A method of degrading antibiotics and a composition for degrading antibiotics for microalgae

Abstract

This application pertains to the field of microalgae applications, and particularly relates to a method for degrading antibiotics and a composition for antibiotic degradation using microalgae. This application provides a method for degrading antibiotics, comprising the following steps: mixing and culturing an antibiotic sample to be treated, a degradation additive, and algal culture until the antibiotics in the algal culture are degraded to discharge standards; the degradation additive is selected from one or more of sodium bicarbonate, glycerol, sodium acetate, and glucose. This application provides a method for degrading antibiotics and a composition for antibiotic degradation using microalgae, which can significantly improve the efficiency of antibiotic degradation by microspheres and increase the yield of high-value-added active products from microalgae, possessing broad environmental remediation and commercial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microalgae application, and particularly relates to a method for degrading antibiotics and a composition for degrading antibiotics for microalgae. BACKGROUND

[0002] In order to treat and prevent bacterial infections occurring in humans and animals, various types of antibiotics have been developed. The massive use of antibiotics has led to an increasing amount of antibiotic residues discharged into aquatic and terrestrial environments through sewage and waste, and the overuse or misuse of antibiotics has led to the emergence of drug-resistant superbugs in humans, making subsequent bacterial treatment more and more difficult. Antibiotic resistance has become one of the global health threats, and thus the conditions for the consumption and use of antibiotics have become more and more stringent.

[0003] Fluoroquinolones (such as ofloxacin and enrofloxacin) are widely used as broad-spectrum antibacterial drugs in hospitals, families, livestock, and aquaculture, and are the third largest antibiotic in global sales. Due to the acute and chronic toxicity of ofloxacin and enrofloxacin to aquatic organisms, even at very low concentrations, they will eventually cause serious damage to aquatic ecosystems and even human health. In addition, more seriously, trace amounts of antibiotics can induce the evolution and development of drug-resistant bacteria and drug-resistant genes, thereby causing potential environmental risks. Therefore, it is necessary to explore a sustainable and efficient method for removing fluoroquinolone antibiotics in water environments.

[0004] Although some physical and chemical technologies have developed efficient methods for removing antibiotics, specific limitations and defects (such as adverse byproducts and expensive costs) limit their practical application. Biotechnological methods are a sustainable method for enhancing antibiotic dissipation and even mineralizing antibiotics. Microalgae-based biological treatment technology can effectively remove antibiotics at low cost and has the advantages of low carbon and renewability. In addition, the microalgae system produces high-value active products for commercial products while degrading antibiotics, achieving waste-to-resource. However, in practical applications, the efficiency of removing fluoroquinolone antibiotics by microalgae technology is not high. SUMMARY

[0005] In view of this, the present application provides a method for degrading antibiotics and a composition for degrading antibiotics for microalgae, which can significantly improve the efficiency of microalgae in degrading antibiotics and increase the yield of high-value active products of microalgae, and has wide environmental remediation and commercial application value.

[0006] The first aspect of the present application provides a method for degrading antibiotics, comprising the following steps:

[0007] mixing and culturing a to-be-treated antibiotic sample, a degradation additive, and an algal liquid until the antibiotic in the algal liquid is degraded to a discharge standard;

[0008] The degradation additive is selected from one or more of sodium bicarbonate, glycerol, sodium acetate, and glucose.

[0009] More preferably, the degradation additive is glycerol.

[0010] Specifically, the degradation additive of the present application is sterilized in advance, and the sterilization method used is conventional sterilization methods such as 0.22 μm membrane filtration sterilization, high-temperature sterilization, and ultraviolet sterilization.

[0011] Preferably, the concentration of the degradation additive in the algal liquid is 0.01 M to 0.2 M.

[0012] More preferably, the concentration of the degradation additive in the algal liquid is 0.05 M to 0.1 M.

[0013] Preferably, the concentration of the antibiotic in the algal liquid is 0.01 to 10 mg / L.

[0014] More preferably, the concentration of the antibiotic in the algal liquid is 5 to 10 mg / L.

[0015] Preferably, the antibiotic sample to be treated is dissolved in a solvent in advance.

[0016] Specifically, the antibiotic sample to be treated is dissolved in a solvent in advance, and ultrasonic dissolution is used to ensure sufficient dissolution.

