Alkali precipitation harvesting and re-culture utilization process for microalgae

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a microalgae alkali precipitation harvesting and re-culture utilization process which comprises the following steps: (1) adding an HEPES buffer solution into a microalgae culture solution to adjust the pH value of the microalgae culture solution to 7.5-8.0 to obtain a microalgae culture solution A; (2) adding a mixed solution of triethanolamine and sodium citrate into the microalgae culture solution A in the step (1) to adjust the pH value of the microalgae culture solution A to 8.0-9.0 to obtain a microalgae culture solution B; (3) adding an alkaline solution into the microalgae culture solution B in the step (2) to adjust the pH value of the microalgae culture solution B to 10.2-13.0 to obtain a microalgae culture solution C; and (4) standing and layering the microalgae culture solution C, discarding the supernatant, collecting the lower concentrated algae solution, transferring into the culture medium, and re-culturing. According to the microalgae alkali precipitation harvesting and re-culture utilization process, the microalgae culture process can be efficiently carried out, and the microalgae alkali precipitation harvesting and re-culture utilization process is suitable for various culture modes of autotrophy, heterotrophy and heterotrophy-autotrophy.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, specifically to a microalgae cultivation process, and more specifically to a microalgae alkali precipitation harvesting and re-cultivation utilization process. Background Art

[0002] Microalgae cultivation, like other microbial cultures, requires a high inoculum ratio to achieve a high initial cell concentration, thus creating a dominant population. This not only inhibits the growth of other bacteria and algae, but also allows for a rapid entry into the logarithmic growth phase, leading to a rapid increase in biomass. However, since the scale-up process generally requires an inoculum ratio of at least 1:10 and a constant initial algae cell concentration, otherwise growth is slow and the culture cycle is excessively long. Existing cultivation methods are generally unsatisfactory for industrial production, and efficiency is limited in the early stages of cultivation. Furthermore, microalgae culture fluids contain microalgae cell debris, metabolites, and other impurities. These substances increase the viscosity of the culture fluid, affect the distribution and movement of microalgae cells within the culture fluid, and interfere with light scattering and absorption. Consequently, reuse of the microalgae culture fluid can inhibit normal microalgae growth. Alkaline flocculation further increases the viscosity of the culture fluid, impairing light absorption and nutrient absorption by the algae cells. Furthermore, high alkaline concentrations can damage algae cells, thereby affecting their physiological activity and dramatically reducing their growth and proliferation capacity, severely impacting the effectiveness of inoculation. Currently, alkaline flocculation is usually used to harvest microalgae after cultivation and then prepare downstream products. However, no research has shown that algae cells after alkaline flocculation can be used for re-cultivation.

[0003] In order to improve the efficiency of industrial cultivation and reduce the cost of cultivation, researchers have successively developed semi-continuous cultivation processes and two-stage cultivation processes. Patent CN202010160532.7 provides a two-stage low-nitrogen and low-phosphorus stress microalgae cultivation method, in which the microalgae culture solution in the logarithmic growth period is centrifuged to obtain algae cells for inoculation. The accumulation of polyunsaturated fatty acids (PUFAs) is significantly improved, and the microalgae cells grow rapidly. However, the cost of obtaining algae cells for re-cultivation by centrifugation is high, and large-scale industrial centrifugal devices are expensive. Patent CN101696389B provides a microalgae cultivation method and a photobioreactor system thereof. After filtration and concentration by 50%, the algae cell concentrate is transported to a nitrogen-stressed culture medium for continued cultivation, thereby achieving rapid accumulation of oils and pigments. However, filtration and concentration are also costly and require additional equipment. Patent CN201310031846.7 provides a semi-continuous fermentation method for heterotrophic Chlorella. This method involves partially harvesting the heterotrophic Chlorella in a fermenter and feeding the culture medium, ensuring that the heterotrophic Chlorella in the culture medium remains in a logarithmic growth phase, thereby maintaining a high growth rate. Existing culture processes all focus on optimizing and improving a single nutritional method, limited by the expansion of culture volume at the same level. Currently, there is no universal culture process that can improve culture efficiency for both heterotrophic and autotrophic algae cultivation. These processes primarily utilize centrifugation or filtration to harvest algal cells, or partially harvest the culture medium for re-cultivation, to achieve higher algal cell growth rates and product accumulation.

