Microalgae culture method and optimization method and application thereof

Abstract

The invention discloses a method for culturing microalgae with swine wastewater and an optimization method and application thereof, and belongs to the field of microalgae culture, the culture method comprises the following steps: (1) diluting culture wastewater, and adding a carbon source and a phosphorus source, the addition amount of the carbon source being 0-5000 mg / L; the addition amount of the phosphorus source is 0-100 mg / L; then adjusting the pH value of the wastewater; (2) inoculating microalgae into the wastewater in the step (1), and culturing; the initial algae density of the microalgae is 0.05 to 1.0 OD660. By adding a specific carbon source and a phosphorus source, the problems of C / N and N / P imbalance and the like in the culture wastewater are solved, and the culture conditions of algae are optimized, so that the protein content in microalgae cells and the microalgae biomass are improved, and the cost of algae culture is reduced. According to the method, in the algae biomass harvesting process, pollutants such as ammonia nitrogen, phosphate and total organic carbon in the wastewater can be synchronously removed, and deep purification of the wastewater is achieved.

Description

Technical Field

[0001] The present invention relates to the field of algae cultivation, and more specifically, to a method for cultivating microalgae and an application thereof, and a method for optimizing conditions for cultivating microalgae by using a response surface methodology. Background Art

[0002] Microalgae are considered as a potential alternative source of protein due to their rapid growth, rich nutrition and high intracellular protein content. For example, the cells of Chlorella pyrenoidosa contain a large amount of protein, which is equivalent to twice that of peanut kernels or five times that of eggs. In addition, the protein in algae cells is high-quality protein, and its amino acid composition is very similar to that of many animal proteins (such as eggs). However, compared with animal proteins, the oil content in algae cells is relatively low and does not contain cholesterol. In addition, algae cells are also rich in nutrients such as carotene and vitamins. These characteristics make microalgae not only a high-quality source of food and feed, but also can be used to produce cosmetics, with extremely high commercial value.

[0003] However, existing algae cultivation technology has the disadvantages of high material costs, and the use of chemicals in the cultivation process can cause secondary pollution; these factors limit the industrial development of algae cultivation.

[0004] We found that aquaculture wastewater contains rich nutrients such as nitrogen, phosphorus and inorganic salts, which are exactly what algae need for growth. However, actual aquaculture wastewater has problems such as high ammonia nitrogen content, C / N and N / P imbalance, which are not conducive to the growth of algae and also affect the protein content in algae cells. Therefore, unadjusted aquaculture wastewater cannot be directly used for microalgae cultivation.

[0005] In view of the above problems and needs, the present invention aims to provide a method for cultivating algae using aquaculture wastewater, which has the advantages of low cost, high growth efficiency of microalgae and high protein content in the cultured microalgae cells. Summary of the invention

[0006] In view of the problems existing in the prior art, the first purpose of the present invention is to provide a method for culturing microalgae, which can reduce the cultivation cost of microalgae and increase the protein content in microalgae cells; the second purpose of the present invention is to provide a method for optimizing the cultivation conditions of microalgae using response surface methodology, which can optimize the addition amount of carbon source and phosphorus source, thereby improving the growth efficiency and protein content of microalgae; the third purpose is to provide the application of the method for culturing microalgae in purifying aquaculture wastewater.

[0007] To solve the above problems, the present invention adopts the following technical solutions.

[0008] A method for culturing microalgae comprises the following steps:

[0009] (1) After diluting the aquaculture wastewater, a carbon source and a phosphorus source are added, wherein the amount of the carbon source added is 0 to 5000 mg / L; the amount of the phosphorus source added is 0 to 100 mg / L; and then the pH of the wastewater is adjusted;

[0010] (2) Inoculating microalgae in the aquaculture wastewater in step (1) for cultivation; the initial algae density of the microalgae is 0.05 to 1.0 OD 660 .

