A Chlorella extracellular fluid, its preparation method and application

Abstract

The present invention belongs to the technical field of biological culture, and particularly relates to a Chlorella extracellular fluid, a preparation method thereof and an application. The preparation method of the Chlorella extracellular fluid comprises the following steps: inoculating the Chlorella algal solution cultured to the logarithmic growth phase into the BG11 medium, adding oxcarbazepine, and growing it 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, centrifuge the Chlorella to obtain the supernatant, filter and subpackage it, and then store it frozen to obtain the Chlorella extracellular fluid. By adding oxcarbazepine, the Chlorella extracellular fluid prepared under specific culture medium and specific growth environment in the present invention has strong oxidation activity, excellent stability and excellent degradation ability, providing a new idea for the treatment of current environmental pollutants such as pharmaceutical products, polycyclic aromatic hydrocarbons and dyes.

Description

Technical Field

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

[0002] Chlorella ( Chlorella ) is a single-celled spherical green alga with a diameter of 3-8 micrometers, rich in high-value products such as proteins, lipids, amino acids and trace elements. It has the advantages of high photosynthetic efficiency, strong nitrogen and phosphorus removal ability, and not competing with crops for land and fertilizers. It grows and reproduces by photosynthetic autotrophy and is extremely widely distributed. It has important application potential in the fields of water purification, bioenergy production and food industry.

[0003] During the growth process, 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 various extracellular substances such as enzymes and organic compounds such as polysaccharides, proteins, and lipids to the outside.

[0004] These extracellular substances of Chlorella not only contribute to its own growth and metabolism, but also have potential application value in environmental remediation. For example, extracellular polysaccharides can be used as adsorbents for heavy metal ions, while extracellular proteins and lipids may play a role in bioenergy production. In addition, the oxygen produced by Chlorella during photosynthesis can provide essential oxygen for other organisms in the water, thus improving the ecological environment of the water.

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

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

[0007] The specific technical solutions are as follows:

[0008] The first object of the present invention is to provide a preparation method of a Chlorella extracellular fluid, comprising the following steps: inoculating the Chlorella algal solution cultured to the logarithmic growth phase into BG11 medium, adding oxcarbazepine, and growing it 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, centrifuge the Chlorella to obtain the supernatant, filter and subpackage it, and then store it frozen to obtain the Chlorella extracellular fluid.

[0009] In the technical solution of the present invention, the extracellular fluid of Chlorella prepared by adding oxcarbazepine under specific culture media and specific growth environments has strong oxidation activity, excellent stability and outstanding degradation ability, providing a new idea for the treatment of current environmental pollutants such as pharmaceutical products, polycyclic aromatic hydrocarbons and dyes.

[0010] Further, the final concentration of the oxcarbazepine is 5 - 30 mg / L.

[0011] Further, the initial inoculation amount of the Chlorella algal solution is 5 - 10% of the volume of the culture medium.

[0012] Further, the BG11 culture medium comprises the following components: sodium nitrate 1 - 2 g / L, anhydrous disodium hydrogen 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, copper sulfate anhydrous 0.000078 - 0.000080 g / L, sodium molybdate dihydrate 0.00038 - 0.00040 g / L, cobalt nitrate hexahydrate 0.000048 - 0.000050 g / L.

[0013] Further, the rule of the intermittent illumination is 12 h darkness and 12 h light.

[0014] Furthermore, the illumination intensity is 1000 - 3000 lux.

[0015] Further, the supernatant is filtered through a 0.22 - 0.45 μm filter membrane, and after being aliquoted, it is stored at -20 °C.

[0016] The second object of the present invention is to provide an extracellular fluid of Chlorella, which is prepared by using the preparation method of the extracellular fluid of Chlorella as described above.

[0017] The third object of the present invention is to provide an application of the extracellular fluid of Chlorella as described above in the field of pollutant treatment.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1)The extracellular fluid of Chlorella obtained by the preparation method provided by the present invention has strong oxidation activity and can oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol, causing the color change of ABTS and guaiacol. This not only verifies the wide range of oxidation substrates of the extracellular fluid of Chlorella, broadens the understanding of the biological functions of microalgae, but also opens up new ways for its applications in the fields such as bioenergy development;

[0020] (2)The extracellular fluid of Chlorella obtained by the preparation method provided by the present invention has extremely strong stability, can withstand high temperatures, acids and alkalis, and maintains stable oxidation activity in the presence of most metal ions and organic reagents;