[0004] Alkaline flocculation is commonly used for algal cell harvesting or culture medium reuse. The study "Research on the Harvest of Synechococcus 7002 by Alkali Flocculation" investigated the effects of different pH values, settling times, and algal cell concentrations on flocculation, but did not use the algal solution for re-cultivation. The study "Alkaline Flocculation Harvest and Recycling of Dunaliella Salina Cultured in Bicarbonate" investigated the use of alkaline flocculation to harvest Dunaliella salina. The study then used physical and chemical methods to treat the spent culture medium, reusing it to achieve the same effect as fresh culture medium for Dunaliella salina cultivation. This reuse of the culture medium saved water and nutrients, but also did not involve re-cultivation of algal cells. Therefore, microalgae cultivation requires a low-cost algal cell harvesting process that does not affect algal cell activity and allows for re-cultivation. Summary of the Invention

[0005] In view of the problems and shortcomings in the prior art, the present invention aims to provide a process for the harvesting and re-cultivation of microalgae by alkali precipitation.

[0006] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a microalgae alkali precipitation harvesting and re-cultivation utilization process, comprising the following steps:

[0008] (1) adding HEPES buffer to a microalgae culture solution to adjust the pH of the microalgae culture solution to 7.5-8.0, thereby obtaining a microalgae culture solution A;

[0009] (2) adding a mixed solution of triethanolamine and sodium citrate to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 8.0-9.0, thereby obtaining a microalgae culture solution B;

[0010] (3) adding an alkaline solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 10.2-13.0, thereby obtaining a microalgae culture solution C;

[0011] (4) The microalgae culture solution C is allowed to stand for stratification, the upper clear liquid is discarded, and the lower concentrated algae solution is collected. The concentrated algae solution is transferred to the culture medium for re-cultivation.

[0012] Preferably, the molar ratio of triethanolamine to sodium citrate in the triethanolamine and sodium citrate composite solution in step (2) is 3:1 to 1:3.

[0013] More preferably, the concentration of the mixed solution of triethanolamine and sodium citrate in step (2) is 0.1 mol / L.

[0014] Preferably, the alkaline solution in step (3) is sodium hydroxide solution or potassium hydroxide solution.

[0015] Preferably, the standing time in step (4) is 10 min to 21 h.

[0016] Preferably, in step (1), the microalgae culture solution is a microalgae culture solution obtained by adding microalgae to a culture solution for culturing; or microalgae are added to a culture solution for culturing, the culture solution is centrifuged to collect the precipitate, water is added to the precipitate, and the mixture is mixed to obtain the microalgae culture solution.

[0017] More preferably, in step (1), the microalgae are added to the culture medium for culture, the culture medium is centrifuged to collect the precipitate, the precipitate is washed with water 1 to 2 times, water is added to the precipitate, and the mixture is mixed to obtain the microalgae culture medium.

[0018] Preferably, the microalgae are autotrophic algae or heterotrophic algae.

[0019] Preferably, the microalgae is one of autotrophic Chlorella sorokinensis, heterotrophic Chlorella sorokinensis, Cladosporium tenuifolium, and Spirulina.

[0020] More preferably, the microalgae is one of autotrophic Chlorella sorokinensis and heterotrophic Chlorella sorokinensis.

[0021] Preferably, the culture medium in step (4) is one of BG11 culture medium, heterotrophic culture medium, SP culture medium, and Zarrouk culture medium.

[0022] More preferably, the specific composition of the heterotrophic culture medium is: 10-50 g / L glucose, 1-5 g / L yeast powder or peptone, and 1.7 g / L BG11 culture medium.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) When the prior art uses alkaline flocculation to harvest microalgae, a strong alkaline solution is used for direct flocculation. The pH fluctuates violently over a wide range, causing the enzyme activity of the algae cells to decrease and damage the activity, resulting in reduced cell membrane permeability and reduced ability to absorb and utilize nutrients. The alkaline solution can also form precipitates with metal ions, drastically changing the osmotic pressure, causing damage to the cells and even cell death. The present invention can accurately, gently and gradually increase the pH value of the culture solution by using HEPES buffer, a mixed solution of triethanolamine and sodium citrate, and an alkaline solution, and does not cause drastic changes in the living environment of the algae cells during the adjustment process. The method of harvesting and re-cultivating microalgae by alkaline precipitation of the present invention can provide the nutrients such as carbon source, nitrogen source and trace elements required for the growth of microalgae on the basis of protecting microalgae cells, stabilizing the culture environment and accurately controlling the pH, and promote the conversion of carbon source and nitrogen source nutrients. At the same time, it has no toxic residues and causes little damage to the microalgae cells.

