CN109305726B - Application of phycocyte secretion in degrading estrogen - Google Patents
Application of phycocyte secretion in degrading estrogen Download PDFInfo
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- CN109305726B CN109305726B CN201811093716.5A CN201811093716A CN109305726B CN 109305726 B CN109305726 B CN 109305726B CN 201811093716 A CN201811093716 A CN 201811093716A CN 109305726 B CN109305726 B CN 109305726B
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Abstract
The invention discloses an application of algal secretion in degrading estrogen. Aiming at the difficult degradability of estrogen in aquatic environment, organic substances secreted in the growth cycle of algae, particularly protein parts in the organic substances are applied to the degradation of estrogen, and the intermediate products after the degradation of estrogen are substances with lower toxicity. The invention provides a simpler, more time-saving and better-effect method for processing environmental estrogen and bioremediation and utilization of microalgae, natural algae secretion is adopted to degrade the estrogen in the environment, secondary pollution is not caused, and natural materials are fully utilized, so that the method is very environment-friendly.
Description
Technical Field
The invention belongs to the technical field of application of phycocyte secretion, and particularly relates to application of the phycocyte secretion in degrading estrogen.
Background
Estrogen (Estrogen) is a substance that promotes the development of secondary sexual characteristics and the maturation of sexual organs in female animals, and is secreted by the ovaries and placenta of female animals. The natural estrogen mainly comprises estradiol, estrone and estriol, most of the estrogen drugs commonly used in clinic at present are derivatives artificially synthesized by taking the estradiol as a matrix, and the 17 beta-estradiol (E2) has particularly strong endocrine interference and is one of the important hormones for regulating the endocrine system. The natural estrogen can be discharged into sewage treatment plants, soil, farmlands and other environments along with the excrement of animals and human bodies, and the artificially synthesized estrogen can also be discharged into the sewage treatment plants and other environments. The conventional active sewage treatment system cannot effectively remove estrogen in raw water, and the estrogen is discharged into rivers along with the effluent of a sewage treatment plant and accumulated in the environment. Estrogen, which is a typical endocrine disrupting substance in the environment, is accumulated and enriched in organisms in a direct or indirect way; after the estrogen is combined with hormone receptors in vivo, environmental estrogen can cause nervous system disorder, endocrine disturbance, immunity reduction, reproductive disorder and the like of organisms, and has strong carcinogenicity and pathogenicity.
Although the estrogen concentration in the environment is at trace levels, it is still difficult to remove. Even at very low concentrations, E2 can also affect the vital activity of aquatic animals. Algae are a group of eukaryotes of the protist kingdom (and some are also prokaryotes, such as algae of the phylum cyanobacteria). Algae use sunlight and nutrients in the surrounding environment to perform photosynthesis during their growth cycle, and also metabolize Algae Organic Matter (AOM) due to the progress of respiration or massive death. Algal organisms belong to the Natural Organic Matter (NOM) which is a natural component of natural surface water and includes various compounds such as oligosaccharides, polysaccharides, proteins, peptides, amino acids and trace amounts of other organic acids. Algae organic matter AOM is generally divided into two parts, organic matter released into water environment by algae due to metabolism during the growth cycle is called Extracellular Organic Matter (EOM), organic matter existing inside algae cells and discharged out of the body due to cell death cell membrane rupture is called Intracellular Organic Matter (IOM).
Chlorella (Chlorella) is a kind of unicellular freshwater algae of the genus Chlorella of the phylum Chlorophytum, is spherical unicellular freshwater algae, 3-8 microns in diameter, one of the earliest lives on the earth, appears more than 20 hundred million years ago, is a high-efficiency photosynthetic plant, grows and breeds by photoautotrophic, has extremely wide distribution, has very high contents of protein, fat and carbohydrate in cells, contains various vitamins, can be eaten and used as bait. The utilization of proteins, polysaccharides, lipids and the like contained in living chlorella cells has been widely carried out, but the collection, concentration and utilization of the secretion of chlorella cells have not been regarded as important. Algae, as a bioremediation agent, can respond to external influencing factors due to the diversity and expressive properties of various metabolisms, and thus can be well used for the remediation of various pollutants. Biodegradation in water is the major route for steroid estrogen removal. At present, the application of phycocyte secretion in degrading estrogen is not reported.
