Cotton adsorbent for removing cyanobacteria whose surface is modified with chitosan and urea

Abstract

The present invention relates to a cotton adsorbent for removing cyanobacteria with a surface modified with chitosan and urea, and a method for removing green algae using the same. The cotton adsorbent with a surface modified with chitosan and urea according to the present invention is not only environmentally friendly because it utilizes natural materials such as chitosan and cotton, but also contains a large amount of amine groups by modifying the surface of the cotton material with chitosan polymer and urea, thereby enabling effective adsorption of negatively charged harmful cyanobacterial cells. In particular, the cotton adsorbent with a surface modified with chitosan and urea according to the present invention does not cause secondary pollution by cyanotoxins resulting from the destruction of cyanobacterial cells due to the use of the adsorbent and is free from ecotoxicity; thus, it has the advantage of minimizing various negative effects, such as toxicity to the aquatic ecosystem, resulting from actual use. In addition, the surface-modified cotton adsorbent of the present invention, which is modified with chitosan and urea, can be reused through a desorption process after adsorbing cyanobacteria; therefore, when used for industrial purposes, it can be usefully employed as an eco-friendly and economical material.

Description

Technology Field

[0001] The present invention relates to a cotton adsorbent for removing cyanobacteria with a surface modified with chitosan and urea, and a method for removing green algae using the same. Background Technology

[0002] Algal bloom refers to cyanobacteria in freshwater environments such as rivers or lakes ( Cyanobacteria This refers to the phenomenon where the color of water turns a deep green due to the abnormal proliferation of cyanobacteria. Recently, along with the rise in average temperatures caused by global warming, the influx of pollutants from various sources located near freshwater environments has led to a significant increase in the severity and frequency of algal blooms, with the damage becoming increasingly serious year after year. Cyanobacteria cause blooms in aquatic environments, increasing water turbidity and negatively affecting the aesthetics; furthermore, they rapidly consume underwater oxygen, hindering the growth of other organisms, and induce foul odors and toxins, thereby causing environmental and ecological problems. Currently, this phenomenon is not limited to Korea but is receiving attention as a global issue, and various solutions are being researched in diverse fields.

[0003] The main types of cyanobacteria causing algal blooms in Korea are the genus Microcystis ( Microsystis ), Anabaena genus ( Anabaena ), genus Aphanizomenon ( Aphanizomenon ) and the genus Oscillatoria ( Oscillatoria As such, the above four species are designated and managed as harmful cyanobacteria by the Ministry of Environment because they can have harmful effects on the environment by releasing toxic substances. They typically multiply very actively at water temperatures above 22℃, and among the four types mentioned above, Microcystis eruginosa in particular Microsystis aeruginosa ...) belongs to the cyanobacteria that most commonly cause algal blooms.

[0004] Microcystes eruginosa is known to release a toxic substance called microcystin. Although there are over 70 variants of microcystin, Microcystin-LR is the most toxic; it is known to cause hepatotoxicity and neurotoxicity when ingested by mammals. For this reason, the World Health Organization (WHO) limits the concentration of algal toxins in drinking water to 1 μg / L. The increase in algal toxins poses a significant threat, as it is closely linked to our daily lives and can lower our quality of life by causing not only direct toxicity issues but also increased water treatment costs and restrictions on hydrophilic activities. Therefore, to sustainably maintain water resources, it is necessary to remove and control Microcystes eruginosa from water systems.

[0005] Against this background, while researching eco-friendly materials with algae removal functionality, the inventors confirmed that cotton adsorbents with surfaces additionally modified in a Daphnia culture solution along with chitosan showed improved cyanobacteria adsorption performance compared to cotton adsorbents with surfaces modified in chitosan alone, and were the first to identify that the main component contained in the Daphnia culture solution is urea. Accordingly, a cotton adsorbent was developed by modifying degreased cotton with a mixed solution of chitosan and urea, and the said material [addresses] Microcystis eruginosa, a major cyanobacteria that causes algae blooms ( Microsystis aeruginosa The present invention was completed by confirming that ) can be effectively removed (adsorbed). Prior art literature

[0006] Korean Registered Patent No. 10-2176159 The problem to be solved

[0007] Therefore, the objective of the present invention is to provide a cotton adsorbent surface-modified with chitosan and urea that can effectively remove harmful cyanobacteria causing algal blooms.

[0008] Another objective of the present invention is to provide a method for removing algae using a cotton adsorbent whose surface is modified with chitosan and urea. means of solving the problem

[0009] In order to achieve the objectives of the present invention as described above,

[0010] The present invention provides a cotton adsorbent for removing cyanobacteria with a surface modified with chitosan and urea.

[0011] In one embodiment of the present invention, the cotton adsorbent for removing cyanobacteria can be manufactured through a process comprising: a) a step of pre-treating cotton fibers in a sodium hydroxide solution with a concentration of 3M; and b) a step of modifying the surface of pre-treated cotton fibers by immersing them in a mixed solution of chitosan and urea and then reacting them.

