A seawater chlorella with high ammonia-nitrogen removal rate and high starch accumulation capacity, and a culture method and application thereof

Abstract

The application discloses seawater chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity and a culture method and application thereof. In view of the problem that the growth rate is reduced due to salt stress when traditional biological remediation technology is applied to a tidal area, thereby leading to reduction of ammonia nitrogen treatment efficiency. The method uses seawater chlorella with strong adaptability, significantly improves the ammonia nitrogen absorption capacity and starch accumulation amount of seawater chlorella through salt synergistic effect and combined with immobilization technology, thereby realizing effective governance of ammonia nitrogen pollution in the tidal area. Under the condition of 5‰ salinity and 3 mg / L ammonia nitrogen concentration, the ammonia nitrogen removal rate of seawater chlorella reaches 93.60%, and the starch accumulation amount reaches 10.06 μg / 10 6 cells, and the ammonia nitrogen removal rate and starch accumulation amount are increased by 23.2% and 58.4% respectively. The method has the advantages of simple process, high efficiency, strong environmental adaptability and the like, and is suitable for ammonia nitrogen pollution governance and microalgae resource utilization in the tidal area.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microalgae, and particularly relates to a seawater chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity, and a culture method and application thereof. BACKGROUND

[0002] Tidal river refers to an area with significant salinity fluctuations. Influenced by hydrological conditions and seasonal factors, the upstream range of saltwater intrusion in these areas varies greatly, forming different salinity regions: low salinity region (0.5‰-5‰), medium salinity region (5‰-18‰), and high salinity region (18‰-30‰). As a transitional area between terrestrial and marine ecosystems, tidal rivers harbor rich biodiversity, but are sensitive to environmental changes, especially the impact of nutrients such as ammonia and phosphorus.

[0003] Ammonia nitrogen generally refers to nitrogen in the form of free ammonia nitrogen (NH3) and ammonium ion (NH4 + ), which is present in high concentrations in domestic wastewater, industrial wastewater, and aquaculture wastewater. Inorganic nutrients discharged into estuaries cannot be effectively removed, leading to the accumulation of inorganic nutrients. Currently, in tidal rivers, inorganic nitrogen exceeds the standard, and ammonia nitrogen is one of the main pollutants in tidal rivers.

[0004] In the water remediation technology of tidal rivers, biological technology is widely used. Common organisms include bacteria, fungi, microorganisms, aquatic plants, macroalgae, and microalgae. However, due to the characteristics of salinity fluctuations and low carbon-nitrogen ratio in tidal rivers, microorganisms are not suitable for use in tidal rivers due to their insufficient salt tolerance and high demand for carbon sources. Canna and reed are widely used in low salinity regions (0.5‰-5‰), but when the salinity exceeds 5‰, the growth of reed and canna is significantly inhibited, thereby significantly affecting their effectiveness in water remediation. Ulva mainly grows in tidal rivers such as estuaries and bays, and has strong salt tolerance, capable of adapting to salinity changes of 10‰-30‰. It also has strong inorganic nitrogen assimilation capacity, but its growth is significantly inhibited when the salinity is below 10‰, such as at 5‰ salinity, its ammonia nitrogen removal efficiency is only 52.8%.

[0005] Microalgae are a highly diverse group of photosynthetic organisms that can use light energy to split water into molecular oxygen and protons, and convert inorganic carbon (CO2) into carbohydrates, ultimately forming beneficial biomass - starch. As the oldest group of photosynthetic organisms, microalgae grow rapidly using sunlight, carbon and nitrogen sources. Among them, the ammonia nitrogen absorption capacity of Chlorella vulgaris is extremely outstanding, and it also has the advantages of fast growth rate, not occupying agricultural land, strong environmental adaptability, and rich in various bioactive molecules, and is widely concerned worldwide as a raw material for biofuel production, functional food and feed supplement.

[0006] In view of the problem that the traditional biological remediation technology is subjected to the stress effect caused by the decrease of salinity in the process of treating nutrient salt in a tidal area, thereby resulting in poor ammonia nitrogen removal efficiency, the application utilizes a culture method combining salt synergistic effect and immobilization technology, thereby obtaining Chlorella vulgaris with high ammonia nitrogen removal rate and high starch accumulation capacity, which can be applied to the ammonia nitrogen removal and co-production of starch in a tidal area and a salt-containing water body, and realizes the organic integration of wastewater treatment and biological resource technology. SUMMARY

[0007] In view of the problem that the traditional biological remediation technology is subjected to the stress effect caused by the decrease of salinity in the process of treating nutrient salt in a tidal area, thereby resulting in poor ammonia nitrogen removal efficiency, the application utilizes a culture method combining salt synergistic effect and immobilization technology, thereby obtaining Chlorella vulgaris with high ammonia nitrogen removal rate and high starch accumulation capacity, which can be applied to the ammonia nitrogen removal and co-production of starch in a tidal area and a salt-containing water body, and realizes the organic integration of wastewater treatment and biological resource technology.