[0017] Preferably, the antibiotic sample to be treated is derived from the sea, rivers, municipal sewage, hospital wastewater, livestock and poultry manure, or a landfill.

[0018] Preferably, the antibiotic of the antibiotic sample to be treated includes one or more of fluoroquinolones, macrolides, lincomycins, polypeptides, β-lactams, aminoglycosides, and sulfonamides.

[0019] Specifically, the antibiotic of the antibiotic sample to be treated is ofloxacin and enrofloxacin, the solvent of the antibiotic sample to be treated is dimethyl sulfoxide, and ultrasonic dissolution is used to ensure sufficient dissolution.

[0020] Preferably, the algal liquid includes microalgae and a microalgae culture medium; the microalgae are selected from one or more of Haematococcus pluvialis, Chlorella vulgaris, Scenedesmus, Dunaliella salina, Phaeodactylum tricornutum, and Euglena gracilis, and the microalgae culture medium is selected from one or more of BBM medium, BG11 medium, f / 2 medium, and HUT medium.

[0021] More preferably, the algal liquid includes Haematococcus pluvialis and BBM medium.

[0022] More preferably, the optimal concentration of glycerol added is 0.05 M when the Haematococcus pluvialis degrades ofloxacin in the algal broth.

[0023] More preferably, the optimal concentration of glycerol added is 0.75 M when the Haematococcus pluvialis degrades enrofloxacin in the algal broth.

[0024] Preferably, the culture conditions of the mixed culture include:

[0025] The initial density of the microalgae in the algal broth is 1 x 10 4 individuals / mL; the culture is carried out in a sterile aeration manner, the culture temperature is 20-30℃, the light intensity during the exponential growth phase of the microalgae is 50-200 μmol·m -2 ·s -1 , the light intensity during the stationary phase of the microalgae is 300-600 μmol·m -2 ·s -1 , and the light / dark cycle is 12h / 12h.

[0026] Specifically, the microalgae can be cultured in an artificial incubator, a photobioreactor or an industrialized culture pond.

[0027] More preferably, the culture period of the mixed culture from inoculation to the end of the culture is 16 days.

[0028] More specifically, when the microalgae is Haematococcus pluvialis, the parameters of the mixed culture include:

[0029] the light intensity during 0-12 days is 120 μmol·m -2 ·s -1 , the light intensity during 12-16 days is 400 μmol·m -2 ·s -1 , and the light / dark cycle during the whole culture period is 12h / 12h. The whole culture period is equipped with aeration system, the aeration is filtered through a 0.22 μm membrane to achieve sterile conditions, and the aeration amount is 15 mL / min.

[0030] Preferably, the method further includes collecting the microalgae and discharging the treated liquid.

[0031] Specifically, the collected microalgae are subjected to physical, chemical or biological techniques to obtain high-value products, such as astaxanthin, oil, unsaturated fatty acids, etc. which can be extracted from Haematococcus pluvialis.

[0032] The second aspect of the present application provides a composition for degrading antibiotics for microalgae, the composition comprising a degradation additive and a microalgae culture medium; the degradation additive is selected from one or more of sodium bicarbonate, glycerol, sodium acetate and glucose.

[0033] Specifically, when in use, the microalgae are inoculated into the composition, and then the antibiotic sample to be treated is added, so that the microalgae can efficiently degrade the high-dose antibiotic.