[0011] Furthermore, the carbon source added in step (1) is glucose, and the added amount is 200-800 mg / L; the phosphorus source added is dipotassium hydrogen phosphate, and the added amount is 0-8 mg / L;

[0012] In step (2), the initial algae density of the microalgae is 0.1 to 0.9 OD 660 .

[0013] Furthermore, in step (2), the culture temperature is 15-35°C, the light intensity is 3000-4000 lux, and the light-dark ratio is 12h:12h. These parameters are based on the simulation of the microalgae growth environment under natural conditions; to ensure that the growth cycle of the microalgae under light and dark conditions is consistent with the natural environment. The culture temperature is preferably 25°C

[0014] Furthermore, the types and concentrations of the main pollutants in the aquaculture wastewater in step (1) are: 68-680 mg / L total nitrogen, 50-547 mg / L ammonia nitrogen, 3.24-25.6 mg / L total phosphorus, 0.74-18.7 mg / L phosphate, 36.4-1160 mg / L total organic carbon (TOC), 532-682 mg / L potassium, 314-410 mg / L sodium, 79.7-114 mg / L calcium, 15.2-20.7 mg / L magnesium, 1.30-4.52 mg / L iron, 0.02-0.10 mg / L zinc, 0.02-0.08 mg / L copper, 0.06-0.16 mg / L molybdenum and 0.01-0.05 mg / L cobalt.

[0015] Furthermore, in step (1), the aquaculture wastewater is pig farming wastewater, the dilution multiple is 0 to 10 times, and the pH is 4.0 to 8.0.

[0016] Furthermore, the types and concentrations of major pollutants in pig wastewater are: 680 mg / L total nitrogen, 547 mg / L ammonia nitrogen, 3.24 mg / L total phosphorus, 0.740 mg / L phosphate, 36.4 mg / L TOC, 532 mg / L potassium, 314 mg / L sodium, 79.7 mg / L calcium and 15.2 mg / L magnesium.

[0017] Furthermore, in step (2), the microalgae is a Chlorophyta algae, and the Chlorophyta algae is one of Chlorella pyrenoidosa, Chlorella vulgaris and Scenedesmus, preferably Chlorella pyrenoidosa.

[0018] Furthermore, in step (2), the culture period is 3 to 14 days, preferably 8 days.

[0019] The invention also provides a method for optimizing microalgae culture conditions by using a response surface methodology.

[0020] Furthermore, the phosphorus source addition amount, carbon source addition amount and initial algae density were selected as independent variables, and the protein content in algae cells was selected as the response value. The Box-Behnken design was used to arrange the experiment and collect experimental data. The following RSM regression equation was established:

[0021]

[0022] Among them, x1 is the amount of phosphorus source added, x2 is the amount of carbon source added, x3 is the initial algae density, and y is the content of protein in algae cells;

[0023] b0 is the intercept term, b1, b2, b3, b 12 、b 13 、b 23 、b 11 、b 22 and b 33 is the regression coefficient.

[0024] Furthermore, taking Chlorella pyrenoidosa as the experimental object, the specific steps of optimization using response surface methodology are as follows:

[0025] (1) Single factor experiment: Chlorella pyrenoidosa was inoculated into aquaculture wastewater. The temperature, pH, light intensity, light-dark ratio and culture days were fixed. The initial algae density, phosphorus source addition and carbon source addition were selected as variables for single factor experiment. The biomass of Chlorella pyrenoidosa and the content of protein in algae cells were used as the investigation indicators to screen out the investigation range of initial algae density, phosphorus source addition and carbon source addition in RSM response surface experiment.