[0008] The above object of the application is achieved by the following technical scheme.

[0009] The application provides a culture method of Chlorella vulgaris with high ammonia nitrogen removal rate and high starch accumulation capacity, which comprises the following steps:

[0010] In the batch culture mode, first, Chlorella vulgaris is cultured under the conditions of suitable salinity (30 ‰-33 ‰) and sufficient nitrogen source (ammonia nitrogen concentration 8-12 mg / L), and then the Chlorella vulgaris is immobilized by sodium alginate after growing to the logarithmic growth phase, and then the immobilized Chlorella is cultured under the conditions of salt synergy (salinity 5 ‰-15 ‰) and nitrogen source limitation (ammonia nitrogen concentration 2-3 mg / L), and finally Chlorella vulgaris with high ammonia nitrogen removal rate and high starch accumulation capacity is obtained.

[0011] Further, the culture method specifically comprises the following steps:

[0012] (1) First-stage culture of Chlorella maris: inoculate Chlorella maris seed liquid into artificial seawater medium with sufficient nitrogen source (ammonia nitrogen concentration of 8-12 mg / L) and salinity of 30‰-33‰, and perform first-stage culture in a conical flask. Place the conical flask on a shaker, maintain appropriate light intensity and temperature, and collect Chlorella maris liquid in the logarithmic growth phase after 7-10 days of culture;

[0013] (2) Mix the Chlorella maris liquid obtained in step (1) with a sodium alginate solution, and drop the mixed solution into a calcium chloride solution to obtain algae balls. After completion, place the sodium alginate-Chlorella maris balls in a refrigerator for 24-48 h to shape the algae balls, and obtain immobilized algae balls;

[0014] (3) Place the immobilized algae balls obtained in step (2) in a continuous water inlet baffled reactor to perform second-stage culture in artificial seawater medium, while changing the salinity of the artificial seawater medium to 5‰-15‰ and reducing the ammonia nitrogen concentration to 2-3 mg / L, and maintaining the light intensity and temperature in step (1) for culture;

[0015] (4) Dissolve the immobilized algae balls obtained in step (3) in a sodium citrate solution, and centrifuge to obtain Chlorella maris with high ammonia nitrogen removal rate and high starch accumulation capacity.

[0016] Further, in step (1), the Chlorella maris seed liquid, i.e., the initial algae liquid, is cultured in the above artificial seawater medium for 7-10 days to obtain Chlorella maris liquid, and the OD 680 of the Chlorella maris liquid is 0.25-0.3.

[0017] Further, in step (1), the initial inoculation volume ratio of the Chlorella maris seed liquid is 10%-20%.

[0018] Further, in steps (1) and (3), the light intensity is 4000-8000 lux.

[0019] Further, in steps (1) and (3), the temperature is 25±1℃.

[0020] Further, in steps (1) and (3), the light time is controlled to be 10-12 h per day during the culture process, preferably with a light-dark ratio of 12h:12h, the pH is 7.0-7.5, and the culture time is 7-10 days.

[0021] Further, in step (1), the formula of the artificial seawater medium used in the first stage culture comprises 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, CH3COONa 15-25 mg / L, NH4HCO3 45-67 mg / L, and KH2PO4 18-27 mg / L.

[0022] Further, in step (2), the mass fraction of the sodium alginate solution is 1.5%-2%, the volume ratio of the Nannochloropsis oculata 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.

[0023] Further, in step (3), the formula of the artificial seawater medium used in the second stage culture comprises 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, CH3COONa 15-25 mg / L, NH4HCO3 12-17 mg / L, and KH2PO4 4.4-6.6 mg / L.

[0024] Further, in step (3), the second stage culture is carried out in a baffle reactor, and the hydraulic retention time of the continuous water feeding is 24-48 h. More preferably, the hydraulic retention time is 48 h.

