Chlorella extracellular fluid as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological culture, and particularly relates to a chlorella extracellular fluid and a preparation method and application thereof.The preparation method of the chlorella extracellular fluid comprises the following steps that a BG11 culture medium is inoculated with a chlorella solution cultured to the logarithmic phase, oxcarbazepine is added, and the chlorella extracellular fluid is obtained; the preparation method comprises the following steps: placing chlorella in an intermittent illumination environment with the temperature of 20-30 DEG C and the rotating speed of 100-200 rpm for growth, centrifuging the chlorella to obtain supernate after the chlorella grows to the 10th-16th day, filtering, sub-packaging, and performing cryopreservation to obtain the chlorella extracellular fluid. By adding oxcarbazepine, the chlorella extracellular fluid prepared in a specific culture medium and a specific growth environment has high oxidation activity, excellent stability and excellent degradation capacity, and a new thought is provided for solving the pollution treatment problem of current environmental pollutants such as pharmaceutical products, polycyclic aromatic hydrocarbons and dyes.

Description

Technical Field

[0001] The invention belongs to the technical field of biological culture, and particularly relates to an extracellular solution of Chlorella and a preparation method and application thereof. Background Art

[0002] Chlorella ( Chlorella ) is a single-celled, spherical green algae with a diameter of 3-8 microns. It is rich in high-value-added products such as protein, lipids, amino acids, and trace elements. It has the advantages of high photosynthetic efficiency, strong nitrogen and phosphorus removal capabilities, and does not compete with crops for land or fertilizer. It grows and reproduces photosynthetically and autotrophically, and is extremely widespread. It has significant application potential in water purification, bioenergy production, and the food industry.

[0003] During their growth, microalgae can absorb external inorganic salts and organic substances to maintain their physiological processes, including carbon sources, nitrogen sources, phosphorus sources, etc., and at the same time secrete a variety of enzymes and organic compounds such as polysaccharides, proteins, lipids and other extracellular substances to the outside world.

[0004] These extracellular substances of Chlorella not only aid its own growth and metabolism but also have potential applications in environmental remediation. For example, exopolysaccharides can act as adsorbents for heavy metal ions, while extracellular proteins and lipids may play a role in bioenergy production. Furthermore, the oxygen produced by Chlorella during photosynthesis can provide essential oxygen for other organisms in the water, thereby improving the aquatic ecosystem.

[0005] Therefore, in-depth research on the composition, properties and functions of the extracellular fluid of Chlorella is of great significance for the development of new biotechnology applications. Summary of the Invention

[0006] In view of the current status of the above-mentioned prior art, the present invention provides an extracellular fluid of Chlorella and a preparation method and application thereof. The extracellular fluid of Chlorella has strong oxidative activity, excellent stability and excellent degradation ability.

[0007] The specific technical solutions are as follows: A first object of the present invention is to provide a method for preparing an extracellular fluid of Chlorella vulgaris, comprising the following steps: inoculating a Chlorella fluid cultured to a logarithmic growth phase into a BG11 culture medium, adding oxcarbazepine, and growing the culture medium in an intermittent light environment at a temperature of 20-30°C and a rotation speed of 100-200 rpm; after the Chlorella vulgaris grows to the 10th to 16th day, centrifuging the Chlorella vulgaris to obtain a supernatant, filtering the supernatant, aliquoting the supernatant, and freezing the supernatant to obtain the Chlorella vulgaris extracellular fluid.

[0008] In the technical solution of the present invention, the extracellular fluid of Chlorella vulgaris prepared in a specific culture medium and a specific growth environment by adding oxcarbazepine has strong oxidative activity, excellent stability and outstanding degradation ability, providing a new approach to the pollution treatment of current environmental pollutants such as pharmaceutical products, polycyclic aromatic hydrocarbons and dyes.

[0009] Furthermore, the final concentration of oxcarbazepine is 5-30 mg / L.

[0010] Furthermore, the initial inoculation amount of the Chlorella algae liquid is 5-10% of the volume of the culture medium.

[0011] Furthermore, the BG11 medium comprises the following components: sodium nitrate 1-2 g / L, anhydrous dimethyl phosphate 0.02-0.06 g / L, magnesium phosphate heptahydrate 0.06-0.09 g / L, calcium chloride dihydrate 0.030-0.042 g / L, ammonium ferric citrate 0.004-0.008 g / L, citric acid 0.004-0.008 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.01-0.03 g / L, boric acid 0.00284-0.00288 g / L, manganese chloride monohydrate 0.00180-0.00183 g / L, zinc sulfate heptahydrate 0.000220-0.000224 g / L, anhydrous copper sulfate 0.000078-0.000080 g / L, sodium molybdate dihydrate 0.00038-0.00040 g / L, cobalt nitrate hexahydrate 0.000048-0.000050 g / L.

