Seawater chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity as well as culture method and application thereof

Abstract

The invention discloses seawater chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity as well as a culture method and application of the seawater chlorella. The problems that the growth rate is reduced and the ammonia nitrogen treatment efficiency is reduced due to the salt stress effect when a traditional bioremediation technology is applied to a tidal basin are solved. According to the method, the seawater chlorella with high adaptability is utilized, the ammonia nitrogen absorption capacity and the starch accumulation amount of the seawater chlorella are remarkably improved through the salt synergistic effect in combination with the immobilization technology, and therefore effective treatment on the ammonia nitrogen pollution of the tidal basin is achieved. Under the conditions that the salinity is 0.5% and the ammonia nitrogen concentration is 3 mg / L, the ammonia nitrogen removal rate of the seawater chlorella reaches 93.60%, the starch accumulation amount reaches 10.06 mu g / 10 < 6 > cells, and the ammonia nitrogen removal rate and the starch accumulation amount are increased by 23.2% and 58.4% respectively. The method has the advantages of simple and convenient process, high efficiency, strong environmental adaptability and the like, and is suitable for ammonia nitrogen pollution treatment and microalgae resource utilization in tidal basins.

Description

Technical Field

[0001] The present invention belongs to the technical field of microalgae, and specifically relates to seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity, as well as a cultivation method and application thereof. Background Art

[0002] Tidal basins are areas with significant salinity fluctuations. Influenced by hydrological conditions and seasonal factors, the upstream range of saltwater intrusion in these areas varies significantly, forming distinct salinity zones: low salinity (0.5‰-5‰), medium salinity (5‰-18‰), and high salinity (18‰-30‰). As transitional zones between terrestrial and marine ecosystems, tidal basins harbor rich biodiversity but are sensitive to environmental changes, particularly the impacts of nutrients such as ammonia and phosphorus.

[0003] Ammonia nitrogen usually refers to free ammonia nitrogen (NH3) and ammonium ions (NH4 + Nitrogen, present in the form of nitrogen, is present at high levels in domestic sewage, industrial wastewater, and aquaculture wastewater. This causes inorganic nutrients to be discharged into estuaries and cannot be effectively removed, leading to their accumulation. Currently, inorganic nitrogen levels in tidal waters exceed standards, and ammonia nitrogen is also a major pollutant in tidal waters.

[0004] Biotechnology is widely used in water remediation in tidal basins. Commonly used organisms include microorganisms such as bacteria and fungi, aquatic plants, large seaweed, and microalgae. However, due to the fluctuating salinity and low carbon-nitrogen ratios of water in tidal basins, microorganisms are not suitable for this purpose due to their limited salt tolerance and high carbon source requirements. Emergent aquatic plants such as canna and lily of the valley are widely used in low-salinity areas (0.5‰-5‰). However, when salinity exceeds 5‰, their growth is significantly inhibited, significantly affecting their effectiveness in water remediation. Ulva, which primarily grows in tidal basins such as estuaries and bays, has a strong tolerance to salinity, adapting to salinity fluctuations of 10‰-30‰ and possesses a strong ability to assimilate inorganic nitrogen. However, its growth is significantly inhibited at salinity levels below 10‰. For example, at a salinity of 5‰, its ammonia nitrogen removal efficiency is only 52.8%.

[0005] Microalgae are a highly diverse group of photosynthetic organisms that harness light energy to decompose water into molecular oxygen and protons, and convert inorganic carbon (CO2) into carbohydrates, ultimately forming the beneficial biomass starch. As the oldest group of photosynthetic organisms, microalgae rapidly grow using sunlight, carbon, and nitrogen sources. Among them, Chlorella vulgaris is particularly well-known for its exceptional ammonia nitrogen absorption capacity. Its rapid growth rate, lack of agricultural land occupation, strong environmental adaptability, and rich content of bioactive molecules have garnered widespread global attention as a raw material for biofuel production, functional foods, and feed supplements.

[0006] In response to the problem that traditional bioremediation technology is subject to stress caused by reduced salinity during nutrient salt management in tidal waters, resulting in poor ammonia nitrogen removal efficiency, the present invention utilizes a cultivation method that combines salt synergistic effect and immobilization technology to obtain seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity. It can be used for ammonia nitrogen removal and co-production of starch in tidal waters and saline water bodies, realizing the organic integration of wastewater treatment and bioresource technology. Summary of the Invention

[0007] To address the problem that traditional bioremediation technologies, when applied to tidal waters, are subject to salt stress, resulting in reduced growth rates and, consequently, reduced ammonia nitrogen treatment efficiency, the present invention utilizes a cultivation method that combines salt synergy with immobilization technology to produce marine Chlorella with high ammonia nitrogen removal rates and high starch accumulation capabilities. The resulting marine Chlorella is suitable for the treatment of ammonia nitrogen pollution in tidal waters and for the resource utilization of microalgae.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions.

[0009] The present invention provides a method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity, comprising: A batch culture method was adopted. First, marine Chlorella was cultured under conditions of suitable salinity (30‰-33‰) and sufficient nitrogen source (ammonia nitrogen concentration 8-12 mg / L). After the marine Chlorella grew to the logarithmic growth period, it was immobilized and embedded in sodium alginate. The immobilized algae balls were then cultured under conditions of salt synergy (salinity 5‰-15‰) and nitrogen source limitation (ammonia nitrogen concentration of 2-3 mg / L). Finally, marine Chlorella with high ammonia nitrogen removal rate and high starch accumulation ability was obtained.