[0025] Further, in step (1), the first stage culture condition is that the ammonia nitrogen concentration is 8-12 mg / L, the total phosphorus concentration is 4-6 mg / L, and the salinity is 30‰-33‰. In step (3), the second stage culture condition is that the ammonia nitrogen concentration is reduced to 2-3 mg / L, the total phosphorus concentration is reduced to 1.0-1.5 mg / L, and the salinity is reduced to 5‰-15‰, thereby generating nitrogen limitation and salt synergistic effect. In the present application, the nitrogen to phosphorus ratio (N:P) of the artificial seawater medium is always constant at 2:1, and the total phosphorus concentration is changed with the adjustment of the nitrogen source concentration.

[0026] Further, in step (4), the mass fraction of the sodium citrate solution is 4%-6%.

[0027] The present application effectively solves the problems of low growth rate and high oxidation-reduction pressure of seawater Chlorella applied to tidal flow area ecological restoration, thereby leading to low and unstable ammonia nitrogen removal rate, by utilizing the salt synergistic effect and combining with the immobilization embedding technology, so that the seawater Chlorella has high and stable ammonia nitrogen absorption capacity and starch accumulation amount. Meanwhile, the 5‰-15‰ salt synergistic effect also promotes the up-regulation of starch synthesis pathway related genes, and compared with the culture under the constant condition of suitable salinity 30‰, the salt synergistic effect makes the ammonia nitrogen removal rate and starch accumulation amount increase by 23.2% and 58.4% respectively.

[0028] The seawater Chlorella cultured by the method has an ammonia nitrogen removal efficiency of 93.60% and a starch accumulation amount of 10.06 μg / 10 6 cells under the condition of ammonia nitrogen concentration of 3 mg / L and salinity of 5‰.

[0029] The seawater Chlorella cultured by the method has good application prospect in the ammonia nitrogen pollution control in tidal flow area.

[0030] The present application has the following beneficial effects:

[0031] (1) The seawater Chlorella cultured by the method can significantly improve the ammonia nitrogen absorption capacity of seawater Chlorella, and obtain immobilized seawater Chlorella with high ammonia nitrogen removal rate, and the ammonia nitrogen removal rate is more than 93% within seven days under the condition of ammonia nitrogen concentration of 3 mg / L and salinity of 5‰. Compared with the culture under the constant salinity of 30‰, the ammonia nitrogen removal rate is increased by 23.2% under the 5‰ salt synergistic culture.

[0032] (2) The seawater Chlorella cultured by the method can significantly improve the starch accumulation capacity of seawater Chlorella, and the starch accumulation amount is 10.06 μg / 10 6 cells under the condition of ammonia nitrogen concentration of 3 mg / L and salinity of 5‰. Compared with the culture under the constant salinity of 30‰, the starch accumulation amount is increased by 58.4% under the 5‰ salt synergistic culture. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Fig. 2 is a diagram showing the change trend of the ammonia nitrogen removal rate of seawater Chlorella in the baffling reactor culture period, wherein (a) is the hydraulic retention time of 24 h, and (b) is the hydraulic retention time of 48 h.

[0034] Figure 2 Fig. 3 is a diagram showing the total starch content of seawater Chlorella on the first day and the seventh day in the culture period.

[0035] Figure 3Effect of HRT and salt synergy on the highest ammonia removal rate of seawater Chlorella within 7 days.

[0036] Figure 4 Effect of HRT and salt synergy on the starch accumulation of seawater Chlorella.

[0037] Figure 5 Effect of salt synergy and initial ammonia concentration on the ammonia removal rate of seawater Chlorella.

[0038] Figure 6 Effect of salt synergy and initial ammonia concentration on the starch accumulation of seawater Chlorella.

[0039] Figure 7 Effect of salt synergy on the ammonia removal rate of seawater Chlorella under no-salt condition.

[0040] Figure 8 Effect of salt synergy on the ammonia removal rate of seawater Chlorella under 45‰ salinity condition.

[0041] Figure 9 Effect of different salinity conditions on the ammonia removal rate of seawater Chlorella.

[0042] Figure 10 Effect of different salinity conditions on the starch accumulation of seawater Chlorella.

[0043] Figure 11 Schematic diagram of nitrogen metabolism pathway showing the effect of salt synergy on the expression of key genes in nitrogen metabolism in the examples.

[0044] Figure 12 Graph of differential expression of key genes in nitrogen metabolism in 5‰ salinity vs. 30‰ salinity in the examples.

[0045] Figure 13 Schematic diagram of starch synthesis pathway showing the effect of salt synergy on the expression of key genes in starch synthesis in the examples.

