Method for flocculating microalgae

Abstract

To provide means for easily flocculating microalgae growing and multiplying in treated water by using a flocculant.SOLUTION: It is confirmed that microalgae such as cyanobacteria and green algae of the genus Chlorella can be flocculated in water, when chitosan and sodium hydroxide are added.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for flocculating microalgae, and more particularly to a method for flocculating microalgae by adding chitosan and sodium hydroxide to treatment water containing microalgae and stirring the mixture. [Background technology]

[0002] In areas where the population is increasing, wastewater from residential areas flows in, causing eutrophication, leading to deterioration of water quality such as abnormal algae blooms and the resulting foul odors (see, for example, Non-Patent Document 1).

[0003] For this reason, a method for separating and recovering microalgae (see, for example, Patent Document 1) has been proposed, which comprises an adjustment step of adjusting the salt concentration of raw water so that the surface zeta potential is such that microalgae are likely to aggregate, an aggregation step of adding an inorganic coagulant to the raw water containing the microalgae to cause an aggregation reaction, and a solid-liquid separation step of separating the aggregated flocs produced in the aggregation step into solid-liquid.It is also well known that algae are likely to grow inside cooling towers, and a method for preventing algae growth or adhesion in a water tank (see, for example, Patent Document 2) has been proposed, which involves scraping off algae adhering to the inner wall surface to suppress the growth of algae on this inner wall surface, and a cleaning rolling element (see, for example, Patent Document 3) that purifies the inner surface of the water tank by moving in conjunction with the water flow.

[0004] In addition, microbial control agents containing imidazolium salt derivatives have been proposed as microbial control agents (see, for example, Patent Document 4), but the use of control agents containing such chemical substances can sometimes pose problems such as the toxicity of the control agents themselves.

[0005] In addition, a method has been proposed in which, with the ultimate goal of producing fuel oil from microalgae or producing useful organic matter using microalgae, non-floating halophilic microalgae are moved to near the surface of the seawater or artificial seawater by the buoyancy of oxygen bubbles that adhere to the non-floating halophilic microalgae during cultivation, and the non-floating halophilic microalgae that have moved to near the surface are then recovered.However, it has also been stated that the method of recovering microalgae from culture tanks, etc., in which the microalgae are pumped out and then centrifuged, filtered, etc., is still common in recent years (see, for example, Patent Document 5).

[0006] Chitosan is a type of polymer, and is known to be a polysaccharide produced in nature when chitin, contained in the shells of crustaceans and the cell walls of fungi such as mushrooms, is decomposed by enzymes such as chitinase when crabs and insects molt or when mushrooms divide. Furthermore, a technique for treating turbid water generated at construction sites, such as tunnel construction sites, using a chitosan flocculant has been proposed (see, for example, Non-Patent Document 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-179578 [Patent Document 2] Japanese Patent Application Laid-Open No. 2001-311184 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-126256 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-67573 [Patent Document 5] Japanese Patent Publication No. 2023-166103 [Non-patent literature]

[0008] [Non-Patent Document 1] Environmental Technology Vol.23 No.6 (1994)332-335 [Non-patent document 2] http: / / library.jsce.or.jp / jsce / open / 00984 / 2010 / 2010-0093.pdf Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a means for easily flocculating microalgae growing and proliferating in treatment water using a less toxic flocculant. [Means for solving the problem]

[0010] The present inventors investigated whether the addition of chitosan would cause flocculation using Synechocystis sp. PCC 6803 (hereinafter also referred to as "Synechocystis 6803"), a cyanobacterium that has long been used in research as a model organism. They found that the addition of chitosan caused flocculation in a liquid medium suitable for the growth of cyanobacteria, but not in MilliQ water. They then added chitosan and sodium hydroxide (NaOH) and found that Synechocystis 6803 flocculated in water. Furthermore, they confirmed that the addition of chitosan and sodium hydroxide also caused flocculation in Chlorella algae, a type of green algae, in water, leading to the completion of the present invention.

[0011] That is, the present invention is as specified by the following invention-specifying matters. [1] A method for flocculating microalgae, comprising adding chitosan and sodium hydroxide to treatment water containing microalgae and stirring the mixture to flocculate the microalgae. [2] The method for flocculating microalgae according to [1] above, wherein chitosan is added to the treatment water as a chitosan flocculating reagent containing acetic acid. [3] The method for flocculating microalgae according to [1] above, wherein sodium hydroxide is added to the treatment water as a sodium hydroxide solution containing ethanol. [4] The method for flocculating microalgae according to [1] above, wherein the chitosan concentration in the treatment water containing microalgae is 5 to 50 mg / L. [5] The flocculation method according to [1] above, wherein the concentration of sodium hydroxide in the treated water containing microalgae is 28 to 280 mg / L. [6] The method for flocculating microalgae according to [1] above, wherein the microalgae are cyanobacteria or green algae. [7] The method for flocculating microalgae according to [6] above, wherein the cyanobacteria are cyanobacteria belonging to the genus Synechocystis. [8] The method for flocculating microalgae according to [6] above, wherein the green algae are green algae belonging to the genus Chlorella.

[0012] The present invention is also defined by the following invention-specific matters. [9] A method for purifying the environment by recovering the flocculated microalgae using the method for flocculating microalgae according to any one of [1] to [8] above.

[10] A method for culturing microalgae aggregated by the method for aggregating microalgae according to any one of [1] to [8] above, using an antifoaming agent. [Effects of the Invention]

[0013] According to the present invention, the environment can be purified by recovering the microalgae that have aggregated in the treatment water, and by culturing the recovered microalgae, the proliferated microalgae can be used for various purposes. [Brief explanation of the drawings]

