A method for promoting lipid production of Scenedesmus obliquus and purifying wastewater by utilizing nitrogen and phosphorus in wastewater

Abstract

This invention belongs to the field of organic wastewater treatment technology. To address current issues such as carbon emission reduction and energy shortages, it provides a method for promoting lipid production and wastewater purification using nitrogen and phosphorus in wastewater from *Scenedesmus obliquus*. *Scenedesmus obliquus* is inoculated into artificial wastewater and cultured. While adding salicylic acid at a concentration of 20-40 mg / L, low-intensity ultrasound (200 W, 40 kHz frequency) is applied for 5-10 minutes. Cultivation continues until nitrogen and phosphorus in the wastewater are completely consumed. The nitrogen and phosphorus removal rate is measured, and the algae are collected to produce algal powder for oil extraction. Coupled with artificial wastewater, this method achieves wastewater resource recovery while saving costs. The nitrogen and phosphorus removal rate in the wastewater is as high as approximately 90%, the oil content of *Scenedesmus obliquus* is higher than 60%, and the oil yield is as high as 40 mg / L·d. ‑1 .

Description

Technical Field

[0001] The present invention belongs to the technical field of organic wastewater treatment, and specifically relates to a method for utilizing nitrogen and phosphorus in wastewater to promote lipid production by Scenedesmus obliquus and purify wastewater. Low-intensity ultrasound combined with salicylic acid is used to promote Scenedesmus obliquus to recover nitrogen and phosphorus in wastewater, while increasing lipid production by Scenedesmus obliquus, thereby purifying wastewater and promoting lipid production by microalgae. Background Art

[0002] The development of clean and renewable energy has become the focus of attention, including hydropower, solar energy, wind energy, biomass energy, geothermal energy and ocean energy. Among them, biomass energy is energy with biomass as a carrier, that is, solar energy is stored in the form of chemical energy in organic biomass that can grow, with the characteristics of less pollution, easy combustion and low ash content. At present, the problem of insufficient global energy supply is getting worse and worse. The development and utilization of biomass energy can not only alleviate the problem of global energy shortage, but also reduce CO2 emissions and help alleviate the greenhouse effect.

[0003] Biomass energy has now developed into the third generation of raw materials - microalgae, which has been widely used due to its unique advantages. Microalgae has higher photosynthetic efficiency than other oil-producing plants, has a short cultivation cycle, and can grow all year round; it does not occupy land, avoiding competition with crops for land space; microalgae can fix CO2, and each ton of microalgae can fix 1.83 tons of CO2, and the oil produced is carbon neutral, which helps reduce carbon emissions and achieve zero CO2 emissions. Although microalgae has many advantages, its high cultivation cost and the problem of the need to improve the output value rate hinder the process of large-scale industrialization.

[0004] Existing research has induced microalgae to synthesize high-added substances such as carbohydrates, proteins, oils, and astaxanthin by adjusting nutritional parameters and culture conditions. Summary of the invention

[0005] In order to solve the current problems of carbon emission reduction and energy shortage, the present invention uses microalgae to purify wastewater and produce lipids at the same time, thereby achieving the purpose of reducing the cost of microalgae cultivation and recovering nitrogen and phosphorus in wastewater, and provides a method for promoting lipid production of Scenedesmus obliquus and purifying wastewater by using nitrogen and phosphorus in wastewater. The method promotes the rapid growth of Scenedesmus obliquus to increase the oil yield, purify wastewater, and finally realizes the combination of wastewater treatment and resource utilization.

[0006] The present invention is realized by the following technical scheme: a method for promoting lipid production of Scenedesmus obliquus and purifying wastewater by utilizing nitrogen and phosphorus in wastewater, Scenedesmus obliquus ( Scenedesmus obliquus)FACHB-12 was inoculated into artificial wastewater for cultivation. Low-intensity ultrasound was applied while salicylic acid was added. The concentration of salicylic acid in the artificial wastewater was 20-40 mg / L. The ultrasonic power of the low-intensity ultrasound was 200 W, and the ultrasonic frequency was 40 kHz. The ultrasonic time was 5-10 min. The culture was carried out until nitrogen and phosphorus substances in the wastewater were consumed. The nitrogen and phosphorus removal rate in the wastewater was determined, the algae species were collected, algae powder was made, and oil was extracted.

[0007] The specific steps are as follows:

[0008] (1) Preparation of algae seed solution: Add Scenedesmus obliquus to BG11 culture medium and culture at 25°C, light intensity of 3000 lux, light-dark ratio = 12h:12h. Shake 2-3 times a day on a constant temperature shaker for 10-15 min each time to ensure uniform distribution of the microalgae. Cultivate the microalgae to the logarithmic growth phase and use it as the algae seed solution for subsequent experiments.