[0012] In one embodiment of the present invention, step b) can modify the surface by immersing the pretreated cotton fiber in a solution mixed with chitosan and urea in a weight ratio of 1:05 to 1:1 and reacting for 3 to 5 hours.

[0013] In one embodiment of the present invention, the cyanobacteria is Microcystis eruginosa, which causes green algae ( Microcystis aeruginosa It can be.

[0014] In addition, the present invention provides a method for removing algae, comprising the step of treating a cotton adsorbent for removing cyanobacteria, whose surface is modified with chitosan and urea, in a body of water where algae have occurred or where signs of occurrence have been observed. Effects of the invention

[0015] The cotton adsorbent surface-modified with chitosan and urea according to the present invention is not only environmentally friendly because it utilizes natural materials such as chitosan and cotton, but also contains a large amount of amine groups by modifying the surface of the cotton material with chitosan polymers and urea, thereby enabling effective adsorption of negatively charged harmful cyanobacterial cells. In particular, the cotton adsorbent surface-modified with chitosan and urea according to the present invention does not cause secondary pollution by cyanotoxins resulting from the destruction of cyanobacterial cells due to the use of the adsorbent and is free from ecotoxicity; thus, it has the advantage of minimizing various negative effects, such as toxicity to the aquatic ecosystem, resulting from actual use. Furthermore, since the cotton adsorbent surface-modified with chitosan and urea according to the present invention can be reused through a desorption process after adsorption of cyanobacteria, it can be usefully employed as an environmentally friendly and economical material when used for industrial purposes. Brief explanation of the drawing

[0016] Figure 1 shows the GC-MS analysis results of the Daphnia culture medium. Figures 2a and 2b show the results of a urea detection reaction conducted to confirm and verify urea in a Daphnia culture (2a: color development, 2b: absorbance). Figure 2c shows the calibration curve of the linear relationship between absorbance and urea concentration in an aqueous solution. Figure 2d shows the results of measuring changes in urea concentration under various culture conditions. Figure 3 shows the results of measuring changes in surface characteristics of 'cotton' and 'chitosan-urea cotton' adsorbents ((a) XPS analysis results of cotton, (b) N of cotton 1S Spectrum, (c) XPS analysis results of the chitosan-urea plane, (d) N of the chitosan-urea plane 1S spectrum). Figure 4a is a graph showing the cell density of Microcystis eruzinosa over time after the addition of 'cotton', 'chitosan-cotton', and 'chitosan-urea cotton' adsorbents (control group: adsorbent-free group). Figure 4b shows the results of measuring the removal rate (%) of Microcystis eruzinosa over time after the addition of 'cotton', 'chitosan-cotton', and 'chitosan-urea cotton' adsorbents. Figure 4c shows the results of measuring changes in absorbance over time after the addition of 'cotton', 'chitosan-cotton', and 'chitosan-urea cotton' adsorbents (control group: adsorbent-free group). Figures 5a to 5c are FE-SEM scan images showing the surface changes of 'cotton', 'chitosan-cotton', and 'chitosan-urea cotton' adsorbents before and after the adsorption of harmful cyanobacteria (5a: cotton, 5b: chitosan-cotton, 5c: chitosan-urea cotton). Figure 6 shows the results of measuring the removal rate (%) of Microcystis eruzinosa over time after the addition of 'citric acid chitosan-urea cotton adsorbent', 'acetic acid chitosan-urea cotton adsorbent', 'acetic acid (microwave) chitosan-urea cotton adsorbent' and 'acetic acid (glutaraldehyde) chitosan-urea cotton adsorbent' (6a: 24 hours elapsed, 6b: 1 to 6 hours elapsed). Figure 7 shows images of changes in the surface of the adsorbent before and after the removal of Microcystis eruginosa cells, observed via FE-SEM scans ((A): 'Citrate chitosan-urea cotton adsorbent' before cyanobacteria removal, (A-1) and (A-2): 'Citrate chitosan-urea cotton adsorbent' after cyanobacteria removal, (B): 'Acetic chitosan-urea cotton adsorbent' before cyanobacteria removal, (B-1) and (B-2): 'Acetic chitosan-urea cotton adsorbent' after cyanobacteria removal, (c): 'Acetic (microwave) chitosan-urea cotton adsorbent' before cyanobacteria removal, (C-1) and (C-2): 'Acetic (microwave) chitosan-urea cotton adsorbent' after cyanobacteria removal, (D): 'Acetic (glutaraldehyde) chitosan-urea cotton adsorbent' before cyanobacteria removal 'Adsorbent', (D-1) and (D-2): 'Acetic acid (glutaraldehyde) chitosan-urea cotton adsorbent' after cyanobacteria removal). Figure 8 shows the results of measuring the removal rate (%) of Microcystis eruzinosa over time for each adsorbent prepared with different mixing ratios of chitosan and urea (1:0.01, 1:0.05, 1:0.1, 1:0.25, 1:0.5, 1:0.75). Specific details for implementing the invention

[0017] The present invention relates to a cotton adsorbent for removing cyanobacteria with a surface modified with chitosan and urea.