[0026] (2) Optimization of RSM response surface method: According to the experimental parameters selected in step (1), the temperature was fixed at 25°C, the pH was 6.5, the light intensity was 4000 lux, the light-dark ratio was 12h:12h, and the number of culture days was fixed at 8 days. The phosphorus source (dipotassium hydrogen phosphate) addition amount (x1), the carbon source (glucose) addition amount (x2) and the initial algae density (x3) were used as response factors, and the protein content in the algae cells was used as the response value (y). According to the Box-Behnken design, a three-level three-factor response surface experiment was arranged, a multiple regression equation was established, and the optimal combination conditions of initial algae density, phosphorus source addition amount and carbon source addition amount were optimized;

[0027] (3) According to step (2), the simplified multiple regression equation for the protein content in algae cells is: According to the equation, the best conditions for protein production were optimized as follows: phosphorus source addition of 8 mg / L, carbon source addition of 200 mg / L, initial algae density of 0.1 OD 660 When the protein content in the cells of Chlorella pyrenoidosa reached a maximum of 52.7%, the dry weight of protein was 346 mg / L, and the biomass of Chlorella pyrenoidosa was 658 mg / L;

[0028] (4) At the end of the cultivation according to step (2), under the optimal conditions for protein production, the contents of ammonia nitrogen, phosphate and TOC in the aquaculture wastewater were only 125 mg / L, 0.28 mg / L and 40 mg / L, respectively, and the removal efficiencies were 77.2%, 96.8% and 83.1%, respectively.

[0029] The invention also provides an application of the method for culturing microalgae in wastewater purification.

[0030] Compared with the prior art, the advantages of the present invention are:

[0031] 1. The method of culturing microalgae using wastewater provided in this scheme solves the problems of C / N and N / P imbalance in pig farm wastewater by accurately selecting specific carbon source additives and phosphorus source additives for addition, so that it can be used for the cultivation of microalgae, effectively optimizing the cultivation conditions of algae, thereby reducing the cost of algae cultivation; and the microalgae cultivated by this method have a higher growth rate and a higher protein content in the algae cells. The harvested algae can be used to produce algae products such as food, feed and cosmetics, and have certain economic value. In addition, the method of culturing microalgae can effectively remove pollutants such as ammonia nitrogen, total nitrogen, phosphate, total phosphorus and total organic carbon (TOC) in wastewater, thereby achieving efficient purification of wastewater.

[0032] 2. This program accurately adjusts the amount of phosphorus source and carbon source added by constructing a regression equation between specific response factors and response values, thereby increasing the protein content in microalgae cells and then determining the optimal culture conditions.

[0033] 3. This method utilizes the photosynthesis process of algae to convert CO2 in the atmosphere into polysaccharides, which not only provides oxygen to the environment, but also reduces the impact of greenhouse gases on the environment, and promotes carbon recycling and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram showing the effect of the number of culture days on the biomass of Chlorella pyrenoidosa and the protein content in algal cells in the present invention;

[0035] Figure 2 Schematic diagram of the effect of pH on the biomass of Chlorella pyrenoidosa and the protein content in algal cells in the present invention;

[0036] Figure 3 Schematic diagram of the effect of temperature on the biomass of Chlorella pyrenoidosa and the protein content in algal cells in the present invention;

[0037] Figure 4 Schematic diagram showing the effect of the amount of carbon source added on the biomass of Chlorella pyrenoidosa and the protein content in algal cells in the present invention;

[0038] Figure 5 Schematic diagram showing the effect of the amount of phosphorus source added on the biomass of Chlorella pyrenoidosa and the protein content in algal cells in the present invention;

[0039] Figure 6 Schematic diagram of the effect of initial algae density on the biomass of Chlorella pyrenoidosa and the protein content in algae cells in the present invention;

[0040] Figure 7 It is a schematic diagram of the growth status and biomass of Chlorella pyrenoidosa after the Chlorella culture environment is optimized by using the response surface methodology in the present invention;

[0041] Figure 8 The response surface diagram and contour diagram of the effect of the phosphorus source addition amount and carbon source addition amount on the protein content in the present invention;

[0042] Fig. 9 The response surface diagram and contour diagram of the effects of the phosphorus source addition amount and the initial algae density on the protein content in the present invention;

[0043] Fig.10 The response surface diagram and contour diagram of the effects of the amount of carbon source added and the initial algae density on the protein content in the present invention;

[0044] Fig.11 It is a schematic diagram of the content of pollutants in wastewater after the cultivation in the present invention. DETAILED DESCRIPTION

