Culture method for improving metal-containing acid wastewater purification efficiency of hot spring red algae

Abstract

The invention discloses a culture method for purifying AMD wastewater and improving the yield of phycocyanin by using hot spring red algae, relates to the technical field of algae culture, and aims to improve the yield of phycocyanin while treating wastewater by using the characteristics of acid resistance and heavy metal resistance of the hot spring red algae so as to realize improvement in two aspects of environmental protection and production efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of algal culture, and particularly to a method for culturing microalgae with AMD wastewater to recover metals and co-produce phycocyanin. Background Art

[0002] Galdieria sulphuraria is a single-celled eukaryotic microalgae sourced from extreme environments, widely distributed in highly acidic and hot environments such as acidic sulfuric volcanic hot springs and volcanic craters, capable of tolerating strong acids (pH 0.5–4), high temperatures (up to 56 °C), and heavy metals. Galdieria sulphuraria has a strong ability to survive under extreme conditions and may have certain advantages in the purification of acidic heavy metal wastewater.

[0003] Galdieria sulphuraria copes with low pH environments through different mechanisms such as replacing cellular phospholipids with betaine lipids and the biosynthesis of polar lipids. Galdieria sulphuraria can not only tolerate strong acid environments, but also has a better adsorption effect on heavy metals such as Au under acidic conditions. Research shows that the metabolism of Galdieria sulphuraria is diverse and can utilize more than 50 different carbon sources such as glucose and glycerol, and can grow in autotrophic, heterotrophic, and mixotrophic growth modes. Based on this, the mixotrophic method is usually adopted when treating wastewater with Galdieria sulphuraria. In addition, Galdieria sulphuraria can accumulate proteins (especially phycocyanin) and carbohydrates (such as glycogen) during growth, and phycocyanin can be accumulated in autotrophic, heterotrophic, and mixotrophic growth modes. Phycocyanin has anti-cancer activity, antioxidant activity, anti-inflammatory activity, and immunomodulatory activity, and has broad application prospects in fields such as feed, food, and cosmetics. The main type of phycocyanin contained in Galdieria sulphuraria is R-phycocyanin, which has the characteristic of high temperature resistance (73 °C), and because of its low pH culture environment, Galdieria sulphuraria can prevent the contamination of organisms such as bacteria. Therefore, phycocyanin extracted from Galdieria sulphuraria has its advantages.

[0004] Currently, there are studies on the application of Galdieria sulphuraria in various types of wastewater treatment at home and abroad, and it has been confirmed that it can remove ammonia nitrogen, total nitrogen, total phosphorus, Au 3+ and Pd 2+ and other substances in wastewater. It was also found in the research that both live cells and dead cells have an adsorption effect on Au 3+ and Pd 2+The adsorption rates are the same, inferring that the adsorption process of heavy metal ions by hot spring red algae does not depend on cell metabolism, while the adsorption process of most microalgae for heavy metals involves cell metabolism. In addition, existing research has linked the treatment of industrial high-ammonia-nitrogen wastewater with the production of phycocyanin, confirming the application value of hot spring red algae in the production of phycocyanin using wastewater. However, current research on the purification of wastewater by hot spring red algae mostly focuses on the removal of nitrogen and phosphorus in urban wastewater and industrial wastewater. There is a lack of research on the purification of acidic heavy metal wastewater by hot spring red algae, and the feasibility of its purification of acidic heavy metal wastewater is not clear. The optimal conditions for purifying acidic heavy metal wastewater, the highest tolerated heavy metal concentration, and the ability to synthesize phycocyanin have not been clearly elucidated.

[0005] Therefore, it is necessary to seek a more effective method for culturing microalgae in AMD wastewater to recover metals and co-produce phycocyanin. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for culturing microalgae in AMD wastewater to recover metals and co-produce phycocyanin to overcome the deficiencies of the prior art. This method promotes the efficiency of metal recovery and the accumulation of phycocyanin by adding sufficient low-cost organic carbon sources in the cultivation of microalgae in AMD wastewater.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a cultivation method for improving the purification efficiency of hot spring red algae in AMD wastewater, including the following steps:

[0008] 1) Pretreatment of AMD wastewater, adjusting the pH to 1 - 3, and inoculating hot spring red algae into the pretreated AMD wastewater;

[0009] 2) Adding small molecule organic carbon sources such as glucose, glycerol, sodium acetate, etc. to the AMD wastewater, and controlling the cultivation temperature at 30 - 40 °C;

[0010] 3) After the cultivation is completed, harvesting the microalgae to obtain algal bodies rich in phycocyanin.

[0011] Preferably, the pretreatment method of the AMD wastewater in step 1 is sedimentation and filtration to remove particulate matter in the wastewater to obtain clarified wastewater.

[0012] Preferably, in the pretreatment of the AMD wastewater in step 1, the suitable specific concentration ranges of various metal ions in the AMD wastewater are as follows: Fe 2+ Concentration 50 - 400 mg / L, Ni 2+ Concentration 1.5 - 5 mg / L, Zn 2+ Concentration 10 - 40 mg / L, Cu 2+ Concentration 5 - 20 mg / L, Mg 2 + Concentration 50 - 120 mg / L, Al 3+Concentration 80 - 200 mg / L, Cd 2+ Concentration 0.05 - 0.2 mg / L, Pb 2+ Concentration 0.2 - 0.5 mg / L; Use sulfuric acid to adjust the pH of AMD wastewater to 1 - 3.