[0018] The cotton adsorbent for removing cyanobacteria according to the present invention can be manufactured through a process comprising: a) a step of pre-treating cotton fibers in a sodium hydroxide solution with a concentration of 3M; and b) a step of modifying the surface of pre-treated cotton fibers by immersing them in a mixed solution of chitosan and urea and then reacting them.

[0019] Step a) of the present invention is a step of pre-treating cotton fibers in a sodium hydroxide solution with a concentration of 3M, through which hydroxyl groups (-OH) can be introduced into the cotton fibers.

[0020] Step b) of the present invention is a step of modifying the surface of a cotton fiber with chitosan, and more specifically, a step of modifying the surface by immersing the cotton fiber pretreated through step a) in a solution mixed with chitosan and urea in a weight ratio of 1:05 to 1:1 and reacting for 3 to 5 hours.

[0021] The 'chitosan-urea cotton' adsorbent of the present invention manufactured through the above process can effectively remove (adsorb) cyanobacteria, and preferably, Microcystis eruginosa, which causes algal blooms ( Microcystis aeruginosa ) can be removed.

[0022] The 'chitosan-cotton' adsorbent of the present invention is not only environmentally friendly because it utilizes natural materials such as chitosan and cotton, but also contains a large amount of amine groups by modifying the surface of the cotton material with chitosan polymer and urea, so it can effectively adsorb negatively charged harmful cyanobacteria cells.

[0023] While researching eco-friendly materials with algae removal functionality, the inventors confirmed that cotton adsorbents with surfaces additionally modified in a Daphnia culture solution along with chitosan showed improved cyanobacteria adsorption performance compared to cotton adsorbents with surfaces modified in chitosan alone, and were the first to identify that the main component contained in the Daphnia culture solution is urea (see Figures 1 and 2). Accordingly, a cotton adsorbent was developed by modifying degreased cotton with a mixed solution of chitosan and urea, and the said material [addresses] Microcystis eruginosa, a major cyanobacteria that causes algae blooms ( Microsystis aeruginosa It was confirmed that ) was effectively removed (adsorbed) (see Fig. 4). It was confirmed that these results were due to the aggregation effect of a compound derived from natural enemy organisms (Urea) (see Fig. 5).

[0024] In addition, the present invention relates to a method for removing algae, comprising the step of treating a 'chitosan-urea cotton' adsorbent for adsorbing cyanobacteria in a body of water where algae have occurred or where signs of occurrence have been observed.

[0025] In addition, the present invention relates to a method for recovering cyanobacteria from a 'chitosan-urea cotton' adsorbent, comprising the steps of: treating a 'chitosan-urea cotton' adsorbent for cyanobacteria adsorption in a water body where algal blooms occur to adsorb cyanobacteria; and adding the 'chitosan-urea cotton' adsorbent with adsorbed cyanobacteria to a basic solution of a specific concentration and then treating it with ultrasound.

[0026] In the following examples of the present invention, cells can be detached from a 'chitosan-urea cotton' adsorbent while maintaining their cell structure without destroying the cyanobacterial cells through a 0.1N sodium hydroxide solution and 40kHz ultrasonic treatment. Accordingly, the harmful cyanobacterial cells recovered through the detachment process of the present invention can be utilized industrially, such as for biofuel.

[0027] In addition, the present invention relates to a method for regenerating a 'chitosan-urea cotton' adsorbent for removing cyanobacteria, comprising: i) a step of treating a 'chitosan-urea cotton' adsorbent for removing cyanobacteria in a water body where green algae have occurred to adsorb cyanobacteria; ii) a step of adding the 'chitosan-urea cotton' adsorbent with adsorbed cyanobacteria to a 0.1N sodium hydroxide solution and then treating it with 40kHz ultrasound to desorb the cyanobacteria; and iii) a step of washing the 'chitosan-urea cotton' adsorbent with desorbed cyanobacteria using distilled water and then freeze-drying it.

[0028] In the following examples of the present invention, the 'chitosan-urea cotton' adsorbent of the present invention, regenerated through steps i) to iii), can efficiently adsorb cyanobacteria without losing functionality even when used repeatedly. Accordingly, since the 'chitosan-urea cotton' adsorbent of the present invention can be regenerated (recycled) through the above process, it can be usefully utilized as an eco-friendly and economical material.

[0030] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0032] <Example>

[0034] Preparation of ingredients

[0035] Chitosan (Manufacturer: SHOWA, Chitosan, [C6H 11 NO4] n , FW161.16), urea (Manufacturer: Deoksan Science, Urea, CO(NH2)2, FW60.06) and anhydrous citric acid (Manufacturer: Deoksan Science, citric acid anhydrous, C3H4(OH)(COOH)3, FW192.12) were purchased from Deoksan Science; absorbent cotton was purchased from Daehan Medical; water fleas ( Daphnia magna ) was purchased from Joyhaus (https: / / smartstore.naver.com / joyhaus); glutaraldehyde (GA, 25wt%) was purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan).