[0045] Embodiment 1:

[0046] 1. Single Factor Experiment

[0047] 1. Effects of culture days on biomass and protein content in Chlorella pyrenoidosa

[0048] The specific operation process of the culture day optimization experiment is as follows:

[0049] (1) inoculating Chlorella vulgaris into a culture medium and culturing it for 3 to 4 days to allow it to enter a logarithmic phase;

[0050] (2) Add a certain volume of water to the piggery wastewater to dilute the piggery wastewater 1:1, then add hydrochloric acid and sodium hydroxide, and adjust the pH to 6.5;

[0051] (3) The pyrenoid chlorella in the logarithmic growth phase in step (1) is inoculated into the piggery wastewater in step (2), with an initial inoculation amount of 0.7 OD 660 , equivalent to the dry weight of algae is 161 mg / L.

[0052] (4) Placing the inoculated Chlorella pyrenoidosa in step (3) in an incubator, culturing for 2, 5, 8, 11 and 14 days respectively at a temperature of 25° C., a light intensity of 3000 to 4000 lux, and a light-dark ratio of 12 h:12 h. After the culturing, measuring the biomass of Chlorella pyrenoidosa and the protein content in the algal cells.

[0053] The biomass of Chlorella pyrenoidosa Figure 1 As shown in part (1), the protein content in algal cells is Figure 1 As shown in part (2) of the figure. Figure 1 It can be seen that with the extension of the number of culture days, the biomass of Chlorella pyrenoidosa and the protein content in algal cells gradually increased. On the 11th day of culture, the growth of Chlorella pyrenoidosa entered a stable period, and the biomass and protein content in algal cells reached the maximum, which were 423 mg / L and 240 mg / L, respectively. When the number of culture days exceeded 8 days, the daily growth rate of the biomass of Chlorella pyrenoidosa and the protein content in algal cells began to slow down. Taking into account the culture cycle and breeding costs, the final preferred culture days are 8 days. Beyond this culture period, the growth rate and the growth rate of protein content in algal cells will not continue to increase, indicating that further extending the culture time will not further improve the growth efficiency and protein accumulation.

[0054] 2. Effect of pH on biomass and protein content in Chlorella pyrenoidosa cells

[0055] (1) inoculating Chlorella vulgaris into a culture medium and culturing it for 3 to 4 days to allow it to enter a logarithmic phase;

[0056] (2) adding a certain volume of water to the piggery wastewater to dilute the piggery wastewater 1:1, then adding hydrochloric acid and sodium hydroxide to the piggery wastewater to adjust the pH of the piggery wastewater to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 respectively;

[0057] (3) Inoculate the pyrenoid chlorella in the logarithmic phase in step (1) into the piggery wastewater in step (2) at an inoculation amount of 0.1 OD 660 , which is equivalent to 23.0 mg / L of dry weight of algae;

[0058] (4) Placing the pyrenoid Chlorella inoculated in step (3) in an incubator, culturing for 8 days at a temperature of 25° C., a light intensity of 3000 to 4000 lux, and a light-dark ratio of 12 h:12 h. After the culturing, measuring the biomass of the pyrenoid Chlorella and the protein content in the algae cells.

[0059] The biomass of Chlorella pyrenoidosa Figure 2 As shown in part (3); the protein content in the pyrenoid Chlorella cell is as follows Figure 2 As shown in part (4) of the figure. Figure 2 It can be seen that with the gradual increase of pH value, the biomass of Chlorella pyrenoidosa and the protein content in algal cells both increased first and then decreased. When the pH was 7, the biomass of Chlorella pyrenoidosa reached the highest value; when the pH was 6, the protein content in Chlorella pyrenoidosa reached the highest value.