[0009] (2) Cultivating algae liquid and purifying wastewater at the same time: the algae liquid obtained in step (1) is centrifuged, the supernatant is discarded, and the algae liquid is washed twice with deionized water, inoculated into artificial wastewater and cultured for 2 days to allow it to adapt to the new environment. After 2 days, salicylic acid SA solution is added thereto and low-intensity ultrasound is applied at the same time. After the treatment is completed, the liquid is placed in a constant temperature incubator and cultured until the nutrients are completely consumed. The culture conditions are as follows: light intensity of 3000 lux, temperature of 25°C, initial pH of 6.8-7.2, light-dark ratio of 12h:12h, and shaking on a shaker 2-3 times a day for 10-15min each time. The liquid is cultured until the nitrogen and phosphorus substances in the wastewater are completely consumed and then harvested;

[0010] (3) Wastewater purification degree detection: The algae liquid mixed evenly in step (2) was collected the next day after harvesting, and the algae liquid was filtered through a 0.45 μm microporous filter membrane. The clear liquid was retained and the wastewater indicators were measured according to the national standard; TN was measured by potassium persulfate oxidation-ultraviolet spectrophotometry, TP was measured by molybdenum antimony anti-spectrophotometry, and NH4 + -N was determined by Nessler's reagent spectrophotometry;

[0011] (4) Extracting oil: Extracting oil using the improved Bligh method: Collect the algae liquid that has been grown in step (2), centrifuge it, and use a freeze dryer to precipitate it to make dry algae powder. Use an organic solvent to extract the oil in the algae cells; the organic solvent is a mixed solution of chloroform, methanol and water, and the volume ratio of chloroform, methanol and water in the mixed solution is 1:1:0.9.

[0012] In step (2), the concentration of salicylic acid SA is 20 mg / L or 40 mg / L; the ultrasonic power of the low-intensity ultrasonic wave is 200 W, the ultrasonic frequency is 40 kHz, and the ultrasonic duration is 5 min or 10 min.

[0013] Preferably, the concentration of salicylic acid SA in step (2) is 40 mg / L.

[0014] In the artificial wastewater in step (2): COD: 200 mg / L, TN: 60 mg / L, TP: 5 mg / L, NH4 + -N: 20mg / L.

[0015] In step (1), Scenedesmus obliquus is cultured in a 250 ml conical flask and shaken three times a day on a constant temperature shaker at 150 r / min and 25° C. for 15 minutes each time.

[0016] Under the culture method, the TN removal rate ranged from 95.67 to 96.89%, the TP removal rate ranged from 89.74 to 96.23%, and the NH4 + The range of -N removal rate was 99.05-99.58%, the range of absorbance was 2.18-2.65, the range of dry weight was 460-660 mg / L, the range of oil content was 57.34-73.81%, and the range of oil yield was 42.40-50.11 mg / L·d -1 .

[0017] Scenedesmus obliquus used in the present invention Scenedesmus obliquus (FACHB-12) was purchased from the freshwater algae seed bank of Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. During the microalgae cultivation process, the shaker was shaken every day to ensure that the microalgae were evenly distributed and to prevent the microalgae from growing on the wall. The present invention found that a higher concentration of salicylic acid had a better effect on promoting the growth and lipid production of Scenedesmus obliquus.

[0018] Under the culture method, the TN removal rate ranged from 95.67 to 96.89%, the TP removal rate ranged from 89.74 to 96.23%, and the NH4 + The range of -N removal rate was 99.05-99.58%, the range of absorbance was 2.18-2.65, the range of dry weight was 460-660 mg / L, the range of oil content was 57.34-73.81%, and the range of oil yield was 42.40-50.11 mg / L·d -1 .

[0019] The present invention uses low-intensity ultrasound combined with salicylic acid because low-intensity ultrasound generates mechanical effects through external energy waves, which can improve the permeability of cell membranes, increase the contact between nutrients and microalgae, and the cavitation effect improves enzyme activity and activates the defense mechanism of microalgae; the plant hormone salicylic acid is applied exogenously to induce the synthesis of endogenous salicylic acid in microalgae, affecting the synthesis of proteins and photosynthetic pigments in the algae to increase its growth rate and regulate its growth and development. In addition, it works on the antioxidant system, improves the gene expression of antioxidant active substances to generate antioxidant active substances, thereby accumulating a large amount of oil.

