Chlorella and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to chlorella and application thereof. The chlorella provided by the invention is separated from Tengchong Shuishui River in Yunnan, and is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M 20242807. The chlorella LBV13 disclosed by the invention can grow in original culture sewage and has efficient purification capacity on the culture sewage, and detection shows that the highest removal rates of COD (Chemical Oxygen Demand), TN (Total Nitrogen), NH4 < + >-N and TP (Total Phosphorus) of the chlorella for the culture sewage are 48.28 percent, 91.73 percent, 98.15 percent and 88.99 percent respectively; therefore, the chlorella disclosed by the invention can effectively remove nitrogen, phosphorus and ammonia nitrogen in the sewage, so that the purpose of purifying the sewage is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to chlorella and application thereof. Background Art

[0002] With the development of large-scale and intensive aquaculture, the amount of sewage discharged has increased rapidly. Especially in areas with high aquaculture density, such as rural areas around farms, due to the lack of effective sewage treatment facilities, untreated sewage is often discharged directly into rivers, lakes or groundwater. Aquaculture sewage contains a large amount of nitrogen, phosphorus and organic matter, which leads to the rapid deterioration of the water quality of surrounding water bodies and causes a series of environmental problems.

[0003] The advantages of using microalgae to treat aquaculture wastewater are multifaceted, including efficient removal of nitrogen and phosphorus, resource recovery, low energy consumption, and environmental friendliness. Microalgae have unique advantages, especially in dealing with nitrogen and phosphorus pollution. Microalgae can not only effectively remove nitrogen and phosphorus from water, but also use nutrients in sewage to grow and further convert them into high-value biomass, which can be used as organic fertilizer, biofuel, etc. However, the main problems in the current application of microalgae in sewage treatment are poor tolerance to high-concentration sewage, limited removal of pollutants, and difficulty in collecting microalgae.

[0004] Some Chlorella species have a higher tolerance to complex environmental conditions (such as high salt, high ammonia nitrogen, heavy metal ions, etc.), can resist changes in the external environment and maintain their own growth. They are also rich in nutrients, with high protein content, rich lipids and essential fatty acids, vitamins and functional substances. They have been included in the "Feed Raw Materials Catalogue" and can be used as an important raw material in the feed field.

[0005] Immobilized microalgae form a higher cell concentration through the carrier, which provides a stable environment for the microalgae to attach, helps to adsorb and metabolize nitrogen and phosphorus in sewage, and can treat more sewage per unit volume, with a higher pollutant removal rate. In addition, immobilized microalgae are attached to the carrier, which is easy to separate and collect from sewage and can be recycled, which improves the controllability of the operation and reduces the operating cost. Summary of the invention

[0006] The purpose of the present invention is to provide a kind of Chlorella and application thereof.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a kind of Chlorella, wherein two nucleic acid sequences of the Chlorella contain the sequence shown in Sequence 1 in the sequence table.

[0009] According to the above-mentioned Chlorella, preferably, the Chlorella is Chlorella sorokiniana LBV13, the preservation number of Chlorella LBV13 is CCTCCNO:M 20242807, and it is preserved in China Center for Type Culture Collection (CCTCC), and the address of the preservation institution is China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is December 12, 2024.

[0010] According to the above-mentioned Chlorella, preferably, the Chlorella is morphologically green and spherical, has a cell diameter of about 1-8 μm under normal growth conditions, grows as a single cell, and has a 18s rDNA sequence similarity of 99.34% with that of Chlorella sorokiniana, indicating the closest relationship.

[0011] The above-mentioned separation and purification method of Chlorella is as follows: collect water samples from Dieshuhe Waterfall in Tengchong City, Yunnan Province, transfer the collected water samples to centrifuge tubes, centrifuge at 8000 rpm for 10 minutes at 4°C to precipitate microalgae, then carefully remove the supernatant and retain the precipitate; add an appropriate amount of BG11 culture medium to the precipitate, stir well to obtain a concentrated solution; press the concentrated solution for 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The gradient was diluted with sterile water to obtain gradient dilutions. 100 μL of each gradient dilution was spread on the surface of solid BG11 medium containing 5% agar powder and placed under light culture conditions until a single algae colony appeared. Finally, a single algae colony was selected according to its morphology, size and color, and streaked on solid BG11 medium for purification culture.

[0012] The second aspect of the present invention provides a seed culture medium for culturing the Chlorella described in the first aspect, wherein the seed culture medium is obtained by diluting the following solutes to 1 liter with water: straw hydrolyzate, NaNO3 1.5g, K2HPO4·3H2O 0.012g, MgSO4·7H2O 0.075g, CaCl2·2H2O 0.036g, citric acid 0.006g, ammonium ferric citrate 0.006g, EDTA 0.001 g, Na2CO3 0.02 g, H3BO4 0.00286g, MnCl2·H2O 0.00181g, ZnSO4·7H2O 0.000222g, CuSO4·5H2O 0.000079g, Na2MoO4·2H2O 0.00039g, Co(NO3)2·6H2O 0.000049g; wherein, the amount of the straw hydrolyzate in the seed culture medium is determined by the glucose content in the straw hydrolyzate, and the glucose content in the seed culture medium is 2-5g / L.

[0013] According to the above-mentioned seed culture medium, preferably, the seed culture medium is obtained by diluting the following solutes to 1 liter with water: straw hydrolyzate, NaNO3 1.5g, K2HPO4·3H2O 0.012g, MgSO4·7H2O 0.075g, CaCl2·2H2O0.036g, citric acid 0.006g, ammonium ferric citrate 0.006g, EDTA 0.001 g, Na2CO3 0.02 g, H3BO4 0.00286g, MnCl2·H2O 0.00181g, ZnSO4·7H2O 0.000222g, CuSO4·5H2O 0.000079g, Na2MoO4·2H2O 0.00039g, Co(NO3)2·6H2O 0.000049g; wherein, the amount of the straw hydrolyzate in the seed culture medium is determined by the glucose content in the straw hydrolyzate, and the glucose content in the seed culture medium is 5g / L.