Disclosure of Invention
Aiming at the problems, the invention provides an application of an algae cell secretion in degrading estrogen.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the application of phycocyte secretion in degrading estrogen is provided.
The algae cell secretion includes extracellular organic matter and/or intracellular organic matter of algae.
The phycocyte secretion is derived from Chlorella vulgaris.
The estrogen is 17 beta-estradiol.
The invention has the beneficial effects that: aiming at the difficult degradability of estrogen in aquatic environment, organic substances secreted in the growth cycle of algae, particularly protein parts in the organic substances are applied to the degradation of estrogen, and the intermediate products after the degradation of estrogen are substances with lower toxicity. The invention provides a simpler, more time-saving and better-effect method for processing environmental estrogen and bioremediation and utilization of microalgae, natural algae secretion is adopted to degrade the estrogen in the environment, secondary pollution is not caused, and natural materials are fully utilized, so that the method is very environment-friendly.
Drawings
FIG. 1 is a graph showing the results of the biodegradation of AOM at various E2 concentrations, wherein A is E2 concentration of 1.0mg/L, B is E2 concentration of 0.5mg/L, and C is E2 concentration of 0.2 mg/L.
FIG. 2 is a comparison of fluorescence signals of IOM and EOM before and after 10 days of culture in 1mg/L E2 solution, wherein a is before EOM treatment, b is after EOM treatment, c is before IOM treatment, and D is after IOM treatment.
FIG. 3 shows the GC-MS detection results of E2 and its major biodegradation product at day 3.
FIG. 4 shows the GC-MS detection results of E2 and its major biodegradation product at day 6.
FIG. 5 shows the GC-MS detection results of E2 and its major biodegradation product at day 9.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The experimental procedures in the following examples are conventional unless otherwise specified.
Example 1 extraction of algal cell secretions
Culture of chlorella
BG-11 Medium composition (mg. L)-1)：NaNO3，150.0；MgSO4·7H2O，15.0；K2HPO4·3H2O，8.0；CaCl2·2H2O, 7.0; citric acid, 7.0; ammonium ferric citrate, 7.0; EDTA, 0.2; NaSiO3·9H2O，12；H3BO3，2.8；MnCl2·4H2O，1.8；CuSO4·5H2O，0.09；Na2MoO4·2H2O，0.05；ZnSO4·7H3O，0.3；Na2CO3，4.0。
Initial seed density was about 3.8X 107The cell/ml, the illumination intensity is 500-8000 Lux, the light/dark time ratio is 12: 12, 14: 10, 16: 8, the algae density can reach about 5.8 multiplied by 10 after illumination culture for 10 days7The yield of the obtained algae cell secretion is large.
The culture method mainly comprises the steps of adjusting the illumination intensity and the light-dark ratio, adopting the optimized BG-11 culture medium, culturing the chlorella without adding other organic matters, and then respectively collecting and concentrating the intracellular and extracellular organic matters. Through experiments with different illumination intensities and times, the experimental conditions which can meet the requirements of high and fast protein, polysaccharide and lipid yields are comprehensively screened out.
Second, extraction of phycocyte secretion
Extracting EOM: chlorella in logarithmic phase (450 ml of Chlorella cultured in light for 10 days in this example) was taken, and the cell suspension was first centrifuged at 10000r/min for 10 minutes to obtain a supernatant, which was then resuspended in ultrapure water and centrifuged at 12000r/min for 10 minutes, and 3 times of washing were repeated to obtain a supernatant (i.e., EOM) for further use. The settled algae mud is left for standby.
And (3) extraction of IOM: the algal mud obtained by the centrifugal precipitation was resuspended in ultrapure water, and disrupted by a 200W low-frequency cell disruptor for 0.5 hour while being immersed in ice water. Then, the disrupted algae cells were centrifuged at 16000r/min for 10 minutes, and the supernatant was IOM.