[0020] Compared with the prior art, the cultivation method of Scenedesmus obliquus described in the present invention is a mixed culture mode, which adopts a combination of physical and chemical methods to cultivate Scenedesmus obliquus by low-intensity ultrasound combined with salicylic acid, shortening the microalgae cultivation time, greatly improving the oil yield, recovering nitrogen and phosphorus in wastewater, and achieving the purpose of reducing microalgae cultivation costs and wastewater resource utilization. The removal rate of nitrogen and phosphorus in wastewater is as high as more than 90%, and the oil yield of Scenedesmus obliquus is as high as 40mg / L·d -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the effect of Scenedesmus obliquus on TN removal under combined treatment in an embodiment of the present invention;

[0022] Figure 2 This is a diagram showing the effect of Scenedesmus obliquus removing TP under combined treatment in an embodiment of the present invention;

[0023] Figure 3 The results of the combined treatment of Scenedesmus obliquus against NH4 + -N removal effect diagram;

[0024] Figure 4 This is a graph showing the growth of Scenedesmus obliquus under combined treatment in an embodiment of the present invention;

[0025] Figure 5 is a graph showing the specific growth rate of Scenedesmus obliquus under combined treatment in an embodiment of the present invention;

[0026] Figure 6 is a graph of the dry weight of Scenedesmus obliquus under the combined treatment in an embodiment of the present invention;

[0027] Figure 7 is a chlorophyll diagram of Scenedesmus obliquus under combined treatment in an embodiment of the present invention;

[0028] Figure 8 This is a diagram showing the oil content and yield of Scenedesmus obliquus under combined treatment in an embodiment of the present invention;

[0029] Fig. 9 This is a diagram showing the changes in functional groups of Scenedesmus obliquus under combined treatment in an example of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0032] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0033] The experimental methods in the following examples are all conventional methods unless otherwise specified; the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified. Scenedesmus obliquus , FACHB-12), purchased from the freshwater algae seed bank of Wuhan Institute of Hydrobiology, Chinese Academy of Sciences, salicylic acid was purchased from Tianjin Guangfu Technology Development Co., Ltd. and was of high purity, and the low-intensity ultrasonic sound source was generated in an ultrasonic cleaning machine.

[0034] Example 1: A method for utilizing nitrogen and phosphorus in wastewater to promote lipid production by Scenedesmus obliquus and purify wastewater, Scenedesmus obliquus ( Scenedesmus obliquus )FACHB-12 was inoculated into artificial wastewater for cultivation. Low-intensity ultrasound was applied while salicylic acid was added. The concentration of salicylic acid in the artificial wastewater was 20-40 mg / L. The ultrasonic power of the low-intensity ultrasound was 200 W, and the ultrasonic frequency was 40 kHz. The ultrasonic time was 5-10 min. The culture was carried out until nitrogen and phosphorus substances in the wastewater were consumed. The nitrogen and phosphorus removal rate in the wastewater was determined, the algae species were collected, algae powder was made, and oil was extracted.

[0035] The specific steps include:

[0036] (1) Preparation of algae seed solution: S. obliquus was cultured in BG11 medium and cultured in a constant temperature incubator with a light intensity of 3000 lux, 25°C, and a light-dark ratio of 12h:12h until the logarithmic growth phase, which was used as the algae seed solution for subsequent experiments;

[0037] (2) Cultivating algae liquid and purifying wastewater at the same time: The algae liquid obtained in step (1) is centrifuged, the supernatant is discarded, and the microalgae is washed twice with deionized water. It is then inoculated into 150 ml of artificial wastewater and cultured for 2 days to allow it to adapt to the artificial wastewater environment. The inoculation amount of the algae is 10-15% v / v, so that the initial absorbance value of the microalgae is between 0.3 and 0.4. The inoculation amount of microalgae was determined according to the absorbance value of the microalgae inoculated into 150ml culture medium by inoculating the algae seed solution, so that the initial absorbance value of the microalgae inoculated into the culture medium could reach between 0.3 and 0.4, and the inoculation amount of the algae seed was determined to be 10-15% v / v; 20mg / L salicylic acid was added after 2 days, and ultrasonic waves were applied for 5 minutes, the ultrasonic power of low-intensity ultrasonic waves was 200W, and the ultrasonic frequency was 40kHz; the culture conditions were 3000lux, 25℃, and light-dark ratio = 12h:12h, and the culture was carried out until the nitrogen and phosphorus substances in the wastewater were consumed, and the cell absorbance and specific growth rate were measured every other day. At the end of the experiment, the dry weight, lipid content and yield, and cell functional groups were measured;

[0038] (3) Wastewater purification degree test: The algae solution mixed evenly in step (2) was collected every other day, filtered through a 0.45 μm microporous filter membrane, the algae residue was discarded and the clear liquid was retained, which was diluted to several different times and TN, TP, and NH4 were measured in accordance with the "National Environmental Protection Standards of the People's Republic of China". + -Determination of N.