[0060] 3. Effects of temperature on biomass and protein content in Chlorella pyrenoidosa cells

[0061] (1) inoculating Chlorella vulgaris into a culture medium and culturing it for 3 to 4 days to allow it to enter a logarithmic phase;

[0062] (2) adding a certain volume of water to the pig farm wastewater to dilute the pig farm wastewater at a ratio of 1:1, and then adding hydrochloric acid and sodium hydroxide to the pig farm wastewater to adjust the pH of the pig farm wastewater to 6.5;

[0063] (3) Inoculate the pyrenoid chlorella in the logarithmic phase in step (1) into the piggery wastewater in step (2) at an inoculation amount of 0.1 OD 660 , which is equivalent to 23.0 mg / L of dry weight of algae;

[0064] (4) The pyrenoid chlorella inoculated in step (3) was placed in an incubator and cultured for 8 days at temperatures of 15, 25 and 35° C., light intensity of 3000 to 4000 lux, and a light-dark ratio of 12h:12h. After the culture was completed, the biomass of the pyrenoid chlorella and the protein content in the algal cells were measured.

[0065] The biomass of Chlorella pyrenoidosa Figure 3 As shown in part (5); the protein content in the pyrenoid Chlorella cell is as follows Figure 3 As shown in part (6) of the figure. Figure 3 It can be seen that when the temperature is 25°C, the biomass and protein content of Chlorella pyrenoidosa reach the maximum, which are 217 mg / L and 121 mg / L respectively. Based on the above results, the temperature range for RSM response surface experiment is preferably 25°C.

[0066] 4. Effect of the amount of carbon source added on the biomass and protein content of Chlorella pyrenoidosa (carbon source is glucose)

[0067] (1) inoculating Chlorella vulgaris into a culture medium and culturing it for 3 to 4 days to allow it to enter a logarithmic phase;

[0068] (2) A certain volume of water was added to the pig farm wastewater to dilute the pig farm wastewater 1:1, and then glucose was added to the pig farm wastewater. The addition amounts of glucose were 0, 100, 200, 500, 1000, 5000, 10000 and 50000 mg / L respectively; hydrochloric acid and sodium hydroxide were then added to the pig farm wastewater, and the pH was adjusted to 6.5;

[0069] (3) Inoculate the pyrenoid chlorella in the logarithmic phase in step (1) into the piggery wastewater in step (2) at an inoculation amount of 0.1 OD 660 , which is equivalent to 23.0 mg / L of dry weight of algae;

[0070] (4) Placing the pyrenoid Chlorella inoculated in step (3) in an incubator, culturing for 8 days at a temperature of 25° C., a light intensity of 3000 to 4000 lux, and a light-dark ratio of 12 h:12 h. After the culturing, measuring the biomass of the pyrenoid Chlorella and the protein content in the algae cells.

[0071] The biomass of Chlorella pyrenoidosa Figure 4 As shown in part (7); the protein content in the pyrenoid Chlorella cell is as follows Figure 4 As shown in part (8) of Figure 4 It can be seen that when the carbon source addition amount is 10000 mg / L, the biomass of Chlorella pyrenoidosa reaches a maximum of 585 mg / L; when the carbon source addition amount is 100 mg / L, the protein content in the algae cells reaches a maximum of 215 mg / L. When the carbon source addition amount is greater than 1000 mg / L, the biomass of Chlorella pyrenoidosa almost remains unchanged. Based on the above results, the addition amount of the carbon source is preferably 200-800 mg / L.

[0072] 5. Effect of the amount of phosphorus added on the biomass and protein content of Chlorella pyrenoidosa (phosphorus source is potassium dihydrogen phosphate)

[0073] (1) inoculating Chlorella vulgaris into a culture medium and culturing it for 3 to 4 days to allow it to enter a logarithmic phase;

[0074] (2) A certain volume of water was added to the pig farm wastewater to dilute the pig farm wastewater 1:1, and potassium dihydrogen phosphate was added to the pig farm wastewater. The addition amount of potassium dihydrogen phosphate was 0, 1, 4, 6, 8, 20 and 100 mg / L respectively; then hydrochloric acid and sodium hydroxide were added to the pig farm wastewater, and the pH was adjusted to 6.5;

[0075] (3) Inoculate the pyrenoid chlorella in the logarithmic phase in step (1) into the piggery wastewater in step (2) at an inoculation amount of 0.1 OD 660 , which is equivalent to 23.0 mg / L of dry weight of algae;

[0076] (4) Placing the inoculated Chlorella pyrenoidosa in step (3) in an incubator, culturing for 8 days at a temperature of 25° C., a light intensity of 3000 to 4000 lux, and a light-dark ratio of 12 h:12 h. After the culturing, measuring the biomass of Chlorella pyrenoidosa and the protein content in the algal cells.

