Chlorella vulgaris and applications thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a Chlorella and application thereof. The Chlorella is isolated from a river in Dianchi Lake, Tengchong, Yunnan, and is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 20242807. The Chlorella LBV13 of the present application can grow in original aquaculture wastewater, and has a high purification capacity for the aquaculture wastewater. It is detected that the Chlorella (Chlorella sorokiniana) has a highest removal rate of 48.28% for COD, 91.73% for TN, 98.15% for NH4 + N and 88.99% for TP; therefore, the Chlorella of the present application can effectively remove nitrogen, phosphorus and ammonia nitrogen in wastewater, and achieve the purpose of wastewater purification.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a type of Chlorella and its applications. Background Technology

[0002] With the development of large-scale and intensive aquaculture, wastewater discharge has increased rapidly. Especially in areas with high livestock density, such as rural areas surrounding farms, the lack of effective wastewater treatment facilities often leads to untreated wastewater being directly discharged into rivers, lakes, or groundwater. Aquaculture wastewater contains large amounts of nitrogen, phosphorus, and organic matter, causing rapid deterioration of the surrounding water quality and triggering a series of environmental problems.

[0003] Microalgae offer numerous advantages in treating aquaculture wastewater, including efficient nitrogen and phosphorus removal, resource recovery, low energy consumption, and environmental friendliness. Microalgae are particularly effective in addressing nitrogen and phosphorus pollution. They not only effectively remove nitrogen and phosphorus from water but also utilize nutrients in the wastewater for growth, further converting them into high-value biomass that can be used as organic fertilizer and biofuel. However, the main challenges currently facing the application of microalgae in wastewater treatment are their poor tolerance to high-concentration wastewater, limited pollutant removal capabilities, and difficulty in collection.

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

[0005] Immobilized microalgae achieve higher cell concentrations through carriers, which provide a stable environment for microalgae attachment. This facilitates the adsorption and metabolism of nitrogen and phosphorus in wastewater, enabling the treatment of more wastewater per unit volume and achieving higher pollutant removal rates. Furthermore, the immobilized microalgae attached to the carrier are easily separated and collected from the wastewater, allowing for recycling, improved operational controllability, and reduced operating costs. Summary of the Invention

[0006] The purpose of this invention is to provide Chlorella and its applications.

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0008] The first aspect of the present invention provides a Chlorella, wherein the Chlorella nucleic acid sequence contains two sequences shown in Sequence 1 of the sequence listing.

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

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

[0011] The above-mentioned method for isolating and purifying Chlorella is as follows: Water samples were collected from the Dieshuihe Waterfall in Tengchong City, Yunnan Province. The collected water samples were transferred to centrifuge tubes and centrifuged at 8000 rpm for 10 min at 4℃ to precipitate the microalgae. The supernatant was then carefully removed, and the precipitate was retained. An appropriate amount of BG11 culture medium was added to the precipitate and stirred thoroughly to obtain a concentrated solution. The concentrated solution was then divided into 10... -1 10 -2 10 -3 10 -4 10 -5 The gradient dilutions were prepared by diluting the algae in a gradient with sterile water. 100 μL of each gradient dilution was spread onto the surface of solid BG11 medium containing 5% agar powder and incubated under light until individual algal colonies appeared. Finally, individual colonies were selected based on their morphology, size, and color, and then streaked onto solid BG11 medium for purification.

[0012] A second aspect of the present invention provides a seed culture medium for culturing the *Chlorella* described in the first aspect above. The seed culture medium is obtained by bringing the volume of the following solutes to 1 liter with water: straw hydrolysate, NaNO3 1.5 g, K2HPO4·3H2O 0.012 g, MgSO4·7H2O 0.075 g, CaCl2·2H2O 0.036 g, citric acid 0.006 g, ferric ammonium 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; wherein, the amount of straw hydrolysate used in the seed culture medium is determined by the glucose content in the straw hydrolysate, 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 with water to a final volume of 1 liter: straw hydrolysate, NaNO3 1.5 g, K2HPO4·3H2O 0.012 g, MgSO4·7H2O 0.075 g, CaCl2·2H2O 0.036 g, citric acid 0.006 g, ferric ammonium 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; wherein, the amount of straw hydrolysate used in the seed culture medium is determined by the glucose content in the straw hydrolysate, and the glucose content in the seed culture medium is 5g / L.