[0010] Furthermore, the culture method specifically comprises the following steps: (1) The first stage of culturing Chlorella vulgaris: The seed liquid of Chlorella vulgaris is inoculated into an artificial seawater culture medium with sufficient nitrogen source (ammonia nitrogen concentration 8-12 mg / L) and salinity of 30‰-33‰. The first stage of culturing is carried out in a conical flask. The conical flask is placed on a shaker and shaken. The appropriate light intensity and temperature are maintained. After culturing for 7-10 days, the Chlorella vulgaris liquid in the logarithmic growth phase is collected. (2) Mixing the seawater chlorella algae solution obtained in step (1) with the sodium alginate solution, and dripping the mixed solution into the calcium chloride solution to obtain algae balls. After completion, the sodium alginate-seawater chlorella algae balls are placed in a refrigerator for 24-48 hours to allow the algae balls to solidify, thereby obtaining immobilized algae balls; (3) placing the immobilized algae balls obtained in step (2) in a baffled flow reactor with continuous water inflow for second-stage cultivation in an artificial seawater culture medium, while changing the salinity of the artificial seawater culture medium to 5‰-15‰ and reducing the ammonia nitrogen concentration to 2-3 mg / L, while maintaining the light intensity and temperature in step (1) for cultivation; (4) The immobilized algae balls obtained in step (3) are dissolved in a sodium citrate solution, and centrifuged to obtain the seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity.

[0011] Furthermore, in step (1), the seawater Chlorella seed liquid, i.e., the initial algae liquid, is cultured in the artificial seawater medium for 7-10 days to obtain the seawater Chlorella liquid, and its OD 680 It is 0.25-0.3.

[0012] Furthermore, in step (1), the initial inoculation volume ratio of the seawater Chlorella seed liquid is 10%-20%.

[0013] Furthermore, in step (1) and step (3), the light intensity is 4000-8000 lux.

[0014] Furthermore, in step (1) and step (3), the temperature is 25±1°C.

[0015] Furthermore, in step (1) and step (3), the daily illumination time during the culture process is controlled to be 10-12 h, preferably the light-dark ratio is 12h:12h, the pH is 7.0-7.5, and the culture time is 7-10 days.

[0016] Furthermore, in step (1), the formula of the artificial seawater culture medium used in the first stage culture includes NaCl 20-22 g / L, MgSO4·7H2O 4.5-5.0 g / L, MgCl2·6H2O 4.0-4.5 g / L, CaCl21.0-1.2 g / L, KCl0.5-0.6 g / L, NaHCO30.2-0.3 g / L, SrCl20.01-0.02 g / L, KBr 5-10 mg / L, MnSO43.0-3.3 mg / L and CH3COONa 15-25 mg / L, NH4HCO345-67 mg / L, KH2PO418-27 mg / L.

[0017] Furthermore, in step (2), the mass fraction of the sodium alginate solution is 1.5%-2%, and the volume ratio of the seawater Chlorella solution to the sodium alginate solution is 1:1-1.5, preferably 1:1.5; the mass fraction of the calcium chloride solution is 2%-3%, and the volume ratio of the mixed solution to the calcium chloride solution is 1:1-1.5, preferably 1:1.5.

[0018] Furthermore, in step (3), the formula of the artificial seawater culture medium used for the second stage culture includes NaCl 3.3-11 g / L, MgSO4·7H2O 1.5-2.5 g / L, MgCl2·6H2O 1.3-2.3 g / L, CaCl2 0.33-0.6 g / L, KCl 0.2-0.3 g / L, NaHCO3 0.2-0.3 g / L, SrCl2 0.01-0.02 g / L, KBr 5-10 mg / L, MnSO4 3.0-3.3 mg / L and CH3COONa 15-25 mg / L, NH4HCO3 12-17 mg / L, KH2PO4 4.4-6.6 mg / L.

[0019] Furthermore, in step (3), the second stage of cultivation is carried out in a baffled flow reactor, and the hydraulic retention time of continuous water inflow is 24-48 hours. More preferably, the hydraulic retention time is 48 hours.

[0020] Furthermore, the first stage culture conditions in step (1) are: ammonia nitrogen concentration of 8-12 mg / L, total phosphorus concentration of 4-6 mg / L, and salinity of 30‰-33‰. The second stage culture conditions in step (3) are: ammonia nitrogen concentration reduced to 2-3 mg / L, total phosphorus concentration reduced to 1.0-1.5 mg / L, and salinity reduced to 5‰-15‰, thereby generating nitrogen limitation and salt synergistic effects. In the present invention, the nitrogen-to-phosphorus ratio (N:P) of the artificial seawater culture medium is always constant at 2:1, and the total phosphorus concentration changes with the adjustment of the nitrogen source concentration.

[0021] Furthermore, in step (4), the mass fraction of the sodium citrate solution is 4%-6%.

[0022] This invention utilizes a synergistic salt effect combined with immobilization and embedding technology to effectively address the low growth rate and high redox stress faced by marine Chlorella in tidal basin ecological restoration, resulting in low and unstable ammonia nitrogen removal rates. This results in Chlorella having efficient and stable ammonia nitrogen absorption capacity and starch accumulation. Furthermore, the synergistic effect of 5‰-15‰ salt concentrations also promotes the upregulation of genes related to the starch synthesis pathway. Compared to cultivation under a constant salinity of 30‰, this synergistic effect increases ammonia nitrogen removal rates and starch accumulation by up to 23.2% and 58.4%, respectively.

[0023] The seawater chlorella cultured by the method of the present invention has an ammonia nitrogen removal efficiency of 93.60% at an ammonia nitrogen concentration of 3 mg / L and a salinity of 5‰, and the starch accumulation of the seawater chlorella is 10.06 μg / 10 6 cells.