[0077] The biomass of Chlorella pyrenoidosa Figure 5 As shown in part (9); the protein content in the pyrenoid Chlorella cell is as follows Figure 5 As shown in part (10) of Figure 5 It can be seen that when the phosphorus source addition amount is 4 mg / L, the biomass and protein content of Chlorella pyrenoidosa both reach the maximum value, which are 388 mg / L and 219 mg / L respectively. Based on the above results, the optimal range of phosphorus source addition in RSM response surface experiment is 0-8 mg / L.

[0078] 6. Effect of initial algal density on the biomass and intracellular protein content of Chlorella pyrenoidosa

[0079] (1) inoculating Chlorella vulgaris into a culture medium and culturing it for 3 to 4 days to allow it to enter a logarithmic phase;

[0080] (2) adding a certain volume of water to the pig farm wastewater to dilute the pig farm wastewater 1:1, then adding hydrochloric acid and sodium hydroxide to the pig farm wastewater and adjusting the pH to 6.5;

[0081] (3) inoculating the pyrenoid globules in the logarithmic phase in step (1) into the pig farm wastewater in step (2); during inoculation, different initial inoculation amounts were set, and the initial algae densities of the pyrenoid globules were 0.1OD 660 、0.3OD 660 , 0.5OD 660 , 0.7OD 660 and 1.0OD 660 These inoculum amounts correspond to dry weight algal concentrations of 23 mg / L, 69 mg / L, 115 mg / L, 161 mg / L and 230 mg / L, respectively.

[0082] (4) The pyrenoid chlorella inoculated in step (3) is placed in an incubator and cultured for 8 days at a temperature of 25° C., a light intensity of 3000 to 4000 lux, and a light-dark ratio of 12 h:12 h. After the culture is completed, the biomass of the pyrenoid chlorella and the protein content in the algae cells are measured.

[0083] The biomass of Chlorella pyrenoidosa Figure 6 As shown in part (11); the protein content in the pyrenoid Chlorella cell is as follows Figure 6 As shown in part (12) of the figure. Figure 6 It can be seen that the biomass and protein content of Chlorella pyrenoidosa increased with the increase of initial algae density. 660 When the biomass and protein content of Chlorella pyrenoidosa reached 419 mg / L and 218 mg / L respectively, the initial algae density was 1.0 OD 660 At OD 200, the biomass and protein content of Chlorella pyrenoidosa were 434 mg / L and 229 mg / L, respectively, with the increments being less than 5%. 660 Continuing to increase the algae density above 0.9 is no longer significant in terms of economic benefits and biomass output, that is, it has no substantial contribution to increasing the biomass of microalgae and the protein content in algae cells. Based on the above results, the initial algae density in the RSM response surface experiment is preferably in the range of 0.1 to 0.9 OD 660 .

[0084] 2. RSM response surface optimization method for Chlorella pyrenoidosa

[0085] Step 1: Based on the results of the single factor experiment, the amount of phosphorus source added (preferably dipotassium hydrogen phosphate) (x1), the amount of carbon source added (preferably glucose) (x2) and the initial algae density (x3) were used as independent variables, and the content of protein in algae cells was used as the response value (y). A three-level three-factor response surface experiment was arranged according to the Box-Behnken design. The values ​​of each factor and level of the response surface experiment are shown in Table 1.