[0021] (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 extremely broad application prospects of the extracellular fluid of Chlorella in the field of environmental governance;

[0022] (4)The cultivation process of Chlorella can absorb carbon dioxide, which helps to reduce greenhouse gas emissions, and thus, in cooperation with the degradation effect of the extracellular fluid of Chlorella, plays a dual role in environmental protection. Description of the Drawings

[0023] Figure 1 It is a diagram showing the color change of the reaction of the extracellular fluid of Chlorella in the present invention to oxidize ABTS and guaiacol;

[0024] Figure 2 It is a curve graph showing the change of the oxidation activity of the extracellular fluid of Chlorella in the present invention;

[0025] Figure 3 It is a test diagram showing the stability of the oxidation activity of the extracellular fluid of Chlorella in the present invention at different temperatures;

[0026] Figure 4 It is a test diagram showing the stability of the oxidation activity of the extracellular fluid of Chlorella in the present invention at different pH values;

[0027] Figure 5 It is a test diagram showing the stability of the oxidation activity of the extracellular fluid of Chlorella in the present invention in different organic reagents;

[0028] Figure 6 It is a test diagram showing the stability of the oxidation activity of the extracellular fluid of Chlorella in the present invention in different metal ions;

[0029] Figure 7 It is a test diagram showing the degradation performance of the extracellular fluid of Chlorella in the present invention on different pharmaceutical products;

[0030] Figure 8 It is a test diagram showing the degradation performance of the extracellular fluid of Chlorella in the present invention on different polycyclic aromatic hydrocarbons;

[0031] Figure 9 This is a test chart of the degradation performance of the extracellular fluid of Chlorella vulgaris on different dyes in the present invention. Specific embodiments

[0032] The principles and features of the present invention will be described below in conjunction with examples. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0033] In the specific embodiments, the Chlorella vulgaris Chlorella sp. FACHB-9 used was purchased from the FACHB-9 of the Freshwater Algae Culture Collection (FACHB), Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan. In addition, various chemical reagents are all commercially available products.

[0034] In the detection of the oxidation activity of the extracellular fluid of Chlorella vulgaris, an ultraviolet spectrophotometer was used to measure the ABTS oxidation activity of the extracellular fluid of Chlorella Chlorella sp.FACHB-9, and the measurement wavelength was 420 nm.

[0035] Example 1

[0036] A method for preparing the extracellular fluid of Chlorella vulgaris includes the following steps: inoculating the Chlorella vulgaris algal solution cultured to the logarithmic growth phase into the BG11 medium, adding oxcarbazepine, and placing it in an intermittent light environment with a temperature of 20 °C, a light intensity of 1000 lux (12 h darkness, 12 h light), and a rotation speed of 100 rpm to grow. When the Chlorella vulgaris grows to the 16th day, the Chlorella vulgaris is centrifuged to obtain the supernatant, which is filtered through a 0.22 μm filter membrane, subpackaged, and stored at -20 °C to obtain the extracellular fluid of Chlorella vulgaris;

[0037] Among them, the initial inoculation amount of the Chlorella vulgaris algal solution is 5% of the volume of the medium;

[0038] The final concentration of the oxcarbazepine is 5 mg / L;

[0039] The BG11 medium includes the following components: sodium nitrate 1 g / L, anhydrous disodium hydrogen phosphate 0.02 g / L, magnesium phosphate heptahydrate 0.06 g / L, calcium chloride dihydrate 0.030 g / L, ammonium ferric citrate 0.004 g / L, citric acid 0.004 g / L, EDTA 0.0005 g / L, sodium carbonate 0.01 g / L, boric acid 0.00284 g / L, manganese chloride monohydrate 0.00180 g / L, zinc sulfate heptahydrate 0.000220 g / L, copper sulfate anhydrous 0.000078 g / L, sodium molybdate dihydrate 0.00038 g / L, cobalt nitrate hexahydrate 0.000048 g / L.