[0024] The seawater chlorella cultured by the method of the present invention has good application prospects in the treatment of ammonia nitrogen pollution in tidal waters.

[0025] Beneficial effects of the present invention: (1) The method of the present invention can significantly improve the ammonia nitrogen absorption capacity of marine Chlorella, and obtain immobilized marine Chlorella with a high ammonia nitrogen removal rate. At an ammonia nitrogen concentration of 3 mg / L and a salinity of 5‰, the ammonia nitrogen removal rate exceeded 93% within seven days. Compared with the culture at a constant salinity of 30‰, the 5‰ salinity co-culture increased the ammonia nitrogen removal rate by 23.2%.

[0026] (2) The method of the present invention can significantly improve the starch accumulation capacity of marine Chlorella. At an ammonia nitrogen concentration of 3 mg / L and a salinity of 5‰, the starch accumulation amount is 10.06 μg / 10 6 Compared with the culture at a constant salinity of 30‰, the 5‰ salt co-culture increased starch accumulation by 58.4%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 3 shows the changing trend of ammonia nitrogen removal efficiency of Chlorella vulgaris during the culture period of the baffled flow reactor, where (a) the hydraulic retention time is 24 h and (b) the hydraulic retention time is 48 h.

[0028] Figure 2 Total starch content of Chlorella vulgaris on the first and seventh days of the culture cycle, where (a) the hydraulic retention time is 24 h and (b) the hydraulic retention time is 48 h.

[0029] Figure 3 This is the effect of hydraulic retention time and salt synergy on the maximum ammonia nitrogen removal rate of seawater Chlorella within seven days.

[0030] Figure 4 The effects of hydraulic retention time and salt synergy on starch accumulation of marine Chlorella.

[0031] Figure 5 The effects of salt synergy degree and initial ammonia nitrogen concentration on the ammonia nitrogen removal rate of seawater Chlorella.

[0032] Figure 6 The effects of salt synergy and initial ammonia nitrogen concentration on starch accumulation in seawater Chlorella vulgaris.

[0033] Figure 7 This is the effect of salt-free conditions on the removal rate of ammonia nitrogen by Chlorella vulgaris in seawater.

[0034] Figure 8 This is the effect of 45‰ salinity on the removal rate of ammonia nitrogen by Chlorella in seawater.

[0035] Figure 9 The effect of different salinity conditions on the ammonia nitrogen removal rate of Chlorella vulgaris in seawater.

[0036] Figure 10 The effect of different salinity conditions on the starch accumulation of Chlorella vulgaris in seawater.

[0037] Figure 11 Schematic diagram of the nitrogen metabolism pathway showing the effect of salt synergy on the expression of key nitrogen metabolism genes in the examples.

[0038] Figure 12 This is a graph showing the differential expression of key nitrogen metabolism genes at 5‰ salinity vs. 30‰ salinity in the example.

[0039] Figure 13 Schematic diagram of the starch synthesis pathway showing the synergistic effects of salt on key genes for starch synthesis.

[0040] Figure 14 This is a graph showing the differential expression of key genes for starch synthesis at 5‰ salinity vs. 30‰ salinity in the example. DETAILED DESCRIPTION

[0041] The following, combined with specific examples, further illustrates the present invention's method for cultivating marine Chlorella with high ammonia nitrogen removal rates and high starch accumulation capabilities. It should be understood that the materials and methods used in the examples, unless otherwise noted, are conventional in the art, and these materials can be purchased from commercial sources.

[0042] The Chlorella vulgaris seed solution described in the examples of the present invention was obtained from the Yunkewei Experimental Supplies Business Department of the Chinese Academy of Tropical Agricultural Sciences-Haikou Qiongshan, No. 4, Xueyuan Road, Longhua District, Haikou City, Hainan Province.

[0043] The present invention adopts a batch culture method to cultivate marine Chlorella. First, in the first stage, the marine Chlorella is cultured under the conditions of sufficient nitrogen source (ammonia nitrogen concentration 8-12 mg / L) and appropriate salinity (30‰-33‰) to the logarithmic growth phase. The culture time is generally 7-10 days. At this time, the OD of the marine Chlorella is 0.05. 680 is about 0.3, and its cell density is about 10 6 cells / ml; then, a 1.5%-2% sodium alginate solution is mixed with a seawater Chlorella solution at a ratio of 1:1-1.5, and then slowly and continuously dripped into a 2%-3% calcium chloride solution using a peristaltic pump. The immobilized algae balls in the calcium chloride solution are then stored in a 4°C refrigerator for 24-48 hours. During this process, the volume ratio of the mixed solution to the calcium chloride solution is 1:1-1.5. In the second stage of cultivation, the immobilized algae balls are placed in a baffled flow reactor with continuous water inflow and cultured at an ammonia nitrogen concentration of 2-5 mg / L and salinity of 5‰, 15‰, 25‰, or 35‰. Cultivating seawater Chlorella in a baffled flow reactor effectively simulates wastewater treatment systems and can effectively reflect the ammonia nitrogen removal efficiency of seawater Chlorella in actual applications.

[0044] The culture conditions for the first stage of culturing Chlorella vulgaris in the embodiments of the present invention are as follows: Chlorella vulgaris is suspended in a conical flask at a temperature of 25±1°C, placed in a shaker at 180 rpm (12 hours apart) for 5 minutes in the morning and evening, with a light intensity of 6000 lux, a light-dark ratio of 12h:12h, a pH of 7.0-7.5, and a culture time of 7-10 days.

[0045] The culture conditions for the second stage of culturing Chlorella vulgaris in the embodiment of the present invention are as follows: the Chlorella vulgaris is in an immobilized algae ball state, cultured in a baffled flow reactor, at a temperature of 25±1°C, with air aerated for 5 minutes in the morning and evening (12 hours apart), a light intensity of 6000 lux, a light-dark ratio of 12h:12h, a pH of 7.0-7.5, and a culture time of 7-10 days.