[0036] The target contaminant is Microcystis eruginosa ( M. aeruginosa The KW) strain used was identified from Wangsong Reservoir and was distributed by Dr. Chiyong Ahn of the Korea Institute of Bioscience and Biotechnology (KRIBB).

[0038] <Example 1>

[0039] Microcystis eruginosa at the laboratory scale for harmful cyanobacteria control experiments ( Microcystis aeruginosa ) culture

[0040] In this experiment, for the control of harmful cyanobacteria, Microcystis eruginosa ( Microcystis aeruginosa) species were used. BG11 medium was used as the culture medium, and 40 mg K2HPO4, 75 mg MgSO4·7H2O, 36 mg CaCl2·2H2O, 6 mg Ferric Ammonium Citrate, 1 mg Na2EDTA·2H2O, 0.22 mg ZnSO4·7H2O, 20 mg Na2CO3, 2.86 mg H3BO3, 1.81 mg MnCl2·4H2O, 1,500 mg NaNO3, 6 mg Citric acid·H2O, 0.39 mg Na2MoO4·2H2O, 0.0494 mg Co(NO3)2·6H2O, and 0.079 mg CuSO4·5H2O were added to 1 L of triple-distilled water. The medium was sterilized at 121°C for 20 minutes before use.

[0042] <Example 2>

[0043] Analysis of natural enemy-derived analog compounds for identification and verification of natural enemy-derived analog compounds

[0044] In this experiment, to further enhance control performance in controlling harmful cyanobacteria, water fleas, a representative natural enemy organism coexisting in the aquatic ecosystem ( Daphnia magna We conducted an analysis of the substances released during the process of the culture medium of ). Natural enemy organism ( Daphnia magna ) was cultured on M4 medium, and the food source was Chlorella vulgaris ( Chlorella vulgaris ) or Microcystis eruginosa ( Microcystis aeruginosa ) 5×10 5The sample was supplied repeatedly at cells / mL. To perform GC-MS analysis of the natural enemy culture medium, the culture medium of the natural enemy (Daphnia magna) was prepared by primary filtration using a 0.45 µm filter and secondary filtration using a 0.22 µm filter. The prepared sample was concentrated from 500 mL of culture medium to 25 mL using a rotary evaporator. GC-MS analysis was performed using the concentrated sample. An Agilent 7890A, 5975C GC-MS was used, and 1 µL of the sample was injected at a 10:1 split ratio using a DB-5MS UI column. He was injected as the carrier gas at 1.0 mL / min, and the molecular weight range for the library scan was set to 30–500 m / z. As a result of GC-MS analysis, urea was finally selected as the compound derived from a natural enemy organism with the highest similarity (see Fig. 1).

[0045] To determine the emission concentration of urea, identified as a compound derived from natural enemies by GC-MS analysis, in actual Daphnia culture medium according to culture conditions, an analysis of urea emission concentration was conducted using a spectroscopic method involving chemical reactions. The method for analyzing urea through chemical reactions is as follows.

[0046] 1) Preparation of Urea Detection Reagent A; Prepare a solution by mixing 100 ml of phosphoric acid (85%), 300 ml of sulfuric acid (95-98%), 600 ml of triple-distilled water, and 100 mg of ferric chloride. 2) Preparation of Urea Detection Reagent B; Prepare a solution by mixing 500 mg of DAMO (Diacetyl monoxime), 10 mg of TSC (Thiosemicarbazide), and 100 ml of triple-distilled water. 3) Preparation of the Final Urea Detection Reagent; Prepare the urea detection reagent by mixing the prepared Urea Detection Reagent A and Urea Detection Reagent B in a 1:1 ratio. The procedure for measuring urea in an aqueous solution using the prepared solutions is as follows: Measurement of changes in urea concentration in an aqueous solution; 1) Mix 5 µl of the measurement sample, 3 ml of urea detection reagent, and 0.1 ml of Brij-35 solution (Sigma Aldrich, Korea). 2) After mixing the solutions, heat in a water bath for 5 minutes. 3) After cooling, measure the absorbance at a wavelength of 525 nm. 4) For the blank sample, prepare 5 µl of distilled water, 3 ml of urea detection reagent, and 0.1 ml of Brij-35 solution in the same manner as the previous step. 5) For the calibration curve, prepare an aqueous urea solution and proceed with the measurement. Additionally, to confirm the detection specificity of the urea detection method through a chemical reaction in an aqueous solution, a 0.1 M PEI solution (polyetherimide, sigma-aldrich) containing a large amount of amine groups and an aqueous solution of urea dissolved in it were prepared at a concentration of 1 g / L, and the detection specificity was confirmed by proceeding with the urea detection reaction.

[0047] As a result, as shown in Figures 2a and 2b, it was confirmed that a color change occurred specifically with urea, without reacting with the same dose of PEI. This confirmed the specificity of the urea detection method based on the spectroscopic analysis via chemical reaction. To proceed with analysis based on these results, calibration curves were constructed by preparing aqueous urea at various concentrations (1, 10, 50, 100, 500, 1,000 mg). As a result, as shown in Figure 2c, a calibration curve was derived showing a linear relationship between absorbance and urea concentration in aqueous solution, Y = 0.0125 + 0.0005 * X, and R 2 The value was measured as 0.9976.