[0046] Figure 14 Graph of differential expression of key genes in starch synthesis in 5‰ salinity vs. 30‰ salinity in the examples. DETAILED DESCRIPTION

[0047] The seawater Chlorella cultivation method with high ammonia removal rate and high starch accumulation ability of the present application is further described below in conjunction with specific examples. It should be understood that the materials and methods used in the examples are conventional in the art unless otherwise specified, and these materials can be obtained commercially.

[0048] The Chlorella vulgaris seed solution described in the examples of the present invention was obtained from the Experimental Supplies Management Department of the Chinese Academy of Tropical Agricultural Sciences-Haikou Qiongshan Yunkewei, No. 4 Xueyuan Road, Longhua District, Haikou City, Hainan Province.

[0049] The present invention adopts a batch culture method to cultivate seawater Chlorella. First, in the first stage, the seawater 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 seawater 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.

[0050] The culture conditions for the first stage of culturing Chlorella vulgaris in the embodiment of the present invention are as follows: Chlorella vulgaris is in a suspended state, cultured in a conical flask, at a temperature of 25±1°C, placed in a shaker at 180 rpm (12 hours interval) for 5 minutes twice a day, 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.

[0051] The culture conditions for the second stage culture of marine Chlorella in the embodiment of the present invention are as follows: the marine Chlorella is in an immobilized algae ball state, cultured in a baffled flow reactor, the temperature is 25±1°C, air is aerated for 5 minutes in the morning and evening (with an interval of 12 hours), the light intensity is 6000 lux, the light-dark ratio is 12h:12h, the pH is 7.0-7.5, and the culture time is 7-10 days.

[0052] 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 ultimately achieving ammonia nitrogen removal and biomass accumulation.

[0053] Example 1 5‰ salt synergistic effect enhances the ability of immobilized seawater chlorella to absorb ammonia nitrogen and accumulate starch

[0054] A culture method of seawater chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity, comprising the following steps:

[0055] (1) First stage culture of seawater chlorella

[0056] The composition of the artificial seawater medium is: 100 ml of seawater chlorella seed liquid, OD 680 0.25-0.3, 900 ml of artificial seawater, the concentrations of KH2PO4 solution, NH4HCO3 solution and CH3COONa solution in the medium are 22 mg / L, 56.4 mg / L and 20 mg / L respectively, and the ammonia nitrogen concentration and total phosphorus concentration are 10 mg / L and 5 mg / L respectively. Culture in a conical flask, collect seawater chlorella in the logarithmic growth phase after 7-10 days of culture, at this time the OD 680 of seawater chlorella is about 0.3, and the cell density is about 10 6 cells / mL;

[0057] The artificial seawater is prepared by dissolving 33 g of sea salt in 1 L of deionized water to obtain artificial seawater with a salinity of 30‰, and the formula of sea salt (per 100 g) is 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, MnSO4 0.0094 g.

[0058] Culture conditions: seawater chlorella is in a suspended state, cultured in a conical flask, temperature 25±1℃, placed in a shaker twice a day at 180r / min (interval 12h), oscillation 5min, pH=7.0-7.5, light intensity 6000lux, light-dark ratio 12h:12h.

[0059] Seawater chlorella seed liquid, i.e. initial algal liquid, is cultured in the above-mentioned artificial seawater medium for 7-10 days to obtain seawater chlorella culture solution, which has an OD 680 of about 0.3 and a cell density of about 10 6 cells / ml.

[0060] (2) Preparation of immobilized algal balls

[0061] 400 ml of seawater chlorella culture solution is mixed with 400 ml of 2% sodium alginate solution in a 1:1 ratio.

[0062] Preparation of sodium alginate solution: The preparation of sodium alginate solution needs to be carried out under the condition of 95℃ boiling water bath, 400ml deionized water is placed in a graduated beaker, heated to 95℃ in a water bath, then 8g sodium alginate is slowly added under constant stirring, after the sodium alginate is completely dissolved, stop heating; add appropriate amount of deionized water to make the sodium alginate solution constant volume to 400ml.

[0063] Preparation of immobilized algae ball: the seawater chlorella culture solution obtained in the above first stage culture is mixed with 2% sodium alginate solution cooled at a ratio of 1:1, then slowly and continuously dripped into 3% calcium chloride solution by using peristaltic pump, and the immobilized algae ball in the calcium chloride solution is placed in 4℃ refrigerator for 24h to make it fully set.