[0046] The baffled flow reactor described in the embodiments of the present invention is designed with two sedimentation tanks within the reactor to hold the immobilized algae pellets. Water flows upward through these two tanks. As the water rises, the immobilized algae pellets come into full contact with the water flow, allowing the algae within the pellets to effectively absorb ammonia nitrogen from the water, enabling rapid growth and thus achieving ammonia nitrogen removal and biomass accumulation.

[0047] Example 1: The synergistic effect of 5‰ salt enhances the ability of immobilized seawater Chlorella to absorb ammonia nitrogen and accumulate starch A method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity comprises the following steps: (1) The first stage of cultivation of Chlorella vulgaris Artificial seawater culture medium consists of: 100 ml seawater Chlorella seed solution, OD 680 The concentration of KH2PO4 solution, NH4HCO3 solution and CH3COONa solution in the culture medium is 22 mg / L, 56.4 mg / L and 20 mg / L respectively. The concentration of ammonia nitrogen and total phosphorus are 10 mg / L and 5 mg / L respectively. The chlorella in the logarithmic growth phase is collected after 7-10 days of culture. At this time, the OD of the chlorella is 0.25-0.3. 680 About 0.3, cell density is about 10 6 cells / mL; Artificial seawater was prepared by dissolving 33 g of sea salt in 1 L of deionized water to obtain artificial seawater with a salinity of 30‰. The formula of sea salt (per 100 g) was NaCl 62.26 g, MgSO4·7H2O 14.39 g, MgCl2·6H2O 12.76 g, CaCl2 3.2656 g, KCl 1.74 g, NaHCO3 0.49 g, SrCl2 0.046 g, KBr 0.026 g, and MnSO4 0.0094 g.

[0048] Culture conditions: Chlorella vulgaris was suspended in a conical flask at 25±1°C, shaken twice a day at 180 rpm (12h interval) for 5 min, pH 7.0-7.5, light intensity 6000 lux, and a light-dark ratio of 12h:12h.

[0049] After the seawater Chlorella seed solution, i.e. the initial algae solution, is cultured in the artificial seawater medium for 7-10 days, the seawater Chlorella culture solution is obtained, and its OD 680 is about 0.3, and its cell density is about 10 6 cells / ml.

[0050] (2) Preparation of immobilized algae balls Take 400 ml of seawater Chlorella culture medium and mix it with 400 ml of 2% sodium alginate solution in a ratio of 1:1.

[0051] Preparation of sodium alginate solution: The preparation of sodium alginate solution needs to be carried out in a 95℃ boiling water bath. Take 400ml of deionized water and place it in a beaker with a scale line. Place it in a water bath and heat it to 95℃. Then, slowly add 8g of sodium alginate while stirring continuously. After the sodium alginate is completely dissolved, stop heating; add an appropriate amount of deionized water to make the volume of sodium alginate solution 400ml.

[0052] Preparation of immobilized algae balls: The seawater Chlorella culture fluid obtained from the first stage of cultivation was mixed with the cooled 2% sodium alginate solution in a ratio of 1:1, and then the mixture was slowly and continuously dripped into a 3% calcium chloride solution using a peristaltic pump. The immobilized algae balls in the calcium chloride solution were placed in a 4°C refrigerator for 24 hours to allow them to fully set.

[0053] (3) Second stage culture of baffled reactor The immobilized algae pellets were added to a 2-liter baffled flow reactor at a rate of 2,000 pellets per liter of artificial seawater. By replacing the artificial seawater with nitrogen-restricted artificial seawater, the ammonia nitrogen concentration decreased from 10 mg / L to 2 mg / L, and the total phosphorus concentration decreased from 5 mg / L to 1 mg / L.

[0054] The mass of sea salt in the preparation of artificial seawater is 5.45g, making the salinity 5‰. In addition, the NH4HCO3 concentration is reduced to 12mg / L, making the ammonia nitrogen concentration of artificial seawater 2mg / L; the KH2PO4 concentration is reduced to 4.4mg / L, making the total phosphorus concentration of artificial seawater 1mg / L.

[0055] Culture conditions: Chlorella vulgaris was immobilized in the marine reactor and continuously fed with water. The temperature was 25±1℃. Air was pumped into the reactor every 12 hours (once in the morning and once in the evening) for 5 minutes each time. The algae balls were turned over twice to ensure uniform illumination as much as possible. The illumination intensity was 6000 lux and the light-dark ratio was 12h:12h.

[0056] Reactor operating conditions: The reactor is continuously fed with water and the hydraulic retention time is 24 h.

[0057] (4) Determination of ammonia nitrogen in effluent Sampling was performed at the outlet of the baffled flow reactor every other day. The water samples were centrifuged at 4000 r / min for 5 min, and the supernatant was removed. The supernatant was filtered through a 0.45 μm microporous filter membrane to obtain the test sample. The ammonia nitrogen concentration in the test water sample was determined by Nessler's reagent spectrophotometry.

[0058] (5) Dissolution of immobilized algae balls Take 50 algae pellets, add 25 ml of 5% sodium citrate solution, place on a homogenizer, and mix at 700 rpm for 30 minutes to fully dissolve the pellets. Centrifuge the dissolved pellet mixture at 10,000 rpm for 5 minutes, remove the supernatant, and retain the algal cells. Rinse three times with PBS solution, then add 10 ml of PBS solution to the algal cells to obtain the test solution. To prepare the PBS solution, add 8.0 g of NaCl, 0.24 g of KH2PO4, and 1.44 g of Na2HPO4 to 1 L of steam-sterilized deionized water. Mix thoroughly and adjust the pH to 7.0-7.5 to obtain the PBS solution.