[0025] (2) HEPES is a commonly used cell culture buffer. It can stabilize the pH value of the culture medium and provide a suitable ion environment. At the same time, HEPES buffer also has a certain antioxidant capacity, which can remove some active oxygen in the cell culture system and protect cells from the influence of the external environment, thereby improving the culture efficiency. Sodium citrate is a carbon source that can be used by microalgae. It can also provide sodium ions to regulate water activity and the osmotic pressure of the culture medium. Sodium citrate can combine with metal ions such as calcium, magnesium, and zinc in the culture medium to form stable chelates, which can be better absorbed and utilized by microalgae. At the same time, chelates such as calcium citrate and magnesium citrate can also prevent calcium and magnesium ions from precipitating and losing under subsequent high-concentration alkaline conditions. Triethanolamine is produced by the reaction of ethylene oxide and ammonia water, and can provide carbon and nitrogen sources for aquatic organisms. Triethanolamine has excellent buffering properties and can gently and accurately adjust pH. At the same time, triethanolamine is also a chelating agent that can combine with metal ions such as copper, iron, aluminum, and manganese to prevent precipitation and loss under subsequent high-concentration alkaline conditions. Sodium citrate and triethanolamine are both emulsifiers that can maintain the stability of microalgae culture solution.

[0026] (3) The prior art uses centrifugation and filtration methods to obtain algae cells for re-cultivation, which puts the algae cells in a water-deficient and high-osmotic pressure environment, changes the living environment of the algae cells, and causes damage to the activity of the algae cells. The present invention can accurately, gently, and gradually increase the pH value of the culture solution by using HEPES buffer, triethanolamine and sodium citrate mixed solution, and alkaline solution, and can avoid the damage to the algae cells caused by the direct use of strong alkaline substances to adjust the pH, which causes the pH of the culture solution to fluctuate violently. At the same time, HEPES buffer can protect cells from the influence of the external environment due to antioxidant properties and provide cells with a stable and suitable ion environment. Sodium citrate can adjust the osmotic pressure of the culture solution. In addition, sodium citrate and triethanolamine also have an emulsifying effect. The comprehensive use of these solutions has the effect of maintaining the stability of the culture solution and protecting the microalgae cells, so that the algae cells are always in a relatively suitable environment before and after alkali precipitation. The method of harvesting and re-cultivating microalgae by alkali precipitation of the present invention can enrich microalgae cells, meet the high-proportion inoculation amount required for microalgae expansion, reduce the inadaptability of cells after inoculation, shorten the growth stagnation period, and enable the cultivation process to be carried out efficiently.

[0027] (4) The present invention is adaptable to various cultivation modes such as autotrophic, heterotrophic, and heterotrophic-autotrophic, and can be used for both same-level expansion and step-by-step expansion. It does not require large-scale microalgae seed liquid inoculation, does not require new processing equipment, and does not have special requirements for cultivation equipment. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] Example 1

[0030] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0031] (1) adding a mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) with a HEPES buffer concentration of 0.1 mol / L to a culture solution of Chlorella vulgaris autotrophicus to adjust the pH of the culture solution to 7.5 to obtain a microalgae culture solution A, wherein the OD680 of the culture solution of Chlorella vulgaris autotrophicus is 0.630 (measured by a spectrophotometer);

[0032] (2) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 9.0 to obtain a microalgae culture solution B;

[0033] (3) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 10.6 to obtain microalgae culture solution C;

[0034] (4) The microalgae culture solution C was allowed to stand for 30 minutes to allow the microalgae culture solution C to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 3 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 medium for cultivation. The pH was adjusted to 6.8 and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 medium containing microalgae was measured using a spectrophotometer and was 1.469.

[0035] In order to compare the culture effect, Example 1 also set up a control group without inoculation of concentrated algae liquid. The control group was inoculated with unconcentrated algae liquid (that is, 3 mL of the autotrophic Chlorella sorokinensis culture liquid in step (1) of Example 1 was inoculated into BG11 medium). After 10 days of culture, the OD680 of the microalgae culture liquid using BG11 medium was 0.252. From the OD680 value of the control group, it can be seen that the microalgae concentrate of the present invention can be used for re-cultivation of microalgae.

[0036] Example 2

[0037] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0038] (1) The culture solution of Chlorella vulgaris autotrophicensis was centrifuged (rotating speed 12000 rpm, centrifugation time 3 min), and the precipitate was collected after centrifugation. Water was added to the precipitate to restore the original volume, wherein the OD680 of the culture solution of Chlorella vulgaris autotrophicensis was 0.630 (measured by spectrophotometer);

[0039] (2) adding a mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) with a HEPES buffer concentration of 0.1 mol / L to the centrifuged Chlorella vulgaris autotrophicus culture solution to adjust the pH of the microalgae culture solution to 8.0 to obtain microalgae culture solution A;

[0040] (3) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (2) to adjust the pH of the microalgae culture solution A to 8.5 to obtain a microalgae culture solution B;

[0041] (4) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (3) to adjust the pH of the microalgae culture solution B to 12.9 to obtain microalgae culture solution C;

[0042] (5) The microalgae culture solution C was allowed to stand for 10 minutes to allow the microalgae culture solution C to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 medium for cultivation. The pH was not adjusted and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 medium containing microalgae was measured using a spectrophotometer and was 0.762.