[0039] TN is determined by potassium persulfate oxidation-ultraviolet spectrophotometry. Take a sample and dilute it with water to 10 ml. Then add 5 ml of alkaline potassium persulfate solution, plug the tube tightly, and tie the tube with gauze and string to prevent it from popping out. Place the colorimetric tube in a high-temperature and high-pressure sterilizer and digest it at 120°C for 30 minutes. After digestion, add 1 ml of hydrochloric acid solution, dilute it with water to the 25 ml mark, cover the stopper and mix well. Use a 10 mm quartz colorimetric dish on a UV spectrophotometer with water as a reference to measure the absorbance at wavelengths of 220 nm and 275 nm, respectively. The calculation formula is as follows: A b =A b220 -2A b275 ; A s =A s220 -2A s275 ; A r =A s -A b ; Where: A b ——corrected absorbance of blank solution; A b220 ——Absorbance of blank solution at wavelength 220nm; A b275 ——Absorbance of blank solution at wavelength 275nm; A s ——corrected absorbance of standard solution; A s220 ——Absorbance of the standard solution at a wavelength of 220nm; A s275——Absorbance of the standard solution at a wavelength of 220nm; A r ——The difference between the corrected absorbance of the standard solution and the corrected absorbance of the blank solution.

[0040] TP is determined by the molybdenum antimony spectrophotometric method. Take a sample and add 4 ml of potassium persulfate, plug the tube tightly, and tie the tube tightly with gauze and string to prevent it from popping out. Place the colorimetric tube in a high-temperature and high-pressure sterilizer and digest at 120°C for 30 minutes. After digestion, add 1 ml of ascorbic acid solution and mix well. After 30 seconds, add 2 ml of molybdate solution and mix well. After standing at room temperature for 15 minutes, use a 30 mm colorimetric dish to measure the absorbance at a wavelength of 700 nm with water as a reference. The total phosphorus content is expressed as C (mg / L) and the calculation formula is as follows: ; Where: m——measured phosphorus content of the sample, μg; V——volume of the sample used for measurement, ml.

[0041] NH4 + -N is determined by Nessler's reagent spectrophotometry. Take a sample and add water to the mark. Add 1ml of potassium sodium tartrate solution and shake well. Then add 1ml of Nessler's reagent and shake well. After leaving it for 10 minutes, use a 20mm cuvette at a wavelength of 420nm and water as a reference to measure the absorbance. The calculation formula is as follows: ; Where: ρ N ——Mass concentration of ammonia nitrogen in water (in N), mg / L; A s ——absorbance of water sample; A b ——Absorbance of blank sample; a——Intercept of calibration curve; b——Slope of calibration curve; V——Sample volume, ml.

[0042] (4) Extraction of oil: The microalgae cultured to the stable period in step (2) were harvested and vacuum freeze-dried to prepare algae powder. 0.0100 g of algae powder was placed in a dry and clean centrifuge tube, 0.8 mL of distilled water was added, and then 1 mL of chloroform and 2 mL of methanol were added. After oscillation for 2 minutes to mix evenly, ultrasonic crushing was performed for 1 minute. Then 1 ml of chloroform was added to the solution, oscillated for 1 minute to mix evenly, and then 1 ml of distilled water was added. After oscillation for another 1 minute, the mixture was centrifuged at 4000 rpm for 5 minutes. After centrifugation, the bottom chloroform phase was extracted with a syringe and transferred to another dry, clean, constant-weight centrifuge tube. Then 2 mL of chloroform was added to the original tube, oscillated for 1 minute, and centrifuged at 4000 rpm for 5 minutes. The chloroform phase was transferred to the previous chloroform phase, and the operation was repeated twice. The chloroform phases transferred three times were dried at 80°C until the mass was constant, and the weight of the extracted oil was obtained by weighing.

[0043] When organic solvents are used to extract oils and fats, stratification will be formed after the organic solvents are added. The chloroform layer is at the bottom, water and methanol are at the top, and the middle layer is algae. After each extraction, the chloroform layer at the bottom is sucked out, and 2 ml of chloroform is added to continue the extraction. Therefore, the overall volume ratio of the organic solvent is basically maintained at 1:1:0.9.