[0048] Based on the calibration curve, changes in urea concentration in the culture medium of natural enemy organisms under various culture conditions were measured. The conditions under which measurements were taken are shown in Table 1 below, and for the case of feed administration, a single test was conducted to monitor changes in urea concentration for 24 hours. As a result, as shown in Figure 2d, urea was detected only in the feeding group in which Daphnia were fed Chlorella vulgaris and Microcystis eruzinosa. This confirmed that urea is produced through the feeding relationship between Daphnia and Chlorella vulgaris and Microcystis eruzinosa, which serve as food for Daphnia. Furthermore, the urea concentration in the feeding group supplied with Microcystis eruzinosa was measured to be higher than in the Chlorella vulgaris feeding group, and these results are expected to be due to the interaction between the natural enemy organisms and the algae supplied as food.

[0050] Various culture conditions Experimental Example Culture conditions 1 M4 badge 2 M4 medium with added Chlorella vulgaris ( Chlorella vulgaris 5×10 5 cells / mL) 3 M4 medium supplemented with Microcystis eruginosa ( Microcystis aeruginosa 5×10 5 cells / mL) 4 Water flea (no food supply) 5 Water flea (Food source: Chlorella vulgaris) Chlorella vulgaris 5×10 5 cells / mL) 6 Water flea (Food source: Microcystis eruginosa)( Microcystis aeruginosa 5×10 5 cells / mL)

[0052] <Example 3>

[0053] Preparation of the chitosan-urea modified cotton adsorbent of the present invention

[0055] <3-1> Pretreatment of cotton wool using a basic solution

[0056] To modify existing commercially available cotton wool into cationic substances for the development of a material for controlling harmful algae, pretreatment was performed using a basic solution of a specific concentration (3M NaOH). 50g of cotton wool was placed in a 2L tall beaker, and the pretreatment process was carried out for 2 hours by stirring (magnetic stirring and manual stirring) with 1.5L of 3M NaOH solution. After completing the pretreatment process through stirring, the cotton wool was washed 6 to 7 times with triple-distilled water to remove any remaining 3M NaOH solution.

[0058] <3-2> Preparation of Adsorbents through Surface Modification of Cationic Substances

[0059] A chemical modification process was carried out on the degreased cotton pretreated through Example <3-1> using a mixed solution of chitosan, a cationic biopolymer, and urea, a compound similar to a natural enemy organism. The process described above is as follows.

[0060] A 5% citric acid solution (20 g / L) was prepared by mixing anhydrous citric acid with 1,000 ml of triple-distilled water. A 1% chitosan solution (w / w, %) was prepared by dissolving chitosan in the citric acid solution, and a solution for surface modification of the material was prepared by mixing the 1% chitosan solution with urea in a 1:1 ratio based on the weight of the chitosan. Surface modification was carried out using the above chitosan and urea mixed solution for 6 hours. Stirring (magnetic stirring and manual stirring) was performed in parallel, and after the process, a washing process was carried out three times in triple distilled water to remove the chitosan and urea remaining without binding.

[0061] The washed cotton adsorbent mixed and modified with chitosan and urea was freeze-dried. The adsorbent prepared in this way is abbreviated as “Chitosan-urea cotton adsorbent” below.

[0063] <Comparative Example 1>

[0064] Manufacturing of chitosan-modified cotton adsorbent

[0065] Physical and chemical modification of the cotton support was performed using a chitosan solution having positive charge characteristics on the cotton pretreated through Example <3-1> above. The process described above is as follows.

[0066] Pre-treated cotton pads were immersed in a 1% chitosan solution (w / w, %) prepared by dissolving chitosan in a 5% (v / v) acetic acid solution to react with chitosan on the surface. The chitosan surface modification process was carried out by magnetic stirring for 3 hours, and after the reaction was completed, the pads were washed three times with triple-distilled water. The chitosan-modified cotton adsorbents that had been washed were freeze-dried. The adsorbent prepared in this way is referred to as the “chitosan-cotton adsorbent” below.

[0068] <Example 4>

[0069] Observation of surface changes of chitosan-urea cotton adsorbent

[0070] Changes in surface characteristics of the “chitosan-urea cotton adsorbent” prepared through the above <Example 3> were measured.

[0071] As a result, as shown in Figure 3, a change in cationic functional groups (N group) was observed on the surface of the cotton wool mixed and modified with chitosan and urea. Therefore, it was confirmed that chemical modification proceeds stably through the chemical modification presented in the present invention.

[0073] <Example 5>

[0074] Performance evaluation of harmful cyanobacteria control at the laboratory scale

[0075] To evaluate the harmful cyanobacteria control performance of the “chitosan-urea cotton adsorbent” of the present invention for its application in controlling harmful cyanobacteria, a laboratory-scale evaluation was conducted.