[0013] Preferably, the wastewater pretreatment in step 1 is carried out by mixing the wastewater through stirring, aeration, etc.

[0014] Preferably, the device for treating the concentration of AMD wastewater in step 1 is a shaker.

[0015] Preferably, the specific range of the suitable concentration of the small - molecule organic carbon source in step 2 is as follows: glycerol concentration 1 - 10 g / L, glucose concentration 10 - 20 g / L, sodium acetate 10 - 20 g / L;

[0016] Preferably, the thermophilic red alga is Galdieria sulphuraria;

[0017] Preferably, in step 3, the algal bodies in the wastewater are harvested by means of membrane filtration, sedimentation, centrifugation, etc.

[0018] Another technical solution of the present invention is: a cultivation method for improving the efficiency of thermophilic red alga in purifying AMD wastewater, including the following steps:

[0019] 1) Pretreatment of AMD wastewater, adjust the pH to 1 - 3, and use an inductively coupled plasma optical emission spectrometer (ICP - OES)

[0020] to measure the concentration of each heavy metal ion in AMD;

[0021] 2) According to the measurement results, prepare a metal ion compound mixed solution with a corresponding concentration as an AMD wastewater simulation solution, and inoculate the thermophilic red alga into the simulation solution;

[0022] 3) Add small - molecule organic carbon sources such as glucose, glycerol, sodium acetate, etc., and control the cultivation temperature at 30 - 40 °C;

[0023] 4) After the cultivation is completed, harvest the microalgae to obtain algal bodies rich in phycocyanin.

[0024] Specifically, the ICP - OES measurement method is as follows:

[0025]

[0026] Furthermore, on the basis of the metal ion compound mixed solution prepared according to the concentration of each metal ion in AMD wastewater, the present invention further prepares low - AMD and high - AMD concentration wastewater simulation solutions, and cultivates the thermophilic red alga according to steps 2) - 4).

[0027] The beneficial effects of the present invention are as follows:

[0028] Before the start of cultivation, an organic carbon source is added to the culture medium. Through a period of cultivation, the cultivated Thermophilic red algae can obtain a relatively high content or concentration of phycocyanin during the required cultivation stage, and the heavy metal recovery efficiency is also significantly improved, thereby achieving the purpose of cultivating microalgae with AMD wastewater to recover metals and co-producing phycocyanin. Through the reasonable selection of the culture medium composition formula, the type and dosage of the organic carbon source, the culture conditions, etc., after adding a sufficient amount of low-cost organic carbon source, on the basis of purifying and absorbing AMD wastewater, it can better promote the growth of Thermophilic red algae and the accumulation of phycocyanin. The present invention puts forward new insights into the cultivation technology of Thermophilic red algae. There are technological improvements in terms of biomass, the efficiency of recovering metals from AMD wastewater, and the concentration of phycocyanin, etc., opening up a broader market for the production and application of cultivating microalgae with AMD wastewater to recover metals and phycocyanin. Brief Description of the Drawings

[0029] Figure 1 Growth curve of Example 1 of the present invention and Comparative Examples 1-2 in AMD wastewater;

[0030] Figure 2 Schematic diagram of Fv / Fm values of Example 1 of the present invention and Comparative Example 2 in AMD wastewater;

[0031] Figure 3 Schematic diagrams of growth curve (a), specific growth rate (b), dry weight (c) and Fv / Fm value (d) after adding organic carbon source and culturing for 22 days in Examples 2-3 and Comparative Examples 3-4;

[0032] Figure 4 Schematic diagrams of phycocyanin content and concentration after adding organic carbon source and culturing for 22 days in Examples 2-3 and Comparative Examples 3-4;

[0033] Figure 5 Schematic diagrams of phycocyanin content and concentration in Examples 3-5. Detailed Embodiments

[0034] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only examples, and those skilled in the art can think of other obvious variations.

[0035] Detection of metal ion concentration in AMD wastewater samples of the present invention and preparation of simulated solutions:

[0036] An inductively coupled plasma optical emission spectrometer (ICP-OES) is used to measure the concentration of each heavy metal ion in AMD wastewater. The operation and calculation parameters are as shown in the specification part. The detection results and the corresponding ion compound preparation schemes are as follows:

[0037] Table 1 Configuration of AMD Simulated Solution

[0038]

[0039]

[0040] On this basis, considering the differences in the metal ion concentrations of actual industrial AMD wastewater, two solutions with high and low concentrations were further designed in order to cover the concentration ranges of most actual industrial AMD wastewater. The specific test results and the corresponding ion compound configuration schemes are as follows:

[0041] (1) Low AMD Concentration

[0042] Table 2 Configuration of Low-Concentration AMD Simulated Solution

[0043]

[0044] (2) High AMD Concentration

[0045] Table 3 Configuration of High-Concentration AMD Simulated Solution

[0046]

[0047]

[0048] Culture Group without Additional Carbon Source in Example 1

[0049] The medium formula is as follows: 992.89 mg / L ferrous sulfate heptahydrate, 13.43 mg / L nickel sulfate hexahydrate, 15.39 mg / L zinc sulfate heptahydrate, 19.64 mg / L copper sulfate pentahydrate, 618.10 mg / L sodium sulfate, 335.07 mg / L potassium sulfate, 871 mg / L magnesium sulfate heptahydrate, 666.43 mg / L aluminum sulfate octadecahydrate, 0.081 mg / L chromium chloride, 0.27 mg / L lead chloride.