[0014] According to the above-mentioned seed culture medium, preferably, the pH of the seed culture medium is 6.5-7.5.

[0015] According to the above-mentioned seed culture medium, preferably, the glucose content in the straw hydrolyzate is 60 g / L to 70 g / L.

[0016] According to the above seed culture medium, preferably, the method for preparing the straw hydrolyzate is: pre-treating the straw with alkali solution, washing the pre-treated straw, removing the residual alkali solution, and then enzymolyzing the pre-treated straw with cellulase to obtain the straw hydrolyzate. More preferably, the method for preparing the straw hydrolyzate is: mixing the straw with alkali solution, reacting at 65°C to 90°C for 1h to 4h, separating the solid and the liquid, and obtaining the pre-treated straw; washing the pre-treated straw with water, removing the residual alkali solution, and drying the washed pre-treated straw, adding the dried pre-treated straw to a buffer containing cellulase, and reacting at 40°C to 60°C for 24h to 96h to obtain the corn straw hydrolyzate. Furthermore, the alkali solution is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 2% (w / v); during the pretreatment, the mass ratio of the straw to the alkali solution is 1:20; the buffer solution is a HAc-NaAc buffer solution, and the concentration of the HAc-NaAc buffer solution is 0.2 mol / L.

[0017] According to the above-mentioned seed culture medium, preferably, the straw hydrolyzate is corn straw hydrolyzate.

[0018] The third aspect of the present invention provides a method for preparing Chlorella seed liquid, the method comprising: culturing Chlorella in the seed culture medium of the second aspect to obtain the seed liquid.

[0019] According to the above method for preparing Chlorella seed liquid, preferably, the Chlorella is the Chlorella described in the above first aspect.

[0020] According to the above method for preparing Chlorella seed solution, preferably, the temperature for culturing Chlorella is 25° C. to 28° C., and the pH condition for culturing Chlorella is pH 6.5 to 7.5.

[0021] According to the above method for preparing Chlorella seed solution, preferably, the light intensity for culturing Chlorella is 6000 Lux to 10000 Lux, the humidity is 50% to 75%, the light-dark ratio is 12h:12h, and ventilation is required during the cultivation process, and the ventilation rate is 0.5 to 1.5 L / min.

[0022] The fourth aspect of the present invention provides the use of the Chlorella described in the first aspect in sewage treatment.

[0023] According to the above application, preferably, the sewage treatment is sewage nitrogen removal, sewage ammonia nitrogen removal and / or sewage phosphorus removal.

[0024] According to the above application, preferably, the sewage is aquaculture sewage.

[0025] A fifth aspect of the present invention provides a sewage treatment method, comprising: inoculating the Chlorella described in the first aspect into sewage for culturing.

[0026] According to the above-mentioned sewage treatment method, preferably, before inoculating Chlorella into sewage for cultivation, Chlorella is first inoculated into the seed culture medium described in the above-mentioned second aspect for pre-culture, and then the pre-cultured Chlorella is inoculated into sewage for cultivation.

[0027] According to the above sewage treatment method, preferably, the pre-culture time is 3 days to 9 days; and the pre-culture temperature is 25° C. to 28° C.

[0028] According to the above sewage treatment method, preferably, the sewage contains a Chlorella immobilized carrier; after the Chlorella is inoculated into the sewage, the Chlorella cells can be adsorbed and grown on the surface of the immobilized carrier material to form a biofilm. More preferably, the immobilized carrier is a sheet-like porous carrier, and most preferably, the immobilized carrier is a sheet-like corn cob.

[0029] According to the above sewage treatment method, preferably, the content of the immobilized carrier in the sewage is 0.5% (w / v).

[0030] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0031] (1) The Chlorella LBV13 of the present invention can grow in raw aquaculture wastewater and has a high efficiency in purifying aquaculture wastewater. The results show that Chlorella sorokiniana has a high efficiency in purifying COD, TN, and NH4 in aquaculture wastewater. + The highest removal rates of -N and TP are 48.28%, 91.73%, 98.15% and 88.99% respectively; therefore, the Chlorella of the present invention can effectively remove nitrogen, phosphorus and ammonia nitrogen in sewage, thereby achieving the purpose of sewage purification.

[0032] (2) The Chlorella vulgaris LBV13 of the present invention can be adsorbed and immobilized on the surface of a porous carrier. After being immobilized, the maximum removal rates of TN and TP in sewage by the algae strain are increased by 4.99% and 3% respectively compared with the free algae strain.

[0033] (3) The Chlorella screened by the present invention can be cultured heterotrophically with corncob hydrolyzate as a carbon source. Moreover, the present invention also studies a phosphorus-starved seed culture medium for culturing Chlorella, wherein the seed culture medium contains straw hydrolyzate and has a low phosphorus content. The use of the phosphorus-starved seed culture medium to culture Chlorella is helpful to improve the concentration of microalgae cells and the phosphorus absorption capacity of the microalgae cells. It was found through testing that after the Chlorella of the present invention was cultured in the phosphorus-starved seed culture medium supplemented with corncob hydrolyzate for 7 days, it was inoculated in aquaculture wastewater for fixed culture. The removal rate of the total phosphorus in the aquaculture wastewater by the Chlorella reached 54.39% on the first day, and the total phosphorus was completely removed on the ninth day, with a removal rate of 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The photos of the collected water sample (A), the streaking photos (B) and the morphological characteristics of Chlorella sorokiniana LBV13 under a 100X optical microscope (C);