[0044] The results showed that the removal rates of TN and TP in the experimental group treated with 20 mg / L salicylic acid and 5 min ultrasonic wave were 96.89%, 89.74% and NH4 + The -N removal rate was 99.58%, the absorbance of Scenedesmus obliquus was 2.20, the dry weight was 480 mg / L, and the obtained oil content and yield were 73.56% and 44.07 mg / L·d, respectively. -1 .

[0045] Example 2: A method for promoting lipid production by Scenedesmus obliquus and purifying wastewater by utilizing nitrogen and phosphorus in wastewater, wherein the specific method is as follows: when culturing algae liquid and purifying wastewater at the same time, 40 mg / L salicylic acid is added, and ultrasonic waves are applied for 5 minutes. Other methods are the same as those described in Example 1. The method for determining pollution indicators in wastewater is the same as that described in Example 1. The method for determining oil content is the same as that described in Example 1.

[0046] The results showed that the removal rates of TN and TP were 95.67% and 92.17% respectively in the experimental group treated with 40 mg / L salicylic acid and 5 min ultrasonic wave. + The -N removal rate was 99.05%, the absorbance of Scenedesmus obliquus was 2.65, the dry weight was 590 mg / L, and the obtained oil content and yield were 67.94% and 50.11 mg / L·d -1 .

[0047] Comparative Example 1: When culturing algae liquid and purifying wastewater at the same time, 10-15% of the algae liquid in step (1) was inoculated into 150 ml of artificial wastewater without joint treatment. Other methods were the same as those described in Example 1. The method for determining the pollution index in the wastewater was the same as that described in Example 1. The method for determining the oil content was the same as that described in Example 1.

[0048] The results showed that the removal rates of TN, TP and NH4 in the control group without combined treatment were 13.93%, 38.04% and 38.04%, respectively. + -N removal rate was 26.03%, the absorbance of Scenedesmus obliquus was 1.30, the dry weight was 120 mg / L, and the obtained oil content and yield were 63.37% and 9.50 mg / L·d -1 .

[0049] Comparative Example 2: When culturing algae liquid and purifying wastewater at the same time, 10-15% of the algae species in step (1) were inoculated into 150 ml of artificial wastewater, and 20 mg / L of salicylic acid was added, and ultrasonic waves were applied for 10 min. Other methods were the same as those described in Example 1. The method for determining the pollution index in the wastewater was the same as that described in Example 1. The method for determining the oil content was the same as that described in Example 1.

[0050] The results showed that the removal rates of TN and TP were 95.67% and 92.17% respectively in the experimental group treated with 20 mg / L salicylic acid and 10 min ultrasonic wave. + The -N removal rate was 99.05%, the absorbance of Scenedesmus obliquus was 2.18, the dry weight was 460 mg / L, and the obtained oil content and yield were 73.81% and 42.40 mg / L·d, respectively. -1 .

[0051] Comparative Example 3: When culturing algae liquid and purifying wastewater at the same time, 10-15% of the algae species in step (1) were inoculated into 150 ml of artificial wastewater, and 40 mg / L of salicylic acid was added, and ultrasonic waves were applied for 10 min. Other methods were the same as those described in Example 1. The method for determining the pollution index in the wastewater was the same as that described in Example 1. The method for determining the oil content was the same as that described in Example 1.

[0052] The results showed that the removal rates of TN and TP were 95.67% and 92.17% respectively in the experimental group treated with 40 mg / L salicylic acid and 10 min ultrasonic wave. + The -N removal rate was 99.05%, the absorbance of Scenedesmus obliquus was 2.56, the dry weight was 660 mg / L, and the obtained oil content and yield were 57.34% and 47.25 mg / L·d, respectively. -1 .

[0053] Result analysis: The effects of combined treatment and cultivation of Scenedesmus obliquus on nitrogen and phosphorus, dry weight, oil content and yield are shown in Table 1.

[0054] Table 1 Effects of combined treatment and cultivation of Scenedesmus obliquus on nitrogen and phosphorus, dry weight, oil content and yield

[0055]

[0056] In Comparative Example 1, the Scenedesmus obliquus cultured without combined treatment grew slowly, and it was difficult to remove nitrogen and phosphorus in the wastewater in a short time. The TN removal rate was only 13.93%, the TP removal rate was 38.04%, and the NH4 + -N removal rate was 26.03%; secondly, the growth, dry weight, oil content and yield of Scenedesmus obliquus were the lowest, which was difficult to meet the basic requirements for large-scale production of bio-oil.

[0057] It can be seen from Examples 1 and 2 and Comparative Examples 2 and 3 that the nitrogen and phosphorus removal rate, growth and lipid production of Scenedesmus obliquus after combined treatment and cultivation are significantly improved.