[0034] The present application finds that adding a specific degradation additive to the microalgae culture medium can effectively improve the degradation efficiency of the microalgae in biodegrading antibiotics. Antibiotics are drugs that threaten aquatic ecosystems and even human life and health in the environment. Adding a specific degradation additive to the microalgae culture medium can improve the degradation efficiency of antibiotics, which can reduce the damage of antibiotics to the environment and humans on the one hand, and can reduce the culture cost of microalgae and increase the yield of high-value active products on the other hand. Experimental results show that the addition of a specific degradation additive to the Haematococcus pluvialis culture medium significantly improves the biodegradation efficiency of Haematococcus pluvialis on ofloxacin and enrofloxacin. Experimental results show that when the concentration of ofloxacin is 10 mg / mL, the optimal addition amount of glycerol is 0.075 M; when the concentration of enrofloxacin is 10 mg / mL, the optimal addition amount of glycerol is 0.075 M. Experimental results show that the addition of glycerol can improve the yield of lipids and astaxanthin while degrading ofloxacin and enrofloxacin by Haematococcus pluvialis. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0036] Figure 1 The removal efficiency of ofloxacin (A) and enrofloxacin (B) by Haematococcus pluvialis provided in the embodiments of the present application in the BBM culture medium at 1 mg / L, 5 mg / L and 10 mg / L, the abscissa is the concentration of ofloxacin and enrofloxacin (unit: mg / L), and the ordinate is the removal efficiency (unit: %), and the color blocks at the bottom of the figure are the proportions of photolysis, hydrolysis, biological adsorption by Haematococcus pluvialis, biodegradation and residues in the culture medium of ofloxacin and enrofloxacin; Figure 1 Figure 1 The degradation efficiency of ofloxacin and enrofloxacin by Haematococcus pluvialis under the influence of different degradation additives, the abscissa is the concentration of ofloxacin and enrofloxacin (unit: mg / L), and the ordinate is the removal ratio (unit: %), wherein, A is the degradation efficiency of 5 mg / L and 10 mg / L ofloxacin in the presence of different degradation additives, and B is the degradation efficiency of 5 mg / L and 10 mg / L enrofloxacin in the presence of different degradation additives;

[0037] Figure 2

[0038] Figure 3 ​​The removal rates of ofloxacin and enrofloxacin by Haematococcus pluvialis under the cooperation of different concentrations of glycerol provided by the embodiments of the present application are shown in the figure, wherein the abscissa is the removal rate of ofloxacin and enrofloxacin (unit: %), and the ordinate is the concentration of glycerol (unit: mol / L), wherein A is the removal rate of ofloxacin by Haematococcus pluvialis under different glycerol concentrations, and B is the removal rate of enrofloxacin by Haematococcus pluvialis under different glycerol concentrations;

[0039] Figure 4 The concentrations of residual glycerol in the culture medium at different time periods provided by the embodiments of the present application are shown in the figure, wherein the abscissa is the culture time (unit: day), and the ordinate is the concentration of residual glycerol in the culture medium (unit: g / L), OFL represents the ofloxacin group, OFL+Glycerol represents the ofloxacin and glycerol group, ENR represents the enrofloxacin group, and ENR+Glycerol represents the enrofloxacin and glycerol group;

[0040] Figure 5 The proportions of carbohydrates, proteins and total lipids in dry weight of Haematococcus pluvialis at different culture times provided by the embodiments of the present application are shown in the figure, wherein the abscissa is the culture time (unit: day), and the ordinate is the proportion in dry weight (unit: %). Among them, A is the percentage of carbohydrates in dry weight of Haematococcus pluvialis at different culture times, B is the percentage of proteins in dry weight of Haematococcus pluvialis at different culture times, C is the percentage of lipids in dry weight of Haematococcus pluvialis at different culture times, OFL represents the ofloxacin group, OFL+Glycerol represents the ofloxacin and glycerol group, ENR represents the enrofloxacin group, and ENR+Glycerol represents the enrofloxacin and glycerol group;

[0041] Figure 6 The proportions of fatty acid and astaxanthin in dry weight of Haematococcus pluvialis in different experimental groups and the composition of astaxanthin provided by the embodiments of the present application are shown in the figure, wherein the abscissa is different experimental groups, and from left to right, they are A, B and C figures. A figure is the content multiple of total fatty acid in different experimental groups, B figure is the percentage of astaxanthin in dry weight, and C figure is the proportion of monoester, diester and free astaxanthin in total astaxanthin. The bottom of the figure shows that OFL represents ofloxacin (+ for adding, - for not adding), ENR represents enrofloxacin (+ for adding, - for not adding), and Glycerol represents glycerol (+ for adding, - for not adding); DETAILED DESCRIPTION

[0042] The present application provides a method for degrading antibiotics and a composition for degrading antibiotics for microalgae, which solves the technical defects of low efficiency of removing fluoroquinolone antibiotics by microalgae process in the prior art.