[0076] To measure cyanobacteria density (mg / L), first 0.15L min in BG11 medium -1 air injection rate, 50 μmol m -2 Microcystis eruginosa was cultured under light intensity and conditions of 25°C. After separating the cultured Microcystis eruginosa from the medium by centrifugation, the separated cells were counted using a hemocytometer, and subsequently mixed with 150 ml of BG11 medium to obtain an initial cell density of 1 × 10⁶. 6 It was adjusted to cells / ml. The initial cell density for each was 1×10⁶ 6 Three flasks adjusted to cells / ml were prepared for each material, and 2ml samples were taken from each sample, including a control, over time following the application of the prepared adsorbent. Cell counts were performed on the sampled samples using a hemocytometer. Three cell counts were conducted for each material, and cell density was measured by calculating the mean and standard deviation of the counts.

[0077] To measure the removal rate (%) of cyanobacteria, the control rate for each material sample was calculated using the formula ((initial cell count - average of the count of cells remaining after control) / 100)×100 to verify the control rate after cell counting, and the average and standard deviation for each material were calculated to derive the removal rate measurement value.

[0078] Meanwhile, for absorbance measurement, first 0.15L min in BG11 medium -1 air injection rate, 50 μmol m -2Microcystis eruginosa was cultured under light intensity and conditions of 25°C. The cultured Microcystis eruginosa was isolated from the medium by centrifugation. The isolated Microcystis eruginosa cells were mixed with 150 ml of BG11 medium to achieve an initial cell density of 1 × 10⁶. 6 Adjust to cells / ml, and each initial cell density is 1×10 6 Three flasks adjusted to cells / ml were prepared for each material, and 2ml samples were taken from each sample, including a control, over time following the application of the prepared adsorbent. The culture medium was analyzed for each sample using a UV spectrophotometer at an OD of 680nm, and the following results were derived by averaging three replicates in which the same material was added. The reason for measuring at an OD of 680nm is to verify the growth and survival of cells such as *Microcystis eruginosa*. The 680nm wavelength is the wavelength at which cells absorb light most effectively due to their characteristics.

[0079] As a result, when the cotton material modified with the developed cationic substance was introduced for the control of harmful cyanobacteria, the control performance showed approximately 97% control performance over 24 hours (see Fig. 4b). Through this, it was confirmed that the control of harmful cyanobacteria is possible using the material developed through optimal cationic substance modification. This control phenomenon is expected to be the result of the introduction of positively charged functional groups on the surface of the cotton material modified with the cationic substance, which allow harmful cyanobacteria to attach, thereby enabling the attachment and control of harmful cyanobacteria cells that carry a negative charge in the aquatic environment. Consequently, the attachment of harmful cyanobacteria cells to the surface of the material and the control resulting from this reaction can serve as supporting evidence.

[0081] Cell density (cells / mL) of Microcystis eruginosa over time after use of clean cotton, chitosan-cotton adsorbent, and chitosan-urea cotton adsorbent comparison Hours (hr) 1 3 6 9 12 24 control group 103.3 ± 2.12 109.3 ± 0.96 109.3 ± 1.71 121.1 ± 0.25 120.3 ± 3.06 120.8 ± 0.77 noodle 107.3 ± 1.51 99.8 ± 1.19 93.3 ± 0.38 91 ± 1.72 88.8 ± 0.83 89.8 ± 2.63 Chitosan-cotton 38.5 ± 2.92 34 ± 2.66 22.5 ± 2.36 24.3 ± 2.2 21.1 ± 1.61 9.6 ± 0.89 Chitosan-urea cotton 17.17 ± 2.02 17.16 ± 0.21 9.3 ± 1.24 2.3 ± 0.21 5.1 ± 0.64 2.6 ± 0.57

[0083] Removal rate (%) of Microcystis eruginosa over time after use of clean cotton, chitosan-cotton adsorbent, and chitosan-urea cotton adsorbent comparison Hours (hr) 1 3 6 9 12 24 noodle -7.33 ± 1.51 0.16 ± 1.19 6.66 ± 0.38 9 ± 1.72 11.16± 0.83 10.16 ± 2.64 Chitosan-cotton 61.5 ± 2.92 66 ± 2.66 77.5 ± 2.36 75.66 ± 2.2 78.83 ± 1.62 90.33 ± 0.9 Chitosan-urea cotton 82.83 ± 2.02 82.83 ± 0.21 90.66 ± 1.24 94.83 ± 0.21 97.33 ± 0.64 97.66 ± 0.57

[0085] In addition, the surfaces of the harmful cyanobacteria control materials were observed using FE-SEM as cotton, chitosan-cotton, and chitosan-urea cotton adsorbents, respectively. As shown in Figure 5, it was confirmed that harmful cyanobacteria cells aggregated and were adsorbed on the chitosan-urea cotton adsorbent. It was anticipated that these results were derived from the aggregation efficacy of urea, a compound derived from natural enemies, against harmful cyanobacteria. Furthermore, it was anticipated that the control performance against harmful cyanobacteria would be doubled as additional binding sites were secured on the surface due to the aggregated adsorption of harmful cyanobacteria cells.