[0012] Furthermore, the intermittent lighting pattern is 12 hours of darkness and 12 hours of light.

[0013] Furthermore, the light intensity is 1000-3000 lux.

[0014] Furthermore, the supernatant was filtered through a 0.22-0.45 μm filter membrane and stored at -20 °C after aliquoting.

[0015] The second object of the present invention is to provide a Chlorella extracellular fluid prepared by the above-mentioned method for preparing the Chlorella extracellular fluid.

[0016] The third object of the present invention is to provide a use of the extracellular fluid of Chlorella as described above in the field of pollutant treatment.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The extracellular fluid of Chlorella obtained by the preparation method provided by the present invention has strong oxidative activity and can oxidize 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and guaiacol, causing ABTS and guaiacol to change color. This not only verifies the wide range of oxidative substrates of the extracellular fluid of Chlorella, broadens the understanding of the biological functions of microalgae, but also opens up new avenues for its application in fields such as bioenergy development; (2) The extracellular fluid of Chlorella obtained by the preparation method provided by the present invention has extremely strong stability, is resistant to high temperatures, acids and alkalis, and maintains stable oxidation activity in the presence of most metal ions and organic reagents; (3) The extracellular fluid of Chlorella obtained by the preparation method provided by the present invention has excellent degradation ability and has good degradation effects on different pharmaceutical products, polycyclic aromatic hydrocarbons and dyes, indicating that the extracellular fluid of Chlorella has extremely broad application prospects in the field of environmental governance; (4) The cultivation process of Chlorella can absorb carbon dioxide, which helps to reduce greenhouse gas emissions, thereby synergizing with the degradation of Chlorella's extracellular fluid and playing a dual role in environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a color change diagram of the reaction between ABTS and guaiacol oxidized by the extracellular fluid of Chlorella vulgaris in the present invention; Figure 2 This is a curve diagram showing changes in the extracellular fluid oxidation activity of Chlorella in the present invention; Figure 3 This is a test chart of the oxidation activity stability of the extracellular fluid of Chlorella vulgaris at different temperatures; Figure 4 This is a graph showing the stability of oxidative activity of the extracellular fluid of Chlorella vulgaris at different pH values. Figure 5 This is a test chart of the oxidative activity stability of the extracellular fluid of Chlorella vulgaris under different organic reagents; Figure 6 This is a graph showing the oxidative activity stability test of the extracellular fluid of Chlorella vulgaris under different metal ions; Figure 7 This is a graph showing the degradation performance test of the extracellular fluid of Chlorella vulgaris on different pharmaceutical products; Figure 8 This is a graph showing the degradation performance of the extracellular fluid of Chlorella vulgaris on different polycyclic aromatic hydrocarbons; Figure 9 This is a test chart of the degradation performance of the extracellular fluid of Chlorella vulgaris on different dyes. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0020] In a specific embodiment, the chlorella used is Chlorella sp. FACHB-9 was purchased from the Freshwater Algae Culture Bank (FACHB) of the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. All chemical reagents were commercially available.

[0021] In the detection of oxidative activity of extracellular fluid of Chlorella, UV spectrophotometer was used to measure the oxidative activity of Chlorella. Chlorella The ABTS oxidation activity of the extracellular fluid of sp.FACHB-9 was determined at a wavelength of 420 nm.

[0022] Example 1 A method for preparing an extracellular fluid of Chlorella vulgaris comprises the following steps: inoculating a Chlorella vulgaris fluid cultured to a logarithmic growth phase into a BG11 culture medium, adding oxcarbazepine, and growing the culture medium in an intermittent lighting environment with a temperature of 20°C, a light intensity of 1000 lux (12 hours of darkness, 12 hours of light), and a rotation speed of 100 rpm; after the Chlorella vulgaris grows to the 16th day, centrifuging the Chlorella vulgaris to obtain a supernatant, filtering the supernatant through a 0.22 μm filter membrane, aliquoting the supernatant, and storing the supernatant at -20°C to obtain the Chlorella vulgaris extracellular fluid; Wherein, the initial inoculation amount of the Chlorella algae liquid is 5% of the volume of the culture medium; The final concentration of oxcarbazepine is 5 mg / L; The BG11 culture medium includes the following components: 1 g / L sodium nitrate, 0.02 g / L anhydrous dimethyl phosphate, 0.06 g / L magnesium phosphate heptahydrate, 0.030 g / L calcium chloride dihydrate, 0.004 g / L ammonium ferric citrate, 0.004 g / L citric acid, 0.0005 g / L EDTA, 0.01 g / L sodium carbonate, 0.00284 g / L boric acid, 0.00180 g / L manganese chloride monohydrate, 0.000220 g / L zinc sulfate heptahydrate, 0.000078 g / L anhydrous copper sulfate, 0.00038 g / L sodium molybdate dihydrate, and 0.000048 g / L cobalt nitrate hexahydrate.