[0014] [Figure 1] (a) Chitosan was added to a final concentration of 15 mg / L to cyanobacterial solution B1 prepared from Synechocystis 6803 suspended in BG-11 medium, resulting in flocculation. (b) Chitosan was added to a final concentration of 100 mg / L to cyanobacterial solution Q prepared from Synechocystis 6803 suspended in MilliQ water, resulting in unagglutination. [Figure 2](a) Cyanobacterial solution B1 prepared from Synechocystis 6803 suspended in BG-11 liquid medium was added with chitosan at a final concentration of 15 mg / L and 0.7 M sodium hydroxide solution, resulting in flocculation. (b) Cyanobacterial solution Q prepared from Synechocystis 6803 suspended in MilliQ water was added with chitosan at a final concentration of 15 mg / L and 0.7 M sodium hydroxide solution, resulting in flocculation. [Figure 3] (a) Figure 1 shows the state of aggregation when chitosan and a NaOH-EtOH mixed solution at a final concentration of 15 mg / L are added to cyanobacterial solution B1 prepared from Synechocystis 6803 suspended in BG-11 liquid medium. (b) Figure 1 shows the state of aggregation when chitosan and a NaOH-EtOH mixed solution at a final concentration of 15 mg / L are added to cyanobacterial solution B1 prepared from Synechocystis 6803 suspended in MilliQ water. [Figure 4] (a) Synechocystis 6803 (OD730=1) aggregated by adding chitosan and a NaOH-EtOH mixture at a final concentration of 15 mg / L. (b) Synechocystis 6803 (OD730=3) aggregated by adding chitosan and a NaOH-EtOH mixture at a final concentration of 20 mg / L. [Figure 5] This shows a graph of the absorbance values ​​at OD730nm of the supernatants after centrifugation for cyanobacterial solution B1 and cyanobacterial solution Q, each containing different concentrations of added chitosan. [Figure 6] The figures show the state of Synechocystis 6803: (a) precipitate without chitosan addition, (b) precipitate with chitosan addition (50 mg / L), (c) supernatant without chitosan addition, and (d) supernatant with chitosan addition (50 mg / L). [Figure 7] (a) Chlorella sorokiniana (OD730=1) flocculated by adding chitosan at a final concentration of 10 mg / L. (b) Chlorella sorokiniana (OD730=3) flocculated by adding chitosan at a final concentration of 10 mg / L. [Figure 8]The graph shows the absorbance values ​​at OD730nm of the supernatants after centrifugation for green algae solution B1 and green algae solution Q, each containing different concentrations of added chitosan. [Figure 9] The figures show the state of Chlorella sorokiniana (a) precipitate without chitosan addition, (b) precipitate with chitosan addition (50 mg / L), (c) supernatant without chitosan addition, and (d) supernatant with chitosan addition (50 mg / L). [Figure 10] (a) Chlorella vulgaris (OD730=1) flocculated by adding chitosan at a final concentration of 25 mg / L. (b) Chlorella vulgaris (OD730=3) flocculated by adding chitosan at a final concentration of 55 mg / L. [Figure 11] This graph shows the absorbance values ​​at OD730nm of the supernatants after centrifugation for Chlorella Solution C1 and Chlorella Solution Q, each containing different concentrations of added chitosan. [Figure 12] The figures show the state of Chlorella vulgaris (a) precipitate without chitosan addition, (b) precipitate with chitosan addition (50 mg / L), (c) supernatant without chitosan addition, and (d) supernatant with chitosan addition (50 mg / L). [Figure 13] This shows the culture of Chlorella sorokiniana in media containing antifoaming agents. (a) KM72, (b) KM72S, (c) KM72F, and (d) KM72FS are the antifoaming agents. [Figure 14] This shows Synechocystis 6803 cultured in a medium containing an antifoaming agent. (a) KM72 and (b) KM72F are the antifoaming agents. [Figure 15] This shows Synechocystis 6803 cultured in a medium without an antifoaming agent. [Figure 16] This shows Synechocystis 6803 and Chlorella sorokiniana cultured in media containing an antifoaming agent. DETAILED DESCRIPTION OF THE INVENTION

[0015] The method for flocculating microalgae of the present invention is not particularly limited as long as it involves adding chitosan and sodium hydroxide to and stirring treated water containing microalgae to flocculate the microalgae. Examples of treated water containing microalgae include domestic wastewater, agricultural wastewater, wastewater from garbage disposal plants, wastewater from power plants, sewage treatment water, and cooling water in cooling towers.

[0016] In the present invention, aggregation refers to the aggregation of microalgae dispersed in the treatment water to form larger aggregates called flocs. When the density and size of the flocs increase, they sink to the bottom of the treatment water, etc., and the sedimentation is sometimes referred to as sediment.

[0017] The microalgae referred to above refer to algae present in water such as freshwater and seawater, the size of which is mainly about 1 μm to 100 μm. The microalgae in the present invention are not particularly limited as long as they are algae that can be aggregated by the aggregation method of the present invention, but examples include organisms that perform photosynthesis and generate oxygen that can grow and proliferate in the treated water, excluding mosses, ferns, and spermatophytes, and for example, prokaryotic algae such as cyanobacteria, and eukaryotic algae.

[0018] The cyanobacteria can be one type of bacteria that performs photosynthesis accompanied by oxygen production, and examples of such cyanobacteria include those of the orders Synechococcales, Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, Stigonematales, and Gloeobacterales, with cyanobacteria of the order Synechococcales being preferred. Examples of cyanobacteria in the order Synechococcales include cyanobacteria from the genera Synechocystis, Synechococcus, Aphanocapsa, Coelosphaerium, Merismopedia, Prochlorococcus, and Acaryochroris, with preferred examples being cyanobacteria belonging to the genus Synechocystis.

[0019] Examples of the eukaryotic algae include green algae, euglena algae, red algae, glaucophyte algae, cryptophyte algae, dinoflagellates, goldenrod algae, diatoms, brown algae, xanthophyte algae, haptophyte algae, raphidophyte algae (green flagellates), and prasinophyte algae.

[0020] Examples of the green algae include algae belonging to the Chlorophyceae, Trebouxiophyceae, Prasinophyceae, Ulvophyceae, and the like of the division Chlorophyta.

[0021] Examples of algae belonging to the Treboxiophyceae include algae of the genus Chlorella, and examples of algae belonging to the Chlorophyceae include algae of the genus Chlamydomonas.

[0022] Examples of algae belonging to the genus Chlorella include Chlorella vulgaris, Chlorella sorokiniana, Chlorella regularis, Chlorella pyrenoidosa, Chlorella ellipsidea, and Chlorella minutissima, with Chlorella vulgaris and Chlorella sorokiniana being preferred. For convenience, they may also include protozoa that have Chlorella living intracellularly as an organelle, such as Paramecium bursaria, Stentor basalis, and Greenhydra.

[0023] Examples of the Euglena algae include Euglena (Euglena), a species of the genus Euglena belonging to the order Euglenales of the class Euglenophyceae of the phylum Euglenozoa. Furthermore, the microalgae of the present invention are not limited to a specific type of microalgae, but may also include phytoplankton, which is a group of many types of microalgae.