[0043] Example 3

[0044] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0045] (1) adding HEPES buffer to a culture solution of Chlorella vulgaris autotrophica to adjust the pH of the culture solution to 8.0 to obtain a microalgae culture solution A, wherein the OD680 of the culture solution of Chlorella vulgaris autotrophica was 0.630 (measured using a spectrophotometer);

[0046] (2) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 9.0 to obtain a microalgae culture solution B;

[0047] (3) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 11.9 to obtain microalgae culture solution C;

[0048] (4) The microalgae culture solution C was allowed to stand for 20 minutes to allow the microalgae culture solution C to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized heterotrophic culture medium (the components of the heterotrophic culture medium were: 20 g / L glucose, 2 g / L yeast powder, and 1.7 g / L BG11 culture medium) for cultivation. The pH was not adjusted and the culture was maintained at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the heterotrophic culture medium containing microalgae was measured using a spectrophotometer and was 4.67.

[0049] Example 4

[0050] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0051] (1) centrifuging the culture solution of Chlorella vulgaris autotrophica, collecting the precipitate after centrifugation, and adding water to the precipitate to restore the original volume, wherein the OD680 of the culture solution of Chlorella vulgaris autotrophica is 0.630 (measured using a spectrophotometer);

[0052] (2) adding HEPES buffer to the centrifuged Chlorella vulgaris culture solution to adjust the pH of the microalgae culture solution to 7.5 to obtain microalgae culture solution A;

[0053] (3) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (2) to adjust the pH of the microalgae culture solution B to 9.0 to obtain microalgae culture solution B;

[0054] (4) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (3) to adjust the pH of the microalgae culture solution B to 10.2 to obtain microalgae culture solution C;

[0055] (5) The microalgae culture solution C was allowed to stand for 30 minutes to allow the microalgae culture solution C to separate into layers. The supernatant was discarded, and the lower concentrated algae solution was collected. 3 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized heterotrophic culture medium (same as in Example 3) for cultivation. The pH was not adjusted and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the heterotrophic culture medium containing microalgae was measured using a spectrophotometer and was 7.35.

[0056] Example 5

[0057] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0058] (1) adding HEPES buffer to a culture solution of heterotrophic Chlorella sorokinensis to adjust the pH of the microalgae culture solution to 7.5 to obtain a microalgae culture solution A, wherein the OD680 of the heterotrophic Chlorella sorokinensis culture solution is 13.58 (measured using a spectrophotometer);

[0059] (2) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 8.5 to obtain a microalgae culture solution B;

[0060] (3) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 10.9 to obtain a microalgae culture solution C;

[0061] (4) The microalgae culture solution C was allowed to stand for 21 hours to allow the microalgae culture solution C to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 medium for cultivation. The pH was not adjusted and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 medium containing microalgae was measured using a spectrophotometer and was 1.026.

[0062] Example 6

[0063] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0064] (1) The heterotrophic Chlorella Sorokinensis culture solution was centrifuged (speed 12000 rpm, centrifugation time 3 min), the lower algal mud was collected, water was added to the lower algal mud to the original volume and then centrifuged again (speed 12000 rpm, centrifugation time 3 min), the lower algal mud was collected, the operation was repeated twice, and water was added to restore to the original volume. The OD680 of the heterotrophic Chlorella Sorokinensis culture solution was 13.58 (measured by spectrophotometer);

[0065] (2) adding HEPES buffer to the centrifuged heterotrophic Chlorella sorokinensis culture solution to adjust the pH of the microalgae culture solution to 8.0 to obtain microalgae culture solution A;

[0066] (3) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (2) to adjust the pH of the microalgae culture solution A to 9.0 to obtain a microalgae culture solution B;

[0067] (4) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (3) to adjust the pH of the microalgae culture solution B to 13.0 to obtain microalgae culture solution C;

[0068] (5) The microalgae culture solution C was allowed to stand for 60 minutes to allow the microalgae culture solution C to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 medium containing microalgae was measured using a spectrophotometer and was 1.613.