[0058] Figures 1 to 3 Cultivation of Scenedesmus obliquus for combined treatment of TN, TP and NH4 in wastewater + -N removal rate diagram, the embodiment and the comparative example show different downward trends. In the embodiment, TN and NH4 + -N content reached the minimum on the 6th day of the experiment, and TP content reached the minimum on the 8th day. The degradation effect of Example 1 was good overall, with the lowest TN concentration of 2.03 mg / L and a removal rate of 96.89%, and NH4 + -N was reduced from 20mg / L to 0.08mg / L, with a removal rate of up to 99.58%, while the best removal effect of TP was in Example 2, which was reduced from 5mg / L to 0.19mg / L, with a removal rate of up to 96.23%; in the early stage of the experiment, the degradation effect of TN was not good, which may be due to the low initial algae density and the fact that according to NH4 + -N degradation changes ( Figure 3 ), throughout the entire experimental process, NH4 + -N content showed a linear downward trend, indicating that Scenedesmus obliquus preferentially used NH4 + -N, which was also found in Przutocka's research. Algae will preferentially use NH4 + -N and other reduced nitrogen, since algae do not produce active nitrate reductase, they react with NO3 - -N absorption and utilization only occurs in NH4 + -N concentration is very low or depleted, which results in a low degree of TN degradation in the early stage. In the middle of the experiment, the utilization rate of nitrogen by Scenedesmus obliquus was faster, the nitrogen-phosphorus ratio increased, the growth rate of microalgae slowed down, and the utilization rate of phosphorus decreased slightly. The experiment continued with a downward trend. The combined treatment method was used to recover nitrogen and phosphorus in wastewater. Examples 1 and 2 had better effects, with a nitrogen and phosphorus treatment rate of more than about 90%.

[0059] Effects of combined treatment on the growth of Scenedesmus obliquus Figure 4 , 5 It is obvious that the combined treatment has a promoting effect on the growth of S. obliquus and the effect is obvious. The highest biomass is 2.65 in Example 2, which is about 2.0 times that of Example 1. In terms of the promotion of microalgae growth, salicylic acid is more dominant and contributes more to the growth of microalgae, while ultrasound plays a smaller role. Under the same salicylic acid concentration, the biomass of Examples 1 and 2 with 5 minutes of ultrasound is higher than that of Comparative Examples 2 and 3 with 10 minutes of ultrasound, which is also reflected in the specific growth rate. In terms of comparative growth rate ( Figure 5), high concentration of salicylic acid has a hysteresis effect on the growth of Scenedesmus obliquus. The promotion effect is weak in the early stage when the biomass of the microalgae is low, but the hysteresis effect weakens with the increase of the biomass of the microalgae. The growth rate of 40 mg / L exceeds that of 20 mg / L and remains at the highest state for a period of time.

[0060] The changes in dry weight of Scenedesmus obliquus after combined treatment were shown in Figure 6 Compared with CO2 as the carbon source alone, the dry weight of the oblique Scenedesmus in the artificial wastewater with the addition of organic carbon after the combined treatment is greatly improved compared with the comparative example 1. The dry weight improvement effect of comparative example 3 is the best, which is 660 mg / L, which is 4.5 times higher than that of comparative example 1; the other embodiments and comparative examples are 480, 460, and 590 mg / L, respectively, which is 3 to 4 times higher than that of the control group. In the mixed culture mode, the oblique Scenedesmus can use light source, inorganic carbon, and organic carbon to carry out carbon fixation and biosynthesis through photosynthesis and glycolysis. At the same time, the combined treatment stimulates the oblique Scenedesmus to accelerate cell reproduction, greatly increasing the dry weight of the oblique Scenedesmus.

[0061] After combined treatment, the contents of chlorophyll and chlorophyll a were significantly increased. Figure 7 , Example 1 had the highest chlorophyll content at the end of the week, which was 0.926 mg / L, 1.95 times higher than that of Comparative Example 1 (0.314 mg / L). The chlorophyll content of the 20 mg / L group was higher than that of the 40 mg / L group. Combined with the changes in dry weight and oil content, it was shown that chlorophyll may have been transformed. Algae cells use chlorophyll as a nitrogen source under nitrogen starvation and nitrogen depletion conditions to cleverly convert proteins or polypeptides into oils or carbohydrates. The activity of lipid products is well correlated with chlorophyll composition. Higher chlorophyll b in the mixed culture stage is conducive to lipid accumulation, while higher chlorophyll a promotes dry weight growth.