[0059] (6) Determination of the number of Chlorella cells in seawater Take 5ml of the algae solution to be tested and measure its OD using a UV spectrophotometer 680 The number of Chlorella vulgaris cells was calculated according to the following formula (1): Seawater Chlorella (10 6 cells / mL) =3.43×OD 680 -0.0182 (R 2 =0.994) Formula (1) Where R 2 Represents the degree of curve fitting.

[0060] (7) Determination of starch content Take 5ml of the algae solution to be tested, centrifuge at 10000r for 5min, remove the supernatant, and retain the algae cells; rinse the algae cells 3 times with 95% anhydrous ethanol to remove interfering substances; add 2ml PBS solution to the treated algae cells, heat in an 80℃ water bath for 30 minutes, further extract the soluble sugar in the cells, centrifuge again (12360g, 5min) to take the precipitate, and obtain algae cells without soluble sugar and photosynthetic pigment interference; use 10% hydrochloric acid to heat in a 95℃ boiling water bath for 30min to extract starch and fully convert the starch into soluble sugar. Use the Solebo kit to determine the glucose concentration. The determination process is as follows: 200μL of the solution to be tested, 200μL of distilled water, 100μL of anthrone working solution and 1ml of concentrated sulfuric acid are added to the determination tube in sequence, heated in a 95℃ water bath for 10min, and then the A 620 (i.e. absorbance at 620nm), and then according to the standard curve, the glucose concentration was obtained.

[0061] Soluble sugar content (mg / L) = 120.07×A 620 -1.2551(R 2 =0.9996) formula (2) Where R 2 Represents the degree of curve fitting.

[0062] Example 2: The synergistic effect of 15‰ salt concentration on the ability of immobilized seawater Chlorella to absorb ammonia nitrogen and accumulate starch A method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity comprises the following steps: (1) First stage cultivation of Chlorella vulgaris. Same as Example 1; (2) Preparation of immobilized algae balls. Same as Example 1; (3) The immobilized algae balls are cultured in the baffled flow reactor for the second stage: The immobilized algae pellets were added to a 2-liter baffled flow reactor at a rate of 2,000 pellets per liter of artificial seawater. By replacing the artificial seawater with nitrogen-restricted artificial seawater, the ammonia nitrogen concentration decreased from 10 mg / L to 2 mg / L, and the total phosphorus concentration decreased from 5 mg / L to 1 mg / L.

[0063] The mass of sea salt in the preparation of artificial seawater is 16.36g, and the artificial seawater salinity is prepared to be 15‰. In addition, the amount of NH4HCO3 added is reduced to 12mg / L, so that the ammonia nitrogen concentration of the artificial seawater is 2mg / L; the amount of KH2PO4 added is reduced to 4.4mg / L, so that the total phosphorus concentration of the artificial seawater is 1mg / L.

[0064] Culture conditions: Chlorella vulgaris was immobilized in the marine reactor and continuously fed with water. The temperature was 25±1℃. Air was pumped into the reactor every 12 hours (once in the morning and once in the evening) for 5 minutes each time. The algae balls were turned over twice to ensure uniform illumination as much as possible. The illumination intensity was 6000 lux and the light-dark ratio was 12h:12h.

[0065] (4) Determination of effluent ammonia nitrogen: same as in Example 1; (5) Dissolution of immobilized algae balls: same as in Example 1; (6) Determination of the number of Chlorella vulgaris cells: Same as in Example 1; (7) Determination of starch content: Same as Example 1.

[0066] Example 3: 25‰ salt concentration synergistically enhances the ability of immobilized seawater Chlorella to absorb ammonia nitrogen and accumulate starch A method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity comprises the following steps: (1) First stage cultivation of Chlorella vulgaris: same as in Example 1; (2) Preparation of immobilized algae balls: same as in Example 1; (3) The immobilized algae balls are cultured in the baffled flow reactor for the second stage: The immobilized algae pellets were added to a 2-liter baffled flow reactor at a rate of 2,000 pellets per liter of artificial seawater. By replacing the artificial seawater with nitrogen-restricted artificial seawater, the ammonia nitrogen concentration decreased from 10 mg / L to 2 mg / L, and the total phosphorus concentration decreased from 5 mg / L to 1 mg / L.

[0067] The mass of sea salt in the preparation of artificial seawater is 27.27g, and the artificial seawater salinity is prepared to be 25‰. In addition, the amount of NH4HCO3 added is reduced to 12mg / L, so that the ammonia nitrogen concentration of the artificial seawater is 2mg / L; the amount of KH2PO4 added is reduced to 4.4mg / L, so that the total phosphorus concentration of the artificial seawater is 1mg / L.

[0068] Culture conditions: Chlorella vulgaris was immobilized in the marine reactor and continuously fed with water. The temperature was 25±1℃. Air was pumped into the reactor every 12 hours (once in the morning and once in the evening) for 5 minutes each time. The algae balls were turned over twice to ensure uniform illumination as much as possible. The illumination intensity was 6000 lux and the light-dark ratio was 12h:12h.

[0069] (4) Determination of effluent ammonia nitrogen: same as in Example 1; (5) Dissolution of immobilized algae balls: same as in Example 1; (6) Determination of the number of Chlorella vulgaris cells: Same as in Example 1; (7) Determination of starch content: Same as Example 1.