[0069] Example 7

[0070] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0071] (1) adding HEPES buffer to a culture solution of heterotrophic Chlorella sorokinensis to adjust the pH of the microalgae culture solution to 8.0 to obtain a microalgae culture solution A, wherein the OD680 of the heterotrophic Chlorella sorokinensis culture solution is 13.58 (measured using a spectrophotometer);

[0072] (2) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 9.0 to obtain a microalgae culture solution B;

[0073] (3) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 12.5 to obtain microalgae culture solution C;

[0074] (4) The microalgae culture solution C was allowed to stand for 8 h to allow the microalgae culture solution C to separate into layers. The supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized heterotrophic culture medium (same as in Example 3) for cultivation. The pH was not adjusted and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the heterotrophic culture medium containing microalgae was measured using a spectrophotometer and was 12.9.

[0075] Example 8

[0076] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0077] (1) The heterotrophic Chlorella Sorokinensis culture solution was centrifuged (speed 12000 rpm, centrifugation time 3 min), the lower algal mud was collected, water was added to the lower algal mud to the original volume and then centrifuged again (speed 12000 rpm, centrifugation time 3 min), the lower algal mud was collected, the operation was repeated twice, and water was added to restore to the original volume. The OD680 of the heterotrophic Chlorella Sorokinensis culture solution was 13.58 (measured by spectrophotometer);

[0078] (2) adding HEPES buffer to the centrifuged heterotrophic Chlorella sorokinensis culture solution to adjust the pH of the microalgae culture solution to 7.5 to obtain microalgae culture solution A;

[0079] (3) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (2) to adjust the pH of the microalgae culture solution A to 9.0 to obtain a microalgae culture solution B;

[0080] (4) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (3) to adjust the pH of the microalgae culture solution B to 11.6 to obtain microalgae culture solution C;

[0081] (5) The microalgae culture solution C was allowed to stand for 60 minutes to allow the microalgae culture solution C to separate into layers. The supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized heterotrophic culture medium (same as in Example 3) for cultivation. The pH was not adjusted and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the heterotrophic culture medium containing microalgae was measured using a spectrophotometer and was 15.44.

[0082] Example 9

[0083] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0084] (1) The heterotrophic Chlorella Sorokinensis culture solution was centrifuged (speed 12000 rpm, centrifugation time 3 min), the lower algal mud was collected, water was added to the lower algal mud to the original volume and then centrifuged again (speed 12000 rpm, centrifugation time 3 min), the lower algal mud was collected, the operation was repeated twice, and water was added to restore to the original volume. The OD680 of the heterotrophic Chlorella Sorokinensis culture solution was 13.58 (measured by spectrophotometer);

[0085] (2) adding HEPES buffer to the centrifuged heterotrophic Chlorella sorokinensis culture solution to adjust the pH of the microalgae culture solution to 8.0 to obtain microalgae culture solution A;

[0086] (3) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (2) to adjust the pH of the microalgae culture solution A to 9.0 to obtain a microalgae culture solution B;

[0087] (4) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (3) to adjust the pH of the microalgae culture solution B to 11.6 to obtain microalgae culture solution C;

[0088] (5) The microalgae culture solution C was allowed to stand for 10 minutes to allow the microalgae culture solution C to separate into layers. The supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized heterotrophic culture medium (same as in Example 3) for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 19.02.

[0089] In order to compare the culture effect, Example 9 also set up a control group that was not inoculated with concentrated algae liquid. The control group was inoculated with unconcentrated algae liquid (that is, 1 mL of the heterotrophic Chlorella sorokinensis culture liquid in step (1) of Example 9 was inoculated into BG11 medium). After 10 days of cultivation in the control group, the OD680 of the microalgae culture liquid in the heterotrophic medium was 11.89.

[0090] Example 10

[0091] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0092] (1) adding HEPES buffer to the culture solution of the microalgae to adjust the pH of the microalgae culture solution to 7.5, thereby obtaining a microalgae culture solution A, wherein the OD680 of the culture solution of the microalgae is 1.56 (measured using a spectrophotometer);

[0093] (2) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 8.5 to obtain a microalgae culture solution B;

[0094] (3) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 11.5 to obtain microalgae culture solution C;

[0095] (4) The microalgae culture solution C was allowed to stand for 10 minutes to allow the microalgae culture solution C to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 3 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized SP culture medium for cultivation. The pH was not adjusted and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the SP culture medium containing microalgae was measured using a spectrophotometer and was 1.27.