[0086] Table 1 Factors and levels of response surface experiment

[0087]

[0088] Step 2: According to the experimental design in step 1, the microalgae cultivation plan was carried out under the conditions of temperature of 25°C, pH of 6.5, light intensity of 3000-4000 lux, light-dark ratio of 12h:12h and cultivation days of 8 days. The experimental data obtained are shown in Table 2.

[0089] Table 2 Response surface experimental design and experimental results

[0090]

[0091]

[0092] Step 3: Minitab software was used to conduct a detailed analysis of the response values ​​of each experimental point in Table 2, thereby establishing a response model for the protein content in Chlorella cells with phosphorus source addition x1, carbon source addition x2 and initial algae density x3 as independent variables. By performing significance tests on the parameters of the regression equation, the parameters that were not statistically significant were removed, and finally a simplified regression model was obtained; among them, the phosphorus source was dipotassium hydrogen phosphate and the carbon source was glucose.

[0093] The expression of the regression model is as follows:

[0094]

[0095] Where y represents the content of protein in algal cells, and x1 and x2 represent the added amounts of dipotassium hydrogen phosphate and glucose, respectively.

[0096] Step 4: Minitab was used to perform variance analysis on the regression model to determine the influence of each factor on the protein content in Chlorella pyrenoidosa cells, and also to test the validity of the regression equation; the analysis structure is shown in Table 3.

[0097] Table 3 Analysis of variance table of response surface regression equation

[0098]

[0099]

[0100] Note: *: indicates significant difference (P<0.05); **: indicates extremely significant difference (P<0.01).

[0101] As can be seen from Table 3, the regression model is highly significant (P<0.001), the P value of the lack of fit test is 0.21 (>0.05), and the coefficient of determination (R 2 ) is 0.959, indicating that the model has a good fit with the true value and can explain 95.9% of the changes. According to the ratio of the sum of squares of the regression equation (Adj SS) to the sum of squares of the residual (Adj MS), the signal-to-noise ratio of the model is 23.4, which is much greater than 4. These data show that the model has sufficient signal strength and is effective.

[0102] Step 5: Randomly select three groups of optimized process parameters to carry out verification experiments; the verification experimental results are shown in Table 4.

[0103] Table 4 Verification test results after optimizing process parameters

[0104]

[0105] As shown in Table 4, the measured protein content in algae cells is slightly different from the predicted protein content in algae cells, which is basically consistent with the theoretical prediction results, indicating that the optimization of the aquaculture process based on the response surface methodology is accurate and reliable.

[0106] Step 6: According to the principle of maximizing the protein content in pyrenoid algae cells, the experimental data were further analyzed using Minitab software, and the optimal conditions for the protein content in pyrenoid algae cells were obtained as follows: the phosphorus source addition amount was 8 mg / L, the carbon source addition amount was 200 mg / L, and the initial algae density was 0.1 OD 660 , the protein content in the cells of Chlorella pyrenoidosa was 53.0%. At this time, the biomass of Chlorella pyrenoidosa was 658mg / L and the dry weight of protein was 346mg / L. The verification experiment showed that the protein content in the cells of Chlorella pyrenoidosa was 54.1%, which was not much different from the theoretical prediction value, indicating that the model had a good fit. The analysis and prediction of the use of aquaculture wastewater to cultivate microalgae by regression equation was reliable. The process conditions can be used to optimize the use of aquaculture wastewater to cultivate microalgae; and the protein content in microalgae cells was significantly increased.

[0107] The growth of Chlorella pyrenoidosa in each experimental group in step 2 is as follows: Figure 7 As shown in part (13), the biomass of Chlorella pyrenoidosa is Figure 7 As shown in part (14) of the figure. (Since the conditions of experimental groups 13 to 17 in step 2 are the same, the growth conditions and protein content of these groups of protein core Chlorella are the same, so Figure 7 Only the experimental results of the 1st to 13th groups of experiments are shown. )

[0108] In order to intuitively show the effect of the interaction between various factors on the protein content in algae cells, response surface diagrams and contour diagrams were drawn based on the fitted regression equation. Figure 8 As shown in part (15); the contour lines of the effect of phosphorus source addition and carbon source addition on protein content are shown in Figure 8 As shown in part (16); the response surface diagram of the effect of phosphorus source addition and initial algae density on protein content is shown in Fig. 9 As shown in part (17), the contour lines of the effect of phosphorus source addition and initial algae density on protein content are as follows Fig. 9 As shown in part (18); the response surface diagram of the effect of carbon source addition and initial algae density on protein content is shown in Fig.10 As shown in part (19), the contour lines of the effect of carbon source addition and initial algae density on protein content are as follows Fig.10 As shown in part (20).

