A method for extracting and separating high-carbon and low-nitrogen and phosphorus organic components in blue algae

Abstract

The application discloses a method for extracting and separating high-carbon and low-nitrogen and phosphorus organic components in blue algae. The method adopts a process of step-by-step extraction by using an alkaline solution, and when necessary, a mild wall breaking is assisted by adding a low-concentration oxidizing agent, and the interaction between biological molecules is weakened by using sodium chloride, so that high-carbon organic components such as polysaccharides and lipids are effectively decomposed and extracted, and the outflow of high-nitrogen and high-phosphorus components such as proteins and nucleic acids is largely prevented, and the high-carbon and low-nitrogen and phosphorus organic components in the blue algae are successfully obtained. The reagents used in the application are cheap, safe and environmentally friendly, the operation condition is mild, the energy consumption is low, the application is easy to implement, and the application is suitable for large-scale industrial application. The carbon, nitrogen and phosphorus compositions of the extract meet the requirements of directional acid production or other conversion and utilization modes, and the remaining algal cell precipitate is rich in nitrogen and phosphorus, and can be used for further extraction of nucleic acids, proteins, polypeptides or amino acids, and can also be directly used as a fertilizer.

Description

Technical Field

[0001] This invention relates to the field of resource utilization of cyanobacteria, and more specifically, to a method for extracting high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria. Background Technology

[0002] Cyanobacteria are prokaryotic microorganisms that can convert atmospheric carbon dioxide into biomass through photosynthesis, releasing oxygen in the process. Due to the influx of large amounts of nutrients, many lakes have experienced explosive growth of cyanobacteria in recent years, causing serious water quality deterioration. If cyanobacteria can be harvested and processed, and its carbon-containing components can be rationally utilized, it will be of great significance in helping to achieve carbon peaking and carbon neutrality goals, and in mitigating water pollution problems caused by the massive proliferation of cyanobacteria in lakes such as Taihu Lake and Dianchi Lake. Currently, the main methods for treating harvested cyanobacteria are drying and incineration, which convert carbon back into carbon dioxide and release it into the atmosphere—an energy-intensive and inefficient method for carbon reduction. However, extracting the high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria can be used to convert them into small-molecule organic acids, creating biomass carbon sources for denitrification treatment in wastewater treatment plants. It can also be used to further extract polysaccharides and lipids, providing upstream raw materials for biopharmaceutical products and new energy development. The components in the algal cell precipitate remaining after extraction, which are high in nitrogen and phosphorus such as proteins and nucleic acids, can be used as raw materials to synthesize biodegradable materials or to make functional fertilizers.

[0003] Cyanobacteria have much thicker cell walls than most Gram-negative bacteria, and their peptidoglycan layers are highly cross-linked. A major challenge in the development and utilization of biomass from cyanobacteria is how to properly disrupt their cell walls and selectively extract the desired components. Existing methods for cell wall disruption and extraction include bead milling, ultrasonication, microwave methods, high-temperature boiling, high pressure, the addition of chemical reagents such as acids and alkalis, and enzymatic hydrolysis. The first few methods all face problems such as complex equipment and high energy consumption when applied on a large scale, especially for the size range of cyanobacterial single-cell dispersions (0.5–60 μm), where cell wall disruption efficiency is low. Due to the tough and robust cell walls of cyanobacteria, direct extraction methods using acids, alkalis, and enzymes often yield poor results in cell wall disruption.

[0004] Cyanobacteria have a high water content, and their cell density is close to that of water. The biological organic matter of cyanobacteria mainly consists of polysaccharides, lipids, proteins, and nucleic acids. In lakes, they often exist in the form of communities, with extracellular polysaccharide mucus accumulating large numbers of cyanobacterial cells within a gelatinous membrane. The polysaccharides and lipids in cyanobacterial cells have a high carbon content and low nitrogen and phosphorus content, while nitrogen and phosphorus are mainly distributed in proteins, nucleic acids, and intracellular polyphosphates. The content of polysaccharides and proteins varies greatly among different cyanobacterial species and under different growth conditions (Olofsson, M.; Lamela, T.; Nilsson, E.; Bergé, JP; del Pino, V.; Uronen, P.; Legrand, C. Seasonal variation of lipids and fatty acids of the microalgae Nannochloropsis oculata grown in outdoor large-scale photobioreactors. Energies 2012, 5, 1577–1592.). The protein content typically ranges from 6% to 72% of the dry weight of the algae, while the polysaccharide content typically ranges from 8% to 64% (Teuling, E.; Wierenga, PA; Schrama, JW; Gruppen, H. Comparison of protein extracts from various unicellular green sources. J. Agric. Food Chem. 2017, 65(36) 7989–8002.). The lipid content generally does not exceed 20% of the dry weight (Becker, EW. Micro-algae as a source of protein. Biotechnol. Adv. 2007, 25, 207–210.).