[0050] The final pH value is 2.65. The prepared medium is dispensed into conical flasks, 250 mL per flask. After the medium is dispensed into the conical flasks, it is inoculated using a disposable pipette, and the concentration of Galdieria sulphuraria algal cells is adjusted to 10 million cells / mL. After inoculation, it is sealed with a biological breathable sealing film.

[0051] After inoculation and sealing, place it in a light shaker. The shaker parameters are a temperature of 35°C and a rotation speed of 150 rpm. On the 0th, 3rd, 6th, 9th, 14th, 18th, and 22nd days, parameters such as the cell density, the maximum light energy conversion efficiency of PSⅡ (Fv / Fm), and the pH value of the thermophilic red algae are measured. On the 22nd day, the heavy metal content, algal cell concentration, and phycocyanin content in the culture medium are measured. The sampling operation method is the same as inoculation. During the culture process, each time 10 mL is taken out and placed in a centrifuge tube for analysis. After sampling, it should be sealed again and returned to the shaker.

[0052] Cell density determination: Take 100 μl of the thermophilic red algae culture solution and dilute it 10 times. Place the diluted solution on a hemocytometer and count it under a microscope. The counting area consists of 25 large squares. According to the diagonal direction, count the algal cells in the 5 large squares in the upper left, lower left, upper right, lower right, and center, and take the average value. The cell density calculation formula is: algal cell density (cells / mL) = average value * 25 * 10 4 。

[0053] Determination of Fv / Fm: Use a FluorPen FP110 handheld chlorophyll fluorescence meter (Ecotech Ecological Technology Ltd) to measure the chlorophyll fluorescence Fv / Fm of dark-adapted algal cells.

[0054] Experimental group with additional glucose carbon source in Example 2

[0055] The culture medium formula is: 992.89 mg / L ferrous sulfate heptahydrate, 13.43 mg / L nickel sulfate hexahydrate, 15.39 mg / L zinc sulfate heptahydrate, 19.64 mg / L copper sulfate pentahydrate, 618.10 mg / L sodium sulfate, 335.07 mg / L potassium sulfate, 871 mg / L magnesium sulfate heptahydrate, 666.43 mg / L aluminum sulfate octadecahydrate, 0.081 mg / L chromium chloride, 0.27 mg / L lead chloride.

[0056] The final pH value is 2.65. The prepared culture medium is dispensed into conical flasks, 250 mL per flask; after the culture medium is dispensed into the conical flasks, it is sealed with a biological breathable sealing film.

[0057] 5 g / L ammonium sulfate, 0.8 g / L sodium dihydrogen phosphate, and 5 g / L glucose are additionally added to the transferred culture medium. After adding, carry out the inoculation operation. Use a disposable pipette for inoculation and adjust the Galdieria sulphuraria algal cell concentration to 10 million cells / mL. After inoculation, seal it with a sealing film.

[0058] After inoculation and sealing, place it in a light shaker with the shaker parameters of 35°C and 150rpm. The cell density of hot spring red algae, the maximum light energy conversion efficiency of PSⅡ (Fv / Fm) and pH value were measured on days 0, 3, 6, 9, 14, 18 and 22. The heavy metal content, algae cell concentration and phycocyanin content in the culture medium were measured on day 22. The sampling operation method is the same as inoculation. During the culture process, 10mL is taken out each time and placed in a centrifuge tube for analysis. After sampling, the tube should be sealed and put back into the shaker.

[0059] Cell density determination: Take 100μl of hot spring red algae culture solution and dilute it 10 times. Put the dilution on the blood cell counting plate and count it under a microscope. The counting area is composed of 25 large squares. According to the diagonal direction, count the number of algae in the upper left, lower left, upper right, lower right and center squares, and take the average value. The formula for calculating cell density is: Algae cell density (cells / mL) = average value * 25 * 10 4 .

[0060] Determination of Fv / Fm: Chlorophyll fluorescence Fv / Fm was measured on dark-adapted algal cells using FluorPen FP110 handheld chlorophyll fluorescence (Ecotech Ecological Technology Ltd).

[0061] Biomass determination: After the culture is completed, take 40mL of culture solution, centrifuge at 8000rpm for 10min, discard the supernatant and add about 20mL of ultrapure water again, mix well; repeat the centrifugation operation three times. Wash the centrifuged algae mud with a washing bottle filled with ultrapure water into a weighed flat weighing dish. The weighing dish has been dried to constant weight at 90℃ in advance and cooled to room temperature in a dryer. The weighing dish containing algae mud is placed in an oven and dried to constant weight. Subtract the weight of the weighing dish to obtain the dry biomass of hot spring red algae in the taken volume of algae liquid.