[0070] Depend on Figure 1 It can be seen that under HRT=24h, 5‰ salinity, 15‰ salinity, 25‰ salinity and 30‰ salinity (30‰ salinity is the most suitable salinity for seawater Chlorella, so the seawater Chlorella cultured at 30‰ salinity is used as the control group. The culture conditions are basically the same as those in Example 1, except for step (3). In the 30‰ salinity control group, step (3) does not change the artificial seawater culture medium conditions), the highest ammonia nitrogen removal rates are 80.9%, 80.1%, 68.4% and 64.3% respectively. The results show that the synergistic conditions of 5‰ and 15‰ salt have a significant promoting effect on the ammonia nitrogen removal rate of seawater Chlorella. Figure 2 It can be seen that at HRT=24h, 5‰ salinity, 15‰ salinity, 25‰ salinity and 30‰ salinity, the total starch accumulation (i.e. the difference between the starch content on the seventh day and the starch content on the first day) can reach a maximum of 8.52, 8.25, 6.96 and 6.14 μg / 10 6 Under salinity conditions of 5‰, 15‰, and 25‰, the starch accumulation of Chlorella vulgaris increased by 38.8%, 34.4%, and 13.4%, respectively. This result indicates that the synergistic salt conditions of 5‰ and 15‰ have a significant promoting effect on the starch accumulation of Chlorella vulgaris.

[0071] Example 4 The hydraulic retention time of the reactor during the second stage of cultivation was extended to investigate the effect of the extended time on the ability of marine Chlorella to absorb ammonia nitrogen and accumulate starch.

[0072] In this example, the operating steps of the above examples 1-3 were followed, and four types of nitrogen-limited artificial seawater were set, with salinities of 5‰, 15‰, 25‰, and 30‰, respectively. The hydraulic retention time of the reactor during the second stage of cultivation in the baffled flow reactor in step (3) was extended to 48 h, and seawater Chlorella with four different salinities (5‰, 15‰, 25‰, and 30‰) was obtained by culturing at HRT = 48 h.

[0073] The determination of ammonia nitrogen and starch content in the effluent was carried out using the same operating methods as in Example 1.

[0074] Depend on Figure 3 It can be seen that when HRT=48h, compared with HRT=24h, the ammonia nitrogen removal rates at 5‰ salinity, 15‰ salinity, 25‰ salinity and 30‰ salinity increased from 80.9% to 90.5%, 80.1% to 91.5%, 68.4% to 86.6%, and 64.3% to 76.6%, respectively. Figure 4 It can be seen that when HRT=48h, compared with HRT=24h, the total starch content at 5‰ salinity, 15‰ salinity, 25‰ salinity and 30‰ salinity increased from 8.52μg / 10 6 cells increased to 9.53 μg / 10 6 cells, 8.25 μg / 10 6 cells increased to 9.61μg / 10 6 cells, 6.96 μg / 10 6 cells increased to 8.09μg / 10 6 cells, 6.14 μg / 10 6 cells increased to 6.67μg / 10 6 cells.

[0075] The above results show that extending the hydraulic retention time to 48h helps marine Chlorella to fully absorb ammonia nitrogen, while enhancing the nitrogen-limited environment, thereby significantly improving the starch accumulation capacity of marine Chlorella.

[0076] The present invention systematically explored the effects of a series of factors on the ammonia nitrogen removal rate and starch accumulation of marine Chlorella through experiments (Examples 5 to 12). These factors included different initial ammonia nitrogen concentrations, different salt synergy levels, and different time (hydraulic retention time) conditions.

[0077] Example 5 By varying the ammonia nitrogen concentration during the second stage of cultivation, the effect of nitrogen limitation on the ammonia nitrogen absorption capacity and starch production of marine Chlorella was investigated. Following the cultivation method of Example 1, the concentrations of the NH₄HCO₃ solution during the second stage of cultivation were varied to 11.3 mg / L, 17 mg / L, 22.6 mg / L, and 28.2 mg / L, respectively, while the concentrations of the KH₂PO₄ solution were 4.4, 6.6, 8.8, and 11 mg / L, respectively. Specifically, during the second stage of cultivation, the ammonia nitrogen concentrations of the culture medium were 2, 3, 4, and 5 mg / L, respectively, while the total phosphorus concentrations were 1, 1.5, 2, and 2.5 mg / L, respectively. The artificial seawater was supplemented with 5.45 g / L of sea salt, had a salinity of 5‰, and a hydraulic retention time of 48 hours.

[0078] Example 6 Compared with Example 5, this example only adjusts the salinity to 15‰, that is, the amount of sea salt added to the artificial seawater is 16.36 g / L, and other conditions remain unchanged.

[0079] Example 7 Compared with Example 5, this example only adjusts the salinity to 25‰, that is, the amount of sea salt added to the artificial seawater is 27.27 g / L, and other conditions remain unchanged.

[0080] Example 8 Compared with Example 5, this example only adjusts the salinity to 35‰, that is, the amount of sea salt added to the artificial seawater is 38.18 g / L, and other conditions remain unchanged.

[0081] Example 9 Compared with Example 1, this example does not change the state of the artificial seawater culture medium during the first stage of cultivation. The salinity is maintained at 30‰ and the cultivation is continued, while other conditions remain unchanged.

[0082] Example 10 Compared with Example 1, this example does not change the state of the artificial seawater culture medium during the first stage of cultivation, and the salinity is maintained at 30‰ for continued cultivation, but the hydraulic retention time is adjusted to 48 hours. The other conditions are the same as in Example 1.

[0083] Example 11 Compared with Example 1, this example sets the ammonia nitrogen concentration in the second stage of cultivation to 3 mg / L, the salinity to 0‰ (salt-free condition), and the hydraulic retention time to 48 h. The remaining conditions are the same as in Example 1.