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0044] In the following embodiments, the reagents or raw materials used are commercially available or self-made. In the embodiments of the present application, the fluoroquinolone antibiotics degraded by Haematococcus pluvialis are ofloxacin and enrofloxacin. The purity of ofloxacin and enrofloxacin is greater than 98%, and they are purchased from Shanghai Aladdin. The preparation and storage concentration of ofloxacin and enrofloxacin is 5 mg / mL and 10 mg / mL. The solvent of ofloxacin and enrofloxacin is dimethyl sulfoxide, and the dissolution under ultrasonic conditions ensures sufficient dissolution.

[0045] Embodiment 1

[0046] In the embodiments of the present application, 1 mg / L, 5 mg / L and 10 mg / L of ofloxacin and enrofloxacin are added respectively in the BBM culture medium of Haematococcus pluvialis, so as to explore the removal rate of ofloxacin and enrofloxacin by Haematococcus pluvialis. The specific steps are as follows:

[0047] 1. First, ofloxacin and enrofloxacin are dissolved in dimethyl sulfoxide under ultrasonic conditions at a solubility of 5 mg / ml and 10 mg / ml, as a storage solution. Then, Haematococcus pluvialis is exposed to the BBM culture medium of ofloxacin and enrofloxacin with a final concentration of 1 mg / L, 5 mg / L and 10 mg / L.

[0048] 2. The culture of Haematococcus pluvialis in the embodiments of the present application is carried out in a sterile aeration culture in a constant temperature incubator, the sterile aeration rate is 15 ml / min, the culture temperature is 25℃, the light intensity is 120 μmol·m -2 ·s -1 from 0 to 12 days, the light intensity is 400 μmol·m -2 ·s -1 from 12 to 16 days, the light and dark cycle is 12h / 12h, and the initial cell density of Haematococcus pluvialis is 1×10 4 cells / ml.

[0049] 3. Detection of the remaining ofloxacin and enrofloxacin in the culture medium: the content of the remaining ofloxacin and enrofloxacin in the culture medium is detected by high performance liquid chromatography. The chromatographic conditions are as follows: Agilent high performance liquid chromatography (HPLC, Agilent, USA) is equipped with a C18 chromatographic column (4.6×150 mm, 5 μm, Agilent, USA) and an ultraviolet detector, the detection wavelength is 272 nm, and ofloxacin and enrofloxacin are quantified.

[0050] Wherein, after the experiment, the content of the remaining ofloxacin and enrofloxacin in the culture medium was detected. The removal efficiency of Haematococcus pluvialis to different concentrations of ofloxacin and enrofloxacin was as follows Figure 1 When the concentration of ofloxacin and enrofloxacin was 1 mg / L, the removal efficiency of Haematococcus pluvialis to ofloxacin was 100%, and the removal efficiency of Haematococcus pluvialis to enrofloxacin was 89.71% through detection. The removal rate of Haematococcus pluvialis to 5 mg / L ofloxacin and enrofloxacin was 37.83% and 34.44% respectively, and the removal rate of Haematococcus pluvialis to 10 mg / L ofloxacin and enrofloxacin was 21.85% and 22.81% respectively, indicating that high-dose fluoroquinolone antibiotics had toxicity to Haematococcus pluvialis, and Haematococcus pluvialis could not efficiently degrade high-dose fluoroquinolone antibiotics.

[0051] Example 2

[0052] The example of the application provides the removal rate of 5, 10 mg / L ofloxacin and enrofloxacin under the condition of adding 0.05M different degradation additives in the culture medium, and the organic substrate can improve the tolerance and biodegradation ability of Haematococcus pluvialis to antibiotics. 0.05M different degradation additives: sodium bicarbonate, glycerol, sodium acetate and glucose are used to evaluate the removal effect of Haematococcus pluvialis to high-concentration ofloxacin and enrofloxacin. The specific steps are as follows:

[0053] 1. First, dissolve ofloxacin and enrofloxacin in dimethyl sulfoxide under ultrasonic conditions at a solubility of 5, 10 mg / ml as a storage solution. Then expose Haematococcus pluvialis to BBM medium with a final concentration of 5, 10 mg / L ofloxacin and enrofloxacin.

[0054] 2. Add the same concentration (0.05M) of sodium bicarbonate, glycerol, sodium acetate and glucose in the culture medium of Haematococcus pluvialis, and do not add degradation additives as a control group.