[0023] Example 2 A method for preparing an extracellular fluid of Chlorella vulgaris comprises the following steps: inoculating a Chlorella vulgaris fluid cultured to a logarithmic growth phase into a BG11 culture medium, adding oxcarbazepine, and growing the culture medium in an intermittent lighting environment with a temperature of 25°C, a light intensity of 2000 lux (12 hours of darkness, 12 hours of light), and a rotation speed of 150 rpm; after the Chlorella vulgaris grows to the 13th day, centrifuging the Chlorella vulgaris to obtain a supernatant, filtering the supernatant through a 0.22 μm filter membrane, aliquoting the supernatant, and storing the supernatant at -20°C to obtain the Chlorella vulgaris extracellular fluid; Wherein, the initial inoculation amount of the Chlorella algae liquid is 8% of the volume of the culture medium; The final concentration of oxcarbazepine was 18 mg / L; The BG11 culture medium includes the following components: 1.5 g / L sodium nitrate, 0.04 g / L anhydrous dimethyl phosphate, 0.075 g / L magnesium phosphate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.006 g / L ammonium ferric citrate, 0.006 g / L citric acid, 0.001 g / L EDTA, 0.02 g / L sodium carbonate, 0.00286 g / L boric acid, 0.00181 g / L manganese chloride monohydrate, 0.000222 g / L zinc sulfate heptahydrate, 0.000079 g / L anhydrous copper sulfate, 0.00039 g / L sodium molybdate dihydrate, and 0.000049 g / L cobalt nitrate hexahydrate.

[0024] Example 3 A method for preparing an extracellular fluid of Chlorella vulgaris comprises the following steps: inoculating a Chlorella vulgaris fluid cultured to a logarithmic growth phase into a BG11 culture medium, adding oxcarbazepine, and growing the culture medium in an intermittent lighting environment with a temperature of 30°C, a light intensity of 3000 lux (12 hours of darkness, 12 hours of light), and a rotation speed of 200 rpm; after the Chlorella vulgaris grows to the 10th day, centrifuging the Chlorella vulgaris to obtain a supernatant, filtering the supernatant through a 0.45 μm filter membrane, aliquoting the supernatant, and storing the supernatant at -20°C to obtain the Chlorella vulgaris extracellular fluid; Wherein, the initial inoculation amount of the Chlorella algae liquid is 10% of the volume of the culture medium; The final concentration of oxcarbazepine is 30 mg / L; The BG11 culture medium includes the following components: 2 g / L sodium nitrate, 0.06 g / L anhydrous dimethyl phosphate, 0.09 g / L magnesium phosphate heptahydrate, 0.042 g / L calcium chloride dihydrate, 0.008 g / L ammonium ferric citrate, 0.008 g / L citric acid, 0.0015 g / L EDTA, 0.03 g / L sodium carbonate, 0.00288 g / L boric acid, 0.00183 g / L manganese chloride monohydrate, 0.000224 g / L zinc sulfate heptahydrate, 0.000080 g / L anhydrous copper sulfate, 0.00040 g / L sodium molybdate dihydrate, and 0.000050 g / L cobalt nitrate hexahydrate.

[0025] Example 4 A method for preparing extracellular fluid of Chlorella vulgaris is described with reference to Example 1, except that the final concentration of oxcarbazepine is 10 mg / L.

[0026] Example 5 A method for preparing extracellular fluid of Chlorella vulgaris is described with reference to Example 1, except that the final concentration of oxcarbazepine is 15 mg / L.

[0027] Example 6 A method for preparing extracellular fluid of Chlorella vulgaris is described with reference to Example 1, except that the final concentration of oxcarbazepine is 20 mg / L.