[0024] The chitosan to be added to the treatment water containing the microalgae is a polysaccharide that mainly has a structure in which glucosamine is linked by β1,4 bonds and may contain N-acetylglucosamine as a constituent other than glucosamine. The origin of the chitosan used in the present invention is not particularly limited, and for example, chitosan obtained by deacetylating all or part of chitin obtained by removing calcium, protein, etc. from crustacean shells or the like through hydrolysis can be used. Commercially available products can also be used, but chitosan with a deacetylation degree of 50.0 mol% is preferred, 70.0 mol% or more is preferred, and 80.0 mol% or more is more preferred.

[0025] Since the chitosan is insoluble in water, it is preferably used as a solution by dissolving it in an organic acid such as acetic acid or citric acid, or an inorganic acid such as hydrochloric acid or nitric acid. For ease of handling, it is more preferable to use a chitosan solution in which chitosan is dissolved in acetic acid as a chitosan agglutinating reagent.

[0026] The method for preparing the chitosan agglutinating reagent is not particularly limited as long as it achieves the effects of the present invention. An example of a method for preparing the chitosan agglutinating reagent is to add and suspend chitosan in water such as MilliQ water so that the chitosan concentration becomes 0.1 to 1%, preferably 0.3 to 0.7%, and more preferably 0.4 to 0.6%, to prepare chitosan-MilliQ water, and then add an equal amount (w / v) of acetic acid to the chitosan and stir the mixture.

[0027] The concentration of chitosan added to the treatment water containing microalgae is not particularly limited as long as the effects of the present invention are achieved, but can be 5 mg / L or more, preferably 8 mg / L or more, more preferably 10 mg / L or more, and even more preferably 15 mg / L or more. Considering cost and the like, the upper limit can be 50 mg / L, for example.

[0028] The sodium hydroxide (NaOH) to be added and stirred into the treatment water containing the microalgae is preferably a NaOH-EtOH mixed solution, which is a mixture of sodium hydroxide and ethanol. It has been confirmed that the ethanol does not inhibit the coagulation reaction in the microalgae.

[0029] The concentration of sodium hydroxide added to the treatment water containing microalgae is not particularly limited as long as the effects of the present invention are achieved, but an example is 28 mg / L or more. The upper limit is 280 mg / L, preferably 200 mg / L. The mass ratio of chitosan to NaOH is 1:1 to 1:40, preferably 1:2 to 1:20, more preferably 1:3 to 1:10, more preferably 1:4 to 1:7, and even more preferably 1:5 to 1:6.

[0030] The concentration of ethanol (EtOH) added to the treatment water containing microalgae is not particularly limited as long as the effects of the present invention are achieved, but can be 12 mg / L or more, preferably 13.5 mg / L or more. The upper limit can be 140 mg / L, preferably 100 mg / L. The mass ratio of NaOH:EtOH can be 4:1 to 1.2:1, preferably 3:1 to 1.5:1, and more preferably 2.2:1 to 1.8:1.

[0031] The method for purifying the environment of the present invention can be carried out by recovering the aggregated microalgae using the above-mentioned method for flocculating microalgae. Examples of methods for recovering microalgae include known methods such as centrifugation, sedimentation, flotation, and filtration.

[0032] The recovered microalgae can be further cultured. The method for culturing the microalgae is not particularly limited as long as it is a known culture method, such as culturing microalgae in a microalgae culture facility containing a culture medium such as a liquid medium in which the microalgae can be cultured. A method can also be adopted in which a culture medium in which the microalgae can be cultured is added to the treated water such as the wastewater, and the microalgae are cultured without preparing any specific culture facility.

[0033] The medium for culturing the above-mentioned microalgae is not particularly limited as long as it is a medium in which the microalgae can grow and proliferate, and examples thereof include BG-11 medium and C medium. However, it is preferable to cultivate the microalgae using an antifoaming agent to prevent the culture vessel from becoming dirty due to foaming during culture, the microalgae from adhering to the piping of the culture apparatus due to foaming, and the microalgae from flowing out of the culture vessel through the exhaust pipe of the culture apparatus.

[0034] The antifoaming agent is not particularly limited as long as it does not affect the growth of microalgae and has an antifoaming effect in the culture medium, and examples include silicone-based antifoaming agents such as the KM series from Shin-Etsu Chemical Co., Ltd.

[0035] The cultured microalgae can be used, for example, as ingredients for foods, cosmetics, etc., as a food source such as a protein source, and / or as an energy source such as biomass fuel, and can also be used to fix carbon dioxide, which is considered a greenhouse gas.

[0036] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example]

[0037] [Example 1] [Agglomeration test 1] (Preparation of BG-11 medium) BG-11 liquid medium, known to be suitable for culturing cyanobacteria, was prepared according to the instructions at https: / / osanaimeiji.wixsite.com / website / %E8%A4%87%E8%A3%BD-10-sds-page-1. Specifically, to prepare BG-11 liquid medium, six stock solutions (solutions I to V and HEPES solution) were prepared as follows: solution I: 500x, solution II: 20x, solution III: 500x, solution IV: 1000x, solution V: 1000x, and HEPES solution: 50x. These were mixed together before use to prepare the liquid medium.

[0038] [Liquid I] Citric acid 0.6g Ferric ammonium citrate 0.6g Na2EDTA 0.1g / 200mL

[0039] [Liquid II] 330g NaNO K2HPO40.78g MgSO4·7H2O 1.5g / 1L

[0040] [Liquid III] CaCl2·2H2O 3.8g / 200mL [IV fluid] Na2CO3 4g / 200mL

[0041] [V liquid] H3BO3 2.86g MnCl₂·4H₂O 1.81g ZnSO4·7H2O 0.22g CuSO4·5H2O 0.08g Na2MoO4·2H2O 0.021g 10%H2SO4 solution 50uL After making up to 1 L with MilliQ water, 0.0494 g of Co(NO3)2·6H2O was added.

[0042] HEPES liquid 1M HEPES-KOH (pH 7.8)

[0043] To prepare the liquid medium, stock solutions II, HEPES, III, IV, and V were placed in a measuring cylinder in that order, then filled up with MilliQ water, transferred to a medium bottle, and autoclaved. After autoclaving, the stock solution of solution I was added to the solution once it had cooled to prepare the liquid medium.