[0062] Determination of phycocyanin content: After the cultivation is completed, remove 20mL of culture medium, centrifuge at 8000rpm for 10min, discard the supernatant and add about 10mL of ultrapure water again, mix well; repeat the centrifugation operation three times. Add pure water to the algae mud after centrifugation, shake and dilute to 5mL. Take 1mL of it, add liquid nitrogen and use a mortar to grind it thoroughly, then dilute to 3mL, take 1.5mL of it and centrifuge it at 4500×g for 5 minutes. After centrifugation, take 1mL, add 2mL of pure water and mix well, and use a spectrophotometer to measure the absorbance of the solution at 652nm and 615nm. The formula for calculating the concentration of phycocyanin is as follows:

[0063]

[0064] Example 3 Experimental group with additional addition of glycerol carbon source

[0065] The culture medium formula is as follows: 992.89 mg / L ferrous sulfate heptahydrate, 13.43 mg / L nickel sulfate hexahydrate, 15.39 mg / L zinc sulfate heptahydrate, 19.64 mg / L copper sulfate pentahydrate, 618.10 mg / L sodium sulfate, 335.07 mg / L potassium sulfate, 871 mg / L magnesium sulfate heptahydrate, 666.43 mg / L aluminum sulfate octadecahydrate, 0.081 mg / L chromium chloride, 0.27 mg / L lead chloride.

[0066] The final pH value is 2.65. The prepared culture medium is dispensed into conical flasks, 250 mL per flask; after dispensing into conical flasks, the culture medium is sealed with a biological breathable sealing film.

[0067] 5 g / L ammonium sulfate, 0.8 g / L sodium dihydrogen phosphate and 5 g / L glycerol are additionally added to the transferred culture medium. After the addition, inoculation is carried out using a disposable pipette, and the cell concentration of Galdieria sulphuraria is adjusted to 10 million cells / mL. After inoculation, it is sealed with a sealing film.

[0068] After inoculation and sealing, it is placed in an illuminated shaker. The shaker parameters are a temperature of 35 °C and a rotation speed of 150 rpm. On the 0th, 3rd, 6th, 9th, 14th, 18th, and 22nd days, parameters such as the cell density, the maximum light energy conversion efficiency (Fv / Fm) of PSⅡ, and the pH value of the thermophilic red algae are measured. On the 22nd day, the heavy metal content, the algal cell concentration, and the phycocyanin content in the culture medium are measured. The sampling operation method is the same as inoculation. During the culture process, 10 mL is taken out each time and put into a centrifuge tube for analysis. After sampling, it should be sealed again and put back into the shaker.

[0069] Cell density determination: Take 100 μl of the thermophilic red algae culture solution and dilute it 10 times. Place the diluted solution on a hemocytometer and count it under a microscope. The counting area consists of 25 large squares. According to the diagonal direction, count the algal cells in the 5 large squares in the upper left, lower left, upper right, lower right, and center, and take the average value. The cell density calculation formula is: algal cell density (cells / mL) = average value * 25 * 10 4 。

[0070] Determination of Fv / Fm: Use a FluorPen FP110 handheld chlorophyll fluorometer (Ecotech Ecological Technology Ltd) to measure the chlorophyll fluorescence Fv / Fm of dark-adapted algal cells.

[0071] Biomass determination: After the cultivation was completed, 40 mL of the culture solution was taken, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and about 20 mL of ultrapure water was added again and mixed well; the centrifugation operation was repeated three times. The centrifuged algal sludge was washed into a weighed flat weighing dish with a wash bottle filled with ultrapure water. The weighing dish had been dried to a constant weight at 90 °C in advance and cooled to room temperature in a desiccator. The weighing dish containing the algal sludge was placed in an oven and dried to a constant weight, and the weight of the weighing dish was subtracted to obtain the dry biomass of the thermophilic red algae in the taken volume of the algal solution.

[0072] Determination of phycocyanin content: After the cultivation was completed, 20 mL of the culture solution was taken, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and about 10 mL of ultrapure water was added again and mixed well; the centrifugation operation was repeated three times. Pure water was added to the centrifuged algal sludge, shaken well and made up to 5 mL. 1 mL of it was taken, added with liquid nitrogen and ground thoroughly in a mortar and then diluted to 3 mL. 1.5 mL of it was centrifuged at 4500×g for 5 minutes. After centrifugation, 1 mL was taken, 2 mL of pure water was added and mixed well, and the absorbance of the solution at 652 nm and 615 nm was measured using a spectrophotometer. The phycocyanin concentration calculation formula is as follows:

[0073]

[0074] Example 4 High-concentration and low-concentration AMD simulated solution experimental groups

[0075] On the basis of Example 3, the high-AMD-concentration and low-AMD-concentration culture media in Table 2 - Table 3 were used for replacement respectively, the pH value was adjusted to 2.65, and the prepared culture medium was dispensed into conical flasks, 250 mL per flask; after the culture medium was dispensed into the conical flasks, it was sealed with a biological breathable sealing film.