[0055] After the experiment, the content of the remaining ofloxacin and enrofloxacin in the culture medium was detected, and the results were as follows Figure 2, the removal efficiency of antibiotics is obviously higher than that of other degradation additives. In the experiment of degrading 5 mg / L ofloxacin under the promotion of different degradation additives, the degradation efficiency of ofloxacin by Haematococcus pluvialis without any degradation additive is 35.07%, and the degradation efficiencies of ofloxacin by Haematococcus pluvialis under the synergistic action of sodium bicarbonate, glycerol, sodium acetate and glucose are 58.81%, 99.59%, 47.71% and 34.96%, respectively; in the experiment of degrading 10 mg / L ofloxacin under the promotion of different degradation additives, the degradation efficiency of ofloxacin by Haematococcus pluvialis without any degradation additive is 14.99%, and the degradation efficiencies of ofloxacin by Haematococcus pluvialis under the synergistic action of sodium bicarbonate, glycerol, sodium acetate and glucose are 28.35%, 68.65%, 24.18% and 16.29%, respectively.

[0056] In the experiment of degrading 5 mg / L enrofloxacin under the promotion of different degradation additives, the degradation efficiency of enrofloxacin by Haematococcus pluvialis without any degradation additive is 24.81%, and the degradation efficiencies of enrofloxacin by Haematococcus pluvialis under the synergistic action of sodium bicarbonate, glycerol, sodium acetate and glucose are 44.66%, 90.92%, 39.72% and 32.37%, respectively; in the experiment of degrading 10 mg / L enrofloxacin under the promotion of different degradation additives, the degradation efficiency of enrofloxacin by Haematococcus pluvialis without any degradation additive is 12.69%, and the degradation efficiencies of enrofloxacin by Haematococcus pluvialis under the synergistic action of sodium bicarbonate, glycerol, sodium acetate and glucose are 20.4%, 54.37%, 19.42% and 11.88%, respectively.

[0057] It is illustrated that the degradation additive has an assisting effect on the degradation of high-dose fluoroquinolone antibiotics by Haematococcus pluvialis, and glycerol is especially used.

[0058] Example 3

[0059] The example provides the ability of Haematococcus pluvialis to degrade 10 mg / L ofloxacin and enrofloxacin under the synergistic action of different concentrations of glycerol, 0.01M, 0.02M, 0.03M, 0.05M, 0.075M, 0.1M, 0.15M and 0.2M of glycerol are added in the BBM culture medium of Haematococcus pluvialis at the initial stage of culture, and the contents of residual ofloxacin and enrofloxacin in the culture medium are detected after the experiment, the amounts of ofloxacin and enrofloxacin degraded by Haematococcus pluvialis under the synergistic action of glycerol are calculated, and the results are as follows Figure 3When the Haematococcus pluvialis is exposed to 0.05M-0.1M glycerol, the removal efficiency of antibiotics is higher, wherein, under the co-metabolic condition of 0.075M glycerol, the removal efficiency of Haematococcus pluvialis to ofloxacin is the highest, the content of ofloxacin degraded by biological behavior is 7.77mg / L, and the degradation efficiency is 67.14%; under the co-metabolic condition of 0.05M glycerol, the removal efficiency of Haematococcus pluvialis to enrofloxacin is the highest, the content of enrofloxacin degraded by biological behavior is 5.13mg / L, and the degradation efficiency is 42.83%.

[0060] Example 4

[0061] The present application provides the residual amount of glycerol in the culture medium during the whole growth cycle of Haematococcus pluvialis. The experiment is divided into four groups, namely, the ofloxacin group (marked as OFL), the ofloxacin+glycerol group (marked as OFL+Glycerol), the enrofloxacin group (marked as ENR), and the enrofloxacin+glycerol group (marked as ENR+Glycerol). The initial concentration of antibiotics in the four groups is 10mg / L, 0.075M glycerol (i.e. 6.92g / L) is added to the culture medium in the OFL+Glycerol group, and 0.05M glycerol (i.e. 4.60g / L) is added to the culture medium in the ENR+Glycerol group. The residual amount in the culture medium is measured at 0 day, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days and 16 days. Figure 4 The glycerol in the culture medium is consumed at a stable speed during the dormancy stage, and the residual amount of glycerol in the culture medium with ofloxacin is 3.09g / L, and the residual amount of glycerol in the culture medium with enrofloxacin is 1.69g / L on the 12th day; it is rapidly consumed to 0 during the growth stage.