[0028] Example 7 A method for preparing extracellular fluid of Chlorella vulgaris is described with reference to Example 1, except that the final concentration of oxcarbazepine is 25 mg / L.

[0029] Example 8 A method for preparing extracellular fluid of Chlorella vulgaris is described with reference to Example 1, except that the final concentration of oxcarbazepine is 30 mg / L.

[0030] Comparative Example 1 A method for preparing extracellular fluid of Chlorella vulgaris is described with reference to Example 1, except that oxcarbazepine is not added.

[0031] test: 1. Oxidative activity test of Chlorella extracellular fluid: (1) ABTS oxidation activity test of Chlorella extracellular fluid: In a 1 mL reaction system, 100 µL of the extracellular solution of Chlorella prepared in Examples 1-3 and Comparative Example 1 diluted with 100 mM acetate buffer (pH 4.0) was added to a solution having a dilution degree of 0.3X, 0.5X, or 1X the initial concentration of the extracellular solution of Chlorella. 900 µL of 1 mM ABTS-acetate buffer was added, and the color of oxidized ABTS at different dilution degrees was recorded, see FIG. Figure 1 .

[0032] (2) Guaiacol oxidation activity test of Chlorella extracellular fluid: In a 5 mL reaction system, 100 µL of the extracellular fluids of Chlorella prepared in Examples 1-3 and Comparative Example 1 diluted with 50 mM phosphate buffer (pH 5.5) to a dilution level of 0.3X, 0.5X, and 1X the initial concentration of the extracellular fluid of Chlorella was added. 1 mL of 50 mM guaiacol, 1 mL of 2% hydrogen peroxide, and 2.9 mL of 50 mM phosphate buffer (pH 5.5) were added. The color of the oxidized guaiacol at different dilution levels was recorded, see the chart for details. Figure 1 .

[0033] Depend on Figure 1The results of oxidizing ABTS and guaiacol using Chlorella extracellular fluid at different concentrations revealed that the Chlorella extracellular fluid produced color reactions with ABTS and guaiacol, respectively, demonstrating its ability to oxidize ABTS and guaiacol. Furthermore, the color gradually darkened with increasing Chlorella extracellular fluid concentration, demonstrating a gradual increase in oxidizing ability. The colors of Examples 1-3 were significantly darker than those of Comparative Example 1, indicating that the Chlorella extracellular fluids of the present invention had significantly greater oxidizing ability than the Chlorella extracellular fluid of Comparative Example 1.

[0034] (3) Test on the oxidative activity change curve of Chlorella extracellular fluid: During the preparation process of Example 1, Examples 4-8, and Comparative Example 1, samples were taken on the 0th, 2nd, 4th, 6th, 8th, 10th, 12th, 14th, and 16th days of growth of Chlorella to measure ABTS oxidation activity. When sampling, the extracellular fluid of the Chlorella to be tested was shaken to mix, 1 mL of the extracellular fluid of Chlorella was added to a cuvette, and the oxidation activity was tested using the ABTS method. Each sample was tested in triplicate. The oxidation activity of the extracellular fluid of Chlorella was measured every two days, and the oxidation activity change curve was plotted. Figure 2 .

[0035] The calculation formula of ABTS oxidation activity is as follows:

[0036] Where N is the dilution factor, ∆OD 420 V is the difference in absorbance at 420 nm before and after the reaction within 1 min. 总 is the total volume of the reaction system (mL), V 样 is the sample volume added to the reaction (mL), T is the reaction time (min), L is the optical path length of the cuvette (cm), and the molar extinction coefficient ε = 3.6 × 10 4 M -1 cm -1 The unit of calculation of ABTS oxidation activity is μmol·L -1 min -1 .

[0037] Depend on Figure 2 The oxidative activity curve of the extracellular fluid of Chlorella vulgaris at different concentrations of oxcarbazepine shows that on days 10-16, the oxidative activity of the extracellular fluid of Chlorella vulgaris significantly increased when 5-30 mg / L oxcarbazepine was added (Examples 1 and Examples 4-8) compared with no oxcarbazepine addition (Comparative Example 1), and the oxidative capacity gradually increased. When the final concentration of oxcarbazepine was 5 mg / L, the oxidative activity reached a maximum of 930 μmol·L on day 16. -1 min -1 .

