] Figure 2 This is a graph showing the removal effects of different algae on enrofloxacin; where A represents extracellular space and B represents intracellular space.

[0018] Figure 3 These are images showing the effect of different algae on enrofloxacin removal on day 6.

[0019] Figure 4 The OD of different concentrations of enrofloxacin on Nostoc sp. SCAU-13 680 The effect of Chla content and chlorophyll fluorescence parameters is shown in the figure.

[0020] Figure 5 This is a photograph of an experiment using the filter paper filtration method to collect algal cells. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0022] The filamentous microalgae *Nostoc* sp. SCAU-13 (also known as *Nostoc* sp. strain J), isolated, purified, and identified in our laboratory, has been described in the literature "Lu YZ, Zhuo C, Li YJ, Li HS, Yang MY, Xu DN, He HZ". * The 16S RNA sequence of this algae (disclosed in "Evaluation of filamentous heterocystous cyanobacteria for integrated pig-farm biogasslurry treatment and bioenergy production. Bioresource Technology, 2020, 297:122418, DOI:10.1016 / j.biortech.2019.122418") is deposited on BG110 solid agar plates. The accession number is OR874847.1.

[0023] The single-celled algae *Chlamydomonas* sp. GX2 and *Chlorella* sp. HS02 were isolated, purified, and preserved in our laboratory.

[0024] The composition of BG110 liquid culture medium is as follows: K2HPO4 4. 3H₂O 0.04g / L, MgSO₄ 4. 7H2O 0.075g / L, CaCl 2.2H2O 0.036g / L, citric acid 0.006g / L, ferric ammonium citrate 0.006g / L, EDTA 0.001g / L, Na2CO3 0.02g / L, trace element A5 1mL.

[0025] The composition of trace element A5 is as follows: H3BO3 2.860 g / L, NaMoO 4. 2H₂O 0.021g / L, ZnSO₄ . 7H2O 0.222g / L, CuSO4 . 5H₂O 0.079 g / L, MnCl₂ . 4H₂O 1.810 g / L, NiSO₄ . 6H2O 0.479g / L.

[0026] BG11 medium is BG110 liquid medium with 1.6 g / L Na2NO3 added.

[0027] Example 1: Evaluation of the enrofloxacin removal capacity of three algae

[0028] Algal activation: The unicellular algae *Chlamydomonas* sp. GX2 and *Chlorella* sp. HS02, and *Nostoc* sp. SCAU-13 were activated to evaluate their growth and enrofloxacin removal capacity on media containing enrofloxacin. *Chlamydomonas* sp. GX2 and *Chlorella* sp. HS02 were tested using BG11 medium, while *Nostoc* sp. SCAU-13 was tested using BG110 medium.

[0029] (1) SCAU-13 and GX2 and HS02 algae were inoculated into sterile BG110 and BG11 liquid culture media, respectively. The cultures were incubated at a temperature of 28.0±0.5℃, a light intensity of 9000±1000 lux, and a day-night ratio of 12h:12h. The cultures were shaken 4 to 6 times a day to obtain GX2, HS02 and SCAU-13 algae solutions in the logarithmic growth phase.

[0030] (2) BG110 and BG11 liquid culture media were separately placed into 100 mL glass Erlenmeyer flasks, each containing 40 mL of liquid culture media. The flasks were sterilized at 121 °C for 16 min. After cooling, enrofloxacin was added to achieve a final concentration of 200 μg / L, thus creating enrofloxacin-containing culture media. Simultaneously, two experimental groups were set up: a CK-light group (using BG110 liquid culture media) without algae and a CK-dark group (using BG110 liquid culture media) without algae and in the dark. A total of 5 experimental groups were set up, with 3 replicates for each group. Algal cultures of the three algae in the logarithmic growth phase were inoculated into the enrofloxacin-containing culture media, and the final algal concentration was set at OD0.05. 680 =0.1, incubator temperature 28.0±0.5℃, light intensity 9000±1000 lux, day-night ratio = 12h:12h, shake well 4-6 times a day.

[0031] (3) Samples were taken on days 0, 2, 4, and 6 of the experiment to measure the growth indicators (OD) of the three algae. 680 The concentration of enrofloxacin in the culture medium and within the microalgal cells. Growth indicators for the three algae are as follows: Figure 1 As shown, the concentration of enrofloxacin in the culture medium and within the algal cells is as follows: Figure 2 As shown in (a) and (b) in the figure, and from Figure 3 It can be seen that SCAU-13 has the highest ability to remove enrofloxacin from the culture medium, reaching 73.2%. Subsequently, this algal strain was used to conduct enrofloxacin removal experiments.

[0032] Enrofloxacin assay method: Take 1 mL of algal solution, filter it through a 0.22 μm microporous membrane, then filter it through a 0.22 μm aqueous filter, and transfer it to a sample bottle for storage at -20℃ until analysis. Simultaneously, collect the filtered algal cells, add 7 mL of a mixture of methane and methanol (1:2 volume ratio), and treat the mixture in an ultrasonic cleaner at 30℃, 40 kHz, and 2.2 KW for 1 hour to promote cell lysis and release of cell sap. Then, centrifuge the mixture at 8000 rpm for 10 minutes. Collect 1 mL of the supernatant, filter it through a 0.22 μm aqueous filter, and transfer it to a reagent bottle for storage at -20℃ until analysis.