[0076] 5 g / L ammonium sulfate, 0.8 g / L sodium dihydrogen phosphate and 5 g / L glycerol were additionally added to the transferred culture medium. After the addition, inoculation operation was carried out, and a disposable pipette was used for inoculation to adjust the Galdieria sulphuraria algal cell concentration to 10 million cells / mL. After inoculation, it was sealed with a sealing film.

[0077] After inoculation and sealing, it was placed in an illumination shaker. The shaker parameters were a temperature of 35 °C and a rotation speed of 150 rpm. On the 0th, 3rd, 6th, 9th, 14th, 18th, and 22nd days, parameters such as the cell density of the thermophilic red algae, the maximum light energy conversion efficiency (Fv / Fm) of PSⅡ, and the pH value were measured. On the 22nd day, the heavy metal content, algal cell concentration, and phycocyanin content in the culture medium were measured. The sampling operation method was the same as that of inoculation. During the cultivation process, 10 mL was taken out each time and put into a centrifuge tube for analysis. After sampling, it should be sealed again and put back into the shaker.

[0078] Example 5 AMD wastewater solution experimental group

[0079] Take the AMD wastewater sample of the present invention, adjust the pH to 2.65 with sulfuric acid, and dispense it into conical flasks, 250 mL per flask; after dispensing the culture medium into the conical flasks, seal them with a biological breathable sealing film.

[0080] 5 g / L ammonium sulfate, 0.8 g / L sodium dihydrogen phosphate and 5 g / L glycerol were additionally added to the transferred culture medium. After the addition, inoculation was carried out using a disposable pipette, and the concentration of Galdieria sulphuraria algal cells was adjusted to 10 million cells / mL. After inoculation, seal it with a sealing film.

[0081] After inoculation and sealing, place it in a light shaking incubator. The parameters of the shaking incubator are a temperature of 35 °C and a rotation speed of 150 rpm. On the 0th, 3rd, 6th, 9th, 14th, 18th, and 22nd days, parameters such as the cell density of Thermophilic red algae, the maximum light energy conversion efficiency (Fv / Fm) of PSⅡ, and the pH value were measured. On the 22nd day, the heavy metal content, algal cell concentration, and phycocyanin content in the culture medium were measured. The sampling operation method is the same as inoculation. During the culture process, each time 10 mL was taken out and put into a centrifuge tube for analysis. After sampling, it should be sealed again and put back into the shaking incubator.

[0082] Experimental group of Chlorella vulgaris in Comparative Example 1

[0083] The culture medium formula is: 992.89 mg / L ferrous sulfate heptahydrate, 13.43 mg / L nickel sulfate hexahydrate, 15.39 mg / L zinc sulfate heptahydrate, 19.64 mg / L copper sulfate pentahydrate, 618.10 mg / L sodium sulfate, 335.07 mg / L potassium sulfate, 871 mg / L magnesium sulfate heptahydrate, 666.43 mg / L aluminum sulfate octadecahydrate, 0.081 mg / L chromium chloride, 0.27 mg / L lead chloride.

[0084] The final pH value is 2.65. The prepared culture medium is dispensed into conical flasks, 250 mL per flask; after dispensing the culture medium into the conical flasks, seal them with a biological breathable sealing film.

[0085] 5 g / L ammonium sulfate and 0.8 g / L sodium dihydrogen phosphate were additionally added to the transferred culture medium. After the addition, inoculation was carried out using a disposable pipette, and the concentration of Chlorella vulgaris algal cells was adjusted to 10 million cells / mL. After inoculation, seal it with a sealing film.

[0086] After inoculation and sealing, place it in an illuminated shaker. The shaker parameters are a temperature of 35°C and a rotation speed of 150 rpm. On days 0, 3, 6, 9, 14, 18, and 22, measure parameters such as the cell density of Chlorella, the maximum light energy conversion efficiency of PSⅡ (Fv / Fm), and the pH value. The sampling operation method is the same as inoculation. During the cultivation process, each time 10 mL is taken out and put into a centrifuge tube for analysis. After sampling, it should be sealed again and returned to the shaker.

[0087] Cell density determination: Take 100 μl of Chlorella culture solution and dilute it 10 times. Place the diluted solution on a hemocytometer and count it under a microscope. The counting area consists of 25 large squares. According to the diagonal orientation, count the algal cells in the 5 large squares in the upper left, lower left, upper right, lower right, and center, and take the average value. The cell density calculation formula is: algal cell density (cells / mL) = average value * 25 * 10 4 。

[0088] Determination of Fv / Fm: Use a FluorPen FP110 handheld chlorophyll fluorometer (Ecotech Ecological Technology Ltd) to measure the chlorophyll fluorescence Fv / Fm of dark-adapted algal cells.

[0089] Scenedesmus experimental group of Comparative Example 2

[0090] The medium formula is: 992.89 mg / L ferrous sulfate heptahydrate, 13.43 mg / L nickel sulfate hexahydrate, 15.39 mg / L zinc sulfate heptahydrate, 19.64 mg / L copper sulfate pentahydrate, 618.10 mg / L sodium sulfate, 335.07 mg / L potassium sulfate, 871 mg / L magnesium sulfate heptahydrate, 666.43 mg / L aluminum sulfate octadecahydrate, 0.081 mg / L chromium chloride, 0.27 mg / L lead chloride.