[0064] (3) Baffled reactor second stage culture

[0065] The above immobilized algae ball is added into a baffled reactor with a volume of 2L, and the amount of immobilized algae ball added is 2000 / 1L artificial seawater. The artificial seawater is replaced by nitrogen-limited artificial seawater, and the ammonia nitrogen concentration is reduced from 10mg / L to 2mg / L, and the total phosphorus concentration is reduced from 5mg / L to 1mg / L.

[0066] The mass of sea salt in the preparation of artificial seawater is 5.45g, so that the salinity is 5‰, in addition, the concentration of NH4HCO3 is reduced to 12mg / L, so that the ammonia nitrogen concentration of artificial seawater is 2mg / L; the concentration of KH2PO4 is reduced to 4.4mg / L, so that the total phosphorus concentration of artificial seawater is 1mg / L.

[0067] Culture conditions: seawater chlorella is in immobilized state, continuous water feeding reactor culture, temperature 25±1℃, air pump is used to blow air into the reactor at intervals of 12h (once in the morning and once in the evening), each time for 5min, at the same time, the algae ball is turned twice, and the light is as uniform as possible, the light intensity is 6000lux, and the light-dark ratio is 12h:12h.

[0068] Reactor operating conditions: the reactor is continuously fed with water, and the hydraulic retention time is 24h.

[0069] (4) Determination of effluent ammonia nitrogen

[0070] Every other day, sampling is carried out at the outlet of the baffled reactor, the water sample is centrifuged at 4000r / min for 5min, then the supernatant is removed, the supernatant is filtered through a 0.45μm microporous filter to obtain a sample to be tested, and the concentration of ammonia nitrogen in the sample to be tested is determined by using Nash reagent spectrophotometry.

[0071] (5) Dissolution of immobilized algae ball

[0072] Take 50 algae balls, add 25 ml of 5% sodium citrate solution, place on a homogenizer, and mix at 700 rpm for 30 minutes to fully dissolve the algae balls. Centrifuge the dissolved algae mixture at 10,000 rpm for 5 minutes, remove the supernatant, retain the algal cells, rinse them three times with PBS solution, and then add 10 ml of PBS solution to the algal cells to obtain the algal solution to be tested. 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.

[0073] (6) Determination of the number of Chlorella vulgaris cells

[0074] Take 5ml of the algae solution to be tested and measure its OD using a UV spectrophotometer 680 The number of seawater Chlorella cells was calculated according to the following formula (1):

[0075] Seawater Chlorella (10 6 cells / mL) = 3.43 × OD 680 -0.0182(R 2 =0.994) formula (1)

[0076] Where R 2 Represents the degree of curve fitting.

[0077] (7) Determination of starch content

[0078] 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: add 200μL of the solution to be tested, 200μL of distilled water, 100μL of anthrone working solution and 1ml of concentrated sulfuric acid to the determination tube in sequence, heat in a 95℃ water bath for 10min, and determine A 620 (i.e. absorbance at 620nm), and then according to the standard curve, the glucose concentration was obtained.

[0079] Soluble sugar content (mg / L) = 120.07 × A 620 -1.2551(R 2 =0.9996) formula (2)

[0080] wherein R 2 represent the degree of curve fitting.

[0081] Example 2 15‰ of salt synergistic conditions enhance the ability of immobilized seawater chlorella to absorb ammonia nitrogen and accumulate starch

[0082] A culture method of seawater chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity, comprising the following steps:

[0083] (1) First stage culture of seawater chlorella. Same as Example 1;

[0084] (2) Preparation of immobilized algal balls. Same as Example 1;

[0085] (3) Second stage culture of immobilized algal balls in a baffling reactor:

[0086] The above immobilized algal balls are added to a baffling reactor with a volume of 2L, and the amount of immobilized algal balls added is 2000 per 1L artificial seawater. Replace the artificial seawater with nitrogen-limited artificial seawater, and reduce the ammonia nitrogen concentration from 10mg / L to 2mg / L, and the total phosphorus concentration from 5mg / L to 1mg / L.

[0087] The mass of sea salt in the preparation of artificial seawater is 16.36g, and the artificial seawater with a salinity of 15‰ is prepared, and in addition, the amount of NH4HCO3 is reduced to 12mg / L, so that the ammonia nitrogen concentration of the artificial seawater is 2mg / L; the amount of KH2PO4 is reduced to 4.4mg / L, so that the total phosphorus concentration of the artificial seawater is 1mg / L.