[0033] The residues of 17 sulfonamides and 15 quinolones in aquatic products were analyzed according to the Ministry of Agriculture Announcement No. 1077-1-2008. Liquid chromatography-tandem mass spectrometry (Thermofisher Ultimate 3000 HPLC + AB 4500) was used. Chromatographic conditions were: Acclaim™ RSLC 120C18 column (2.2 μm, 2.1 × 100 mm); flow rate: 0.30 mL / min; column temperature: 40℃; injection volume: 10 μL; mobile phase: A was 0.1% formic acid aqueous solution, and B was acetonitrile. Gradient elution conditions are shown in Table 1. Mass spectrometry conditions were: electrospray ionization (ESI); multiple reaction monitoring (MRM); positive ion mode; spray voltage: 55 kV; curtain gas: 30 psi; collision gas: High; temperature: 550℃; GS1: 55 psi; GS2: 55 psi.

[0034] Table 1 Gradient elution conditions

[0035]

[0036] Example 2: Evaluation of the removal capacity of filamentous microalgae Nostoc sp. SCAU-13 for different concentrations of enrofloxacin

[0037] 100 mL of BG110 medium was transferred to a 250 mL Erlenmeyer flask and sterilized at 121 °C for 16 min. After cooling, enrofloxacin was added to achieve final concentrations of 0, 25, and 100 μg / L, creating enrofloxacin-containing media. Two experimental groups were also established: a light group (enrofloxacin without algae) and a dark group (enrofloxacin without algae). A total of five experimental groups were set up, with three replicates for each group. SCAU-13 microalgae in the logarithmic growth phase were inoculated into each group, and the final algal concentration was set at OD0.05. 680 =0.2. The specific steps are the same as those in Example 1.

[0038] Samples were taken on days 0, 2, and 4 of the experiment to determine the OD680 and Chla content of filamentous microalgae in the culture medium. The results are as follows: Figure 4 As shown. From Figure 4 It was found that the algal strain Nostoc sp. SCAU-13 grew well in the enrofloxacin-containing culture medium, and the chlorophyll fluorescence parameters Fv / Fm and Pi_Abs values ​​were not significantly different from those in the enrofloxacin-free culture medium. Table 2 shows the concentration and removal rate of enrofloxacin in the media containing 25 μg / L and 100 μg / L. Table 2 shows that the removal rate reached 80.2% on day 4 in the 25 μg / L enrofloxacin medium, and 68.7% on day 4 in the 100 μg / L enrofloxacin medium.

[0039] Method for determining Chla content: Take 6 mL of algal solution and place it in a 10 mL plastic tube. Centrifuge and discard the supernatant. Add 6 mL of ice-cold 80% acetone and extract at 4°C for 24 hours. Then centrifuge the solution at 6000 rpm for 10 minutes to remove cell debris and measure the OD. 663 and OD 646 , of which OD 646 and OD 663 These represent the absorbance at 646 and 663 nm, respectively. The calculation formula is: C Chla =12.21×OD 663 -2.81×OD 646 .

[0040] Table 2. Results of Nostoc sp. SCAU-13's removal effect on enrofloxacin at concentrations of 25 μg / L and 100 μg / L after 4 days of treatment.

[0041]

[0042] Example 3: Collection of enrofloxacin after treatment with filamentous microalgae Nostoc sp. SCAU-13

[0043] The algae treatment experiment was conducted using the same method as in Example 2. After 4 days of treatment, the algae solution was collected and the OD value was measured. 680 The algal solution was then filtered through a layer of qualitative filter paper, and the OD of the filtrate was measured. 680 . with OD 680 Represents algal biomass, expressed as OD before and after filtration. 680 The difference in OD values ​​of unfiltered algal solution 680 The percentage value represents the algae recovery rate using the filter paper filtration method.

[0044] Experiments on collecting algal cells using filter paper filtration method, such as Figure 5 As shown in Table 3, the cell yield reached 90.9 ± 4.5%, indicating that the vast majority of algal cells can be collected by a simple filter paper filtration method.

[0045] Table 3. Results of algal cell collection on day 4 using the filter paper filtration method.

[0046]

[0047] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a filamentous microalga in the removal of enrofloxacin from water, characterized by: The name of the filamentous microalgae is Nostoc sp.SCAU-13.

2. The application of filamentous microalgae according to claim 1 in the removal of enrofloxacin from water, characterized in that... The process includes the following steps: adding filamentous microalgae strains or filamentous microalgae solutions to enrofloxacin-contaminated water for cultivation to remove enrofloxacin.

3. The application of filamentous microalgae according to claim 2 in the removal of enrofloxacin from water, characterized in that: The filamentous microalgae solution mentioned is a filamentous microalgae solution cultured to the logarithmic growth phase or the stationary phase.

4. The application of the filamentous microalgae according to claim 3 in the removal of enrofloxacin from water, characterized in that: The filamentous microalgae solution is obtained through the following steps: inoculating the filamentous microalgae seed into the culture medium and culturing it to the logarithmic growth phase or the stationary phase.

5. The application of the filamentous microalgae according to claim 4 in the removal of enrofloxacin from water, characterized in that: The culture was carried out at 25–30°C, with a light intensity of 8000–10000 lux and a light-dark period of 10–14 h.

6. The application of the filamentous microalgae according to claim 4 in the removal of enrofloxacin from water, characterized in that: The culture medium is BG110 liquid culture medium.

7. The application of filamentous microalgae according to claim 2 in the removal of enrofloxacin from water, characterized in that: The amount of filamentous microalgae species or microalgae solution added is based on the OD of algal cells in the water. 680 Calculated for values ​​between 0.1 and 0.2.

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