[0044] (Preparation of cyanobacterial solution) Synechocystis 6803 was used. BG-11 liquid medium was used as the culture medium, and the medium was continuously irradiated with white light at 30-100 μmol photons / m while aerating with 2% CO2. 2 The cyanobacteria were cultured at 30°C under a light condition of 0.5 s. After sufficient growth was confirmed, they were subjected to an aggregation test. The fully grown Synechocystis 6803 was centrifuged at 1000 rpm for 10 minutes, and the precipitate was collected and resuspended in BG-11 liquid medium to an OD730 of 1 to prepare two types of cyanobacteria solution: Solution B1, in which the precipitate was resuspended in MilliQ water to an OD730 of 1.

[0045] (reagent) Chitosan-MilliQ water was prepared by adding Chitosan 100 (Fujifilm Wako Pure Chemical Industries, Ltd.) to MilliQ water to a chitosan concentration of 0.5% and suspending it. An equal amount (w / v) of acetic acid (special reagent grade) was added to the chitosan-MilliQ water and further stirred to prepare a chitosan agglutination reagent.

[0046] (Agglomeration test method) Cyanobacterial solution B1 and cyanobacterial solution Q were each placed in a 50 mL beaker and vigorously stirred with a stirrer. After that, chitosan flocculation reagent was added to the solution to a chitosan concentration of 5 mg / L and vigorously stirred for 5 minutes. After leaving the solution to stand for 10 minutes, the flocculation state of each cyanobacterial solution was observed. After leaving the solution to stand for 10 minutes, chitosan flocculation reagent was further added to each cyanobacterial solution to a chitosan concentration of 10 mg / L, and the solution was stirred for 5 minutes. After leaving the solution to stand for 10 minutes, the flocculation state was observed. After this, the pattern of adding chitosan in 5 mg / L increments, stirring for 5 minutes, and leaving the solution to stand for 10 minutes was repeated until the final concentration reached 100 mg / L, and the flocculation state was observed.

[0047] Table 1 shows the results of the aggregation state when a chitosan aggregation reagent was added to cyanobacterial solution B1 or cyanobacterial solution Q. Figure 1(a) shows a photograph of the aggregation state when chitosan was added to cyanobacterial solution B1 at a final concentration of 15 mg / L, and Figure 1(b) shows a photograph of the aggregation state when chitosan was added to cyanobacterial solution Q at a final concentration of 100 mg / L.

[0048] [Table 1] *1: No aggregation or precipitation was observed. *2: Not implemented

[0049] (Result 1) As is clear from Table 1, in cyanobacteria solution B1, aggregation was observed up to chitosan concentrations of 5 mg / L and 10 mg / L, and precipitation was observed at 15 mg / L, but in cyanobacteria solution Q, neither aggregation nor precipitation was observed even when chitosan was added at a concentration of 100 mg / L.

[0050] (Result 2) As is clear from Figures 1(a) and (b), flocculation was observed when chitosan was added to cyanobacterial solution B1 at a final concentration of 15 mg / L, but flocculation did not occur when chitosan was added to cyanobacterial solution Q at a final concentration of 100 mg / L, more than six times the 15 mg / L concentration. This suggests that a stable flocculation reaction may not be achieved depending on the salt and pH conditions of the solution containing cyanobacteria and other microalgae. Therefore, we decided to investigate conditions under which a stable flocculation reaction could be achieved even in general treated water such as wastewater.

[0051] [Example 2] (Preparation of cyanobacterial solution) Two types of cyanobacteria solutions, namely, cyanobacteria solution B1 and cyanobacteria solution Q, were prepared using the same procedure as in Example 1 (preparation of cyanobacteria solution).

[0052] (agglutination reagent) The chitosan agglutinating reagent was used as prepared in (Agglutinating Reagent) of Example 1. Furthermore, a 0.7 M sodium hydroxide solution and a NaOH-EtOH mixed solution containing 1 M sodium hydroxide and ethanol at a ratio of 7:3 were prepared using MilliQ water.

[0053] (Agglomeration test method) The cyanobacterial solution B1 and cyanobacterial solution Q were each placed in two 50 mL beakers and vigorously stirred. After vigorously stirring, chitosan flocculation solution was added to each of the cyanobacterial solutions B and Q, respectively, to achieve a chitosan concentration of 5 mg / L. The solution was then vigorously stirred for 5 minutes. After 10 minutes of static stirring, the flocculation state of each cyanobacterial solution was observed. Further chitosan flocculation reagent was then added to each of the cyanobacterial solutions to achieve a chitosan concentration of 10 mg / L. The solution was stirred for 5 minutes, then allowed to stand for 10 minutes. The flocculation state was observed after 10 minutes of static stirring. The chitosan flocculation reagent was then added in 5 mg / L increments, followed by 5 minutes of static stirring, and then 10 minutes of static stirring. The concentrations of NaOH and EtOH were as shown in Table 2. The results are shown in Table 2 and Figures 2 and 3.

[0054] [Table 2]

[0055] (Result 1) As is clear from Table 2, when 0.7 M sodium hydroxide solution was added to the chitosan flocculation reagent, or when NaOH-EtOH mixed solution was added to the chitosan flocculation reagent, flocculation was observed in both cyanobacterial solution B1 and cyanobacterial solution Q when the chitosan concentration was 5 mg / L and 10 mg / L, and precipitation was observed at 15 mg / L.

[0056] (Result 2) Figure 2(a) shows a photograph of the state of cyanobacteria solution B1 after adding chitosan to a final concentration of 15 mg / L and flocculating with 0.7 M sodium hydroxide solution, while Figure 2(b) shows a photograph of the state of cyanobacteria solution Q after adding chitosan to a final concentration of 15 mg / L and flocculating with 0.7 M sodium hydroxide solution. Figure 3(a) shows a photograph of the state of cyanobacteria solution B1 after adding chitosan to a final concentration of 15 mg / L and flocculating with NaOH-EtOH mixed solution, while Figure 3(b) shows a photograph of the state of cyanobacteria solution Q after adding chitosan to a final concentration of 15 mg / L and flocculating with NaOH-EtOH mixed solution.

[0057] Whether using a chitosan flocculating reagent and an aqueous solution of sodium hydroxide alone or a chitosan flocculating reagent and an NaOH-EtOH mixed solution, no difference was observed in the state of precipitation or flocculation in cyanobacterial solution B1 or cyanobacterial solution Q, confirming that a stable flocculation reaction could be obtained. Since it is expected that the addition of ethanol may be necessary depending on the type of microalgae, further investigation was carried out using a NaOH-EtOH mixed solution as the sodium hydroxide solution.