[0087] <Example 6>

[0088] Optimization of cross-linking in the manufacture of chitosan-urea cotton adsorbents

[0090] <6-1> Preparation of Citric Acid Chitosan-Urea Cotton Adsorbent

[0091] A 5% citric acid solution (20 g / L) was prepared by mixing anhydrous citric acid with 1000 ml of triple-distilled water. A 1% chitosan solution (w / w, %) was prepared by dissolving chitosan powder in the citric acid solution, and a solution for surface modification of the material was prepared by mixing the 1% chitosan solution with urea in a 1:1 ratio relative to the weight of the chitosan. Surface modification of the degreased cotton pretreated through Example <3-1> was carried out using the chitosan and urea mixed solution. The mixture was stirred for 3 hours (37°C shaking incubator (120 rpm)), and then washed three times in triple-distilled water to remove any remaining chitosan and urea that had not bonded, followed by freeze-drying to produce the 'citric acid chitosan-urea cotton adsorbent' of the present invention.

[0093] <6-2> Preparation of Acetic Acid Chitosan-Urea Cotton Adsorbent

[0094] Compared to Example <6-1> above, the 'acetic acid chitosan-urea cotton adsorbent' of the present invention was prepared by using 5% acetic acid (v / v) instead of 5% citric acid as the solvent to dissolve chitosan, while carrying out all other processes in the same way.

[0096] <6-3> Preparation of Acetic Acid (Microwave) Chitosan-Urea Cotton Adsorbent

[0097] A 1% chitosan solution (w / w, %) was prepared by dissolving chitosan powder in a 5% acetic acid (v / v) solution, and the 1% chitosan solution and urea were mixed in a 1:1 ratio relative to the weight of the chitosan, followed by 6 repeated treatments with a 700W microwave for 30 seconds each. Surface modification of the degreased cotton pretreated in Example <3-1> was performed using the above mixed solution. The mixture was stirred for 3 hours (37℃ shaking incubator (120rpm)), and subsequently, to remove any remaining chitosan and urea that had not bonded, it was washed three times in triple distilled water and then freeze-dried to produce the 'acetic acid (microwave) chitosan-urea cotton adsorbent' of the present invention.

[0099] <6-4> Preparation of Acetic Acid (Glutaraldehyde) Chitosan-Urea Cotton Adsorbent

[0100] A 1% chitosan solution (w / w, %) was prepared by dissolving chitosan powder in a 5% acetic acid (v / v) solution, and a solution for surface modification of the material was prepared by mixing the 1% chitosan solution with urea in a 1:1 ratio relative to the weight of the chitosan. Surface modification of the degreased cotton pretreated in Example <3-1> was carried out using the above mixed solution. The mixture was stirred for 3 hours (37℃ shaking incubator (120rpm)), and then washed three times in triple distilled water to remove any remaining chitosan and urea that had not bonded. Subsequently, the adsorbent was added to 1L of GA solution (0.6mL GA / L, 0.1M NaOH-based) and crosslinking was carried out at room temperature (25℃) for 1 hour while stirring. To remove residual substances, the 'acetic acid (glutaraldehyde) chitosan-urea cotton adsorbent' of the present invention was prepared by performing a washing process three times in triple distilled water and then freeze-drying.

[0102] <6-5> Comparison of Cyanobacteria Control Performance by Adsorbent

[0103] To verify the removal efficiency of the adsorbent prepared through the above Examples <6-1> to <6-4>, first, 0.15 L min in BG11 medium -1 air injection rate, 50 μmol m -2 Microcystis eruginosa was cultured under light intensity and conditions of 25°C. First, the cultured Microcystis eruginosa was isolated from the medium by centrifugation. Subsequently, the isolated Microcystis eruginosa cells were counted using a hemocytometer and mixed with 150 ml of BG11 medium to achieve an initial cell density of 5 × 10⁶. 6 The concentration was adjusted to cells / ml. Subsequently, 0.3g of adsorbent was added to the culture medium sample containing Microcystis eruginosa. The sample with the added adsorbent was cultured for 24 hours under conditions identical to the primary culture conditions, including air injection rate, light intensity, and temperature. Sampling was performed at fixed time intervals, and the number of Microcystis eruginosa cells present in the solution was measured using a hemacytometer (Hausser Scientific, USA). All adsorption removal evaluations were performed in triplicate.

[0104] First, cell control performance was compared and analyzed based on a 24-hour period, but no clear difference in performance was found (see Fig. 6a).

[0105] Accordingly, the amount of adsorbent was reduced to 1 / 4 (0.075g) and the cyanobacteria cell counting time was adjusted to 1 hour to 6 hours to evaluate the cyanobacteria control performance.

[0106] As a result, it was found that the cyanobacteria control performance was superior when chitosan was dissolved in acetic acid compared to citric acid, and that inducing chemical bonding through microwaves and cross-linking through glutaraldehyde during the treatment process reduced the cyanobacteria removal efficiency of the adsorbent (see Fig. 6b).