[0035] Figure 2 Phylogenetic tree constructed for the 18s rDNA partial sequence of Chlorella vulgaris LBV13;

[0036] Figure 3 This is the growth curve of Chlorella vulgaris LBV13 in pig farm wastewater;

[0037] Figure 4 The removal effect of Chlorella LBV13 on raw aquaculture wastewater: (A) COD, (B) TP, (C) NH4 + -N, TN;

[0038] Figure 5 This is the effect picture of immobilized microalgae on corncob slices;

[0039] Figure 6 The results are the test results of the removal efficiency of aquaculture wastewater by immobilized microalgae and suspended microalgae;

[0040] Figure 7 This is the growth curve of the phosphorus starvation culture of Chlorella vulgaris LBV13; where Autotrophy is the autotrophic control group, Heterotrophy is the heterotrophic control group, and P- is the phosphorus starvation group;

[0041] Figure 8 The chlorophyll fluorescence curve Fv / Fm of the phosphorus starvation culture of Chlorella vulgaris LBV13; Autotrophy is the autotrophic control group, Heterotrophy is the heterotrophic control group, and P- is the phosphorus starvation group;

[0042] Fig. 9 The growth curve of Chlorella LBV13 cultured in aquaculture wastewater after phosphorus starvation; Autotrophy is the autotrophic control group, Heterotrophy is the heterotrophic control group, and P- is the phosphorus starvation group;

[0043] Fig.10 The chlorophyll fluorescence curve Fv / Fm of Chlorella vulgaris LBV13 cultured in aquaculture wastewater after phosphorus starvation; Autotrophy is the autotrophic control group, Heterotrophy is the heterotrophic control group, and P- is the phosphorus starvation group;

[0044] Fig.11The removal effect of Chlorella LBV13 after phosphorus starvation culture on aquaculture wastewater: (A) total nitrogen; (B) total phosphorus;

[0045] Fig.12 The distribution diagram of the physical and chemical properties of Chlorella LBV13 cultured in different culture media and wastewater: (A) protein, (B) lipid, (C) carbohydrate, (D) lutein. DETAILED DESCRIPTION

[0046] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0047] Example 1: Isolation, purification and identification of Chlorella

[0048] Collect water samples from Dieshuhe Waterfall in Tengchong City, Yunnan Province (water sample photos as shown in Figure 1 The collected water sample was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 min at 4°C to precipitate the microalgae. The supernatant was carefully removed, and the precipitate was retained. An appropriate amount of BG11 medium was added and stirred thoroughly to obtain a concentrate. The concentrate was then heated to 10 °C. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The gradient was diluted with sterile water to obtain gradient dilutions. Take 100 μL of each gradient dilution and spread it on the surface of solid BG11 medium containing 5% agar powder, and place it under light culture conditions until a single algae colony appears. Select a single algae colony according to its morphology, size and color, and streak it on solid BG11 medium for purification (see the picture of plate streak purification for details). Figure 1 The results are shown in Figure 2(B) and observed by fluorescence inverted microscope. Figure 1 As shown in C. Figure 1 As shown in Figure C, the microalgae are green and spherical in shape. Under normal growth conditions, the cell diameter is about 1-8 μm and they grow as a single cell.

[0049] Pick a single algae colony and add it to a PCR tube with 20μL sterile water. Repeatedly blow and beat to disperse the algae colony. It can be observed that the sterile water is slightly green, and the solution is uniform without large algae colonies. Then place the PCR tube in a metal bath and heat it at 99℃ for 20min, then cool it in a refrigerator at 4℃ to obtain a DNA template for PCR amplification. Among them, the reaction system for PCR amplification is: 1.4μL 18s-F primer, 1.4μL 18s-R primer, 1.4μL DNA template, 25μL 2×taq enzyme, and add sterile water to 35μL; among them, the sequence of 18s-F is CCAACCTGGTTGATCCTGCCAGTA, and the sequence of 18s-R primer is CCTTGTTACGACTTCACCTTCCTCT. The PCR amplification reaction conditions are: 95℃5min; 95℃30s, 55℃30s, 72℃1.5min, a total of 35 cycles; 72℃ extension for 10min. The PCR amplification product was electrophoresed using 1% agarose gel, and the positive band product with clear and no mixed bands was sent to the company for Sanger sequencing to obtain the 18s rDNA sequencing results. The nucleotide sequence of 18s rDNA is shown in Sequence 1 in the sequence table.

[0050] Sequence 1 is specifically:

[0051] GAGAGCGGAGACCGCCCCCGGTAGCCAATCCGAACACTTCACCAGCACACCCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCAAGCTGATGACTTGCGCTTACTAGGCATTCCTCGTTGAAGATTAATAATTGCAATAATCTATCCCCATCACGATGCAGTTTCAAAGATTACCCGGGCCTCTCGGCCAAGGCTAGGCTCGTTGAATGCATCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCATGCTTCCATTGGCTAGTCGCCAATAGTCCCTCTAAGAAGTCCGCCGGCTGGCGAACCAACCGTGACTATTTAGCAGGCTGAGGTCTCGTTCGTTACCGGAATCAACCTGACAAGGCAACCCACCAACTAAGAACGGCCATGCACCACCACCCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCTCACTATGTCTGGACCTGGTAAGTTTTCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACGCCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCGGAACCCAAAAACTTTGATTTCTCATAAGGTGCCGGCGGAGTCATCGAAGAAACATCCGCCGAT-CCCTAGTCGGCATCGTTTATGGTTGAGACTAGGACGGTATCTAATCGTCTTCGAGCCCCCAACTTTCGTTCTTGATTAATGAAAACATCCTTGGCAAATGCTTTCGCAGTAGTTCGTCTTTCATAAATCCAAGAAATTTCACCTCTGACAATGAAATACGAATGCCCCCGACTGTCCCTTCTTAATTCATTACTCCGGTCCTACAGACCCAACAGGATTAGCCAGAGTCCTATCGTGTTATTCCATGCTAATGTATTCAGAGCGTAGGCCTGCTTTGAACACTCTAAATTTACTCAAAGTAACAGCGCCGACTCCGAGTCCCGGACAGTGAAGCCCAGGAGCCCGTCCCCGGCAACAAGGTGGGCCCTGCCAGTGCACACCGAAGCGGCGGACCGGCAGGCCCCACCCGAAATCCAACTACGAGCTTTTTAACTGCAGCAACTTAAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGATCCTCGTTAAGGGGTTTAGATTGTACTCATTCCAATTACCAGACCTGAAAAGGCCCAGTATTGTTATTTATTGTCACTACCTCCCTGTGTCAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATCCTCCGTCACCCGTTACCACCATGGTAGGCCTCTATCCTACCATCGAAAGTTGATAGGGCAGAAATTTGAATGAAACATCGCCGGCACAAGGCCATGCGATTCGTGAAGTTATCATGATTCACCGCGAGTCGGGCAAGCCCGGTCGGTCTTTTATCTAATAAATACGTCCCTTCCAGAAGTCGGGATTTACGCACGTATTAGCTCTAGATTTACTACGGGTATCCGAGTAGTAGGTACCATCAAATAAACTATAACTGATTTAATGAGCCATTCGCAGTTTCACAGTATAAAGCAGTTTATACTTAGACATGCATGGCTAATCT。

[0052] The above 18s rDNA sequence was compared with the existing homologous sequences by BLAST analysis in the NCBI database, and a phylogenetic tree was constructed. The results are as follows Figure 2 As shown. Figure 2 It can be seen that the sequence similarity between the screened microalgae and Chlorella sorokini reached 99.34%, and the relationship was the closest; further combined with the results of morphological observation, the screened microalgae were identified as belonging to the genus Chlorella, named Chlorella sorokiniana LBV13, and preserved in the China Center for Type Culture Collection (CCTCC), with the preservation number CCTCC NO:M 20242807, and the address of the preservation institution is China Type Culture Collection Center of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is December 12, 2024.

[0053] Example 2: Adaptive cultivation of Chlorella LBV13 and study on its effect on pollutant removal in pig farm wastewater

[0054] The pig farm aquaculture sewage was used to carry out adaptive culture of Chlorella LBV13, and the specific method was as follows: 100 mL of Chlorella LBV13 in the exponential growth period was collected by centrifugation, washed with sterile deionized water, added into 300 mL of aquaculture sewage with a dilution ratio of 20% (the aquaculture sewage with a dilution ratio of 20% is a mixture of aquaculture sewage stock solution and water at a volume ratio of 1:4), and air was introduced for acclimation and culture for 7 days; after 7 days, the cultured Chlorella sewage mixture was stirred evenly, 100 mL was taken and added into 300 mL of aquaculture sewage with a dilution ratio of 40%, and air was introduced for acclimation and culture for 7 days; the above steps were repeated to add the Chlorella sewage mixture into aquaculture sewage with dilution ratios of 60%, 80%, and 100% in sequence for acclimation and culture for 7 days. Among them, the aquaculture wastewater is from a pig farm in Zhengzhou. The COD, ammonia nitrogen, total nitrogen and total phosphorus contents in the raw liquid of aquaculture wastewater are 1516.8 mg / L, 634.2 mg / L, 646.6 mg / L and 14.5 mg / L respectively.

[0055] 200 ml of Chlorella after the above adaptive culture was added into a flat-plate photoreactor filled with 2 L of pig farm aquaculture wastewater for cultivation. The growth of Chlorella in the aquaculture wastewater was observed during the cultivation process. At the same time, the COD, total nitrogen (TN), and ammonia nitrogen (NH4 +-N) and total phosphorus (TP); according to national standards (HJ828-2017, HJ 535-2009, HJ 636-2012, GB 11893-89), COD determination method is potassium dichromate method, ammonia nitrogen determination method is Nessler reagent spectrophotometry, total nitrogen determination method is potassium persulfate oxidation ultraviolet spectrophotometry, and total phosphorus determination method is ammonium molybdate spectrophotometry. Among them, the flat-plate photobioreactor has a length of 0.3m, a width of 0.2m, and a height of 0.1m. Air is introduced to stir the algae liquid. The culture conditions are light intensity 8000Lux, temperature 28±1℃, humidity 50% and light-dark ratio of 12h:12h.

[0056] The growth test results of Chlorella in pig farm wastewater are as follows: Figure 3 shown.

[0057] Depend on Figure 3 It can be seen that within the first day, the biomass of Chlorella showed an obvious downward trend, within 7 days, the biomass of Chlorella increased from 0.43g / L to 1.15g / L, and entered a stable period on the 7th day, eventually reaching 1.25g / L.

[0058] The results of the changes in the contents of COD, total nitrogen (TN), ammonia nitrogen and total phosphorus in the aquaculture wastewater during the cultivation of Chlorella are as follows: Figure 4 shown.

[0059] Depend on Figure 4 It can be seen that the COD in the aquaculture sewage raw liquid showed a wave-like downward trend, and the highest COD removal rate reached 48.28%. The reason is that on the one hand, the microalgae itself uses the organic matter in the biogas slurry, which will cause the COD to decrease. On the other hand, the microalgae's own metabolism will also secrete some organic matter into the biogas slurry, causing the COD to increase. + -N) content decreased by nearly 80% within 3 days, total nitrogen (TN) content decreased by 91.73% within 12 days, and ammonia nitrogen (NH4 + -N) content decreased by 98.15%; the total phosphorus (TP) content in the aquaculture wastewater raw liquid decreased by 88.99% within 9 days.