[0088] Cultivation conditions: seawater chlorella is in an immobilized state, continuous water feeding reactor culture, temperature 25±1℃, air pump is used to blow air into the reactor every 12h (morning and evening each once), each time 5min, at the same time, the algal balls are turned twice, and the light is as uniform as possible, the light intensity is 6000lux, and the light-dark ratio is 12h:12h.

[0089] (4) Determination of effluent ammonia nitrogen: same as Example 1;

[0090] (5) Dissolution of immobilized algal balls: same as Example 1;

[0091] (6) Determination of seawater chlorella cell number: same as Example 1;

[0092] (7) Determination of starch content: same as Example 1.

[0093] Example 3 25‰ of salt synergistic conditions enhance the ability of immobilized seawater chlorella to absorb ammonia nitrogen and accumulate starch

[0094] A method for cultivating seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity comprises the following steps:

[0095] (1) First stage cultivation of Chlorella vulgaris: same as in Example 1;

[0096] (2) Preparation of immobilized algae balls: same as in Example 1;

[0097] (3) The immobilized algae balls are cultured in the baffled flow reactor for the second stage:

[0098] 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.

[0099] 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.

[0100] Culture conditions: Chlorella vulgaris was immobilized in the marine environment and continuously fed into the reactor with water. The temperature was 25±1°C. 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.

[0101] (4) Determination of effluent ammonia nitrogen: same as in Example 1;

[0102] (5) Dissolution of immobilized algae balls: same as in Example 1;

[0103] (6) Determination of the number of Chlorella vulgaris cells: Same as in Example 1;

[0104] (7) Determination of starch content: Same as Example 1.

[0105] 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 the total starch accumulation (i.e. the difference between the starch content on the seventh day and the starch content on the first day) under HRT = 24h, 5‰ salinity, 15‰ salinity, 25‰ salinity and 30‰ salinity can reach 8.52, 8.25, 6.96 and 6.14 μg / 10 6 cells, respectively. The results show that the salinity of 5‰ and 15‰ have a significant promoting effect on the starch accumulation of Nannochloropsis oceanica.

[0106] Example 4

[0107] The reactor hydraulic retention time during the second stage of the culture process was extended, and the effect of the time extension on the ammonia nitrogen absorption and starch accumulation capacity of Nannochloropsis oceanica was investigated.

[0108] In this example, the operating steps of the above-mentioned Examples 1-3 were followed, and four kinds of nitrogen-limited artificial seawater were set, with salinities of 5‰, 15‰ and 25‰ and 30‰. The reactor hydraulic retention time during the second stage of the culture process in the baffled reactor was extended to 48h, and Nannochloropsis oceanica cultured under HRT = 48h at four different salinities (5‰, 15‰ and 25‰ and 30‰) was obtained.

[0109] The effluent ammonia nitrogen and starch content were determined using the same operating method as in Example 1.

[0110] From Figure 3 It can be seen that, under HRT = 48h, compared with HRT = 24h, the ammonia nitrogen removal rates under 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. From Figure 4 It can be seen that, under HRT = 48h, compared with HRT = 24h, the total starch content under 5‰ salinity, 15‰ salinity, 25‰ salinity and 30‰ salinity increased from 8.52 μg / 10 6 cells to 9.53 μg / 10 6 cells, 8.25 μg / 10 6 cells to 9.61 μg / 10 6 cells, 6.96 μg / 10 6 cells to 8.09 μg / 10 6 cells, 6.14 μg / 10 6 cells to 6.67 μg / 10 6 cells.

[0111] The above results show that the extension of the hydraulic retention time to 48 h helps the seawater Chlorella to fully absorb ammonia nitrogen, and at the same time, enhances the nitrogen-limited environment, thereby significantly improving the starch accumulation capacity of the seawater Chlorella.

[0112] The present application systematically explores the influence of a series of factors on the ammonia nitrogen removal rate and starch accumulation amount of seawater Chlorella through experiments (Examples 5-12), including different initial ammonia nitrogen concentrations, different salt synergies, and different time (hydraulic retention time) conditions.

[0113] Example 5

[0114] By changing the ammonia nitrogen concentration in the second stage culture, the promoting effect of the nitrogen limitation degree on the ammonia nitrogen absorption capacity and starch production capacity of the seawater Chlorella is investigated. According to the culture method of Example 1, the concentration of NH4HCO3 solution in the second stage culture is changed to 11.3 mg / L, 17 mg / L, 22.6 mg / L and 28.2 mg / L, and the concentration of KH2PO4 solution is 4.4, 6.6, 8.8 and 11 mg / L, respectively. That is, the ammonia nitrogen concentration of the culture medium in the second stage culture is 2, 3, 4 and 5 mg / L, respectively, and the total phosphorus concentration is 1, 1.5, 2 and 2.5 mg / L, respectively, the sea salt addition amount of artificial seawater is 5.45 g / L, the salinity is 5‰, and the hydraulic retention time is 48 h.