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

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

[0016] According to the above-mentioned seed culture medium, preferably, the preparation method of the straw hydrolysate is as follows: pretreating the straw with an alkaline solution, washing the pretreated straw to remove residual alkaline solution, and then enzymatically hydrolyzing the pretreated straw with cellulase to obtain straw hydrolysate. More preferably, the preparation method of the straw hydrolysate is as follows: mixing the straw with an alkaline solution, reacting at 65℃~90℃ for 1h~4h, separating the solid and liquid to obtain pretreated straw; washing the pretreated straw with water to remove residual alkaline solution, drying the washed pretreated straw, adding the dried pretreated straw to a buffer solution containing cellulase, and reacting at 40℃~60℃ for 24h~96h to obtain corn straw hydrolysate. Further, the alkaline solution is a sodium hydroxide solution with a concentration of 2% (w / v); during the pretreatment, the mass ratio of straw to alkaline solution is 1:20; the buffer solution is an HAc-NaAc buffer with a concentration of 0.2 mol / L.

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

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

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

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

[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 aeration is required during the culturing process at a rate of 0.5 to 1.5 L / min.

[0022] The fourth aspect of the present invention provides the application of Chlorella as described in the first aspect in wastewater treatment.

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

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

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

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

[0027] According to the above-mentioned wastewater treatment method, preferably, the pre-culture time is 3 days to 9 days; the pre-culture temperature is 25℃ to 28℃.

[0028] According to the above-described wastewater treatment method, preferably, the wastewater contains a Chlorella immobilization carrier; after Chlorella is inoculated into the wastewater, the Chlorella cells can adsorb and grow on the surface of the immobilization carrier material to form a biofilm. More preferably, the immobilization carrier is a sheet-like porous carrier; most preferably, the immobilization carrier is a sheet-like corn cob.

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

[0030] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows:

[0031] (1) The Chlorella sorokiniana LBV13 of this invention can grow in raw aquaculture wastewater and has a high efficiency in purifying aquaculture wastewater. Testing showed that Chlorella sorokiniana effectively reduced COD, TN, and NH4 in aquaculture wastewater. + The highest removal rates of nitrogen, phosphorus, and ammonia nitrogen in wastewater are 48.28%, 91.73%, 98.15%, and 88.99%, respectively. Therefore, the Chlorella of this invention can effectively remove nitrogen, phosphorus, and ammonia nitrogen from wastewater, thereby achieving the purpose of wastewater purification.

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

[0033] (3) The Chlorella obtained by screening in this invention can be heterotrophically cultured using corn cob hydrolysate as a carbon source. In addition, this invention also studied a phosphorus-starved seed culture medium for culturing Chlorella. This seed culture medium contains straw hydrolysate and has a low phosphorus content. Using this phosphorus-starved seed culture medium to culture Chlorella helps to improve the concentration of microalgal cells and the phosphorus absorption capacity of microalgal cells. It was found that after culturing Chlorella in the phosphorus-starved seed culture medium with corn cob hydrolysate for 7 days, it was inoculated into aquaculture wastewater for immobilization culture. The removal rate of total phosphorus in aquaculture wastewater by Chlorella reached 54.39% on the 1st day and was completely removed on the 9th day, with a removal rate of 100%. Attached Figure Description

[0034] Figure 1 Photographs of collected water samples of Chlorella sorokiniana LBV13 (A), streak plate photograph (B), and morphological feature diagrams under a 100X optical microscope (C).

[0035] Figure 2 A phylogenetic tree constructed based on a partial 18S rDNA sequence of Chlorella LBV13;

[0036] Figure 3 The growth curve of Chlorella LBV13 in pig farm wastewater raw solution;

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

[0038] Figure 5 Image showing the effect of immobilizing microalgae on corn cob slices;

[0039] Figure 6 The results show the removal rates of aquaculture wastewater by immobilized and suspended microalgae.

[0040] Figure 7 The growth curves of Chlorella LBV13 under phosphorus starvation culture are shown; 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 curves (Fv / Fm) of Chlorella LBV13 culture under phosphorus starvation are shown; where Autotrophy is the autotrophic control group, Heterotrophy is the heterotrophic control group, and P- is the phosphorus starvation group.

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

[0043] Figure 10 The chlorophyll fluorescence curves (Fv / Fm) of Chlorella LBV13 cultured in aquaculture wastewater after phosphorus starvation are shown; where Autotrophy is the autotrophic control group, Heterotrophy is the heterotrophic control group, and P- is the phosphorus starvation group.