[0091] The final pH value is 2.65. The prepared medium is dispensed into conical flasks, 250 mL per flask; after dispensing the medium into the conical flasks, seal them with a biological breathable sealing film.

[0092] 5 g / L ammonium sulfate and 0.8 g / L sodium dihydrogen phosphate are additionally added to the transferred medium. After adding, carry out the inoculation operation. Use a disposable pipette for inoculation and adjust the Scenedesmus cell concentration to 10 million cells / mL. After inoculation, seal it with a sealing film.

[0093] After inoculation and sealing, place it in a light shaker. The shaker parameters are a temperature of 35°C and a rotation speed of 150 rpm. On the 0th, 3rd, 6th, 9th, 14th, 18th, and 22nd days, parameters such as the cell density of Scenedesmus, the maximum light energy conversion efficiency (Fv / Fm) of PSⅡ, and the pH value were measured. The sampling operation method is the same as inoculation. During the culture process, 10 mL is taken out each time and put into a centrifuge tube for analysis. After sampling, it should be sealed again and put back into the shaker.

[0094] Cell density determination: Take 100 μl of Chlorella culture solution and dilute it 10 times. Place the diluted solution on a hemocytometer and count it under a microscope. The counting area consists of 25 large squares. According to the diagonal orientation, count the algal cells in the 5 large squares in the upper left, lower left, upper right, lower right, and central positions, and take the average value. The cell density calculation formula is: algal cell density (cells / mL) = average value * 25 * 10 4 。

[0095] Determination of Fv / Fm: Use a FluorPen FP110 handheld chlorophyll fluorescence meter (Ecotech Ecological Technology Ltd) to measure the chlorophyll fluorescence Fv / Fm of dark-adapted algal cells.

[0096] Experimental group of the conventional medium of Cyanidium caldarium in Comparative Example 3

[0097] Replace the simulated AMD wastewater medium with the conventional medium of Cyanidium caldarium. The medium formula is: 1.20 g / L phosphoric acid, 10.56 g / L ammonium sulfate, 1.6 g / L magnesium sulfate heptahydrate, 0.69 g / L calcium chloride dihydrate, 0.23 g / L sodium ferric (Ⅲ) ethylenediaminetetraacetate, 0.07 g / L disodium ethylenediaminetetraacetate monohydrate, 0.0995 g / L sodium chloride, 0.6035 g / L potassium chloride, 0.0496 g / L boric acid, 0.0160 g / L manganese chloride tetrahydrate, 0.0112 g / L zinc chloride, 0.0080 g / L copper sulfate pentahydrate, 0.0041 g / L sodium molybdate dihydrate, 0.0040 g / L cobalt dichloride hexahydrate.

[0098] The final pH value was adjusted to 2.65 with sulfuric acid. The prepared medium was dispensed into conical flasks, 250 mL per flask; after the medium was dispensed into conical flasks, it was inoculated using a disposable pipette, and the cell concentration of Galdieria sulphuraria was adjusted to 10 million cells / mL. After inoculation, it was sealed with a biological breathable sealing film.

[0099] After inoculation and sealing, place it in a light shaker. The shaker parameters are a temperature of 35 °C and a rotation speed of 150 rpm. On the 0th, 3rd, 6th, 9th, 14th, 18th, and 22nd days, parameters such as the cell density of Thermorubrum sp., the maximum light energy conversion efficiency of PSⅡ (Fv / Fm), and the pH value were measured. On the 22nd day, the heavy metal content, algal cell concentration, and phycocyanin content in the culture medium were measured. The sampling operation method is the same as inoculation. During the culture process, each time 10 mL is taken out and placed in a centrifuge tube for analysis. After sampling, it should be sealed again and returned to the shaker.

[0100] Cell density determination: Take 100 μl of the Thermorubrum sp. culture solution and dilute it 10 times. Place the diluted solution on a hemocytometer and count it under a microscope. The counting area consists of 25 large squares. According to the diagonal orientation, count the algal cells in the 5 large squares in the upper left, lower left, upper right, lower right, and central positions, and take the average value. The cell density calculation formula is: algal cell density (cells / mL) = average value * 25 * 10 4 。

[0101] Determination of Fv / Fm: Use a FluorPen FP110 handheld chlorophyll fluorescence (Ecotech Ecological Technology Ltd) to measure the chlorophyll fluorescence Fv / Fm of dark-adapted algal cells.

[0102] Biomass determination: After the culture is completed, take 40 mL of the culture solution, centrifuge it at 8000 rpm for 10 min, discard the supernatant, and then add about 20 mL of ultrapure water again and mix well; repeat the centrifugation operation three times. Wash the centrifuged algal sludge into a pre-weighed flat weighing dish with a wash bottle filled with ultrapure water. The weighing dish has been dried to a constant weight at 90 °C in advance and cooled to room temperature in a desiccator. The weighing dish containing the algal sludge is placed in an oven and dried to a constant weight. Subtract the weight of the weighing dish to obtain the dry biomass of Thermorubrum sp. in the taken volume of the algal solution.