Example 2 degradation of E2 by algal cell secretions
First, experiment method
1. The EOM and IOM extracts from example 1 were divided in equal portions into 3 groups, each of which was subdivided into 3 replicates (approximately 50ml of supernatant per sample due to the negligible volume of centrifuged algal cells and debris from disrupted algal cells relative to the volume of supernatant). Different amounts of E2 were added to the 3 groups of EOM samples to give final E2 concentrations of 1.0mg/L, 0.5mg/L, 0.2mg/L in the 3 groups of EOM samples, and different amounts of E2 were also added to give final E2 concentrations of 1.0mg/L, 0.5mg/L, 0.2mg/L in the 3 groups of IOM samples. Samples were taken every 24 hours for 10 times in total to test the degradation capacity of algal cell secretions.
Residual E2 in the supernatant was detected by high performance liquid chromatography. The total removal (Pt) of chlorella to E2 was calculated using the following equation:
in the formula, Ct: e2 concentration after a period of time;
C0: initial E2 concentration.
2. Determination of degradation products of E2
Activation of the extraction column: adding 5mL of chromatographic grade ethyl acetate into an Oasis HLB extraction column (purchased from Wolttech technologies (Shanghai)) at a flow rate of 1.0-1.5 mL/min, and performing pre-pretreatment to eliminate impurities on the extraction column so as to avoid interference of the impurities on an analysis substance; soaking in 5mL of methanol for 5min, and vacuumizing the extraction column; and then 5mL of ultrapure water (with the flow rate of 1.0-2.0 mL/min) is used for leaching the extraction column, the extraction column is vacuumized for at least 20min after being repeated for three times, and water is removed as much as possible.
Adsorbing a sample: the sample is passed through the extraction column at a flow rate of less than 5.0 mL/min.
Sample elution: eluting the sample by using 5mL of ethyl acetate in a vacuum environment, repeating the elution for 3-5 times at a flow rate of 1.0-1.5 mL/min, and collecting effluent liquid; continuously vacuumizing for 10min after no liquid flows out; blowing high-purity nitrogen into the collected liquid at 45 ℃ slowly until the liquid is dried; finally, the blow-dried material was dissolved in 1mL of n-hexane.
Derivatization: derivatization was performed to reduce the polarity of the analyte and to provide good chromatographic separation characteristics, and 50. mu.L of pyridine and 100. mu.L of BSTFA (1% TMCS) were added to the re-dissolved sample, and the mixture was reacted at 70 ℃ for 30min, cooled to room temperature, and then analyzed by gas chromatography-mass spectrometry (GC-MS).
Sample introduction conditions are as follows: using DB-5MS column (30 m.times.0.25 mm.times.0.25 μm, J)&W Scientific, USA) and a Thermionic Specific Detector (TSD). GC: high-purity helium is taken as carrier gas, and the constant flow rate is 1.0 mL/min; injecting sample in a non-shunting way, wherein the temperature of a sample injection port is 280 ℃, and the sample injection amount is 1.0 mu L. The temperature rising procedure is as follows: the column is initially warmed at 50 deg.C for 2min, heated to 260 deg.C at 12 deg.C/min for 15.0min, and then heated to 280 deg.C at 3.0 deg.C/min for 5 min. MS: the interface temperature is 280 ℃, the transmission line temperature is 300 ℃, the ionization source is an FID ion source, the electron impact ionization mode of the mass spectrometer is adjusted to 70eV, 250 ℃, and the solvent delay time is 15 min; mass Spectrometry in full Scan mode at 2400min-1Data was collected at a rate in the range of m/z-50-600. The final degradation product of E2 was identified according to the national standard technical research quality spectral library.
3. Determination of the content of the Components in EOM and IOM
Determination of polysaccharide content using anthrone-sulfuric acid method. A specific procedure was to dip the sample solution in a 1mL brown glass bottle into an ice bath. After 6min in ice bath, 4mL anthrone-sulfuric acid solution was added, boiled in boiling water bath for 10min, and then cooled with tap water for 10 min. Finally, the sample was measured at 620nm with an ultraviolet-visible spectrophotometer. And the protein concentration content was analyzed using BCA protein concentration assay kit (enhanced) (Beyotime, china). The algae fat is measured by Nile red method, and the algae liquid is centrifuged at 10000 rmp.min-1Centrifuging for 10min, resuspending in 20% DMSO (dimethylulfoxide), water bathing at 40 deg.C for 20min, adding 200: 3 volume ratio of algae solution and NR (0.1 mg/ml)-1Nile red dye solution) for 5 min. Finally, an F-7000 fluorescence spectrophotometer is used to measure the wavelength of the light emitted by the light source at the excitation wavelength of 500-700nm and the emission wavelength of 575nm with the 10nm interval of 2400 nm-min-1Data is collected.