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

[0045] Figure 12 The distribution of physicochemical properties of Chlorella LBV13 in different culture media and wastewater: (A) protein, (B) lipid, (C) carbohydrate, (D) lutein. Detailed Implementation

[0046] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

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

[0048] Water samples were collected from Dieshuihe Waterfall in Tengchong City, Yunnan Province (photos of the water samples are shown). Figure 1 As shown in Figure A), the collected water sample was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 min at 4°C to precipitate microalgae. The supernatant was carefully removed, retaining the precipitate, and an appropriate amount of BG11 culture medium was added. The mixture was thoroughly stirred to obtain a concentrated solution. The concentrated solution was then divided into 10... -1 10 -2 10 -3 10 -4 10 -5 The gradient dilutions were prepared by diluting the algae in a series of sterile waters. 100 μL of each gradient dilution was spread onto the surface of solid BG11 medium containing 5% agar powder and incubated under light until individual algal colonies appeared. Individual colonies were selected based on their morphology, size, and color, and then streaked onto solid BG11 medium for purification (see image for streak purification). Figure 1 As shown in Figure B), and observed using a fluorescence inverted microscope, the results are as follows. Figure 1 As shown in C. From Figure 1 As shown in C, microalgae are green spherical in shape, and under normal growth conditions, their cell diameter is about 1-8 μm, and they grow as single cells.

[0049] A single algal colony was added to a PCR tube containing 20 μL of sterile water. The colony was repeatedly pipetted to disperse it, resulting in a slightly greenish tint to the sterile water and a homogeneous solution without large algal fragments. The PCR tube was then heated in a metal bath at 99°C for 20 min, followed by cooling at 4°C to obtain the DNA template for PCR amplification. The PCR amplification reaction mixture consisted of: 1.4 μL of 18S-F primer, 1.4 μL of 18S-R primer, 1.4 μL of DNA template, and 25 μL of 2×taq enzyme, with the volume adjusted to 35 μL using sterile water. The 18S-F sequence was CCAACCTGGTTGATCCTGCCAGTA, and the 18S-R sequence was CCTTGTTACGACTTCACCTTCCTCT. The PCR amplification conditions were: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1.5 min, for a total of 35 cycles; and a final extension at 72°C for 10 min. The PCR amplification products were subjected to electrophoresis on a 1% agarose gel. Products with clear, non-spam positive bands were sent to the company for Sanger sequencing to obtain 18S rDNA sequencing results. The nucleotide sequence of the 18S rDNA is shown in Sequence 1 of the sequence listing.

[0050] Sequence 1 is as follows:

[0051] GAGAGCGGAGACCGCCCCCGGTAGCCAATCCGAACACTTCACCAGCACACCCAATCGGTAGGAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCAAGCTGATGACTTGCGCTTACTAGGCATTCCTCGTTGAAGATTAATAATTGCAATAATCTATCCCCATCACGATGCAGTTTCAAAGATTACCCGGGCCTCTCGGCCAAGGCTAGGCTCGTTGAATGCATCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCATGCTTCCATTGGCTAGTCGCCAATAGTCCCTCTAAGAAGTCCGCCGGCTGGCGAACCAACCGTGACTATTTAGCAGGCTGAGGTCTCGTTCGTTACCGGAATCAACCTGACAAGGCAACCCACCAACTAAGAACGGCCATGCACCACCACCCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCTCACTATGTCTGGACCTGGTAAGTTTTCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACGCCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCGGAACCCAAAAACTTTGATTTCTCATAAGGTGCCGGCGGAGTCATCGAAGAAACATCCGCCGAT-CCCTAGTCGGCATCGTTTATGGTTGAGACTAGGACGGTATCTAATCGTCTTCGAGCCCCCAACTTTCGTTCTTGATTAATGAAAACATCCTTGGCAAATGCTTTCGCAGTAGTTCGTCTTTCATAAATCCAAGAAATTTCACCTCTGACAATGAAATACGAATGCCCCCGACTGTCCCTTCTTAATTCATTACTCCGGTCCTACAGACCCAACAGGATTAGCCAGAGTCCTATCGTGTTATTCCATGCTAATGTATTCAGAGCGTAGGCCTGCTTTGAACACTCTAAATTTACTCAAAGTAACAGCGCCGACTCCGAGTCCCGGACAGTGAAGCCCAGGAGCCCGTCCCCGGCAACAAGGTGGGCCCTGCCAGTGCACACCGAAGCGGCGGACCGGCAGGCCCCACCCGAAATCCAACTACGAGCTTTTTAACTGCAGCAACTTAAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGATCCTCGTTAAGGGGTTTAGATTGTACTCATTCCAATTACCAGACCTGAAAAGGCCCAGTATTGTTATTTATTGTCACTACCTCCCTGTGTCAGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATCCTCCGTCACCCGTTACCACCATGGTAGGCCTCTATCCTACCATCGAAAGTTGATAGGGCAGAAATTTGAATGAAACATCGCCGGCACAAGGCCATGCGATTCGTGAAGTTATCATGATTCACCGCGAGTCGGGCAAGCCCGGTCGGTCTTTTATCTAATAAATACGTCCCTTCCAGAAGTCGGGATTTACGCACGTATTAGCTCTAGATTTACTACGGGTATCCGAGTAGTAGGTACCATCAAATAAACTATAACTGATTTAATGAGCCATTCGCAGTTTCACAGTATAAAGCAGTTTATACTTAGACATGCATGGCTAATCT。