[0084] Example 12 Compared with Example 1, this embodiment sets the ammonia nitrogen concentration in the second stage of cultivation to 3 mg / L, the salinity to 45‰, and the hydraulic retention time to 48 hours. The remaining conditions are the same as those in Example 1.

[0085] The ammonia nitrogen absorption capacity and starch production capacity of the marine Chlorella cultured in Examples 5 to 12 are shown in Table 1.

[0086] Table 1

[0087] From Table 1, Figure 5 、 Figure 6 The results showed that the salt synergistic effect of Chlorella vulgaris in the 5‰, 15‰ and 25‰ salinity groups was significant. When the hydraulic retention time was 48h and the ammonia nitrogen concentration was 2mg / L, the ammonia nitrogen removal rate reached 90.5%, 91.5% and 80.6% respectively, and the starch accumulation was 9.53, 9.61 and 7.98μg / 10 6 cells, compared with the control group at 30‰ salinity with an ammonia nitrogen removal rate of 76.6% and starch accumulation of 6.67μg / 10 6 cells, the synergistic effect of salt concentrations of 5‰, 15‰ and 25‰ increased the ammonia nitrogen removal rate by 18.1%, 19.5% and 5.2%, respectively, and increased the starch accumulation by 42.9%, 44.1% and 19.6%, respectively. The synergistic effect of salt concentrations of 5‰ and 15‰ was the most significant. When the ammonia nitrogen concentration was 3 mg / L, the seawater Chlorella vulgaris had the highest ammonia nitrogen removal efficiency of 93.60% and the maximum starch accumulation of 10.06 μg / 10 6 cells, compared with the 30‰ salinity control group, its ammonia nitrogen removal rate and starch accumulation increased by 23.2% and 58.4% respectively. Its salt synergistic effect was significantly better than that of the other salinity groups. As the ammonia nitrogen concentration continued to increase to 5 mg / L, the 5‰ salinity group always had the highest ammonia nitrogen removal rate and starch accumulation. Therefore, 5‰ salinity can enable marine Chlorella to obtain the highest ammonia nitrogen absorption efficiency and starch accumulation capacity. The salinity in the range of 15‰-25‰ also has a certain promoting effect, but the effect is not as good as 5‰ salinity. 35‰ salinity has a certain stress effect on marine Chlorella, and its ammonia nitrogen removal rate is reduced. As the ammonia nitrogen concentration continues to increase, the promoting effect of the 5‰ salt synergistic effect first increases and then decreases. When the ammonia nitrogen concentration is 3 mg / L, the 5‰ salt synergistic effect coupled with the nitrogen limitation effect significantly enhances the ammonia nitrogen removal efficiency and starch accumulation of marine Chlorella.

[0088] Depend on Figure 7 It can be seen that the growth of marine Chlorella stagnates under salt-free conditions (0‰) and there is almost no ammonia nitrogen removal effect.

[0089] Depend on Figure 8 It can be seen that the early growth of Chlorella vulgaris is restricted under the salinity of 45‰, but it can still grow at this salinity, and the highest ammonia nitrogen removal rate within 7 days is only 48.6%.

[0090] Depend on Figure 9 and Figure 10 It can be seen that under the initial ammonia nitrogen concentration of 3 mg / L, the ammonia nitrogen removal rate and starch accumulation of Chlorella in the salinity range of 5‰-15‰ are significantly improved compared with the salinity of 30‰, while salt-free conditions (0‰) and high-salt conditions (greater than 30‰) inhibit its ammonia nitrogen removal rate and starch accumulation.

[0091] In general, the salinity of 30‰ as the control group is consistent with the salinity of the first stage of cultivation, so there is no salt synergistic effect. The salinity of 5‰, 15‰ and 25‰ appropriately reduced the salinity in the second stage of cultivation, and then showed a certain salt synergistic effect, among which the salt synergistic effect in the salinity range of 5‰-15‰ was more significant. When the salinity continued to decrease to no salinity (0‰), it would significantly inhibit the growth of marine Chlorella, and there was almost no ammonia nitrogen removal effect; when the salinity continued to rise to above 35‰, the salt stress effect was more significant, and there was no synergistic effect, and the higher the salinity, the more significant the inhibitory effect on the ammonia nitrogen removal rate of marine Chlorella. Therefore, marine Chlorella can obtain the highest ammonia nitrogen removal rate of 93.6% and starch accumulation of 10.06μg / 10 under the second stage culture conditions of salinity of 5‰ and ammonia nitrogen concentration of 3mg / L. 6 cells.

[0092] Depend on Figure 11 It is known that ammonia nitrogen is the nitrogen source directly used by marine Chlorella, while nitrate nitrogen and nitrite nitrogen need to be converted into ammonia nitrogen in the body of Chlorella before they can be used. Ammonia nitrogen is used in the synthesis of nucleic acids and amino acids in Chlorella. Ammonia nitrogen enters the cell through AMT (ammonium transporter) and is then fixed into glutamine through the gdhA gene. Subsequently, the required amino acids are synthesized under the action of various genes. Figure 12 As shown (* indicates P < 0.05, indicating significant differential gene expression; *** indicates P < 0.001, indicating extremely significant differential gene expression), 5‰ salt exposure increased AMT by 2.49 units and gdhA (nitrogen assimilation) by 4.34 units. The FDR analysis showed that AMT and gdhA showed significant changes under 5‰ salt exposure, indicating that ammonia nitrogen absorption and assimilation were significantly enhanced under salt exposure. This demonstrates at the molecular level that 5‰ salt exposure significantly promotes ammonia nitrogen absorption in Chlorella vulgaris. Although changes in other key genes were not significant, they also provide some data support for the enhanced nitrogen metabolism process.