Second, experimental results
(1) Analysis of AOM contribution to E2 degradation
As shown in figure 1, the biodegradation of E2 is caused by the addition of EOM and IOM, and the removal effect of the extracted intracellular and extracellular secretions on E2 is obvious, particularly the removal effect on low-concentration E2 is obvious, namely the removal rate of 70% is finally achieved after 10 days of reaction. In the 0.2mg/L E2 solution, E2 was eventually removed by 70% and 54% over a 10 day period due to the addition of EOM and IOM, respectively; while in E2 of 0.5mg/L, the removal rates were 51% and 43%, respectively, and in E2 of 1.0mg/L, 38% and 31%. Comparing the degradation effects of EOM and IOM on E2, EOM was found to remove E2 at either concentration initially more slowly than IOM and finally more effectively than IOM. While the concentration of E2 in the white control remained essentially constant throughout the degradation process. It can be seen from the figure that the degradation rate of EOM and IOM is decreasing with increasing concentration of E2, indicating that a high concentration of E2 has a negative effect on its removal, which may be due to too small amount of AOM, and that conditions at the same E2 concentration are not sufficient to completely degrade E2.
(2) Content determination of each component in AOM
EEM fluorescence spectroscopy is a powerful, fast and agile tool to characterize AOMs. AOM fluorescence spectroscopy of Chlorella is performed to verify the presence of organic substances with fluorescent response. The supernatant was subjected to fluorescence scanning after removal of chlorella algal cells, which means that organic substances having a fluorescent signal bound to algal cell walls were leaked out and not detected. Furthermore, polysaccharides are substances that do not have a fluorescent response, and therefore this analysis is mainly intended to detect the presence of proteins, humus and fulvic acid. Fluorescence spectrum data can be interpreted by a fluorescence area integration (FRI) method, and analysis shows that the position where a fluorescence peak appears is unchanged and fluorescence Intensity (IF) is obviously changed. In the chlorella EEM spectrum (fig. 2), two peaks were identified in the EOM spectrum, with the strongest peak appearing at Ex/Em-280/310. As shown in Table 1, the peak reached 89.3mV, which fell exactly in the zone of soluble microbial product. A second peak (15.5mV) was observed at Ex/Em ═ 320/405, representing the humic acid region, consistent with the Villacorte study. In contrast, in FIG. 2, one of the two peaks of IOM was located in a region similar to that of EOM but with a higher fluorescence intensity (214.2mV), and the other peak was located in the region of the Ex/Em ═ 225/330 aromatic protein, and it was found that all the fluorescence-detected substances were significantly reduced in fluorescence signal intensity after 10 days of incubation in 1mg/L E2 solution.
TABLE 1 fluorescence spectra peaks of EOM and IOM before and after 10 days of 1mg/L E2 solution culture
The removal rate of each component in EOM and IOM was calculated by a series of characterization methods (as shown in table 2), and the results showed that IOM contains more soluble microbial products (Ex/Em ═ 280/310) than EOM, and mainly contains high molecular weight substances such as tryptophan proteins, and such substances were significantly reduced before and after the treatment of either IOM or EOM with E2. The change in the AOM content before and after 10 days of E2 treatment is also shown, from which it can be seen that the removal of intracellular protein mass (20.5%) is higher than extracellular (11.1%) while the reduction of extracellular polysaccharide (15.5%) is slightly higher than intracellular (11.2%). Meanwhile, the AOM plays a certain contribution role in the process of degrading E2 by combining with the graph shown in FIG. 2. Although the mechanism of degradation of E2 by algal cell secretion has not been studied, a great deal of experiments have previously found that the water-soluble extracellular polymeric substance (ESP) contains tryptophan components which can be converted into singlet state by irradiation or tryptophan cations are excited into triplet state free radicals, thereby causing the degradation of E2. This also corresponds to the statistics in table 1, in the degradation process of E2, the decomposition of proteins of EOM and IOM produced in chlorella into amino acids, in particular tyrosines, plays an important role in the degradation of E2, and the analysis in the figure shows that the effect of EOM is higher than that of IOM.