[0052] The 18S rDNA sequence was compared with existing homologous sequences in the NCBI database using BLAST, and a phylogenetic tree was constructed. The results are as follows: Figure 2 As shown. By Figure 2 It was found that the screened microalgae had a sequence similarity of 99.34% with Chlorella sorokini, indicating a close phylogenetic relationship. Further morphological observation confirmed that the screened microalgae belonged to the genus Chlorella and was named Chlorella sorokiniana LBV13. It has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20242807. The address of the collection institution is China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is December 12, 2024.

[0053] Example 2: Adaptive cultivation of Chlorella LBV13 and its effect on the removal of pollutants from raw wastewater from pig farms

[0054] Adaptation culture of Chlorella LBV13 was carried out using pig farm wastewater. The specific method was as follows: 100 mL of Chlorella LBV13 in the exponential growth phase was collected by centrifugation and washed with sterile deionized water. It was then added to 300 mL of aquaculture wastewater diluted 20% (the 20% dilution was achieved by mixing the original aquaculture wastewater with water at a volume ratio of 1:4). Air was introduced for acclimatization culture for 7 days. After 7 days, the cultured Chlorella wastewater mixture was stirred evenly, and 100 mL was added to 300 mL of aquaculture wastewater diluted 40%. Air was introduced for acclimatization culture for 7 days. The above steps were repeated, and the Chlorella wastewater mixture was added to aquaculture wastewater diluted 60%, 80%, and 100% for acclimatization culture for 7 days. The wastewater in question is from a pig farm in Zhengzhou. The original wastewater contains 1516.8 mg / L of COD, 634.2 mg / L of ammonia nitrogen, 646.6 mg / L of total nitrogen, and 14.5 mg / L of total phosphorus.

[0055] 200 ml of Chlorella vulgaris, after undergoing the above-mentioned adaptive culture, was added to a flat-plate photoreactor containing 2 L of pig farm wastewater stock solution for cultivation. The growth of Chlorella vulgaris in the wastewater was observed during the cultivation process, and the COD, total nitrogen (TN), and ammonia nitrogen (NH4) in the wastewater stock solution were monitored simultaneously during the Chlorella vulgaris cultivation process. +Changes in COD (nitrogen) and total phosphorus (TP); according to national standards (HJ828-2017, HJ 535-2009, HJ 636-2012, GB 11893-89), the methods for COD determination are potassium dichromate method, ammonia nitrogen determination is Nessler's reagent spectrophotometry, total nitrogen determination is potassium persulfate oxidation ultraviolet spectrophotometry, and total phosphorus determination is ammonium molybdate spectrophotometry. The flat-plate photobioreactor has dimensions of 0.3m long, 0.2m wide, and 0.1m high. Air is introduced to agitate the algal solution. The cultivation conditions are: light intensity 8000 Lux, temperature 28±1℃, humidity 50%, and light-dark ratio 12h:12h.

[0056] The results of the test on the growth of Chlorella in the raw wastewater from pig farms are as follows: Figure 3 As shown.

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

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

[0059] Depend on Figure 4 It can be seen that the COD in the raw aquaculture wastewater exhibits a wave-like downward trend, with the highest COD removal rate reaching 48.28%. This is because: on the one hand, microalgae utilize organic matter in the biogas slurry, causing a decrease in COD; on the other hand, microalgae's own metabolism also secretes some organic matter into the biogas slurry, causing an increase in COD. The total nitrogen (TN) and ammonia nitrogen (NH4) in the raw aquaculture wastewater... + The content of ammonia nitrogen (NH4+) decreased by nearly 80% within 3 days, and the content of total nitrogen (TN) decreased by 91.73% within 12 days. + The total phosphorus (TP) content decreased by 98.15%; the total phosphorus (TP) content in the raw aquaculture wastewater decreased by 88.99% within 9 days.