[0109] 3. After the experiment, the pig wastewater in each group of experiments in Table 2 was tested

[0110] The removal efficiency of waste in piggery wastewater is as follows Fig.11 As shown (because the conditions of experimental groups 13-17 in step 2 are the same, Fig.11 Only the removal efficiency of waste in pig wastewater from the 1st to 13th group of experiments is shown. ): Part (21) in the figure shows the NH4 + The content of NH4 + The removal rate of PO4 in the wastewater after the aquaculture is completed. 3- The content of PO4 3- The removal rate of TOC; Part (25) in the figure is the TOC content in the wastewater after the aquaculture is completed; Part (26) in the figure is the TOC removal rate.

[0111] Depend on Fig.11 It can be seen that the remaining pollutants in the wastewater after cultivation are NH4 + PO4 3- The residual amounts of TOC are 102-150 mg / L, 0.08-3.11 mg / L and 37.3-350 mg / L respectively.

Claims

1. A method for culturing microalgae, characterized in that: The following steps are involved: (1) After diluting the aquaculture wastewater, a carbon source and a phosphorus source are added, wherein the amount of the carbon source added is 0 to 5000 mg / L; the amount of the phosphorus source added is 0 to 100 mg / L; and then the pH of the wastewater is adjusted; (2) Inoculating microalgae in the aquaculture wastewater in step (1) for cultivation; the initial algae density of the microalgae is 0.05 to 1.0 OD 660 .

2. A method for culturing microalgae according to claim 1, characterized in that: The carbon source added in step (1) is glucose, and the added amount is 200-800 mg / L; the phosphorus source added is dipotassium hydrogen phosphate, and the added amount is 0-8 mg / L; In step (2), the initial algae density of the microalgae is 0.1 to 0.9 OD 660 .

3. The method for culturing microalgae according to claim 1, characterized in that: The culture temperature is 15-35°C, the light intensity during the culture process is 3000-40001ux, and the light-dark ratio is 12h:12h.

4. The method for culturing microalgae according to claim 1, characterized in that: In step (1), the aquaculture wastewater is pig farming wastewater, the dilution multiple is 0 to 10 times, and the pH is 4.0 to 8.

0.

5. The method for culturing microalgae according to claim 1, characterized in that: In step (2), the microalgae is a Chlorophyta alga, and the Chlorophyta alga is one of Chlorella pyrenoidosa, Chlorella vulgaris and Scenedesmus.

6. The method for culturing microalgae according to claim 1, characterized in that: In step (2), the culture period is 3 to 14 days.

7. A method for optimizing microalgae culture conditions using response surface methodology, characterized in that: Used to optimize the method for culturing microalgae according to any one of claims 1 to 6.

8. The method for optimizing microalgae culture conditions by response surface methodology according to claim 7, characterized in that: The phosphorus source addition, carbon source addition and initial algae density were selected as independent variables, and the protein content in algae cells was selected as the response value. The Box-Behnken design was used to arrange the experiment and collect experimental data. The following RSM regression equation was established: Among them, x1 is the amount of carbon source added, x2 is the amount of carbon source added, x3 is the initial algae density, and y is the content of protein in algae cells; b0 is the intercept term, b1, b2, b3, b 12 , b 13 , b 23 , b 11 , b 22 and b 33 is the regression coefficient.

9. Use of the method for culturing microalgae according to any one of claims 1 to 6 in wastewater purification.

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