[0060] Example 3: Treatment of aquaculture wastewater by immobilized Chlorella

[0061] The culture fluid of Chlorella LBV13 in the exponential growth phase was collected by centrifugation and washed with sterile deionized water (Chlorella LBV13 was subcultured in BG11 medium), and then inoculated into aquaculture wastewater with and without immobilized carriers for cultivation, and the treatment effects of immobilized Chlorella and suspended Chlorella on aquaculture wastewater were compared.

[0062] The specific experimental method is:

[0063] The experiment set up an immobilization group and a suspension group. The treatment method of the immobilization group was as follows: 500 ml of aquaculture sewage with a dilution ratio of 20% was added to a 1L conical flask (the aquaculture sewage with a dilution ratio of 20% was a mixture of aquaculture sewage stock solution and water at a volume ratio of 1:4; the aquaculture sewage stock solution was the same as that in Example 2), and an immobilization carrier was added, the immobilization carrier was a sheet corn cob (that is, the corn cob was cut into round pieces with a diameter of 3 cm and a thickness of 3 mm, and the immobilization carrier was suspended in the aquaculture sewage). The content of the carrier is 0.5% (w / v), and then the LBV13 culture solution of Chlorella in the logarithmic growth phase is inoculated into the conical flask; the treatment method of the suspension group is: add 500ml of aquaculture wastewater with a dilution ratio of 20% to a 1L conical flask (the aquaculture wastewater with a dilution ratio of 20% is to mix the aquaculture wastewater stock solution with water at a volume ratio of 1:4; the aquaculture wastewater stock solution is the same as that in Example 2), and then the LBV13 culture solution of Chlorella in the logarithmic growth phase is inoculated into the conical flask, and the inoculation amount is the same as that of the immobilized group. The Chlorella in the immobilized group and the suspension group were cultured for 9 days, and the culture conditions were: temperature 25°C, light intensity 8000Lux, humidity 50% and light-dark ratio of 12h:12h. After 9d of culture, the remaining total nitrogen and total phosphorus contents in the aquaculture wastewater were measured, and the determination method was the same as that in Example 2.

[0064] After 9 days of cultivation, the immobilization effect of Chlorella in the immobilization group is shown in the figure Figure 5 As shown. Figure 5 It can be seen that during the cultivation process, Chlorella LBV13 will adhere to the surface of the corn cob flakes to form a biofilm, indicating that Chlorella LBV13 is successfully immobilized.

[0065] After 9 days of cultivation, the removal rates of total nitrogen and total phosphorus in the aquaculture wastewater of the immobilized group and the suspended group were as follows: Figure 6 shown.

[0066] Depend on Figure 6 It can be seen that on the 9th day of Chlorella treatment of sewage, the removal rate of total nitrogen in aquaculture sewage by immobilized group Chlorella reached 71.31%, while that of suspended group Chlorella was only 67.92%; the highest removal rate of total phosphorus in aquaculture sewage by immobilized group Chlorella was 95.28%, while that of suspended group Chlorella was only 92.5%; the removal rates of nitrogen and phosphorus in aquaculture sewage by immobilized Chlorella were higher than those of suspended group Chlorella, and, compared with the suspended group, the maximum removal rates of TN and TP in aquaculture sewage by fixed group Chlorella were increased by 4.99% and 3%, respectively.

[0067] Example 4: Preparation of phosphorus starvation culture medium and phosphorus starvation pre-culture of microalgae

[0068] Chlorella LBV13 was pre-cultured in phosphorus starvation medium to analyze the effect of phosphorus starvation culture on the growth of Chlorella LBV13.

[0069] The specific experimental method is:

[0070] Take the culture medium of Chlorella LBV13 in the exponential growth phase (Chlorella LBV13 was subcultured in BG11 medium) (OD 680 =5) 100ml, transferred to a centrifuge tube, centrifuged at 4°C and 8000rpm for 10min to precipitate the microalgae, discarded the supernatant, retained the precipitate, and resuspended it in sterile water to remove the culture medium remaining on the surface of the microalgae to prevent a small amount of nitrogen and phosphorus from affecting the experiment. The above steps were repeated 3 times, and the microalgae precipitate obtained from the last centrifugation was inoculated into phosphorus starvation medium (recorded as phosphorus starvation group), BG11 medium (recorded as autotrophic control group), and heterotrophic BG11 medium (recorded as heterotrophic control group) respectively, so that the initial OD of the above three culture media was 2.5447 W / cm2. 680 The temperature and humidity of the culture medium were 1, and the culture was carried out for 7 days; wherein the culture conditions were: light intensity of 8000 Lux, humidity of 50%, light-dark ratio of 12h:12h, air was introduced, and the ventilation rate was 1L / min. During the culture process, the growth of Chlorella and the change of chlorophyll fluorescence curve were measured every day.

[0071] Among them, the formula of phosphorus starvation medium is: straw hydrolyzate, NaNO31.5g, K2HPO4·3H2O 0.012g, MgSO4·7H2O 0.075g, CaCl2·2H2O 0.036g, citric acid 0.006g, ammonium ferric citrate 0.006g, EDTA0.001g, Na2CO30.02 g, H3BO40.00286g, MnCl2·H2O 0.00181g, ZnSO4·7H2O 0.000222g, CuSO4·5H2O0.000079g, Na2MoO4·2H2O 0.00039g, Co(NO3)2·6H2O 0.000049g, the above solute is diluted to 1 liter with water to obtain a phosphorus starvation medium; the amount of straw hydrolyzate in the phosphorus starvation medium is determined by the glucose content in the straw hydrolyzate, and the glucose content in the phosphorus starvation medium is 5g / L; the pH of the phosphorus starvation medium is 7.1±0.1.