[0103] Determination of phycocyanin content: After the culture is completed, take 20 mL of the culture solution, centrifuge it at 8000 rpm for 10 min, discard the supernatant, and then add about 10 mL of ultrapure water again and mix well; repeat the centrifugation operation three times. Add pure water to the centrifuged algal sludge, shake well and make up the volume to 5 mL. Take 1 mL of it, add liquid nitrogen and grind it thoroughly in a mortar and then dilute it to 3 mL. Take 1.5 mL of it and centrifuge it at 4500×g for 5 minutes. After centrifugation, take 1 mL, add 2 mL of pure water and mix well, and then use a spectrophotometer to measure the absorbance of the solution at 652 nm and 615 nm. The phycocyanin concentration calculation formula is as follows:

[0104]

[0105] For Comparative Example 4, on the basis of Example 1, the biomass and phycocyanin content were further detected.

[0106] Example 1 compared the growth of different algal types, and Comparative Example 4 further compared the accumulation of phycocyanin on this basis.

[0107] Biomass determination: After the cultivation was completed, 40 mL of the culture solution was taken, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and about 20 mL of ultrapure water was added again and mixed well; the centrifugation operation was repeated three times. The centrifuged algal sludge was washed into a weighed flat weighing dish with a wash bottle filled with ultrapure water. The weighing dish had been dried to a constant weight at 90 °C in advance and cooled to room temperature in a desiccator. The weighing dish containing the algal sludge was placed in an oven and dried to a constant weight, and the weight of the weighing dish was subtracted to obtain the dry biomass of Thermophilic red algae in the taken volume of algal solution.

[0108] Determination of phycocyanin content: After the cultivation was completed, 20 mL of the culture solution was taken, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and about 10 mL of ultrapure water was added again and mixed well; the centrifugation operation was repeated three times. Pure water was added to the centrifuged algal sludge, shaken well and made up to 5 mL. Take 1 mL of it, add liquid nitrogen and grind it thoroughly in a mortar and then dilute it to 3 mL. Take 1.5 mL of it and centrifuge at 4500×g for 5 minutes. After centrifugation, take 1 mL, add 2 mL of pure water and mix well, and then use a spectrophotometer to measure the absorbance of the solution at 652 nm and 615 nm. The phycocyanin concentration calculation formula is as follows:

[0109]

[0110] Analysis of experimental results:

[0111] Analysis of algal cell density: Comparative Example 1 was the most intolerant to the AMD wastewater environment, and algal cells could not be observed on the third day of cultivation. In Comparative Example 2, the number of algal cells also began to decrease until death after the ninth day. However, the number of algal cells in Examples 1-5 and Comparative Examples 3-4 could continue to increase steadily. Taking Example 1 as an example, the algal cell density reached 2.36(10 8 cells / mL at the highest on the 22nd day, indicating that Thermophilic red algae can tolerate the AMD wastewater environment with low pH and grow stably in this environment. Figure 1 The comparison results of algal cell density between some examples and comparative examples are shown.

[0112] The Fv / Fm values of Examples 1-5 showed no obvious change, indicating that the AMD wastewater culture system had no stress on the growth of Thermophilic red algae. However, the Fv / Fm of Comparative Examples 1-2 changed significantly until it dropped to zero, indicating that the AMD wastewater culture environment had a stress on the photosynthetic growth of Chlorella vulgaris and Scenedesmus. Therefore, Thermophilic red algae can be used to treat AMD wastewater, while the other two microalgae are not suitable. The algal species selection is reasonable, and the above results are as Figure 2 shown.

[0113] We further analyzed the additional carbon sources, specifically the comparison of algal cell density, specific growth rate, dry weight, and Fv / Fm between Comparative Examples 3-4 and Examples 2-3. From Figure 3 It can be seen that there were no significant differences in algal cell density, dry weight, and Fv / Fm values between Comparative Example 3 and Comparative Example 4 before 14 days of cultivation (p > 0.05), that is, their growth conditions and physiology remained stable. The algal cell density, specific growth rate, and dry weight of the mixed culture group of Thermosynechococcus sp. in Examples 2 and 3 were significantly higher than those in Comparative Examples 3 and 4 (p < 0.05). After 14 days of cultivation, the algal cell density of Examples 2-3 increased by approximately 91.89% and 144.59% respectively compared to Comparative Example 4, indicating that organic carbon sources (glycerol and glucose) have an obvious effect on promoting the growth of algal cells. In contrast, glycerol does not inhibit the early growth of red algae. In summary, based on the cultivation of Thermosynechococcus sp. in AMD wastewater, Thermosynechococcus sp. can utilize organic carbon sources simultaneously under light conditions and grow through photosynthesis to achieve high-density cultivation.

[0114] From Figure 4 It can be seen that the content of phycocyanin in Comparative Example 3 under different nutrient conditions was 7.58%, which was significantly higher than that of other groups (p < 0.05). Under the nutrient conditions with wastewater, the phycocyanin contents of Comparative Example 4 and Examples 2-3 were relatively low, and Example 2 was the lowest. The reason for this result is that glucose has a certain inhibitory effect on the accumulation of phycocyanin. Considering the phycocyanin concentration, the phycocyanin concentration in Example 3 reached the highest of 244.80 mg / L and the phycocyanin content (4.19%) had little difference compared with other groups, further proving that glycerol is the best carbon source for treating AMD wastewater.