[0062] Figure 8 The figure shows the changes in oil content and yield after combined treatment. Compared with the cultivation of microalgae with inorganic carbon (CO2), the oil content of microalgae can be initially improved by 63.37% when only artificial wastewater containing organic carbon is used without combined treatment. After combined treatment, the oil content is further improved, reaching a maximum of about 73.56% in Example 1, which is 16.08% higher than the control group. The oil yield reaches a maximum of 50.11 mg / L·d in Example 2. -1 , compared with 9.50 mg / L·d in the control group -1 The oil yield of Example 1 was 44.07 mg / L·d. -1 , which was 3.6 times higher than that of the control group.

[0063] In summary, the possible reasons for the large-scale lipid production of microalgae are: 1) the distribution of carbon flow. Less 3PGA is converted into starch and protein, and more 3PGA is directed to lipid synthesis; 2) the conversion of carbon flow. By degrading carbon-containing substances such as starch, carbon flows into lipids.

[0064] Fig. 9 The effect of combined treatment on the functional groups of Scenedesmus obliquus cells. Compared with ultrasound alone, combined treatment increased the -1 The same peaks appear; similarly, the functional groups also show different changes. For example, 3329cm -1 The broad peak at 3371 cm -1 , indicating that the combined treatment affects OH and NH in cell polysaccharides and proteins; 1545cm -1 The peak at 1535 cm -1 1249cm -1 The peak at 1244 cm -1 , indicating that COC related to the sugar backbone has an effect; 1047cm -1 The peak at 1025 cm -1 , from the original small double peak to the current single peak, indicating that the CO of the sugar substance disappears and the CC bond is strengthened. In addition, after the combined treatment, the peaks at 1742 and 1150 cm -1 Two new weak peaks were generated at the ester group's C=O stretching vibration and the CO bond stretching vibration of the sugar substance, which may be related to the accumulation and synthesis of microalgae lipids. The combined treatment and cultivation of Scenedesmus obliquus mainly acted on sugars, lipids, proteins and other substances, and had an impact on amino, carbonyl, carboxyl and other functional groups.

[0065] Therefore, according to Table 1 and Figures 1 to 9 Low-intensity ultrasound combined with salicylic acid to cultivate Scenedesmus obliquus has an excellent effect on the removal of nitrogen and phosphorus from wastewater, and has significantly improved the growth, dry weight, oil content and yield of Scenedesmus obliquus. These results provide favorable data and theoretical support for the combined treatment and cultivation of Scenedesmus obliquus to degrade nitrogen and phosphorus and produce oil. It can be seen that the highest biomass and oil yield were obtained when the salicylic acid addition concentration was 20 and 40 mg / L and the low-intensity ultrasound time was 5 minutes, and the purpose of optimal nitrogen and phosphorus recovery in wastewater was achieved. It can be seen that using the plant hormone salicylic acid to induce Scenedesmus obliquus to increase oil content and yield is a feasible, economical and effective method that meets the requirements of wastewater treatment and resource utilization.