[0062] Example 5

[0063] The present application provides the change of carbohydrates, proteins and lipids during the whole growth process of Haematococcus pluvialis when degrading ofloxacin and enrofloxacin under the cooperation of glycerol.

[0064] The experiment is divided into four groups, namely, the ofloxacin group (marked as OFL), the ofloxacin+glycerol group (marked as OFL+Glycerol), the enrofloxacin group (marked as ENR), and the enrofloxacin+glycerol group (marked as ENR+Glycerol), the initial concentration of antibiotics in the four groups is 10mg / L, 0.075M glycerol (i.e. 6.92g / L) is added to the culture medium in the OFL+Glycerol group, and 0.05M glycerol (i.e. 4.60g / L) is added to the culture medium in the ENR+Glycerol group. The change of carbohydrates, proteins and lipids is measured at 0 day, 3 days, 6 days, 9 days and 12 days.

[0065] Detection of Haematococcus pluvialis carbohydrate: Total sugar content was detected by phenol-sulfuric acid method. Haematococcus pluvialis was collected in a centrifuge tube, and 1 mL of deionized water was added to the tube. After vortexing for 30 s, 1 mL of 5% phenol solution was added to the centrifuge tube, followed by slowly adding 5 mL of 98% concentrated sulfuric acid along the tube wall. Incubate in a water bath at 25 °C for 10 min, and then transfer to a water bath at 30 °C for incubation for 20 min. After the above steps, the standard sample reaction solution and the Haematococcus pluvialis reaction solution were added to a pre-sequenced 96-well plate, and the absorbance at 483 nm was detected by Bio-Tek enzyme marker. The standard curve was drawn according to the hole values of different standard sample concentrations, and the carbohydrate content of Haematococcus pluvialis was calculated according to the standard curve and the collected Haematococcus pluvialis volume.

[0066] Detection of Haematococcus pluvialis protein: Haematococcus pluvialis was collected in a centrifuge tube, 1 mL of RIPA lysis buffer was added, and an appropriate amount of grinding beads was added. Grind for 6 min, and then place on ice for 30 min. Vortex for 30 s every 5 min, repeat for 6 times, and then centrifuge at 12000 rpm for 10 min. Collect the supernatant into a 1.5 mL centrifuge tube. Add the sample to the sample well of the 96-well plate according to the proportion, and supplement with RIPA lysis buffer to 20 μL if the sample is less than 20 μL. Add the standard sample in the BCA protein concentration determination kit to the standard sample well according to the proportion, and supplement with RIPA lysis buffer to 20 μL if the sample is less than 20 μL. Add 200 μL of BCA working solution to the sample well and the standard sample well, and incubate the 96-well plate in a 37 °C incubator for 30 min. Detect the absorbance at 562 nm by Bio-Tek enzyme marker, draw the standard curve according to the standard sample well value, and calculate the protein concentration according to the standard curve and the sample volume.

[0067] Detection of Haematococcus pluvialis lipid: Haematococcus pluvialis was collected in a centrifuge tube, and 3.8 mL of methanol, chloroform and deionized water was added according to the volume ratio of 2:1:0.8. After vortexing for 2 min, centrifuge at 1500 rpm for 5 min at room temperature, and take out the lower chloroform to a weighed centrifuge tube. Add chloroform to the original centrifuge tube, vortex for 2 min, and then centrifuge at 1500 rpm for 5 min at room temperature. Take out the lower chloroform to the previously weighed centrifuge tube, and repeat the extraction once. Dry the collected chloroform by nitrogen blowing instrument, accurately weigh the total lipid-containing tube by analytical electronic balance, and calculate the final total lipid content.