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

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

[0062] The specific experimental method is as follows:

[0063] The experiment included an immobilized group and a suspension group. The immobilized group was treated as follows: 500 ml of aquaculture wastewater diluted 20% (the 20% dilution was achieved by mixing the original aquaculture wastewater with water at a volume ratio of 1:4; this original aquaculture wastewater was the same as in Example 2) was added to a 1 L conical flask, along with an immobilization carrier. The immobilization carrier was a slice of corn cob (corn cob cut into round slices 3 cm in diameter and 3 mm thick, suspended in the aquaculture wastewater). The carrier content was 0.5% (w / v), and then Chlorella LBV13 culture medium in the logarithmic growth phase was inoculated into Erlenmeyer flasks. For the suspension group, 500 ml of 20% diluted aquaculture wastewater (the 20% dilution was achieved by mixing the original aquaculture wastewater with water at a volume ratio of 1:4; this original aquaculture wastewater was the same as in Example 2) was added to a 1 L Erlenmeyer flask, and then Chlorella LBV13 culture medium in the logarithmic growth phase was inoculated into the Erlenmeyer flask, with the inoculation amount being the same as the immobilized group. Both the immobilized and suspension groups of Chlorella were cultured for 9 days under the following conditions: temperature 25℃, light intensity 8000 Lux, humidity 50%, and light-dark ratio 12h:12h. After 9 days of culture, the residual total nitrogen and total phosphorus content in the aquaculture wastewater were measured using the same methods as in Example 2.

[0064] The immobilization effect of Chlorella in the immobilized group after 9 days of cultivation is shown in the figure below. Figure 5 As shown. By Figure 5 It can be seen that during the cultivation process, Chlorella LBV13 will attach to the surface of the corn cob and form a biofilm, indicating that Chlorella LBV13 has been successfully immobilized.

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

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

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

[0068] Chlorella LBV13 was precultured in a phosphorus-starved medium to analyze the effect of phosphorus-starved culture on the growth of Chlorella LBV13.

[0069] The specific experimental method is as follows:

[0070] Take the culture medium of Chlorella LBV13 in the exponential growth phase (all subcultures of Chlorella LBV13 were conducted in BG11 medium) (OD 680 =5) 100ml, transfer to a centrifuge tube, centrifuge at 4℃ and 8000rpm for 10min to precipitate microalgae, discard the supernatant, retain the precipitate, and resuspend it in sterile water to remove residual culture medium on the surface of the microalgae and prevent small amounts of nitrogen and phosphorus from affecting the experiment. Repeat the above steps 3 times. Inoculate the microalgae precipitate obtained from the last centrifugation into phosphorus-starved medium (referred to as phosphorus-starved group), BG11 medium (referred to as autotrophic control group), and heterotrophic BG11 medium (referred to as heterotrophic control group), respectively, so that the initial OD of the above three media is... 680 All samples were cultured for 7 days under the following conditions: light intensity of 8000 Lux, humidity of 50%, light-dark ratio of 12h:12h, and aeration at a rate of 1 L / min. During the culture, the growth of *Chlorella* and changes in its chlorophyll fluorescence curve were measured daily.

[0071] The phosphorus-starved culture medium formula is as follows: straw hydrolysate, NaNO3 1.5g, K2HPO4·3H2O 0.012g, MgSO4·7H2O 0.075g, CaCl2·2H2O 0.036g, citric acid 0.006g, ferric ammonium 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 0.000049g of the above solute was diluted with water to a final volume of 1 liter to obtain the phosphorus-starved medium. The amount of straw hydrolysate in the phosphorus-starved medium was determined by the glucose content in the straw hydrolysate, and the glucose content in the phosphorus-starved medium was 5g / L. The pH of the phosphorus-starved medium was 7.1±0.1.

[0072] The formulation of heterotrophic BG11 medium is as follows: straw hydrolysate, NaNO3 1.5g, K2HPO4·3H2O 0.4g, MgSO4·7H2O 0.075g, CaCl2·2H2O 0.036g, citric acid 0.006g, ferric ammonium 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 0.000049g of the above solute was diluted with water to a final volume of 1 liter to obtain heterotrophic BG11 medium. The amount of straw hydrolysate in the heterotrophic BG11 medium was determined by the glucose content in the straw hydrolysate. The glucose content in the heterotrophic BG11 medium was 5g / L, and the pH of the heterotrophic BG11 medium was 7.1±0.1.