[0058] [Example 3] (Preparation of cyanobacterial solution) Synechocystis 6803 was used as a test strain. It was continuously cultured at 30°C in BG-11 medium under the above light conditions with 2% CO2 aeration. After sufficient cyanobacterial growth was confirmed, it was subjected to an aggregation test. The fully grown Synechocystis 6803 was centrifuged at 1000 rpm for 10 minutes, and the precipitate was collected and resuspended in BG-11 liquid medium to prepare two types of cyanobacterial solution: Cyanobacterial Solution B1, with an OD730 of 1, and Cyanobacterial Solution B3, with an OD730 of 3.

[0059] (agglutination reagent) The chitosan agglutinating reagent prepared in Example 1 (agglutinating reagent) and the NaOH-EtOH mixed solution prepared in Example 2 (agglutinating reagent) were used.

[0060] (Agglomeration test method) The cyanobacterial solution B1 or B3 was placed in a 50 mL beaker and vigorously stirred. After 5 minutes of vigorously stirring, a chitosan flocculation reagent was added to the solution to achieve a chitosan concentration of 5 mg / L. The solution was then stirred vigorously for 5 minutes. The solution was then added with a NaOH-EtOH mixture and stirred vigorously for 5 minutes. The solution was then allowed to stand for 10 minutes, after which the flocculation state of each cyanobacterial solution was observed. The chitosan flocculation reagent was then added to the solution to achieve a chitosan concentration of 10 mg / L. The solution was then stirred for 5 minutes, and the solution was allowed to stand for 10 minutes. The flocculation state of each solution was then observed. The chitosan addition, 5 minutes of stirring, and 10 minutes of standing time were repeated in 5 mg / L increments until the final chitosan concentration reached 40 mg / L. The same volume of NaOH-EtOH mixture was added, i.e., the NaOH-EtOH concentration was increased in increments of 28 mg / L to 13.8 mg / L, and the flocculation state was observed. The results are shown in Figure 4.

[0061] (result) Figure 4(a) shows a photograph of the state of cyanobacteria solution B1 after adding chitosan to a final concentration of 15 mg / L and then adding a NaOH-EtOH mixed solution to cause aggregation. Figure 4(b) shows a photograph of the state of cyanobacteria solution B3 after adding chitosan to a final concentration of 20 mg / L and then adding a NaOH-EtOH mixed solution to cause aggregation.

[0062] As is clear from Figure 4(a), when chitosan was added to a final concentration of 15 mg / L in cyanobacterial solution B1, which was adjusted to an OD730 of 1, aggregation was observed. As is clear from Figure 4(b), when chitosan was added to a final concentration of 20 mg / L in cyanobacterial solution B3, which was adjusted to an OD730 of 3, aggregation was also observed. This confirms that it is possible to aggregate cyanobacteria using low concentrations of chitosan.

[0063] [Example 4] In Example 3 above, since a precipitate was visually confirmed when the chitosan concentration was 15 mg / L, the agglutination reaction product was separated into a supernatant and a precipitate by centrifugation to evaluate the agglutination state more objectively.

[0064] (Preparation of cyanobacterial solution) Two types of cyanobacteria solutions, namely, cyanobacteria solution B1 and cyanobacteria solution Q, were prepared using the same procedure as in Example 1 (preparation of cyanobacteria solution).

[0065] (agglutination reagent) The chitosan agglutinating reagent prepared in Example 1 (agglutinating reagent) and the NaOH-EtOH mixed solution prepared in Example 2 (agglutinating reagent) were used.

[0066] (Agglomeration test method) Eight samples were prepared by placing 50 mL of each of cyanobacterial solution B1 and cyanobacterial solution Q into four beakers. Each sample was vigorously stirred with a stirrer, followed by the addition of chitosan flocculation reagent to a chitosan concentration of 5 mg / L and vigorously stirring for 5 minutes. A NaOH-EtOH mixed solution was then added and vigorously stirred for 5 minutes. After 10 minutes of incubation, the flocculation state of each cyanobacterial solution was observed. Further chitosan flocculation reagent was added to a chitosan concentration of 10 mg / L, followed by 5 minutes of incubation and 10 minutes of incubation. The same chitosan addition, 5 minutes of stirring, and 10 minutes of incubation cycle were repeated, with chitosan flocculation solution added in 5 mg / L increments to achieve final chitosan concentrations of 5 mg / L, 10 mg / L, 25 mg / L, and 50 mg / L. Each time a chitosan flocculation solution was added, the NaOH-EtOH mixed solution was added in the same manner as above, resulting in a total of eight samples. After the agglutination reaction, the sample was placed in a centrifuge tube and centrifuged at 500 rpm for 10 minutes, and the supernatant was measured at OD 730. As negative controls, chitosan-free cyanobacteria solution B1 and chitosan-free cyanobacteria solution Q were also centrifuged in the same manner.

[0067] The absorbance values ​​at OD730nm for the supernatants obtained after centrifugation of the eight samples above after the agglutination reaction and for the negative controls (cyanobacterial solution B1 and cyanobacterial solution Q without chitosan) are shown in Figure 5. Figure 6 also shows the microscopic observations of the precipitates and supernatants of cyanobacterial solution Q above, both with and without chitosan.

[0068] (Result 1) As shown in Figure 5, when chitosan flocculation reagent and NaOH+EtOH solution were added, the absorbance of the supernatant began to decrease at a final chitosan concentration of 5 mg / L. The OD730 value of the supernatant decreased as the chitosan concentration increased. At a chitosan concentration of 5 mg / L, the OD730 values ​​for both cyanobacterial solution B1 and cyanobacterial solution Q decreased by approximately 30–50% compared to the chitosan-free solution. This confirmed that the flocculation reaction already began at a final chitosan concentration of 5 mg / L. Comparing cyanobacterial solution B1 and cyanobacterial solution Q, which was cultured in MilliQ water, showed a lower OD730 value than cyanobacterial solution B, which was cultured in BG-11 medium, indicating that MilliQ water tends to facilitate flocculation. This suggests that the flocculation method of the present invention may be effectively used for wastewater containing no salts.

[0069] (Result 2) As is clear from Figure 6, in the case of cyanobacterial solution Q without chitosan, the precipitate (a) after centrifugation was easily dispersible, and no aggregation or solidification was observed. On the other hand, the precipitate (b) after centrifugation of cyanobacterial solution Q with 50 mg / L of chitosan added was not easily dispersible.