[0072] The formula of heterotrophic BG11 medium is: straw hydrolyzate, NaNO3 1.5 g, K2HPO4·3H2O 0.4 g, MgSO4·7H2O 0.075 g, CaCl2·2H2O 0.036 g, citric acid 0.006 g, ammonium ferric citrate 0.006 g, EDTA 0.001 g, Na2CO3 0.02 g, H3BO4 0.00286 g, MnCl2·H2O 0.00181 g, ZnSO4·7H2O 0.000222 g, CuSO4·5H2O 0.000079 g, Na2MoO4·2H2O 0.00039 g, Co(NO3)2·6H2O 0.000049g, the above solute is diluted to 1 liter with water to obtain heterotrophic BG11 medium; the amount of straw hydrolyzate in the heterotrophic BG11 medium is determined by the glucose content in the straw hydrolyzate, the glucose content in the heterotrophic BG11 medium is 5g / L, and the pH of the heterotrophic BG11 medium is 7.1±0.1.

[0073] The straw hydrolyzate is a corn straw hydrolyzate, and the preparation method of the corn straw hydrolyzate is as follows: weigh 100 g of crushed corn straw into a four-necked flask, add 2% (w / v) NaOH solution (1960 mL of water, 40 g of NaOH) to make the solid-liquid ratio 1:20, and place it in an 80° C. water bath to react for 2 hours; after the pretreatment, use a filter to separate the solid and liquid of the mixture, use 20% acetic acid solution to adjust the pH to neutral, wash with deionized water 2-3 times, and place it in a 60° C. oven to dry to constant weight. 3g of pretreated straw was added, and then an appropriate amount of cellulase (enzyme activity 151.494FPU / mL) and 0.2mol / LHAc-NaAc buffer were added to make the total volume of the reaction system 30mL, and the enzyme was shaken in a constant temperature shaker at 50°C and 200rpm for 72h, and then 2% columnar activated carbon was added thereto for treatment for 2d, filtered, and corn straw hydrolyzate was obtained. The corn straw hydrolyzate was sterilized at 115°C for 15min and then frozen at -20°C for standby use. After testing, the glucose content in the sterilized corn straw hydrolyzate was 69.5g / L.

[0074] During the cultivation process, the method for measuring the growth of Chlorella was to take 1 ml of algae liquid every day and measure its OD value at a wavelength of 680 nm using an ultraviolet spectrophotometer. The chlorophyll fluorescence curve was measured using a portable chlorophyll fluorescence analyzer.

[0075] The test results of Chlorella growth are as follows Figure 7 As shown. Figure 7 It can be seen that the OD of Chlorella in the autotrophic control group was 680 The OD of Chlorella vulgaris in the phosphorus starvation group was 4.152. 680The biomass of Chlorella in the phosphorus starvation group increased by about 1.64 times compared with the autotrophic control group, while the OD of Chlorella in the heterotrophic control group increased by about 1.64 times. 680 The highest value is 13.705.

[0076] The chlorophyll fluorescence curve test results of Chlorella vulgaris are as follows Figure 8 As shown. Figure 8 It can be seen that compared with the autotrophic control group and the heterotrophic control group, the Fv / Fm of Chlorella in the phosphorus starvation group was significantly decreased, which shows that the growth of Chlorella LBV13 was stressed in the phosphorus starvation culture medium.

[0077] Example 5: Study on treatment of aquaculture wastewater by immobilization of Chlorella under phosphorus starvation stress

[0078] The chlorella in the phosphorus starvation group, the autotrophic control group, and the heterotrophic control group in the above Example 4 after pre-culture for 7 days were transferred to centrifuge tubes respectively, and the centrifugation conditions were set to a speed of 8000 rpm, and the centrifugation was performed for 5 minutes to precipitate the chlorella. The supernatant was discarded to obtain a chlorella precipitate, and the chlorella precipitate was resuspended in sterile water to remove the culture medium remaining on the surface of the chlorella. The above steps were repeated 3 times, and the chlorella precipitate obtained by the last centrifugation was inoculated into a flat-plate photobioreactor containing suspended flaky corn cobs (that is, the corn cobs were cut into round pieces with a diameter of 3 cm and a thickness of 3 mm, and the content of flaky corn cobs in the aquaculture wastewater was 0.5% (w / v) and a dilution ratio of 20% aquaculture wastewater (the aquaculture wastewater with a dilution ratio of 20% is to mix the aquaculture wastewater stock solution with water at a volume ratio of 1:4; the aquaculture wastewater stock solution is the same as that in Example 2) so that the initial inoculum amount OD 680 The flat-plate photobioreactor has the specifications of 0.3m in length, 0.2m in width, 0.2m in height, 1L in liquid volume, and the culture conditions are 25℃, 15000Lux in light intensity, 50% in humidity, and 12h:12h in light-dark ratio.

[0079] During the culture process, the growth of each group of Chlorella and the total nitrogen and total phosphorus contents in the aquaculture wastewater were measured every day. The method for measuring the biomass of each group of Chlorella is as follows: the corn cobs loaded with Chlorella are subjected to ultrasonic treatment at 40KHz for 5 minutes in an ultrasonic bath, the carrier is separated after filtration with filter paper, the suspended cells are harvested by centrifugation at 8000rpm for 5 minutes, the cells are rinsed with deionized water 3 times, and then the cells are recovered by centrifugation to measure the biomass of the cells. The method for measuring the chlorophyll fluorescence curve of Chlorella is the same as that in Example 4, and the method for measuring the total nitrogen and total phosphorus contents in the aquaculture wastewater is the same as that in Example 2.