[0040] Example 2

[0041] A method for preparing extracellular fluid of Chlorella vulgaris, comprising the following steps: inoculating the Chlorella vulgaris algal solution cultured to the logarithmic growth phase into BG11 medium, adding oxcarbazepine, and placing it in an intermittent light environment with a temperature of 25 °C, a light intensity of 2000 lux (12 h darkness, 12 h light), and a rotation speed of 150 rpm for growth. When the Chlorella vulgaris grows to the 13th day, centrifuge the Chlorella vulgaris to obtain the supernatant, filter it through a 0.22 μm filter membrane, dispense it, and store it at -20 °C to obtain the extracellular fluid of Chlorella vulgaris;

[0042] Among them, the initial inoculation amount of the Chlorella vulgaris algal solution is 8% of the volume of the medium;

[0043] The final concentration of oxcarbazepine is 18 mg / L;

[0044] The BG11 medium comprises the following components: sodium nitrate 1.5 g / L, anhydrous disodium hydrogen phosphate 0.04 g / L, magnesium phosphate heptahydrate 0.075 g / L, calcium chloride dihydrate 0.036 g / L, ammonium ferric citrate 0.006 g / L, citric acid 0.006 g / L, EDTA 0.001 g / L, sodium carbonate 0.02 g / L, boric acid 0.00286 g / L, manganese chloride monohydrate 0.00181 g / L, zinc sulfate heptahydrate 0.000222 g / L, copper sulfate anhydrous 0.000079 g / L, sodium molybdate dihydrate 0.00039 g / L, cobalt nitrate hexahydrate 0.000049 g / L.

[0045] Example 3

[0046] A method for preparing extracellular fluid of Chlorella vulgaris, comprising the following steps: inoculating the Chlorella vulgaris algal solution cultured to the logarithmic growth phase into BG11 medium, adding oxcarbazepine, and placing it in an intermittent light environment with a temperature of 30 °C, a light intensity of 3000 lux (12 h darkness, 12 h light), and a rotation speed of 200 rpm for growth. When the Chlorella vulgaris grows to the 10th day, centrifuge the Chlorella vulgaris to obtain the supernatant, filter it through a 0.45 μm filter membrane, dispense it, and store it at -20 °C to obtain the extracellular fluid of Chlorella vulgaris;

[0047] Among them, the initial inoculation amount of the Chlorella vulgaris algal solution is 10% of the volume of the medium;

[0048] The final concentration of oxcarbazepine is 30 mg / L;

[0049] The BG11 medium comprises the following components: sodium nitrate 2 g / L, anhydrous disodium hydrogen phosphate 0.06 g / L, magnesium phosphate heptahydrate 0.09 g / L, calcium chloride dihydrate 0.042 g / L, ammonium ferric citrate 0.008 g / L, citric acid 0.008 g / L, EDTA 0.0015 g / L, sodium carbonate 0.03 g / L, boric acid 0.00288 g / L, manganese chloride monohydrate 0.00183 g / L, zinc sulfate heptahydrate 0.000224 g / L, copper sulfate anhydrous 0.000080 g / L, sodium molybdate dihydrate 0.00040 g / L, cobalt nitrate hexahydrate 0.000050 g / L.

[0050] Example 4

[0051] A method for preparing the extracellular fluid of Chlorella, referring to Example 1, is different from Example 1 in that the final concentration of oxcarbazepine is 10 mg / L.

[0052] Example 5

[0053] A method for preparing the extracellular fluid of Chlorella, referring to Example 1, is different from Example 1 in that the final concentration of oxcarbazepine is 15 mg / L.

[0054] Example 6

[0055] A method for preparing the extracellular fluid of Chlorella, referring to Example 1, is different from Example 1 in that the final concentration of oxcarbazepine is 20 mg / L.

[0056] Example 7

[0057] A method for preparing the extracellular fluid of Chlorella, referring to Example 1, is different from Example 1 in that the final concentration of oxcarbazepine is 25 mg / L.

[0058] Example 8

[0059] A method for preparing the extracellular fluid of Chlorella, referring to Example 1, is different from Example 1 in that the final concentration of oxcarbazepine is 30 mg / L.

[0060] Comparative Example 1

[0061] A method for preparing the extracellular fluid of Chlorella, referring to Example 1, is different from Example 1 in that no oxcarbazepine is added.

[0062] Test:

[0063] 1. Oxidation activity test of the extracellular fluid of Chlorella:

[0064] (1) ABTS oxidation activity test of the extracellular fluid of Chlorella:

[0065] In a 1 mL reaction system, it contains 100 μL of the extracellular fluid of Chlorella prepared in Examples 1-3 and Comparative Example 1 diluted with 100 mM acetate buffer (pH 4.0), such that the dilution degree of the extracellular fluid of Chlorella is 0.3X (times), 0.5X, 1X the initial concentration of the extracellular fluid of Chlorella. Then 900 μL of 1 mM ABTS-acetate buffer is added, and the color diagrams of oxidized ABTS at different dilution degrees are recorded. See Figure 1 .