[0070] [Example 5] (Preparation of green algae solution) The green algae Chlorella cf. sorokiniana (hereafter referred to as "Chlorella sorokiniana") newly isolated from wastewater from a pig farm was used as the test medium. It was continuously cultured at 30°C using BG-11 medium with 2% CO2 aeration. After sufficient growth was confirmed, it was subjected to a flocculation test. The sufficiently grown newly isolated strain was centrifuged at 1000 rpm for 10 minutes, and the precipitate was collected. The collected precipitate was resuspended in BG-11 liquid medium to prepare green algae solution B1 with an OD730 of 1 and green algae solution B3 with an OD730 of 3.

[0071] (agglutination reagent) The chitosan agglutinating reagent prepared in Example 1 (agglutinating reagent) and the NaOH-EtOH mixed solution prepared in Example 2 (agglutinating reagent) were used.

[0072] (Agglomeration test method) Green algae solution B1 and green algae solution B3 were each placed in a 50 mL beaker and vigorously stirred with a stirrer. After that, chitosan flocculation reagent was added to the solution to make the chitosan concentration 5 mg / L and vigorously stirred for 5 minutes. A NaOH-EtOH mixed solution was then added and vigorously stirred in the same way. After leaving the solution to stand for 10 minutes, the flocculation state of each green algae solution was observed. Further chitosan flocculation reagent was added to make the chitosan concentration 10 mg / L and stirred for 5 minutes. After leaving the solution to stand for 10 minutes, a NaOH-EtOH mixed solution was added as above. The flocculation results for green algae solution B1 and green algae solution B3 are shown in Figure 7.

[0073] (result) As is clear from Figure 7(a), when chitosan was added to a final concentration of 10 mg / L in green algae solution B1, which was adjusted to an OD730 of 1, flocculation was visually observed. Furthermore, as is clear from Figure 7(b), when chitosan was added to a final concentration of 10 mg / L in green algae solution B3, which was adjusted to an OD730 of 3, flocculation was also observed. For Chlorella sorokiniana, which was examined here, flocculation was visually confirmed at a lower concentration of 10 mg / L of chitosan than for Synechocystis 6803, demonstrating that the chitosan concentration required for flocculation differs depending on the bacterial species.

[0074] [Example 6] In Example 5 above, aggregation was confirmed visually when the chitosan concentration was 10 mg / L, so the aggregated (reacted) product was separated into supernatant and precipitate by centrifugation to evaluate the aggregation state more objectively.

[0075] (Preparation of green algae solution) The above-mentioned Chlorella sorokiniana was used for the test. It was continuously cultured at 30°C using BG-11 medium with 2% CO2 aeration. After sufficient growth was confirmed, it was subjected to a flocculation test. The fully grown Chlorella sorokiniana was centrifuged at 1000 rpm for 10 minutes, and the precipitate was collected and resuspended in BG-11 liquid medium to an OD730 of 1 to prepare green algae solution B1. The precipitate was also resuspended in MilliQ water to an OD730 of 1 to prepare green algae solution Q.

[0076] (agglutination reagent) The chitosan agglutinating reagent prepared in Example 1 (agglutinating reagent) and the NaOH-EtOH mixed solution prepared in Example 2 (agglutinating reagent) were used.

[0077] (Agglomeration test method) Eight samples were prepared by placing 50 mL of each of the above green algae solutions B1 and Q into four beakers. Each sample was vigorously stirred with a stirrer, followed by the addition of a chitosan flocculating reagent to a chitosan concentration of 5 mg / L and vigorously stirring for 5 minutes. A NaOH-EtOH mixed solution was then added and vigorously stirred for 5 minutes. The solution was then allowed to stand for 10 minutes, after which the flocculation state of each green algae solution was observed. The chitosan flocculating reagent was then added to a chitosan concentration of 10 mg / L, followed by stirring for 5 minutes and then allowing to stand for 10 minutes. The flocculation state was then observed. From this point onward, the chitosan flocculating solution was added in 5 mg / L increments, followed by stirring for 5 minutes and allowing to stand for 10 minutes, and the chitosan flocculating solution was added in 5 mg / L increments, followed by stirring for 5 minutes and allowing to stand for 10 minutes, after which the chitosan flocculating solution was added in 5 mg / L increments, followed by chitosan flocculating solution for final chitosan concentrations of 5 mg / L, 10 mg / L, 25 mg / L, and 50 mg / L. Each time the chitosan flocculating solution was added, the NaOH-EtOH mixed solution was added in the same manner as above, resulting in a total of eight samples. After the agglutination reaction, the sample was placed in a centrifuge tube and centrifuged at 500 rpm for 10 minutes, and the supernatant was measured at OD 730. As negative controls, chitosan-free green algae solution B1 and chitosan-free green algae solution Q were also centrifuged in the same manner.

[0078] Figure 8 shows a graph of the absorbance at OD 730 nm for the supernatants obtained after centrifugation of green algae solution B1 and green algae solution Q after the agglutination reaction. Figure 9 also shows the state of the precipitates and supernatants of green algae solution Q observed under a microscope with and without chitosan added.

[0079] (Result 1) As is clear from Figure 8, when the chitosan flocculation reagent and NaOH + EtOH solution were added, the absorbance value of the supernatant began to decrease from a final chitosan concentration of 5 mg / L, and the OD730 value of the supernatant became even lower as the chitosan concentration was further increased. For green algae solution Q, the OD730 value at a chitosan concentration of 5 mg / L was reduced by approximately 80% compared to the solution without chitosan. For green algae solution B1, the OD730 value at 10 mg / L was reduced by approximately 75% compared to the solution without chitosan. Furthermore, when comparing green algae solution B and green algae solution Q, green algae solution Q cultured in MilliQ water tended to flocculate more easily, suggesting the possibility that the present invention may be effectively used in wastewater that does not contain salts.

[0080] (Result 2) As is clear from Figure 9, in the case of green algae solution Q without added chitosan, the precipitate (a) after centrifugation was easily dispersible, and no aggregation or solidification was observed. On the other hand, the precipitate (b) after centrifugation of green algae solution Q to which 50 mg / L of chitosan had been added was not easily dispersible.