[0073] The straw hydrolysate is corn straw hydrolysate. The preparation method of corn straw hydrolysate is as follows: Weigh 100g of crushed corn straw into a four-necked flask, add 2% (w / v) NaOH solution (1960mL water, 40g NaOH) to make the solid-liquid ratio 1:20, and place it in an 80℃ water bath for 2h. After the pretreatment, use a filter screen to separate the solid and liquid components, adjust the pH to neutral with 20% acetic acid solution, wash with deionized water 2-3 times, and place it in a 60℃ oven to dry to constant weight. Add 3g of pretreated straw, then add an appropriate amount of cellulase (enzyme activity 151.494 FPU / mL) and 0.2mol / L HAc-NaAc buffer to make the total reaction volume 30mL. Incubate at 50℃ and 200rpm in a constant temperature shaker for 72h. Then, add 2% columnar activated carbon for 2 days. Filter to obtain corn straw hydrolysate. Sterilize the corn straw hydrolysate at 115℃ for 15min, then freeze at -20℃ for later use. The glucose content in the sterilized corn straw hydrolysate was found to be 69.5g / L.

[0074] During the cultivation process, the growth of Chlorella was measured by taking 1 ml of algal solution daily and measuring 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 results of the Chlorella growth test are as follows: Figure 7 As shown. By Figure 7 It can be seen that, after 7 days of pre-culture, the OD of the autotrophic control group of Chlorella was... 680 The value was 4.152, while the OD of Chlorella vulgaris in the phosphorus-starved group was 4.152. 680The OD200 of Chlorella reached 10.956, representing a 1.64-fold increase in Chlorella biomass in the phosphorus-starved group compared to the autotrophic control group, while the OD200 of Chlorella in the heterotrophic control group was significantly lower. 680 The highest value was 13.705.

[0076] The chlorophyll fluorescence curve detection results of Chlorella are as follows: Figure 8 As shown. By 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 reduced, which indicates that the growth of Chlorella LBV13 was under stress in the phosphorus starvation medium.

[0077] Example 5: Study on the Immobilization Treatment of Aquaculture Wastewater by Chlorella under Phosphorus Starvation Stress

[0078] After 7 days of pre-culturing in the phosphorus-starved group, autotrophic control group, and heterotrophic control group of Example 4, the Chlorella were transferred to centrifuge tubes. Centrifugation was performed at 8000 rpm for 5 minutes to precipitate the Chlorella. The supernatant was discarded, and the Chlorella precipitate was resuspended in sterile water to remove residual culture medium from the surface of the Chlorella. This process was repeated three times. The Chlorella precipitate obtained from the last centrifugation was then inoculated into a plate-type photobioreactor containing suspended flake corn cobs (corn cobs cut into 3 cm diameter, 3 mm thick round slices, with a corn cob content of 0.5% (w / v) in the aquaculture wastewater at a dilution ratio of 20% (the 20% dilution ratio was achieved by mixing the original aquaculture wastewater and water at a volume ratio of 1:4; this original aquaculture wastewater was the same as in Example 2). The initial inoculum size was OD... 680 All values ​​were 0.5, and then cultivation was carried out. The dimensions of the flat-plate photobioreactor were 0.3m long, 0.2m wide, and 0.2m high, with a liquid volume of 1L. The cultivation conditions were: temperature 25℃, light intensity 15000Lux, humidity 50%, and light-dark ratio of 12h:12h.

[0079] During the cultivation process, the growth of *Chlorella* in each group and the total nitrogen and total phosphorus content in the aquaculture wastewater were measured daily. The method for determining the biomass of *Chlorella* in each group was as follows: *Chlorella*-loaded corn cobs were ultrasonically treated at 40 kHz for 5 min in an ultrasonic bath. After filtering through filter paper to separate the carrier, the suspended cells were harvested by centrifugation at 8000 rpm for 5 min, washed three times with deionized water, and then centrifuged again to recover the bacterial cells. The biomass of the bacterial cells was then measured. The method for determining the chlorophyll fluorescence curve of *Chlorella* was the same as in Example 4, and the methods for determining the total nitrogen and total phosphorus content in the aquaculture wastewater were the same as in Example 2.

[0080] The growth results of each group of Chlorella are as follows: Figure 9 As shown. By Figure 9It can be seen that Chlorella cultured under phosphorus starvation pre-processed exhibited higher biomass in immobilized culture treatment of aquaculture wastewater, but the overall difference was not significant.