[0068] The experimental results are as follows Figure 5, the content of carbohydrates of Chlorella vulgaris exposed to ofloxacin and enrofloxacin in the medium added with glycerol was higher, the content of protein of Chlorella vulgaris exposed to ofloxacin and enrofloxacin was significantly reduced, and the content of lipids accumulated in the growth stage of Chlorella vulgaris was higher than that without glycerol added in the medium. On the 14th day, the content of lipids of Chlorella vulgaris reached the highest when degrading ofloxacin and enrofloxacin in the presence of glycerol, which was 17.58% and 16.54% respectively; the content of protein of Chlorella vulgaris on the 12th day when degrading ofloxacin and enrofloxacin in the presence of glycerol accounted for 37.18% and 39.70% of dry weight respectively, and the content of protein on the 16th day accounted for 37.25% and 39.12% of dry weight respectively; the content of carbohydrates of Chlorella vulgaris on the 12th day when degrading ofloxacin and enrofloxacin in the presence of glycerol accounted for 36.44% and 34.34% of dry weight respectively. It is shown that when degrading ofloxacin and enrofloxacin in the presence of glycerol, the biosynthesis direction of Chlorella vulgaris is changed from protein to carbohydrates and lipids.

[0069] Example 6

[0070] The present application provides the content of fatty acids and astaxanthin of Chlorella vulgaris when degrading ofloxacin and enrofloxacin in the presence of glycerol.

[0071] The experiment is divided into four groups, which are ofloxacin group (marked as OFL), ofloxacin + glycerol group (marked as OFL + Glycerol), enrofloxacin group (marked as ENR), and enrofloxacin + glycerol group (marked as ENR + Glycerol). The initial concentration of antibiotics in the four groups is 10 mg / L, 0.075 M glycerol (6.92 g / L) is added in the medium of OFL + Glycerol group, and 0.05 M glycerol (4.60 g / L) is added in the medium of ENR + Glycerol group. The content of total fatty acids, astaxanthin, the proportion of monoester, diester and free astaxanthin in total astaxanthin are determined.

[0072] Fatty acid composition analysis: the following three reagents are prepared in advance: solution A: sodium hydroxide + methanol (2%); solution B: acetyl chloride + methanol (volume ratio 1:10), note that methanol is added first, and acetyl chloride is added along the wall under the condition of magnetic stirring; solution C: 0.6 g of potassium carbonate is dissolved in 10 mL of water.

[0073] Fatty acid composition analysis: the following three reagents are prepared in advance: solution A: sodium hydroxide + methanol (2%); solution B: acetyl chloride + methanol (volume ratio 1:10), note that methanol is added first, and acetyl chloride is added along the wall under the condition of magnetic stirring; solution C: 0.6 g of potassium carbonate is dissolved in 10 mL of water.

[0074] The Haematococcus pluvialis was collected in a 1.5 mL EP tube, 500 μL of toluene was added, and the mixture was transferred to a glass tube; 2 mL of solution A was added, and the mixture was vortexed for 10-20 s and then heated in a water bath at 80°C for 15 min. The heating process was repeated every 5 min. The mixture was cooled at room temperature for 5 min, and then 2 mL of solution B was added. The mixture was heated in a water bath at 80°C for 15 min. The mixture was cooled at room temperature for 5 min, and then 1 mL of a potassium carbonate solution was added, followed by 1 mL of n-hexane and 10 μL of methyl nonadecanoate (10 mg / mL) as an internal standard. The mixture was vortexed for 20 s, and then centrifuged at a low speed to separate the layers. The supernatant was transferred to a new brown sample bottle, dried with high-purity nitrogen, and then 500 μL of n-hexane was added. The composition and content of fatty acids were detected by a GC-MS instrument.

[0075] Detection of astaxanthin: An appropriate amount of Haematococcus pluvialis was collected in a centrifuge tube, 600 μL of a 5% KOH solution (w / v, dissolved in a mixture of methanol: water v / v = 70:30) was added, and the mixture was incubated at 65°C for 10 min, and then centrifuged at 6000 rpm for 5 min. 300 μL of dimethyl sulfoxide and 300 μL of acetic acid were added, and the mixture was incubated at 75°C for 5 min to extract astaxanthin. The free form of astaxanthin was extracted from the saponification of mono- and di- ester astaxanthin, and then incubated with a 21 mM NaOH solution at 23°C in the dark for 3 h. The filtered mixture was analyzed by HPLC. The filtered mixture was identified by HPLC (Waters, UK) equipped with a reversed-phase C18 chromatographic column (Waters, UK), with A phase (a mixture of dichloromethane, methanol, acetonitrile, and water at a volume ratio of 5:85:5.5:4.5) and B phase (a mixture of dichloromethane, methanol, acetonitrile, and water at a volume ratio of 25:28:42.5:4.5) as gradient mobile phases, at a flow rate of 1 mL / min. 10 μL of each sample was analyzed, and the wavelength of the sample was detected at 460 nm by a photodiode array detector. The peak area was quantified according to the normalized peak area of astaxanthin standards.