[0093] Depend on Figure 13 It is known that marine Chlorella fixes carbon dioxide into a usable carbon source through the Calvin cycle, then converts the carbon source into glucose through a series of steps of glycolysis, and finally uses glucose to synthesize starch under the action of a series of genes such as glgC and glgA. Figure 14 It can be seen (** indicates P < 0.01, indicating that the gene differential expression results are very significant, *** indicates P < 0.001, indicating that the gene differential expression results are extremely significant), the 5‰ salt synergistic condition causes rbcS (determines the carbon fixation process) to increase by 4.16 units, and glgA (starch synthase) to increase by 1.03 units. The FDR results of the significance analysis show that the changes in rbcS and glgA are significant under the 5‰ salt synergistic condition. Therefore, the dark reaction of photosynthesis-carbon fixation process is significantly enhanced under salt synergy. At the same time, the regulatory genes pfp and pgm of key precursors in the starch synthesis pathway are all upregulated to a certain extent. Combined with the actual measurement results of starch, this effectively explains the promoting effect of 5‰ salt synergy on starch accumulation in marine Chlorella.

Claims

1. A method for cultivating marine Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity, characterized in that: A batch culture method is adopted. First, marine Chlorella is cultured under the conditions of salinity 30‰-33‰ and ammonia nitrogen concentration 8-12 mg / L. After the marine Chlorella grows to the logarithmic growth period, it is immobilized and embedded in sodium alginate. Then, the immobilized algae balls are cultured under the conditions of salinity 5‰-15‰ and ammonia nitrogen concentration 2-3 mg / L. Finally, marine Chlorella with high ammonia nitrogen removal rate and high starch accumulation ability is obtained.

2. The method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity according to claim 1, characterized in that: The following steps are involved: (1) Inoculating the seawater Chlorella seed liquid into an artificial seawater culture medium with a salinity of 30‰-33‰ and an ammonia nitrogen concentration of 8-12 mg / L for the first stage of cultivation, maintaining the light and temperature for cultivation, and obtaining the seawater Chlorella liquid in the logarithmic growth phase; (2) mixing the seawater chlorella liquid obtained in step (1) with a sodium alginate solution, dripping the mixed solution into a calcium chloride solution to obtain algae balls, and refrigerating the algae balls to solidify them, thereby obtaining immobilized algae balls; (3) The immobilized algae balls obtained in step (2) are cultured in an artificial seawater medium for the second stage, the salinity of the artificial seawater medium is changed to 5‰-15‰, and the ammonia nitrogen concentration is reduced to 2-3 mg / L, and the light and temperature are maintained for cultivation; (4) The immobilized algae balls obtained in step (3) are dissolved in a sodium citrate solution, and centrifuged to obtain the seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity.

3. The method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity according to claim 2, characterized in that: In step (1), the formula of the artificial seawater culture medium used in the first stage culture includes NaCl 20-22 g / L, MgSO4·7H2O 4.5-5.0 g / L, MgCl2·6H2O 4.0-4.5 g / L, CaCl2 1.0-1.2 g / L, KCl 0.5-0.6 g / L, NaHCO3 0.2-0.3 g / L, SrCl2 0.01-0.02 g / L, KBr 5-10 mg / L, MnSO4 3.0-3.3 mg / L and CH3COONa 15-25 mg / L, NH4HCO3 45-67 mg / L, KH2PO4 18-27 mg / L.

4. The method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity according to claim 2, characterized in that: In step (1), the inoculation volume ratio of the seawater Chlorella seed liquid is 10%-20%; the seawater Chlorella seed liquid is cultured in artificial seawater medium for 7-10 days to obtain the seawater Chlorella liquid, and its OD 680 It is 0.25-0.

3.

5. The method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity according to claim 2, characterized in that: In step (1) and step (3), the light intensity is 4000-8000 lux; the temperature is 25±1°C; during the culture process, the daily light duration is controlled to be 10-12 hours, the pH is 7.0-7.5, and the culture time is 7-10 days.

6. The method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity according to claim 2, characterized in that: In step (2), the mass fraction of the sodium alginate solution is 1.5%-2%, and the volume ratio of the seawater Chlorella solution to the sodium alginate solution is 1:1-1.5; the mass fraction of the calcium chloride solution is 2%-3%, and the volume ratio of the mixed solution to the calcium chloride solution is 1:1-1.

5.

7. The method for cultivating marine Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity according to claim 2, characterized in that: In step (3), the formula of the artificial seawater culture medium used for the second stage culture includes NaCl3.3-11 g / L, MgSO4·7H2O 1.5-2.5 g / L, MgCl2·6H2O 1.3-2.3 g / L, CaCl20.33-0.6 g / L, KCl 0.2-0.3 g / L, NaHCO30.2-0.3 g / L, SrCl2 0.01-0.02 g / L, KBr 5-10 mg / L, MnSO43.0-3.3 mg / L and CH3COONa 15-25 mg / L, NH4HCO312-17 mg / L, KH2PO44.4-6.6 mg / L.

8. The method for cultivating marine Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity according to claim 2, characterized in that: In step (3), the second stage of cultivation is carried out in a baffled flow reactor, and the hydraulic retention time of the baffled flow reactor with continuous water inflow is 24-48 hours; in step (4), the mass fraction of the sodium citrate solution is 4%-6%.

9. A marine Chlorella having a high ammonia nitrogen removal rate and a high starch accumulation capacity obtained by the cultivation method according to any one of claims 1 to 8.

10. Use of the marine Chlorella vulgaris with high ammonia nitrogen removal rate and high starch accumulation capacity as claimed in claim 9 in the treatment of ammonia nitrogen pollution in tidal waters.

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