[0066] (2) Test for the guaiacol oxidation activity of the extracellular fluid of Chlorella:

[0067] In a 5 mL reaction system, it contains 100 μL of the extracellular fluid of Chlorella prepared in Examples 1-3 and Comparative Example 1 diluted with 50 mM phosphate buffer (pH 5.5), such that the dilution degree of the extracellular fluid of Chlorella is 0.3X (times), 0.5X, 1X the initial concentration of the extracellular fluid of Chlorella. Then 1 mL of 50 mM guaiacol, 1 mL of 2% hydrogen peroxide are added, and 2.9 mL of 50 mM phosphate buffer (pH 5.5) is added. The color diagrams of oxidized guaiacol at different dilution degrees are recorded. See Figure 1 .

[0068] From Figure 1 the results of the oxidation of ABTS and guaiacol by the extracellular fluid of Chlorella at different concentrations, it is found that the extracellular fluid of Chlorella produces color reactions with ABTS and guaiacol respectively, and has the ability to oxidize ABTS and guaiacol; moreover, as the concentration of the extracellular fluid of Chlorella increases, the color gradually becomes darker, proving that the oxidation ability gradually increases. And the colors of Examples 1-3 are significantly darker than those of Comparative Example 1, indicating that the oxidation ability of the extracellular fluid of Chlorella in the examples of the present invention is significantly stronger than that of the extracellular fluid of Chlorella in Comparative Example 1.

[0069] (3) Test for the oxidation activity change curve of the extracellular fluid of Chlorella:

[0070] During the preparation processes of Example 1, Examples 4-8 and Comparative Example 1, samples are taken at the 0th, 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th days of Chlorella growth to measure the ABTS oxidation activity. When sampling, the extracellular fluid of Chlorella to be tested is shaken well, 1 mL of the extracellular fluid of Chlorella is taken and added to a cuvette, and the oxidation activity is detected by the ABTS method. Three parallels are made for each sample. The oxidation activity of the extracellular fluid of Chlorella is measured every two days, and the oxidation activity change curve is plotted. See Figure 2 .

[0071] The calculation formula for the ABTS oxidation activity is as follows:

[0072]

[0073] 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 .

[0074] Depend on Figure 2 The change curve of the oxidation activity of the extracellular fluid of Chlorella with different concentrations of oxcarbazepine shows that, on the 10th to 16th day, the oxidation activity of the extracellular fluid of Chlorella was significantly improved when 5-30 mg / L oxcarbazepine was added (Example 1, Examples 4-8) compared with no addition of oxcarbazepine (Comparative Example 1), and the oxidation capacity was gradually enhanced. When the final concentration of oxcarbazepine was 5 mg / L, the oxidation activity reached a maximum of 930 μmol·L on the 16th day. -1 ·min -1 .

[0075] 2. Stability test of Chlorella extracellular fluid:

[0076] 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 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 (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.

[0077] The calculation formula of relative activity is as follows:

[0078]

[0079] From Figures 3 - 6 the results, it can be seen that after the extracellular fluid of Chlorella vulgaris in each example and comparative example was treated under various conditions, the relative activities were all relatively high. However, the relative activity of the extracellular fluid of Chlorella vulgaris with the addition of oxcarbazepine in the examples was slightly higher than that of the extracellular fluid of Chlorella vulgaris without the addition of oxcarbazepine in the comparative examples. This shows that the extracellular fluid of Chlorella vulgaris in the examples of the present invention can tolerate high temperatures of 100 °C, has strong acid and alkali resistance, and is less affected by metal ions and organic reagents.

[0080] 3. Degradation performance test of the extracellular fluid of Chlorella vulgaris:

[0081] (1) Degradation performance test of the extracellular fluid of Chlorella vulgaris on pharmaceutical products:

[0082] Add pharmaceutical products (dexamethasone, prednisone, sulfamethazine, sulfamethoxazole, sulfisoxazole, carbamazepine, oxcarbazepine, acetazolamide, promethazine hydrochloride, sulfacetamide, diphenhydramine hydrochloride) to the extracellular fluid of Chlorella vulgaris prepared in Examples 1-3 and Comparative Example 1, and carry out the degradation of the extracellular fluid of Chlorella vulgaris - pharmaceutical product reaction system in an incubator at 28 °C. Sampling is carried out on the 0th and 8th days for high-performance liquid chromatography detection, and the degradation rate of the extracellular fluid of Chlorella vulgaris on different pharmaceutical products on the 8th day is calculated. Among them, the concentration of the pharmaceutical product is 20 mg / L, and the oxidation activity of the extracellular fluid of Chlorella vulgaris is 300 μmol·L -1 ·min -1 , and the results are shown in Figure 7 .