[0066] From the consumption of nutrients of the microalgae and the growth of the microalgae, it can be seen that when the nutrients in the embodiment are exhausted, the growth of the microalgae is about 1.75-2.25, while the growth of the microalgae in the comparative example is about 1.0. Therefore, it takes about 6-8 days for the comparative example to grow to the same absorbance as in the embodiment. Obviously, the present invention significantly shortens the culture time of the microalgae by culturing Scenedesmus obliquus with low-intensity ultrasound combined with salicylic acid.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for promoting lipid production by Scenedesmus obliquus and purifying wastewater by utilizing nitrogen and phosphorus in wastewater, characterized in that: Scenedesmus obliquus Scenedesmus obliquus ) FACHB-12 was inoculated in artificial wastewater for cultivation, and low-intensity ultrasound was applied while adding salicylic acid. The concentration of salicylic acid in the artificial wastewater was 20-40 mg / L, the ultrasonic power of the low-intensity ultrasound was 200 W, and the ultrasonic frequency was 40 kHz. The ultrasonic time was 5-10 min. The culture was carried out until the nitrogen and phosphorus substances in the wastewater were consumed, and the nitrogen and phosphorus removal rate in the wastewater was determined. The algae species were collected, algae powder was prepared, and oil was extracted. The specific steps are as follows: (1) Preparation of algae seed solution: Add Scenedesmus obliquus to BG11 culture medium and culture at 25°C, light intensity of 3000 lux, light-dark ratio = 12h:12h. Shake 2-3 times a day on a constant temperature shaker for 10-15 min each time to ensure uniform distribution of the microalgae. Cultivate the microalgae to the logarithmic growth phase and use it as the algae seed solution for subsequent experiments. (2) Cultivating algae liquid and purifying wastewater at the same time: the algae liquid obtained in step (1) is centrifuged, the supernatant is discarded, and the algae liquid is washed twice with deionized water, inoculated into artificial wastewater and cultured for 2 days to allow it to adapt to the new environment. After 2 days, salicylic acid SA solution is added thereto and low-intensity ultrasound is applied at the same time; after treatment, the liquid is placed in a constant temperature incubator and cultured until the nutrients are exhausted. The culture conditions are as follows: light intensity 3000 lux, temperature 25°C, initial pH 6.8-7.2, light-dark ratio = 12h:12h, shaking 2-3 times a day, each time for 10-15min, and culture until the nitrogen and phosphorus substances in the wastewater are exhausted and harvested; in artificial wastewater: COD: 200mg / L, TN: 60mg / L, TP: 5mg / L, NH4 + -N: 20mg / L; (3) Wastewater purification degree detection: The algae liquid mixed evenly in step (2) was collected the next day after harvesting, and the algae liquid was filtered through a 0.45 μm microporous filter membrane. The clear liquid was retained for the determination of wastewater indicators; TN was determined by potassium persulfate oxidation-ultraviolet spectrophotometry, TP was determined by molybdenum antimony anti-spectrophotometry, and NH4 + -N was determined by Nessler's reagent spectrophotometry; (4) Extracting oil: extracting oil by using the improved Bligh method: harvesting the microalgae cultured to the stable period in step (2), freeze-drying them in vacuum to prepare algae powder, and extracting oil from the algae cells by using an organic solvent; the organic solvent is a mixed solution of chloroform, methanol and water, wherein the volume ratio of chloroform, methanol and water in the mixed solution is 1:1:0.9, extracting with the organic solvent three times, and drying the chloroform phases obtained three times at 80°C until the mass is constant, which is the extracted oil; After cultivation, the TN removal rate ranged from 95.67 to 96.89%, the TP removal rate ranged from 89.74 to 96.23%, and the NH4 + The range of -N removal rate was 99.05-99.58%, the range of absorbance was 2.18-2.65, the range of dry weight was 460-660 mg / L, the range of oil content was 57.34-73.81%, and the range of oil yield was 42.40-50.11 mg / L·d -1 .

2. The method for promoting lipid production by Scenedesmus obliquus and purifying wastewater by utilizing nitrogen and phosphorus in wastewater according to claim 1, characterized in that: In step (2), the concentration of salicylic acid SA is 20 mg / L or 40 mg / L; the ultrasonic power of the low-intensity ultrasonic wave is 200 W, the ultrasonic frequency is 40 kHz, and the ultrasonic duration is 5 min or 10 min.

3. The method for utilizing nitrogen and phosphorus in wastewater to promote lipid production by Scenedesmus obliquus and purify wastewater according to claim 2, characterized in that: The concentration of salicylic acid SA in step (2) is 40 mg / L.

4. The method of utilizing nitrogen and phosphorus in wastewater to promote lipid production by Scenedesmus obliquus and purify wastewater according to claim 1, characterized in that: In step (1), the Scenedesmus obliquus was cultured in a 250 ml conical flask and shaken three times a day on a constant temperature shaker at 150 r / min and 25° C. for 15 minutes each time.

5. The method for promoting lipid production by Scenedesmus obliquus and purifying wastewater by utilizing nitrogen and phosphorus in wastewater according to claim 1, characterized in that: The specific method for extracting oil in step (4) is as follows: take 0.0100g of freeze-dried algae powder into a dry and clean centrifuge tube, add 0.8mL of distilled water, then add 1mL of chloroform and 2mL of methanol, shake for 2min to mix evenly, and then ultrasonically crush for 1min; then add 1ml of chloroform to the solution, shake for 1min to mix evenly, then add 1ml of distilled water, shake for another 1min, and then centrifuge at 4000rpm for 5min; After centrifugation, use a syringe to extract the bottom chloroform phase and transfer it to another dry, clean, constant-weight centrifuge tube, then add 2 mL of chloroform to the original tube so that the volume ratio of chloroform, methanol, and water in the mixed solution is still controlled at 1:1:0.9; after oscillation for 1 minute, centrifuge at 4000 rpm for 5 minutes, transfer the chloroform phase to the previous chloroform phase, and repeat the operation twice; dry the chloroform phases transferred three times at 80°C until the mass is constant, and weigh to obtain the weight of the extracted oil.

Images