[0080] Among them, the growth test results of each group of Chlorella are as follows Fig. 9 As shown. Fig. 9It can be seen that the Chlorella cultured with phosphorus starvation in advance showed a higher biomass in the immobilized culture treatment of aquaculture wastewater, but the overall difference was not significant.

[0081] The chlorophyll fluorescence curve test results of each group of Chlorella are as follows Fig.10 As shown. Fig.10 It can be seen that the Chlorella cultured with phosphorus starvation in advance showed rapid activity recovery in the first 3 days after inoculation into aquaculture wastewater. This may be due to the rapid absorption of nutrients caused by the previous phosphorus deficiency, which increased the utilization rate of pollutants in the wastewater.

[0082] The test results of the removal effect of each group of Chlorella on total nitrogen and total phosphorus in aquaculture wastewater are as follows: Fig.11 shown.

[0083] Depend on Fig.11 It can be seen that for the removal of total phosphorus, the pre-phosphorus-starved Chlorella showed a rapid removal effect after immobilization culture. The total phosphorus removal rate reached 54.39% on the first day. The total phosphorus removal on the first day was about 1.95 times that of the autotrophic control group. After that, the phosphorus removal began to decline slowly. On the 9th day, the total phosphorus was completely removed, and the removal rate was 100%.

[0084] Example 6: Analysis of nutritional components and unsaturated fatty acids of Chlorella LBV13

[0085] Chlorella LBV13 was cultured in BG11 medium, BBM medium, Basal medium and pig farm wastewater (SWW) respectively, and the contents of protein, carbohydrates, lipids and lutein in Chlorella after culture in different culture media were compared, and fatty acid methyl esters in lipids were determined. The specific method is:

[0086] BG11 medium, BBM medium, Basal medium and piggery wastewater stock solution (SWW) (the composition of the aquaculture wastewater stock solution is the same as that of the aquaculture wastewater stock solution in Example 2) were respectively divided into 500mL conical flasks, and 300mL was divided into each bottle for standby after sterilization (wherein, the piggery wastewater stock solution was not sterilized). Chlorella LBV13 in the exponential growth phase was inoculated into the above-mentioned conical flasks. Before inoculation, the algae species were centrifuged and collected and washed with sterile deionized water to avoid the influence of residual culture medium on the experiment. The initial OD of each algae species was controlled to be about 0.2, and the conical flask was placed in a light incubator for cultivation, with a light intensity of 8000Lux, air was introduced at 1L / min, the temperature was 28±1°C, the humidity was 50%, and the light-dark ratio was 12h:12h. After the cultivation was completed, the Chlorella biomass in each culture medium was centrifuged and collected, and its protein, lipid, and carbohydrate content were determined, and fatty acid methyl esters were determined.

[0087] Among them, the formula of BG11 culture medium is: NaNO31.5 g, K2HPO4·3H2O 0.012 g, MgSO4·7H2O0.075 g, CaCl2·2H2O 0.036 g, citric acid 0.006 g, ammonium ferric citrate 0.006 g, EDTA0.001 g, Na2CO30.02 g, H3BO40.00286 g, MnCl2·H2O 0.00181 g, ZnSO4·7H2O 0.000222 g, CuSO4·5H2O 0.000079 g, Na2MoO4·2H2O 0.00039 g, Co(NO3)2·6H2O 0.000049 g, and the above solutes are adjusted to 1L.

[0088] BBM culture medium components (L): NaNO3 0.25 g, CaCl2·2H2O 0.025 g, MgSO4·7H2O 0.075 g, K2HPO4 0.075 g, KH2PO4 0.175 g, NaCl 0.025 g, EDTA 0.05 g, KOH 0.031 g, FeSO4·7H2O 0.00498 g, H3BO4 0.01142 g, ZnSO4·7H2O 0.00882 g, MnCl2·H2O 0.00144 g, MoO3 0.00071 g, CuSO4·5H2O 0.00157 g, Co(NO3)2·6H2O 0.000049 g. The above solutes were adjusted to 1L.

[0089] Basal medium components (L): KNO3 1.5 g, K2HPO4·3H2O 1.25 g, MgSO4·7H2O 1 g, EDTA 0.5 g, H3BO4 0.1142 g, CaCl2·2H2O 0.111 g, FeSO4·7H2O 0.0498 g, ZnSO4·7H2O 0.0882 g, MnCl2·H2O 0.0142 g, MoO3 0.0371 g, CuSO4·5H2O 0.0157, Co(NO3)2·6H2O 0.0049 g. Make the above solutes to 1L.

[0090] Protein determination was carried out by using the national standard Kjeldahl method to determine the total nitrogen content in the algae powder; the carbohydrate content was determined by using the phenol-sulfuric acid method.

[0091] Lipid content determination: n-hexane was used to extract lipids from algae powder and the total lipids in algae powder were obtained by weighing after rotary evaporation. Specific steps: Folch method was used for lipid extraction. Accurately weigh 0.2g algae powder, add 4mL of a mixture of ethyl acetate and ethanol (V / V 1:1), stir magnetically at room temperature for 270min, then add 4mL of deionized water and 2mL of n-hexane in turn, shake and mix, centrifuge at 25℃3500g for 10min, repeat the extraction twice, collect and combine the top layer of solution into a pre-weighed glass tube, and finally remove the solvent with a nitrogen stream and weigh it, and obtain the lipid content according to the difference in weight.