[0096] Example 11

[0097] A microalgae alkali precipitation harvesting and re-cultivation utilization process comprises the following steps:

[0098] (1) adding HEPES buffer to the Spirulina culture solution to adjust the pH of the microalgae culture solution to 8.0 to obtain microalgae culture solution A, wherein the OD680 of the Spirulina culture solution is 0.931 (measured using a spectrophotometer);

[0099] (2) adding a 0.1 mol / L mixed solution of triethanolamine and sodium citrate (the molar ratio of triethanolamine to sodium citrate is 1:2) to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 9.0 to obtain a microalgae culture solution B;

[0100] (3) slowly adding 0.1 mol / L sodium hydroxide solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 11.0 to obtain a microalgae culture solution C;

[0101] (4) The microalgae culture solution C was allowed to stand for 10 minutes to allow the microalgae culture solution C to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 2 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized Zarrouk medium for cultivation. The pH was not adjusted and the culture was carried out at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the Zarrouk medium containing microalgae was measured using a spectrophotometer and was 0.866.

[0102] Comparative Example 1

[0103] Direct alkaline precipitation of autotrophic Chlorella sorokinensis for re-cultivation and utilization:

[0104] (1) Slowly adding 0.1 mol / L sodium hydroxide solution to the culture solution of the microalgae to adjust the pH of the culture solution to 12.9, wherein the OD680 of the culture solution of the autotrophic Chlorella sorokinensis is 0.953 (measured by a spectrophotometer);

[0105] (2) The microalgae culture solution was allowed to stand for 120 minutes to allow the microalgae culture solution to separate into layers. The upper layer of culture solution was discarded, and the lower layer of culture solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.374.

[0106] Compared with Example 2, it can be found that since comparative example 1 directly performs alkaline precipitation, although the OD680 of the culture solution used as seeds is 0.953, which is much higher than that of Example 2, and after a longer period of standing, the pH changes drastically during the alkaline precipitation process and the algae cells are in a high-concentration alkaline solution environment for a long time, which has a greater impact on the activity of the algae cells. The OD after the culture is completed is much lower than that of Example 2.

[0107] Comparative Example 2

[0108] Direct alkaline precipitation of heterotrophic Chlorella soloquina for re-cultivation and utilization:

[0109] (1) Slowly adding 0.1 mol / L sodium hydroxide solution to the heterotrophic Chlorella sorokinensis culture solution to adjust the pH of the microalgae culture solution to 10.9, wherein the OD680 of the heterotrophic Chlorella sorokinensis culture solution is 12.95 (measured using a spectrophotometer);

[0110] (2) The microalgae culture solution was allowed to stand for 21 hours to allow the microalgae culture solution to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.668.

[0111] Compared with Example 5, it can be found that since Comparative Example 2 only performs alkaline flocculation, the pH changes dramatically during the alkaline sedimentation process, and the algae cells are in a high-concentration alkaline solution environment for a long time, which has a great impact on the activity of the algae cells. Therefore, the OD value of Comparative Example 2 after the cultivation is much lower than that of Example 5.

[0112] Comparative Example 3

[0113] Recultivation of autotrophic Chlorella vulgaris using HEPES buffer and sodium hydroxide solution for alkaline precipitation:

[0114] (1) adding HEPES buffer to the culture solution of Chlorella vulgaris autotrophica to adjust the pH of the culture solution to 7.5, and then adjusting the pH of the culture solution to 12.9 using 0.1 mol / L sodium hydroxide solution, wherein the OD680 of the culture solution of Chlorella vulgaris autotrophica was 0.953 (measured by spectrophotometer);

[0115] (2) The microalgae culture solution was allowed to stand for 120 minutes to allow the microalgae culture solution to separate into layers. The upper layer of culture solution was discarded, and the lower layer of culture solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.587.

[0116] Comparison with Example 2 reveals that Comparative Example 3 used HEPES buffer to adjust the pH before alkaline precipitation with sodium hydroxide. Although the OD680 of the seed culture solution, 0.953, was higher than that of Example 2 and was maintained for a longer period, the rapid pH change during alkaline precipitation and the prolonged exposure of the algal cells to a high-concentration alkaline solution significantly impacted their activity, resulting in a lower OD value after the completion of the culture than in Example 2. However, due to the gradual increase in pH and the protective effect of the HEPES buffer on the algal cells, the OD value after the completion of the culture was higher than that of Comparative Example 1.

[0117] Comparative Example 4

[0118] Recultivation of heterotrophic Chlorella vulgaris using HEPES buffer and sodium hydroxide solution for alkaline precipitation:

[0119] (1) HEPES buffer was added to the culture medium of heterotrophic Chlorella sorokinensis to adjust the pH of the microalgae culture medium to 7.5, and then 0.1 mol / L sodium hydroxide solution was slowly added to adjust the pH of the microalgae culture medium to 10.9, wherein the OD680 of the culture medium of heterotrophic Chlorella sorokinensis was 12.95 (measured by spectrophotometer);

[0120] (2) The microalgae culture solution was allowed to stand for 21 hours to allow the microalgae culture solution to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.805.