[0107] Consequently, a chitosan-urea cotton adsorbent manufactured using a process that utilizes acetic acid as a solvent and does not undergo a cross-linking treatment was selected as the final condition.

[0109] Removal rate (%) of Microcystis eruginosa over time for each adsorbent prepared with different solvent types and treatment processes comparison Hours (hr) 1 3 6 12 24 Citric acid chitosan-urea cotton 60.7 ± 0 65.25 ± 0.16 76.4 ± 0.11 - - Acetic acid chitosan-urea cotton 65.5 ± 0.28 68.75 ± 0.09 77.6 ± 0.1 - - Acetic acid (microwave) chitosan-urea cotton 46.3 ± 0.04 36.65 ± 0.27 35.95 ± 0.09 - - Acetic acid (glutaraldehyde) chitosan-urea cotton 32.3 ± 0.21 29.2 ± 0 30.3 ± 0.25 - -

[0111] <6-6> Observation of material surface after cyanobacteria control by adsorbent

[0112] After controlling cyanobacteria according to the adsorbent, changes in the surface of the adsorbent were confirmed using FE-SEM.

[0113] As a result, as shown in Figure 7, it was confirmed that cyanobacterial cells were aggregated and adsorbed on the surface of all adsorbents. Although aggregation was observed in the adsorbents treated with microwave or glutaraldehyde, the ability of the materials to control cyanobacterial cells tended to decrease compared to other manufacturing conditions. Meanwhile, it was confirmed that the adsorbent material prepared using acetic acid instead of citric acid as a solvent had excellent ability to control cyanobacterial cells.

[0115] <Example 7>

[0116] Optimal mixing ratio of chitosan and urea in the manufacture of chitosan-urea cotton adsorbents

[0117] A 1% chitosan solution (w / w, %) was prepared by dissolving chitosan powder in a 5% acetic acid (v / v) solution, and solutions for surface modification of the material were prepared by mixing the 1% chitosan solution with urea at ratios of 1:0.01, 1:0.05, 1:0.25, 1:0.1, 1:0.5, and 1:0.75 relative to the weight of the chitosan, respectively. Surface modification of the degreased cotton pretreated through Example <3-1> was carried out using each of the above chitosan and urea mixed solutions. The mixture was stirred for 3 hours (37℃ shaking incubator (120rpm)), and then washed three times in triple distilled water to remove any remaining unbound chitosan and urea. Afterward, the mixture was freeze-dried to prepare adsorbents with different chitosan and urea mixing conditions (1:0.01, 1:0.05, 1:0.25, 1:0.1, 1:0.5, 1:0.75). For reference, no further experiments were conducted as the control performance was poor after the chitosan and urea mixing ratio of 1:1.

[0118] As a result, it was confirmed that the adsorbent prepared with a chitosan-urea ratio of 1:0.75 (w / w) had the best cyanobacteria removal activity.

[0120] Removal rate (%) of Microcystis eruzinosa over time for each adsorbent prepared with different mixing ratios of chitosan and urea comparison Hours (hr) 1 3 6 Chitosan-urea (1:0.01) cotton 19.95 ± 0.26 44.9 ± 0.32 43.3 ± 0.21 Chitosan-urea (1:0.05) cotton 52 ± 12.5 50.5 ± 0.25 47.65 ± 0.23 Chitosan-urea (1:0.25) cotton 22.7 ± 0.21 49.45 ± 0.02 46.9 ± 0.39 Chitosan-urea (1:0.1) cotton 41.05 ± 0.12 49.65 ± 0.19 52.05 ± 0.27 Chitosan-urea (1:0.5) cotton 86.15 ± 1.47 87.6 ± 0.67 87.45 ± 0.27 Chitosan-urea (1:0.75) cotton 96.95 ± 0.02 96.95 ± 0.02 98.05 ± 0.09

[0122] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 A cotton adsorbent for removing cyanobacteria with a surface modified with chitosan and urea, wherein the cotton adsorbent for removing cyanobacteria is characterized by being manufactured through a process comprising: a) a step of pre-treating cotton fibers in a sodium hydroxide solution with a concentration of 3M; and b) a step of modifying the surface by immersing the pre-treated cotton fibers in a mixed solution of chitosan and urea and then reacting them. Claim 2 delete Claim 3 A cotton adsorbent for removing cyanobacteria with a surface modified with chitosan and urea, wherein in claim 1, step b) modifies the surface by immersing pretreated cotton fibers in a solution mixed with chitosan and urea in a weight ratio of 1:0.5 to 1:1 and reacting for 3 to 5 hours. Claim 4 In paragraph 1, the above cyanobacteria is Microcystis eruginosa ( Microcystis aeruginosa A cotton adsorbent for removing cyanobacteria with a surface modified with chitosan and urea, characterized by being ). Claim 5 A method for removing green algae, comprising the step of treating a cotton adsorbent for removing cyanobacteria, whose surface is modified with chitosan and urea according to any one of claims 1 and 3 to 4, in a water body where green algae have occurred or where signs of occurrence are observed.

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