[0092] Lutein determination method: Weigh 50 mg of algae powder, add 1 mL of acetone and place in an ultrasonic cleaner for 15 minutes, then centrifuge at 4000 rpm for 10 minutes, and take the supernatant. Repeat the extraction step until there is no obvious color change in the supernatant. Combine the obtained supernatants, concentrate by rotary evaporation at 35°C, and make up to 5 mL with methanol and dichloromethane (volume ratio 3:1). Then, centrifuge at 10000 rpm for 2 minutes, take the supernatant and dilute it appropriately, filter it through a 0.22 μm filter membrane, and use liquid chromatography for detection. The standard curve of lutein peak area y and lutein content (x, mg / ml) is: y=33133x-32.307(R 2 =0.9984).

[0093] Fatty acid determination method: introduce the obtained lipid sample into a 10mL glass tube, add 2mL 2% H2SO4-CH3OH and mix well, then hydrolyze at 100℃ for 2h, let it stand and cool to room temperature, add 2mL n-hexane and vortex for 30s, then centrifuge at 3500g for 10min to form two phases. Filter the upper phase rich in FAMEs into a clean bottle and dry it by rotary evaporation. The FAMEs sample obtained after drying is used for component and content analysis. Prepare an internal standard solution of 1mg / mL (solvent is n-hexane) with pentadecanoic acid methyl ester standard. Take 1mL of the internal standard solution and add it to the dried FAMEs sample, fully dissolve it, centrifuge it and perform GC-MS detection according to the detection parameters in Table 1. The percentage of unsaturated fatty acids is the ratio of the sum of the unsaturated fatty acid peak areas to the sum of the fatty acid total areas.

[0094] Table 1 shows the GC-MS detection conditions of fatty acid methyl esters

[0095]

[0096] Among them, the results of protein, lipid, carbohydrate and lutein content test after culturing Chlorella in different culture media and aquaculture wastewater are as follows: Fig.12 shown.

[0097] Depend on Fig.12It can be seen that the Chlorella cultured in Basal medium has the highest protein content of 48.69%, followed by the BG11 medium, where the protein content of Chlorella is 44.84%. The protein content of Chlorella cultured in BBM medium is lower, only 30.23%, but it has the highest lipid content and carbohydrate content, which are 18.15% and 40.8%, respectively. The growth of algae in sewage and the nutritional accumulation levels of protein, lipid, carbohydrate, lutein, etc. are comparable to those in BG11, reaching 42.94%, 18.05%, 17.43% and 6.05 mg / g, respectively.

[0098] The results of the detection of fatty acids in lipids are shown in Table 2.

[0099] Table 2 shows the results of fatty acid content test of Chlorella LBV13 cultured in different culture media and wastewater

[0100]

[0101] As shown in Table 2, the unsaturated fatty acid content of microalgae in BG11 medium, BBM medium and Basal medium was 70.37%, 68.67% and 73.22% respectively, and the unsaturated fatty acid content of microalgae in aquaculture wastewater was 72.85. The types of unsaturated fatty acids in microalgae grown in the four culture media were roughly the same, about 9, including important unsaturated fatty acids such as palmitoleic acid, linolenic acid, and oleic acid.

[0102] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any technician familiar with the profession may use the above technical content as inspiration to make changes or modifications. This is an equivalent embodiment of equivalent changes. However, any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical concept of the present invention still fall within the scope of protection of the claims of the present invention.

Claims

1. A chlorella, characterized in that: The Chlorella nucleic acid sequence contains the sequence shown in Sequence 1 in the sequence table.

2. The Chlorella according to claim 1, characterized in that The chlorella is chlorella ( Chlorella sorokiniana )LBV13, Chlorella LBV13 is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO:M 20242807.

3. A seed culture medium for culturing the Chlorella according to claim 1 or 2, characterized in that: The seed culture medium is obtained by diluting the following solutes to 1 liter with water: straw hydrolyzate, NaNO3 1.5g, K2HPO4·3H2O 0.012g, MgSO4·7H2O 0.075g, CaCl2·2H2O 0.036g, citric acid 0.006g, ammonium ferric citrate 0.006g, EDTA 0.001g, Na2CO3 0.02g, H3BO4 0.00286g, MnCl2·H2O 0.00181g, ZnSO4·7H2O 0.000222g, CuSO4·5H2O 0.000079g, Na2MoO4·2H2O 0.00039g, Co(NO3)2·6H2O 0.000049 g; wherein, the amount of the straw hydrolyzate in the seed culture medium is determined by the glucose content in the straw hydrolyzate, and the glucose content in the seed culture medium is 2 to 5 g / L.

4. A method for preparing Chlorella seed liquid, characterized in that: The method comprises: culturing Chlorella in the seed culture medium of claim 3 to obtain seed liquid.

5. The method according to claim 4, characterized in that The Chlorella is the Chlorella according to claim 1 or 2; the temperature for culturing the Chlorella is 25° C. to 28° C., and the pH condition for culturing the Chlorella is pH 6.5 to 7.

5.

6. Use of the Chlorella described in claim 1 or 2 in sewage treatment.

7. The use according to claim 6, characterized in that: The sewage treatment includes nitrogen removal from sewage, ammonia nitrogen removal from sewage and / or phosphorus removal from sewage.

8. A method for treating sewage, characterized in that: include: The Chlorella according to claim 1 or 2 is inoculated into sewage for cultivation.

9. The sewage treatment method according to claim 8, characterized in that: Before inoculating Chlorella into sewage for culturing, Chlorella is first inoculated into the seed culture medium of claim 3 for pre-culturing, and then the pre-cultured Chlorella is inoculated into sewage for culturing.

10. The sewage treatment method according to claim 9, characterized in that: The pre-culture time is 3 days to 9 days; the pre-culture temperature is 25° C. to 28° C.; and the sewage contains a Chlorella immobilization carrier.

Images