[0121] Comparison with Example 5 reveals that in Comparative Example 4, HEPES buffer was used to adjust the pH before sodium hydroxide was used for alkaline precipitation. Due to the rapid pH change during alkaline precipitation and the prolonged exposure of the algal cells to a high-concentration alkaline solution, the activity of the algal cells was significantly affected, resulting in a lower OD value after the culture than in Example 5. However, due to the gradual increase in pH and the protective effect of the HEPES buffer on the algal cells, the OD value of Comparative Example 4 after the culture was higher than that of Comparative Example 2.

[0122] Comparative Example 5

[0123] Recultivation and utilization of autotrophic Chlorella sorokinensis using triethanolamine and sodium citrate solution and sodium hydroxide solution for alkaline precipitation:

[0124] (1) adding triethanolamine and sodium citrate to the culture solution of the autotrophic Chlorella sorokinensis to adjust the pH of the microalgae culture solution to 9.0, and then adjusting the pH of the microalgae culture solution to 12.9 using 0.1 mol / L sodium hydroxide solution, wherein the OD680 of the culture solution of the autotrophic Chlorella sorokinensis is 0.953 (measured by a spectrophotometer);

[0125] (2) The microalgae culture solution was allowed to stand for 120 minutes to allow the microalgae culture solution to separate into layers. The upper layer of culture solution was discarded, and the lower layer of culture solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.652.

[0126] Compared with Example 2, Comparative Example 1, and Comparative Example 3, it can be found that in Comparative Example 5, before using sodium hydroxide for alkaline precipitation, the pH is adjusted using a mixed solution of triethanolamine and sodium citrate, which can gradually increase the pH. Moreover, compared with Comparative Example 3, the pH gradient change during the alkaline precipitation process is smaller. However, after the pH is adjusted, the algae cells are left to stand for a longer time, and are in a high-concentration alkaline solution environment for a long time, which has a greater impact on the activity of the algae cells. Therefore, the OD value after the culture is lower than that of Example 2 but higher than that of Comparative Example 1 and Comparative Example 3.

[0127] Comparative Example 6

[0128] Recultivation and utilization of heterotrophic Chlorella sorokinensis using triethanolamine and sodium citrate solution and sodium hydroxide solution for alkaline precipitation:

[0129] (1) triethanolamine and sodium citrate were added to the heterotrophic Chlorella vulgaris culture solution to adjust the pH of the microalgae culture solution to 9.0, and then 0.1 mol / L sodium hydroxide solution was slowly added to adjust the pH of the microalgae culture solution to 10.9, wherein the OD680 of the heterotrophic Chlorella vulgaris culture solution was 12.95 (measured using a spectrophotometer);

[0130] (2) The microalgae culture solution was allowed to stand for 21 hours to allow the microalgae culture solution to separate into layers. The upper clear liquid was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.896.

[0131] Compared with Example 5, Comparative Example 2, and Comparative Example 4, it can be found that in Comparative Example 6, the pH is adjusted using a composite solution of triethanolamine and sodium citrate before alkaline precipitation with sodium hydroxide, which can gradually increase the pH. Moreover, compared with Comparative Example 4, the pH gradient changes less during the alkaline precipitation process. Therefore, the OD value after the culture is lower than that of Example 5 but higher than that of Comparative Example 2 and Comparative Example 4.

[0132] Comparative Example 7

[0133] Recultivation of autotrophic Chlorella vulgaris using HEPES buffer and triethanolamine and sodium citrate solution for alkaline precipitation:

[0134] (1) adding HEPES buffer to the culture medium of Chlorella vulgaris autotrophica to adjust the pH of the culture medium to 7.5, and then adding triethanolamine and sodium citrate composite solution to adjust the pH of the microalgae culture medium to 9.0, wherein the OD680 of the culture medium of Chlorella vulgaris autotrophica was 0.953 (measured by spectrophotometer);

[0135] (2) The microalgae culture solution was allowed to stand for 120 minutes to allow the microalgae culture solution to separate into layers. The upper layer of culture solution was discarded, and the lower layer of culture solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.262.