[0038] 2. Stability test of Chlorella extracellular fluid: The extracellular solution of Chlorella prepared in Examples 1-3 and Comparative Example 1 was heated at different temperatures (0°C, 20°C, 40°C, 60°C, 80°C, 100°C, see Figure 3 ), different pH (3, 5, 7, 9, 11, see Figure 4 ), respectively, with a final concentration of 10% of different organic reagents (formic acid, methanol, acetonitrile, ethanol, acetic acid, acetone, n-hexane, xylene, dichloromethane, petroleum ether, isopropanol, DMSO, DMF, see Figure 5 ), different metal ions with a final concentration of 100 mM (Na + , K + 、Zn 2+ 、Cu 2+ 、Mn 2+ , Ca 2+ Mg 2 + 、Fe 3+ , see Figure 6 ) After the mixture was stabilized for 3 hours, samples were taken under each condition to measure the oxidation activity. The oxidation activity determination method was carried out according to the ABTS method.

[0039] The formula for calculating relative activity is as follows:

[0040] Depend on Figure 3-6 The results show that the extracellular fluids of Chlorella in each embodiment and comparative example have high relative activities after being treated under various conditions. However, the relative activity of the extracellular fluids of Chlorella in the embodiments with oxcarbazepine added is slightly higher than the relative activity of the extracellular fluids of Chlorella in the comparative example without oxcarbazepine added. This shows that the extracellular fluids of Chlorella in the embodiments of the present invention can withstand a high temperature of 100°C, have strong acid and alkali resistance, and are less affected by metal ions and organic reagents.

[0041] 3. Degradation performance test of Chlorella extracellular fluid: (1) Degradation performance test of Chlorella extracellular fluid on pharmaceutical products: Pharmaceutical products (dexamethasone, prednisone, sulfamethazine, sulfamethoxazole, sulfisoxazole, carbamazepine, oxcarbazepine, acetazolamide, promethazine hydrochloride, sulfacetamide, and diphenhydramine hydrochloride) were added to the extracellular fluids of Chlorella prepared in Examples 1-3 and Comparative Example 1. The Chlorella extracellular fluid-pharmaceutical product reaction system was degraded in an incubator at 28°C. Samples were taken on days 0 and 8 and analyzed by high-performance liquid chromatography. The degradation rates of the Chlorella extracellular fluid for different pharmaceutical products on day 8 were calculated. The concentration of the pharmaceutical product was 20 mg / L, and the oxidative activity of the Chlorella extracellular fluid was 300 μmol·L -1 min -1 , the results are shown in Figure 7 .

[0042] Depend on Figure 7 The results show that in the degradation system of the extracellular fluid of Chlorella for pharmaceutical products, after eight days of reaction, it was found that the extracellular fluid of Chlorella prepared in the embodiment of the present invention had a high degradation effect on four major categories of drugs: steroid hormones, sulfonamide antibiotics, antiepileptic drugs, and antihistamine agents. Among them, the degradation rates of prednisone, sulfisoxazole, oxcarbazepine, promethazine hydrochloride, and diphenhydramine hydrochloride were all 80% or more, which was significantly higher than the degradation rate of the extracellular fluid of Chlorella for pharmaceutical products in Comparative Example 1 without the addition of oxcarbazepine.

[0043] (2) Degradation performance test of extracellular fluid of Chlorella vulgaris on polycyclic aromatic hydrocarbons: Polycyclic aromatic hydrocarbons (naphthalene, fluorene, phenanthrene, acenaphthene, anthracene, pyrene, fluoranthene, dibenzothiophene, dibenzofuran, biphenyl, and 2-methylnaphthalene) were added to the extracellular fluid of Chlorella prepared in Examples 1-3 and Comparative Example 1. The Chlorella extracellular fluid-polycyclic aromatic hydrocarbon reaction system was degraded in an incubator at 28°C. Samples were taken at 0 h and 24 h for high performance liquid chromatography (HPLC) analysis. The degradation rates of the Chlorella extracellular fluid for different polycyclic aromatic hydrocarbons at 24 h were calculated. The polycyclic aromatic hydrocarbon concentration was 20 mg / L, and the oxidative activity of the Chlorella extracellular fluid was 300 μmol·L -1 min -1 , the results are shown in Figure 8 .

[0044] Depend on Figure 8 The results show that in the degradation system of polycyclic aromatic hydrocarbons by the extracellular fluid of Chlorella, after 24 h of reaction, it was found that, except for anthracene, pyrene, and acenaphthene, the degradation rates of naphthalene, fluorene, phenanthrene, pyrene, fluoranthene, dibenzothiophene, dibenzofuran, biphenyl, and 2-methylnaphthalene by the extracellular fluid of Chlorella prepared in the embodiment of the present invention could reach 90% or above, which was significantly higher than the degradation rate of polycyclic aromatic hydrocarbons by the extracellular fluid of Chlorella without adding oxcarbazepine in Comparative Example 1.