[0081] The chlorophyll fluorescence curve detection results of each group of Chlorella are as follows: Figure 10 As shown. By Figure 10 It can be seen that Chlorella cultured under phosphorus starvation 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 pre-phosphorus deficiency, resulting in higher utilization of pollutants in the wastewater.

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

[0083] Depend on Figure 11 It can be seen that, for the removal of total phosphorus, the immobilized culture of Chlorella after pre-phosphorus starvation showed a rapid removal effect. The total phosphorus removal rate reached 54.39% on the first day, and the total phosphorus removal amount on the first day was about 1.95 times that of the autotrophic control group. After that, the phosphorus removal began to decrease slowly, and the total phosphorus was completely removed on the ninth day, with a removal rate of 100%.

[0084] Example 6: Analysis of nutrient composition and unsaturated fatty acids of Chlorella LBV13

[0085] Chlorella LBV13 was cultured in BG11, BBM, Basal, and septic tank wastewater (SWW) media. The contents of protein, carbohydrates, lipids, and lutein in the chlorella were compared after culture in different media, and the fatty acid methyl ester content in the lipids was determined. The specific methods are as follows:

[0086] BG11 medium, BBM medium, Basal medium, and sludge from pig farms (SWW) (the composition of the sludge was the same as that in Example 2) were dispensed into 500mL Erlenmeyer flasks, with 300mL dispensed into each flask and sterilized for later use (the sludge was not sterilized). Chlorella LBV13, in its exponential growth phase, was inoculated into the Erlenmeyer flasks. Before inoculation, the algae were collected by centrifugation and washed with sterile deionized water to avoid the influence of residual culture medium on the experiment. The initial OD of each algae was controlled to be approximately 0.2. The Erlenmeyer flasks were placed in a light incubator with a light intensity of 8000 Lux, air circulation at 1 L / min, a temperature of 28±1℃, a humidity of 50%, and a light-dark ratio of 12h:12h. After incubation, the Chlorella biomass in each culture medium was collected by centrifugation, and its protein, lipid, and carbohydrate contents were determined. The methyl ester content of fatty acids was also measured.

[0087] The BG11 culture medium formula is as follows: NaNO3 1.5 g, K2HPO4·3H2O 0.012 g, MgSO4·7H2O 0.075 g, CaCl2·2H2O 0.036 g, citric acid 0.006 g, ferric ammonium 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 0.000049 g. The above solutes are brought to a final volume of 1 L.

[0088] BBM medium composition (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 brought to a final volume of 1 L.

[0089] Basal medium composition (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 g, Co(NO3)2·6H2O 0.0049 g. The above solutes were brought to a final volume of 1 L.

[0090] Protein content was determined by the Kjeldahl method according to national standards; carbohydrate content was determined by the phenol-sulfuric acid method.

[0091] Lipid content determination involved extracting lipids from algal powder with n-hexane and weighing the extracted solution after rotary evaporation to obtain the total lipids. Specific steps: Lipid extraction was performed using the Folch method. 0.2 g of algal powder was accurately weighed and added to 4 mL of a mixture of ethyl acetate and ethanol (V / V 1:1). The mixture was magnetically stirred at room temperature for 270 min, followed by the addition of 4 mL of deionized water and 2 mL of n-hexane. After shaking and mixing, the mixture was centrifuged at 3500 g for 10 min at 25°C. The extraction was repeated twice. The combined uppermost layer of the extract was collected into a pre-weighed glass tube. Finally, the solvent was removed using a nitrogen stream, and the mixture was weighed. The lipid content was determined based on the difference in weight.

[0092] Lutein determination method: Weigh 50 mg of algal powder, add 1 mL of acetone, and treat in an ultrasonic cleaner for 15 minutes. Then centrifuge at 4000 rpm for 10 minutes and collect the supernatant. Repeat the extraction steps until the supernatant shows no obvious color change. Combine the obtained supernatants, concentrate by rotary evaporation at 35℃, 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, dilute appropriately, filter through a 0.22 μm filter membrane, and detect by liquid chromatography. The standard curve of lutein peak area y versus lutein content (x, mg / mL) is: y = 33133x - 32.307 (R 2 =0.9984).