[0136] Compared to Comparative Examples 1, 3, and 5, although the pH gradient was increased, the final pH of the culture solution was only 9.0, resulting in poor algal cell sedimentation and a smaller actual inoculum size. Consequently, the OD at the end of culture was much lower than that of the other groups. However, due to a small amount of flocculation and sedimentation of algal cells under alkaline conditions, the actual inoculum size was higher than that of the control group in Example 1, resulting in a higher OD at the end of culture than that of the control group in Example 1.

[0137] Comparative Example 8

[0138] Recultivation of heterotrophic Chlorella vulgaris using HEPES buffer and sodium hydroxide solution for alkaline precipitation:

[0139] (1) adding HEPES buffer to the culture medium of heterotrophic Chlorella sorokinensis to adjust the pH of the culture medium to 75, and then adding triethanolamine and sodium citrate composite solution to adjust the pH of the microalgae culture medium to 9.0, wherein the OD680 of the heterotrophic Chlorella sorokinensis culture medium is 12.95 (measured by spectrophotometer);

[0140] (2) The microalgae culture solution was allowed to stand for 21 hours to allow the microalgae culture solution to separate into layers. The upper supernatant was discarded, and the lower concentrated algae solution was collected. 1 mL of the lower concentrated algae solution was taken and transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterilized BG11 culture medium for cultivation. The pH was adjusted to 6.8 and cultured at 28°C and 200 rpm for 10 days. After 10 days of cultivation, the OD680 of the BG11 culture medium containing microalgae was measured using a spectrophotometer and was 0.417.

[0141] Compared with Comparative Examples 2, 4, and 6, although the pH gradient was increased, the final pH of the culture medium was only 9.0. The heterotrophic Chlorella sorokinensis culture medium had a high organic matter content and complex components. Under low pH conditions, the algal cell sedimentation effect was poor, resulting in a small actual inoculum size. Therefore, the OD after the culture was completed was much lower than that of the other groups.

Claims

1. A process for harvesting and re-cultivating microalgae by alkali precipitation, characterized in that: The following steps are involved: (1) adding HEPES buffer to a microalgae culture solution to adjust the pH of the microalgae culture solution to 7.5-8.0, thereby obtaining a microalgae culture solution A; (2) adding a mixed solution of triethanolamine and sodium citrate to the microalgae culture solution A in step (1) to adjust the pH of the microalgae culture solution A to 8.0-9.0, thereby obtaining a microalgae culture solution B; (3) adding an alkaline solution to the microalgae culture solution B in step (2) to adjust the pH of the microalgae culture solution B to 10.2-13.0, thereby obtaining a microalgae culture solution C; (4) The microalgae culture solution C is allowed to stand for stratification, the upper clear liquid is discarded, and the lower concentrated algae solution is collected. The concentrated algae solution is transferred to the culture medium for re-cultivation.

2. The microalgae alkali precipitation harvesting and re-cultivation utilization process according to claim 1, characterized in that: In the mixed solution of triethanolamine and sodium citrate in step (2), the molar ratio of triethanolamine to sodium citrate is 3:1 to 1:

3.

3. The microalgae alkali precipitation harvesting and re-cultivation and utilization process according to claim 2, characterized in that: The alkaline solution in step (3) is sodium hydroxide solution or potassium hydroxide solution.

4. The microalgae alkali precipitation harvesting and re-cultivation and utilization process according to claim 3, characterized in that: The standing time of step (4) is 10 min to 21 h.

5. The microalgae alkali precipitation harvesting and re-cultivation utilization process according to claim 1, characterized in that: In step (1), the microalgae culture solution is a microalgae culture solution obtained by adding microalgae to a culture solution for culturing; or microalgae are added to a culture solution for culturing, the culture solution is centrifuged to collect the precipitate, water is added to the precipitate, and the mixture is mixed to obtain the microalgae culture solution.

6. The method for collecting and re-cultivating microalgae alkali sediment according to claim 1, characterized in that: The microalgae are autotrophic algae or heterotrophic algae.

7. The microalgae alkali precipitation harvesting and re-cultivation and utilization process according to claim 6, characterized in that: The microalgae is one of autotrophic Chlorella sorokinensis, heterotrophic Chlorella sorokinensis, Cladosporium tenuifolium, and Spirulina.

8. The microalgae alkali precipitation harvesting and re-cultivation and utilization process according to claim 7, characterized in that: The culture medium in step (4) is one of BG11 culture medium, heterotrophic culture medium, SP culture medium, and Zarrouk culture medium.

9. The microalgae alkali precipitation harvesting and re-cultivation and utilization process according to claim 8, characterized in that: The specific composition of the heterotrophic culture medium is: 10-50 g / L of glucose, 1-5 g / L of yeast powder or peptone, and 1.7 g / L of BG11 culture medium.