[0115] Example 6

[0116] Compared with Example 5, the salinity of this embodiment is only adjusted to 15‰. That is, the sea salt addition amount of artificial seawater is 16.36 g / L, and other conditions remain unchanged.

[0117] Example 7

[0118] Compared with Example 5, the salinity of this embodiment is only adjusted to 25‰. That is, the sea salt addition amount of artificial seawater is 27.27 g / L, and other conditions remain unchanged.

[0119] Example 8

[0120] Compared with Example 5, the salinity of this embodiment is only adjusted to 35‰. That is, the sea salt addition amount of artificial seawater is 38.18 g / L, and other conditions remain unchanged.

[0121] Example 9

[0122] Compared with Example 1, the salinity of this embodiment is only adjusted to 25‰. That is, the sea salt addition amount of artificial seawater is 27.27 g / L, and other conditions remain unchanged.

[0123] Example 10

[0124] Compared with Example 1, the artificial seawater medium state in the first stage culture process is not changed in this example, and the salinity is kept at 30‰ for continuous culture, but the hydraulic retention time is adjusted to 48 h, and the rest of the conditions are the same as in Example 1.

[0125] Example 11

[0126] Compared with Example 1, the ammonia nitrogen concentration in the second stage culture process is set to 3 mg / L, the salinity is 0‰ (salt-free condition), and the hydraulic retention time is 48 h in this example. The rest of the conditions are the same as in Example 1.

[0127] Example 12

[0128] Compared with Example 1, the ammonia nitrogen concentration in the second stage culture process is set to 3 mg / L, the salinity is 45‰, and the hydraulic retention time is 48 h in this example. The rest of the conditions are the same as in Example 1.

[0129] The results of the ammonia nitrogen absorption capacity and starch production capacity of the seawater Chlorella cultured in Examples 5-12 are shown in Table 1.

[0130] Table 1

[0131]

[0132] From Table 1, Figure 5 , Figure 6 The results show that the salt synergistic effect of seawater Chlorella in the 5‰, 15‰ and 25‰ salinity groups is significant. When the hydraulic retention time is 48 h and the ammonia nitrogen concentration is 2 mg / L, the ammonia nitrogen removal rates are 90.5%, 91.5% and 80.6%, respectively, and the starch accumulation amounts are 9.53, 9.61 and 7.98 μg / 10 6 cells, respectively, compared with the control group of 30‰ salinity, the ammonia nitrogen removal rate is 76.6% and the starch accumulation amount is 6.67 μg / 10 6 cells. The salt synergistic effect of 5‰, 15‰ and 25‰ increases the ammonia nitrogen removal rate by 18.1%, 19.5% and 5.2%, respectively, and the starch accumulation amount by 42.9%, 44.1% and 19.6%, respectively. The salt synergistic effect of 5‰ and 15‰ is the most significant. When the ammonia nitrogen concentration is 3 mg / L, the seawater Chlorella under 5‰ salinity has the highest ammonia nitrogen removal efficiency of 93.60% and the maximum starch accumulation amount of 10.06 μg / 10 6cells, 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. Salins in the range of 15‰-25‰ also have 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 effect 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.

[0133] 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.

[0134] 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%.

[0135] 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.

[0136] 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 increase 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.

[0137] Depend on Figure 11It can be seen 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 It can be seen (* indicates P < 0.05, indicating that the gene differential expression results are significant, *** indicates P < 0.001, indicating that the gene differential expression results are extremely significant) that the 5‰ salt synergistic condition increases AMT by 2.49 units and gdhA (nitrogen assimilation) by 4.34 units. The FDR results of the significance analysis show that the changes in AMT and gdhA under the 5‰ salt synergistic condition are significant. Therefore, the ammonia nitrogen absorption process and the ammonia nitrogen assimilation process are significantly enhanced under salt synergy. It can be proved from the genetic molecular level that 5‰ salt synergy has a significant promoting effect on the ammonia nitrogen absorption capacity of marine Chlorella. Although the changes in other key genes are not significant, they can also provide certain data support for the enhancement of nitrogen metabolism.