[0076] The results are shown in Table 1. Figure 6, the content of fatty acid produced by Haematococcus pluvialis under the synergistic effect of glycerol is more than that produced without glycerol. The fatty acid produced by Haematococcus pluvialis exposed to ofloxacin under the synergistic effect of glycerol is 1.50 times of that produced without glycerol; the fatty acid produced by Haematococcus pluvialis exposed to enrofloxacin under the synergistic effect of glycerol is 1.41 times of that produced without glycerol. The content of astaxanthin produced by Haematococcus pluvialis under the synergistic effect of glycerol is also higher, and more monoester astaxanthin is accumulated when Haematococcus pluvialis degrades ofloxacin under the synergistic effect of glycerol. The yield of astaxanthin produced by Haematococcus pluvialis degrading ofloxacin under the synergistic effect of glycerol can reach 4.81%, and the yield of astaxanthin produced by Haematococcus pluvialis degrading enrofloxacin under the synergistic effect of glycerol is 4.69%. The proportion of monoester astaxanthin produced by Haematococcus pluvialis degrading ofloxacin under the synergistic effect of glycerol has a significant difference compared with that without glycerol, the proportion of monoester astaxanthin produced by Haematococcus pluvialis degrading ofloxacin under the synergistic effect of glycerol is 75.93%, and the proportion of monoester astaxanthin produced by Haematococcus pluvialis degrading ofloxacin without glycerol is 66.72%.

[0077] In summary, the experimental data of the above examples show that Haematococcus pluvialis can more efficiently biodegrade ofloxacin and enrofloxacin under the synergistic effect of glycerol, and the synergistic effect of glycerol can promote Haematococcus pluvialis to accumulate more carbohydrates and lipids, and the content of astaxanthin is significantly improved, especially monoester astaxanthin.

[0078] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make some improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method of degrading an antibiotic, characterized by, The method comprises the following steps: mixing and culturing an antibiotic sample to be treated, a degradation additive and an algal liquid until the antibiotic in the algal liquid is degraded to a discharge standard; the degradation additive is selected from glycerol; the algal liquid comprises Haematococcus pluvialis and a microalgal culture medium; the culture condition of the mixing and culturing comprises: The initial density of microalgae in the algal liquid is 1×10 4 The algal liquid is cultured by sterile aeration, the culture temperature is 20-30℃, the light intensity is 50-200 μmol·m −2 ·s −1 during the exponential growth phase of the microalgae, and the light intensity is 300-600 μmol·m −2 ·s −1 during the stationary phase of the microalgae, and the light / dark cycle is 12h / 12h.

2. The method of claim 1, wherein, the concentration of the degradation additive in the algal liquid is 0.01 M-0.2 M.

3. The method of claim 1, wherein, the concentration of the antibiotic in the antibiotic sample to be treated in the algal liquid is 0.01-10 mg / L.

4. The method of claim 1, wherein, the antibiotic sample to be treated is previously dissolved in a solvent.

5. The method of claim 1, wherein, the antibiotic sample to be treated is derived from the sea, rivers, municipal sewage, hospital wastewater, livestock and poultry manure or a landfill.

6. The method of claim 1, wherein, the antibiotic of the antibiotic sample to be treated comprises one or more of fluoroquinolones, macrolides, lincomycins, polypeptides, beta-lactams, aminoglycosides and sulfonamides.

7. The method of claim 1, wherein, the microalgal culture medium is selected from one or more of BBM medium, BG11 medium, f / 2 medium and HUT medium.

8. The method according to any one of claims 1 to 7, characterized in that, the method further comprises collecting the microalgae and discharging the treated liquid.

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