[0081] [Example 7] (Preparation of Chlorella Solution) Chlorella vulgaris Beijerinck var. vulgaris (NIES-227, provided by the National Institute for Environmental Studies; hereafter referred to as "Chlorella vulgaris") was used. It was continuously cultured at 25°C with aeration using the C medium shown in Table 3 below. After sufficient growth was confirmed, it was subjected to an agglutination test. The fully grown Chlorella vulgaris was centrifuged at 3000 rpm for 5 minutes, and the precipitate was collected and resuspended in the C medium shown below to prepare Chlorella solution C1 with an OD730 of 1 and Chlorella solution C3 with an OD730 of 3.

[0082] [Table 3]

[0083] (agglutination reagent) The chitosan agglutinating reagent prepared in Example 1 (agglutinating reagent) and the NaOH-EtOH mixed solution prepared in Example 2 (agglutinating reagent) were used.

[0084] (Agglomeration test method) Four 50 mL aliquots of Chlorella Solution C1 and Chlorella Solution C3 were placed in four beakers and vigorously stirred. After vigorously stirring, a chitosan flocculating reagent was added to each solution to achieve a chitosan concentration of 5 mg / L. The mixture was then vigorously stirred for 5 minutes. An equal volume of NaOH-EtOH mixed solution was then added, and the mixture was stirred vigorously again. After 10 minutes of incubation, the flocculation state of each Chlorella solution was observed. Further addition of chitosan flocculating reagent to achieve a chitosan concentration of 10 mg / L was performed. After 5 minutes of incubation, the flocculating state of the Chlorella was observed. After this, the chitosan flocculating solution was added in 5 mg / L increments, followed by 5 minutes of stirring and 10 minutes of incubation. The chitosan flocculating solution was then added in 5 mg / L increments, followed by 5 minutes of incubation, until the final chitosan concentration reached 55 mg / L. Each time the chitosan flocculating solution was added, the NaOH-EtOH mixed solution was added in the same manner. The flocculation results for Chlorella Solution C1 and Chlorella Solution C3 are shown in Figure 10.

[0085] (result) As is clear from Figure 10(a), aggregation was observed when chitosan was added to a final concentration of 25 mg / L in Chlorella solution C1, which was adjusted to OD730 = 1. Furthermore, as is clear from Figure 10(b), aggregation was also visually observed when chitosan was added to a final concentration of 55 mg / L in Chlorella solution C3, which was adjusted to OD730 = 3. Therefore, it was found that the chitosan concentration required for aggregation differs depending on the bacterial species.

[0086] [Example 8] In Example 7 above, aggregation was confirmed visually when the chitosan concentration was 25 mg / L, so the aggregation reaction product was separated into supernatant and precipitate by centrifugation to evaluate the aggregation state more objectively.

[0087] (Preparation of Chlorella Solution) The above-mentioned Chlorella vulgaris was used for the test. It was continuously cultured at 25°C using C medium with 2% CO2 aeration. After sufficient growth was confirmed, it was subjected to an aggregation test. The sufficiently grown Chlorella vulgaris was centrifuged at 3000 rpm for 10 minutes, and the precipitate was collected. The precipitate was resuspended in BG-11 liquid medium to an OD730 of 1 to prepare Chlorella solution C1, and the precipitate was resuspended in MilliQ water to an OD730 of 1 to prepare Chlorella solution Q.

[0088] (agglutination reagent) The chitosan agglutinating reagent prepared in Example 1 (agglutinating reagent) and the NaOH-EtOH mixed solution prepared in Example 2 (agglutinating reagent) were used.

[0089] (Agglomeration test method) 50 mL of each of the above Chlorella Solution C1 and Chlorella Solution Q was placed in four beakers to prepare a total of eight samples. After thorough stirring, chitosan agglutination reagent was added to the samples to achieve a chitosan concentration of 5 mg / L and vigorously stirred for 5 minutes. A NaOH-EtOH mixed solution was then added and vigorously stirred for 5 minutes. After 10 minutes of standing, the flocculation state of each Chlorella solution was observed. Further chitosan agglutination reagent was added to the samples to achieve a chitosan concentration of 10 mg / L, and the solution was stirred for 5 minutes. After 10 minutes of standing, the flocculation state was observed. Subsequently, the chitosan addition, 5-minute stirring, and 10-minute standing cycle was repeated in 5 mg / L increments. Chitosan agglutination solution was added to achieve final chitosan concentrations of 5 mg / L, 10 mg / L, 25 mg / L, and 50 mg / L. Each time a chitosan agglutination solution was added, NaOH-EtOH mixed solution was added in the same manner as above, resulting in a total of eight samples. After the agglutination reaction, the samples were transferred to centrifuge tubes and centrifuged at 500 rpm for 10 minutes. The supernatant was measured at OD730. As negative controls, chlorella solution C1 without chitosan and chlorella solution Q without chitosan were also centrifuged in the same manner.

[0090] Figure 11 shows a graph of the absorbance values ​​at OD 730 nm for the supernatants obtained after centrifuging the eight types of samples after the agglutination reaction, and for the negative controls (Chlorella solution C1 without chitosan and Chlorella solution Q without chitosan). Figure 12 also shows the state of the precipitates and supernatants of Chlorella solution C1 observed under a microscope with and without chitosan.

[0091] (Result 1) As shown in Figure 11, when the chitosan flocculation reagent and NaOH + EtOH solution were added, the absorbance value of the supernatant began to decrease from a final chitosan concentration of 5 mg / L. As the chitosan concentration increased, the OD730 value of the supernatant decreased. For both Chlorella Solution C1 and Chlorella Solution Q, the OD730 value decreased by more than 50% at a chitosan concentration of 10 mg / L compared to the solution without chitosan. Furthermore, when comparing Chlorella Solution C1 and Chlorella Solution Q, Chlorella Solution Q, which was cultured in MilliQ water, tended to flocculate slightly more easily, suggesting the potential for this invention to be effectively used in wastewater containing no salts.

[0092] (Result 2) As is clear from Figure 12, in the case of chlorella solution C1 without added chitosan, the precipitate (a) after centrifugation was easily dispersible, and no aggregation or solidification was observed. On the other hand, the precipitate (b) after centrifugation of chlorella solution C1 with added 50 mg / L of chitosan was not easily dispersible.