[0115] Considering the actual differences in metal ion concentrations in different AMD wastewaters, we further analyzed the influence of metal ion concentrations in AMD wastewater on the accumulation of phycocyanin, and compared the accumulation of phycocyanin in the AMD simulated solutions and AMD wastewater sample groups with different metal ion concentrations in Examples 3-5. Through Figure 5 It can be seen that even if the metal ion concentration in AMD wastewater increases significantly, it will not have an adverse effect on the growth of red algae and the accumulation of phycocyanin, which is consistent with the original growth environment and tolerance reports of Thermosynechococcus sp. A lower metal ion concentration in AMD wastewater will slightly affect the accumulation of phycocyanin, but will not affect the algal cell concentration, and the phycocyanin content still has certain advantages. The AMD wastewater group was slightly inferior to the simulated group, but the phycocyanin accumulation amount also basically reached 90%. It is speculated that the existence form of metal ions may have certain limitations on the nutrient utilization of red algae. In summary, using AMD wastewater (whether it is high-concentration or low-concentration simulated solution or directly using AMD wastewater) can achieve rapid accumulation of phycocyanin and growth of algal cells.

[0116] As can be seen from Table 4, the removal rates of heavy metals Cu, Fe, and Zn in Example 3 were 10.71±0.54 a %, 37.07±2.61 a %, 35.14±2.88 a %, indicating that the hot spring red algae can effectively remove heavy metals in AMD wastewater, and the metal removal rates are higher than those in Comparative Example 3. In particular, the removal rate of Fe in Example 3 is about 28% higher than that in Comparative Example 3. It can be seen that compared with the existing culture medium of red algae, the heavy metal absorption and utilization efficiency of red algae in the AMD wastewater environment is relatively higher, which may be related to the high acidity and high ion concentration of the original growth environment of red algae.

[0117] Table 4 Removal rates of heavy metals Cu, Fe, and Zn by hot spring red algae under different nutrient conditions

[0118]

[0119] The above embodiments are only for explaining the technical concept and characteristics of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for recovering metals and co-producing phycocyanin by cultivating microalgae using AMD wastewater, characterized in that: include: 1) Inoculate hot spring red algae into pretreated AMD wastewater; 2) Adding small molecule organic carbon sources to AMD wastewater; 3) After the cultivation is completed, the microalgae are harvested to obtain algae rich in phycocyanin.

2. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 1, characterized in that: include: 1) Pretreatment of AMD wastewater, adjusting the pH to 1-3, and inoculating hot spring red algae into the pretreated AMD wastewater; 2) Add small molecule organic carbon sources, preferably glucose, glycerol, and sodium acetate, to AMD wastewater and control the culture temperature at 30-40°C; 3) After the cultivation is completed, the microalgae are harvested to obtain algae rich in phycocyanin.

3. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 2, characterized in that: The AMD wastewater pretreatment method is sedimentation filtration to remove particulate matter in the wastewater to obtain clarified wastewater.

4. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 2, characterized in that: In the AMD wastewater pretreatment, the specific concentration ranges of the metal ions in the AMD wastewater are as follows: Fe 2+ Concentration 50-400mg / L, Ni 2+ Concentration 1.5-5mg / L, Zn 2+ Concentration 10-40mg / L, Cu 2+ Concentration 5-20mg / L, Mg 2+ Concentration 50-120mg / L, Al 3+ Concentration 80-200mg / L, Cd 2+ Concentration 0.05-0.2mg / L, Pb 2+ Concentration 0.2-0.5 mg / L; use sulfuric acid to adjust the pH of AMD wastewater to 1-3.

5. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 4, characterized in that: In the AMD wastewater pretreatment, the specific concentration ranges of the metal ions in the AMD wastewater are as follows: Fe 2+ Concentration 200-400mg / L, Ni 2+ Concentration 3-5mg / L, Zn 2+ Concentration 20-40mg / L, Cu 2+ Concentration 10-20mg / L, Mg 2+ Concentration 85-120mg / L, Al 3+ Concentration 150-200mg / L, Cd 2+ Concentration 0.1-0.2mg / L, Pb 2+ Concentration 0.3-0.5 mg / L; use sulfuric acid to adjust the pH of AMD wastewater to 2-3.

6. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 2, characterized in that: Wastewater is mixed by stirring, aeration, etc.

7. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 2, characterized in that: The device for treating AMD wastewater concentration is a shaking table.

8. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 2, characterized in that: The specific concentration range of the small molecule organic carbon source is as follows: glycerol concentration 1-10g / L.

9. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 2, characterized in that: The algae in the wastewater are harvested using membrane filtration, sedimentation, centrifugation and other methods.

10. The method for recovering metals and co-producing phycocyanin by culturing microalgae using AMD wastewater according to claim 1, characterized in that: The hot spring red algae is Galdieria sulphuraria .

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