[0045] (3) Test on the degradation performance of Chlorella extracellular fluid on dyes: Dyes (indigo carmine, reactive blue, bromophenol blue, Congo red, reactive red, Evans blue, malachite green, disperse red, reactive black, crystal violet, and azure B) were added to the extracellular fluid of Chlorella prepared in Examples 1-3 and Comparative Example 1. The Chlorella extracellular fluid-dye reaction system was degraded in an incubator at 28°C. Samples were taken on days 0 and 8 and analyzed by high-performance liquid chromatography. The degradation rates of the Chlorella extracellular fluid for the different dyes on day 8 were calculated. The dye concentration was 100 mg / L, and the oxidative activity of the Chlorella extracellular fluid was 300 μmol·L -1 min -1 , the results are shown in Figure 9 .

[0046] Depend on Figure 9 The results show that in the dye degradation system of the extracellular fluid of Chlorella, after eight days of reaction, the degradation rates of indigo carmine, reactive blue, bromophenol blue, reactive red, malachite green, disperse red, crystal violet, and azure B by the extracellular fluid of Chlorella prepared in the embodiment of the present invention can reach 50% or more, which is significantly higher than the degradation rate of the extracellular fluid of Chlorella in Comparative Example 1 without adding oxcarbazepine.

[0047] In summary, the preparation method of the present invention, by adding oxcarbazepine, the extracellular fluid of Chlorella prepared in a specific culture medium and a specific growth environment has strong oxidative activity, excellent stability and excellent degradation ability, providing a new approach to the current pollution treatment of environmental pollutants such as pharmaceutical products, polycyclic aromatic hydrocarbons and dyes.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing extracellular fluid of Chlorella, characterized in that: The method comprises the following steps: inoculating a chlorella liquid cultured to a logarithmic growth phase into a BG11 culture medium, adding oxcarbazepine, and growing the culture medium in an intermittent light environment at a temperature of 20-30°C and a rotation speed of 100-200 rpm; when the chlorella grows to the 10th to 16th day, centrifuging the chlorella to obtain a supernatant, filtering and packaging the supernatant, and then freezing and storing the supernatant to obtain the chlorella extracellular fluid.

2. The method for preparing the extracellular fluid of Chlorella according to claim 1, characterized in that: The final concentration of oxcarbazepine is 5-30 mg / L.

3. The method for preparing the extracellular fluid of Chlorella according to claim 1, characterized in that: The initial inoculation amount of the Chlorella algae liquid is 5-10% of the volume of the culture medium.

4. The method for preparing the extracellular fluid of Chlorella according to claim 1, characterized in that: The BG11 culture medium comprises the following components: 1-2 g / L sodium nitrate, 0.02-0.06 g / L anhydrous dimethyl phosphate, 0.06-0.09 g / L magnesium phosphate heptahydrate, 0.030-0.042 g / L calcium chloride dihydrate, 0.004-0.008 g / L ammonium ferric citrate, 0.004-0.008 g / L citric acid, 0.0005-0.0015 g / L EDTA, 0.01-0.03 g / L sodium carbonate, 0.00284-0.00288 g / L boric acid, 0.00180-0.00183 g / L manganese chloride monohydrate, 0.000220-0.000224 g / L zinc sulfate heptahydrate, 0.000078-0.000080 g / L anhydrous copper sulfate. g / L, sodium molybdate dihydrate 0.00038-0.00040 g / L, cobalt nitrate hexahydrate 0.000048-0.000050 g / L.

5. The method for preparing the extracellular fluid of Chlorella according to claim 1, characterized in that: The intermittent lighting pattern is 12 hours of darkness and 12 hours of light.

6. The method for preparing the extracellular fluid of Chlorella according to claim 5, characterized in that: The light intensity is 1000-3000 lux.

7. The method for preparing the extracellular fluid of Chlorella according to claim 1, characterized in that: The supernatant was filtered through a 0.22-0.45 μm filter and stored in aliquots at -20 °C.

8. An extracellular fluid of Chlorella, characterized in that: The extracellular fluid of Chlorella is prepared by the method for preparing the extracellular fluid of Chlorella as described in any one of claims 1 to 7.

9. Use of the extracellular fluid of Chlorella as claimed in claim 8 in the field of pollutant treatment.

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