[0083] From Figure 7 the results, it can be seen that in the degradation system of the extracellular fluid of Chlorella vulgaris on pharmaceutical products, after eight days of reaction, it was found that the extracellular fluid of Chlorella vulgaris prepared in the examples of the present invention has a relatively high degradation effect on four major types of drugs, namely steroid hormones, sulfonamide antibiotics, antiepileptic drugs, and antihistamine reagents. Among them, the degradation rates of prednisone, sulfisoxazole, oxcarbazepine, promethazine hydrochloride, and diphenhydramine hydrochloride can all reach 80% and above, which are significantly higher than the degradation rate of the extracellular fluid of Chlorella vulgaris without oxcarbazepine in Comparative Example 1 on pharmaceutical products.

[0084] (2) Degradation performance test of the extracellular fluid of Chlorella vulgaris on polycyclic aromatic hydrocarbons:

[0085] Polycyclic aromatic hydrocarbons (naphthalene, fluorene, phenanthrene, acenaphthene, anthracene, pyrene, fluoranthene, dibenzothiophene, dibenzofuran, biphenyl, 2-methylnaphthalene) were added to the extracellular fluid of Chlorella prepared in Examples 1-3 and Comparative Example 1. The degradation of the extracellular fluid of Chlorella-polycyclic aromatic hydrocarbon reaction system was carried out in an incubator at 28 °C. Samples were taken at 0 h and 24 h for high-performance liquid chromatography detection. The degradation rate of different polycyclic aromatic hydrocarbons by the extracellular fluid of Chlorella at 24 h was calculated. Among them, the concentration of polycyclic aromatic hydrocarbons was 20 mg / L, and the oxidation activity of the extracellular fluid of Chlorella was 300 μmol·L -1 ·min -1 , and the results are shown in Figure 8 .

[0086] From Figure 8 the results, it can be seen 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 examples of the present invention could all reach 90% and 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.

[0087] (3) Degradation performance test of the extracellular fluid of Chlorella on dyes:

[0088] Dyes (indigo carmine, reactive blue, bromophenol blue, congo red, reactive red, evans blue, malachite green, disperse red, reactive black, crystal violet, azure B) were added to the extracellular fluid of Chlorella prepared in Examples 1-3 and Comparative Example 1. The degradation of the extracellular fluid of Chlorella-dye reaction system was carried out in an incubator at 28 °C. Samples were taken at 0 and 8 days for high-performance liquid chromatography detection. The degradation rate of different dyes by the extracellular fluid of Chlorella at 8 days was calculated. Among them, the concentration of dyes was 100 mg / L, and the oxidation activity of the extracellular fluid of Chlorella was 300 μmol·L -1 ·min -1 , and the results are shown in Figure 9 .

[0089] From Figure 9 the results, it can be seen that in the degradation system of dyes by 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 examples of the present invention could all reach 50% and above, which was significantly higher than the degradation rate of dyes by the extracellular fluid of Chlorella without adding oxcarbazepine in Comparative Example 1.

[0090] In summary, in the preparation method of the present invention, the extracellular fluid of Chlorella prepared by adding oxcarbazepine under specific culture media and specific growth environments has strong oxidation activity, excellent stability and outstanding degradation ability, providing new ideas for the treatment of current environmental pollutants such as pharmaceutical products, polycyclic aromatic hydrocarbons and dyes.

[0091] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope 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 liquid under an intermittent light environment at a temperature of 20-30°C and a rotation speed of 100-200 rpm, and 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 liquid; The final concentration of oxcarbazepine is 5-25 mg / L, the initial inoculation amount of the Chlorella algae solution is 5-10% of the volume of the culture medium, and the intermittent lighting pattern is 12 hours of darkness and 12 hours of light.

2. 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.

3. The method for preparing the extracellular fluid of Chlorella according to claim 1, characterized in that: The light intensity is 1000-3000 lux.

4. 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.

5. 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 4.

6. Use of the extracellular fluid of Chlorella as claimed in claim 5 in the field of pollutant treatment.

Images