[0093] [Example 8] [Confirming the effectiveness of antifoaming agents 1] The effects of antifoaming agents on the cultivation of Chlorella sorokiniana were investigated using culture samples to which the following commercially available emulsion-type antifoaming agents, approved under the Food Sanitation Act as food additives for use in aqueous systems, had been added. (1) KM72 (improved fast-acting product) (2) KM72S (product with improved fast-acting properties, contains potassium sorbate) (3) KM72F (product with improved durability) (4) KM72FS (product with improved durability, contains potassium sorbate) (Both manufactured by Shin-Etsu Chemical Co., Ltd.)

[0094] Chlorella sorokiniana (6 × 10E7 cfu / mL) and 350 mL of BG-11 liquid medium were placed in a 500 mL bottle and cultured at 30°C. On the fourth day of culture, when foaming was confirmed to have begun, 58 μg / 350 mL (0.165 g / kg) of each of the antifoaming agents described above in (1) to (4) was added to each culture sample, and observations were made over time. The changes in the condition of the medium bottle for each culture sample are shown in Figure 13.

[0095] (result) As is clear from Figure 13(a), foaming disappeared or weakened in all culture samples one day after the addition of the antifoaming agent. Foaming disappeared in all samples 15 days after the addition.

[0096] [Example 9] [Confirming the effectiveness of antifoaming agents 2] The effect of antifoaming agents on the cultivation of Synechocystis 6803 was confirmed. Synechocystis 6803 at 6 x 10E7 cfu / mL and 200 mL of BG-11 liquid medium were placed in a 350 mL medium bottle and cultured at 30°C. On the 12th day of culture, when sufficient foaming was judged, 33 μg / 200 mL (0.165 g / kg) of the antifoaming agents (1) KM72 and (3) KM72F were added to each culture sample, and observations were made over time. The changes in the condition of the medium bottle for each culture sample are shown in Figure 14.

[0097] (result) As shown in Figure 14, foaming disappeared or weakened in all culture samples 30 minutes after the addition of the antifoaming agent. Foaming disappeared in all samples 8 days after the addition.

[0098] [Comparative Example 1] [Effect of not adding an antifoaming agent on the cultivation of Synechocystis 6803] Synechocystis 6803 at 6 x 10E7 cfu / mL and 200 mL of BG-11 liquid medium were placed in a 350 mL medium bottle and culture was initiated at 30°C without adding an antifoaming agent. Figure 15 shows the state of the medium bottle after 12 days.

[0099] (result) As shown in FIG. 15, when no antifoaming agent was added, foaming caused the culture medium to flow out into the exhaust bottle (area surrounded by an oval).

[0100] [Checking growth status 1] For Synechocystis 6803, the OD730 values ​​were measured 8 days after the addition of the antifoaming agent (1) KM72 or (3) KM72F to the culture medium, and the culture medium without the antifoaming agent of Comparative Example 1. The results are shown in Table 4 below.

[0101] [Table 4]

[0102] As is clear from Table 4, the OD730 value was higher when an antifoaming agent was added than when it was not, confirming that the addition of an antifoaming agent does not have an adverse effect on the growth and proliferation of cyanobacteria.

[0103] [Checking growth status 2] BG-11 solid medium was prepared using the same method as the BG-11 liquid medium, with a final agar concentration of 1.5%. Two autoclaving flasks or medium bottles were prepared. The stock solutions of solutions II, HEPES, III, IV, and V were added to one flask or medium bottle in that order, mixed, and diluted with MilliQ water to a concentration twice the final concentration. Agar and 2 mM sodium thiosulfate were added to the other bottle to prepare a 3% agar concentration (twice the final concentration). The two bottles were autoclaved separately, and after cooling slightly, the two bottles were mixed. Solution I was added to prepare the BG-11 solid medium solution, which was then dispensed onto plates to prepare BG-11 solid medium.

[0104] For Synechocystis 6803, the culture medium 8 days after the addition of the above-mentioned antifoaming agent (1) KM72 or (3) KM72F, and the culture medium without the antifoaming agent (Comparative Example 1) were inoculated onto the above-mentioned agar medium and observed 72 hours later. No colonies were detected in either medium. Therefore, it was confirmed that even when an antifoaming agent was added, there was no generation of bacteria due to damage to the nucleus or cell membrane.

[0105] [Example 10] [Confirming the effectiveness of antifoaming agents 3] The effects of antifoaming agents on the cultivation of Synechocystis 6803 and Chlorella sorokiniana were confirmed. Synechocystis 6803 or Chlorella sorokiniana at 6 x 10E7 cfu / ml were placed in 750 mL of BG-11 liquid medium in a 1 L medium bottle and cultivation was initiated at 30°C. At the start of cultivation, 123 μg / 750 mL (0.165 g / kg) of the antifoaming agent (described above in (1) KM72) was added, and observations were conducted over time. The changes in the condition of the medium bottles for each culture sample are shown in Figure 16.

[0106] (result) As is clear from Figure 16, foam generation was prevented for Synechocystis 6803 up to day 7 after the start of cultivation, and for Chlorella sorokiniana up to day 9. Furthermore, no dirt was observed inside the containers, and no outflow of microalgae was observed outside the containers. [Industrial Applicability]

[0107] The present invention is useful in the field of environmental purification, such as wastewater treatment.

Claims

1. A method for flocculating microalgae, comprising adding chitosan and sodium hydroxide to treatment water containing microalgae and stirring the mixture to flocculate the microalgae.

2. 2. The method for flocculating microalgae according to claim 1, wherein chitosan is added to the treatment water as a chitosan flocculating reagent containing acetic acid.

3. 2. The method for flocculating microalgae according to claim 1, wherein sodium hydroxide is added to the treatment water as a sodium hydroxide solution containing ethanol.

4. 2. The method for flocculating microalgae according to claim 1, wherein the concentration of chitosan in the treatment water containing the microalgae is 5 to 50 mg / L.

5. 2. The flocculation method according to claim 1, wherein the concentration of sodium hydroxide in the treated water containing microalgae is 28 to 280 mg / L.

6. 2. The method for flocculating microalgae according to claim 1, wherein the microalgae are cyanobacteria or green algae.

7. 7. The method for flocculating microalgae according to claim 6, wherein the cyanobacteria is a cyanobacterium belonging to the genus Synechocystis.

8. 7. The method for flocculating microalgae according to claim 6, wherein the green algae belong to the genus Chlorella.

9. A method for purifying the environment by recovering flocculated microalgae using the method for flocculating microalgae according to any one of claims 1 to 8.

10. A method for culturing microalgae aggregated by the method for aggregating microalgae according to any one of claims 1 to 8, using an antifoaming agent.

Images