[0093] Fatty acid determination method: The obtained lipid sample was introduced into a 10 mL glass tube, 2 mL of 2% H2SO4-CH3OH was added, and the mixture was stirred. Hydrolysis was carried out at 100℃ for 2 h, and the mixture was allowed to cool to room temperature. 2 mL of n-hexane was added, and the mixture was vortexed for 30 s. Then, it was centrifuged at 3500 g for 10 min to form two phases. The FAMEs-rich upper phase was filtered into a clean bottle and dried by rotary evaporation. The dried FAMEs sample was used for component and content analysis. A 1 mg / mL internal standard solution (using n-hexane as solvent) was prepared using methyl pentadecanoate as a standard. 1 mL of the internal standard solution was added to the dried FAMEs sample, dissolved completely, centrifuged, filtered, and then analyzed by GC-MS according to the detection parameters in Table 1. The percentage content of unsaturated fatty acids was the ratio of the sum of the peak areas of unsaturated fatty acids to the sum of the total fatty acid peak areas.

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

[0095]

[0096] The results of detecting the content of protein, lipids, carbohydrates, and lutein in Chlorella after cultivation in different culture media and aquaculture wastewater are as follows: Figure 12 As shown.

[0097] Depend on Figure 12It was found that *Chlorella* cultured in Basal medium had the highest protein content at 48.69%, followed by *Chlorella* cultured in BG11 medium at 44.84%. *Chlorella* cultured in BBM medium had a lower protein content of only 30.23%, but the highest lipid and carbohydrate contents, at 18.15% and 40.8%, respectively. The growth of the algal strains in wastewater and the accumulation levels of nutrients such as protein, lipids, carbohydrates, and lutein were comparable to those in BG11, reaching 42.94%, 18.05%, 17.43%, and 6.05 mg / g, respectively.

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

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

[0100]

[0101] Table 2 shows that the unsaturated fatty acid contents of microalgae in BG11, BBM, and Basal media were 70.37%, 68.67%, and 73.22%, respectively, while the unsaturated fatty acid content of microalgae in the aquaculture wastewater was 72.85%. The types of unsaturated fatty acids in microalgae grown on the four media were roughly the same, approximately nine types, 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 merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications. These are equivalent embodiments with variations. However, any simple modifications, 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 protection scope of the claims of the present invention.

Claims

1. A Chlorella characterized by, The Chlorella is Chlorella (Chlorella vulgaris) Chlorella sorokiniana ) LBV13, and the Chlorella LBV13 is preserved in China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 20242807.

2. A method for culturing the Chlorella of claim 1, characterized in that, Chlorella was cultured in a seed culture medium to obtain a seed solution. The seed culture medium was prepared by bringing the volume of the following solutes to 1 liter with water: straw hydrolysate, NaNO3 1.5 g, K2HPO4·3H2O 0.012 g, MgSO4·7H2O 0.075 g, CaCl2·2H2O 0.036 g, citric acid 0.006 g, ferric ammonium 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; wherein, the amount of straw hydrolysate in the seed culture medium is determined by the glucose content in the straw hydrolysate, and the glucose content in the seed culture medium is 2-5 g / L; the Chlorella is the Chlorella described in claim 1.

3. The method of claim 2, wherein, The temperature for culturing Chlorella is 25℃~28℃, and the pH condition for culturing Chlorella is 6.5~7.

5.

4. The application of Chlorella as described in claim 1 in wastewater treatment.

5. Use according to claim 4, characterized in that, The wastewater treatment includes nitrogen removal, ammonia nitrogen removal, and / or phosphorus removal.

6. A method of sewage treatment, characterised in that, include: The Chlorella described in claim 1 is inoculated into sewage for cultivation.

7. The wastewater treatment method according to claim 6, characterized in that, Before culturing Chlorella in wastewater, the Chlorella was first inoculated into a seed culture medium for pre-culture. Then, the pre-cultured Chlorella was inoculated into wastewater for further cultivation. The seed culture medium was obtained by bringing the volume of the following solutes to 1 liter with water: straw hydrolysate, NaNO3 1.5 g, K2HPO4·3H2O 0.012 g, MgSO4·7H2O 0.075 g, CaCl2·2H2O 0.036 g, citric acid 0.006 g, ferric ammonium 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 0.00039g, Co(NO3)2·6H2O 0.000049g; wherein, the amount of straw hydrolysate in the seed culture medium is determined by the glucose content in the straw hydrolysate, and the glucose content in the seed culture medium is 2-5 g / L.

8. The method of sewage treatment according to claim 7, characterized in that, The pre-culture time is 3 to 9 days; the pre-culture temperature is 25°C to 28°C; and the wastewater contains a Chlorella immobilization carrier.

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