TABLE 2 changes in polysaccharide, protein and lipid content of IOM and EOM after 10 days of culture in E2 solution
(3) Analysis of degradation products of E2
The results of GC/MS analysis are shown in FIG. 3, and E2 is indeed converted to other substances, mainly E1 (estrone), by degradation of Chlorella vulgaris. The mass spectrum of the main product is compared and analyzed by adopting a NIST2011 mass spectrum library published by the national technical research institute, and the result shows that the chlorella can convert E2 into E1 with lower estrogen activity in the degradation process, and the phenomenon also accords with the research results of Bram and Yu. As shown in table 3, the detection parameters of the product estrone (E1) with the most obvious and most abundant content in the degradation product of E2 are shown in the table, and the matching degree of the mass spectrum library shows that the substance matching degree of E1 is the highest, so that only the product E1 can be determined, which is also caused by the instability of the degradation product of the organic matter by the microorganism. From fig. 4 and 5, it can be seen that the content of E2 is reduced with time, and gradually, E1 is accumulated, the amount of E1 is gradually increased with the degradation of E2, which indicates that E1 is an intermediate product of chlorella degrading E2, while the content of E1 is also reduced with time, which indicates that E1 is not an end product of E2 degradation, and chlorella may further decompose E1 into other unknown substances, which is also consistent with the previous research results of Weber and Zeng. In addition to the high degree of matching detected by the substance E1, the detection results of other substances are not very good mainly due to the following two reasons: (1) the process of degrading E2 by algae microorganisms is complex, and the conversion product of the original pollutant can be further converted under the action of microorganisms, which brings difficulty to product detection; (2) highly volatile and peroxidized organic compounds are inherently difficult to detect.
TABLE 3 LC-MS data for E2 and its metabolites
Claims (2)
1. The application of chlorella phycocyte secretion in degrading estrogen is characterized in that: the algae cell secretion comprises extracellular organic matters and/or intracellular organic matters of chlorella, and the estrogen is 17 beta-estradiol;
the extraction method of the extracellular organic matter and the intracellular organic matter comprises the following steps: culturing chlorella by using a culture medium, firstly centrifuging a cell suspension to obtain a supernatant after the culture is finished, then re-suspending the cell suspension in water, then centrifuging the cell suspension, and repeatedly washing the cell suspension to obtain a supernatant, wherein the supernatant is an extracellular organic matter obtained by extraction, and the settled algae mud is left for later use; resuspending the centrifuged and precipitated algae mud in water, soaking the algae mud in ice water, crushing the algae mud by using a low-frequency cell crusher, and centrifuging the crushed algae cells to obtain supernatant, namely intracellular organic matters;
the application of the chlorella phycocyte secretion in degrading estrogen adopts the following application mode: mixing extracellular organic matter and/or intracellular organic matter of chlorella with the pollutant to be treated containing 17 beta-estradiol uniformly until the degradation reaction is finished.
2. The use of claim 1, wherein the extraction method of extracellular organic substance and intracellular organic substance comprises: culturing chlorella by adopting a culture medium, taking chlorella in logarithmic growth phase, firstly centrifuging a cell suspension for 10 minutes at 10000r/min to obtain a supernatant, then suspending the supernatant in ultrapure water, centrifuging for 10 minutes at 12000r/min, repeating the washing for 3 times, wherein the supernatant is an extracellular organic matter obtained by extraction, and leaving the precipitated algae mud for later use; and (3) resuspending the algae mud obtained by centrifugal precipitation in ultrapure water, crushing the algae mud for 0.5 hour by using a 200W low-frequency cell crusher under the condition of soaking in ice water, and then centrifuging the crushed algae cells for 10 minutes at 16000r/min to obtain supernatant, namely the intracellular organic matter.
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