[0138] Depend on Figure 13 It is known that marine Chlorella fixes carbon dioxide into an available 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, it effectively explains the promoting effect of 5‰ salt synergy on starch accumulation in marine Chlorella.

Claims

1. A method for culturing a marine Chlorella sp. having high ammonia-nitrogen removal rate and high starch accumulation capacity, characterized by, The seawater Chlorella is cultured in a batch mode, first under the conditions of salinity of 30‰ and ammonia nitrogen concentration of 10 mg / L, and then the seawater Chlorella is embedded by sodium alginate immobilization when the seawater Chlorella grows to the logarithmic growth phase, and the immobilized Chlorella is cultured under the conditions of salinity of 5‰-15‰, ammonia nitrogen concentration of 2-3 mg / L and hydraulic retention time of 48 h, so that the seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity is obtained; the specific steps of embedding the seawater Chlorella by sodium alginate immobilization are as follows: the seawater Chlorella liquid is mixed with a sodium alginate solution, the mixed solution is dropped into a calcium chloride solution to obtain Chlorella balls, and the Chlorella balls are shaped by refrigeration to obtain the immobilized Chlorella balls; the mass fraction of the sodium alginate solution is 2%, the volume ratio of the seawater Chlorella liquid to the sodium alginate solution is 1:1, and the mass fraction of the calcium chloride solution is 3%, and the volume ratio of the mixed solution to the calcium chloride solution is 1:1-1.

5.

2. The method for culturing the seawater chlorella with high ammonia-nitrogen removal rate and high starch accumulation ability according to claim 1, characterized in that, The method comprises the following steps: (1) inoculating a seawater Chlorella seed liquid into an artificial seawater medium with salinity of 30‰ and ammonia nitrogen concentration of 10 mg / L for first-stage culture, maintaining illumination and temperature for culture to obtain seawater Chlorella liquid in the logarithmic growth phase; (2) mixing the seawater Chlorella liquid obtained in step (1) with a sodium alginate solution, dropping the mixed solution into a calcium chloride solution to obtain Chlorella balls, and shaping the Chlorella balls by refrigeration to obtain immobilized Chlorella balls; (3) culturing the immobilized Chlorella balls obtained in step (2) in an artificial seawater medium for second-stage culture, changing the salinity of the artificial seawater medium to 5‰-15‰ and reducing the ammonia nitrogen concentration to 2-3 mg / L, and maintaining illumination and temperature for culture; (4) dissolving the immobilized Chlorella balls obtained in step (3) in a sodium citrate solution, and centrifuging to obtain the seawater Chlorella with high ammonia nitrogen removal rate and high starch accumulation capacity.

3. The method according to claim 2, wherein the Chlorella marina has high ammonia-nitrogen removal rate and high starch accumulation ability. In step (1), the formula of the artificial seawater medium used for the first-stage culture comprises 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, CH3COONa 15-25 mg / L, NH4HCO3 45-67 mg / L and KH2PO4 18-27 mg / L.

4. The method of claim 2, wherein the Chlorella marina has high ammonia-nitrogen removal rate and high starch accumulation ability. In step (1), the inoculation volume ratio of the seed solution of the seawater Chlorella is 10%-20%; the seawater Chlorella liquid is obtained after the seawater Chlorella seed solution is cultured in the artificial seawater medium for 7-10 days, and the OD 680 of the seawater Chlorella liquid is 0.25-0.

3.

5. The method of claim 2, wherein the Chlorella marina has high ammonia-nitrogen removal rate and high starch accumulation capacity. In steps (1) and (3), the illumination is 4000-8000 lux, and the temperature is 25±1 ℃; the illumination time is controlled to be 10-12 h per day, the pH is controlled to be 7.0-7.5, and the culture time is 7-10 days during the culture.

6. The method of claim 2, wherein the Chlorella marina has high ammonia-nitrogen removal rate and high starch accumulation capacity. The formula of the artificial seawater medium used in the second stage culture in step (3) comprises 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, CH3COONa 15-25 mg / L, NH4HCO3 12-17 mg / L and KH2PO4 4.4-6.6 mg / L.

7. The method according to claim 2, wherein the Chlorella marina has high ammonia-nitrogen removal rate and high starch accumulation ability. In step (3), the second stage culture is carried out in a baffled reactor, and the hydraulic retention time of the baffled reactor is 48 h when water is continuously fed; in step (4), the mass fraction of the sodium citrate solution is 4%-6%.

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