[0005] To achieve effective separation of carbon-based biomass from nitrogen and phosphorus components in cyanobacteria, it is necessary to develop a mild, low-energy-consumption method suitable for large-scale industrial applications for the extraction and separation of high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria. The extracted high-carbon, low-nitrogen, and low-phosphorus organic components can be further converted into small-molecule organic acids through targeted transformation reactions, which can be used as a carbon source for denitrifying bacteria in wastewater treatment. In addition, the extract and the remaining algal cell precipitate can also be used as raw materials for further extraction or preparation of other materials or products. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting and separating high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria that is mild, energy-efficient, and suitable for large-scale industrial applications, in order to overcome many shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for extracting and separating high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria involves directly extracting from pre-dehydrated cyanobacteria samples after harvesting or storing them in a sealed container at 4°C. Different extraction solvents are used depending on the storage time. Specifically:

[0009] A. For cyanobacterial samples that have not been stored or have been stored for no more than 3 months, the extraction and separation steps are as follows:

[0010] A1) Using NaOH solution as extraction solvent 1, the sample was stirred and mixed at room temperature. After extraction for 1.5 to 3 hours, the sample was centrifuged and the extract 1 and algal cell precipitate 1 were collected separately.

[0011] A2) Add NaOH solution containing sodium hypochlorite (or hydrogen peroxide) to algal cell precipitate 1 as extraction solvent 2, stir and mix at room temperature for 1.5-3 hours, then centrifuge to separate and collect extract 2 and algal cell precipitate 2 respectively.

[0012] A3) Add NaOH solution containing sodium chloride as extraction solvent 3 to algal cell precipitate 2, stir and mix at 50-70℃ for 1.5-3h, then centrifuge to separate and collect extract 3 and algal cell precipitate 3 respectively.

[0013] A4) Combine supernatants 1 to 3 and separate and purify them using an anion exchange chromatography column to obtain a water-soluble extract of high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria.

[0014] B. For cyanobacteria samples stored for more than 3 months, the extraction and separation steps are as follows:

[0015] B1) Using water as extraction solvent 1, the cyanobacteria sample was stirred and mixed at room temperature. After extraction for 0.5 to 2 hours, the sample was centrifuged and the extract 1 and algal cell precipitate 1 were collected separately.

[0016] B2) Add NaOH solution to algal cell precipitate 1 as extraction solvent 2, stir and mix at room temperature for 0.5-2 hours, then centrifuge to separate and collect extract 2 and algal cell precipitate 2 respectively;

[0017] B3) Add NaOH solution with a concentration of 1 / 3 to 2 / 3 of extraction solvent 2 to algal cell precipitate 2 as extraction solvent 3, stir and mix at room temperature for 0.5 to 2 hours, then centrifuge and collect extract 3 and algal cell precipitate 3 respectively.

[0018] B4) Combine supernatants 1 to 3 to obtain a water-soluble extract of high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria.

[0019] In the above-mentioned method for extracting and separating high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria, the cyanobacteria are not limited to any particular species and are applicable to various cyanobacteria commonly found in lakes and other water bodies, including but not limited to Microcystis wesenbergii, Microcystis aeruginosa, and Microcystis ichthyoblabe.

[0020] Pre-dehydration treatment removes water from the original water body and water adsorbed outside the cells, generally using methods such as air flotation and / or dehydration by a screw press. After pre-dehydration treatment, the water content of the cyanobacteria sample is 87%–90%, and the carbon-to-nitrogen ratio is 4.9.

[0021] In step A1) above, preferably, 100g of cyanobacteria sample is mixed with 50-100mL of extraction solvent 1, and the final NaOH concentration of the mixed system is 0.1-0.2M. This step mainly extracts the extracellular organic matter of cyanobacteria.

[0022] In step A2) above, preferably, 160-320 mL of extraction solvent 2 is added to every 100 g of algal cell precipitate 1; the concentration of NaOH in the extraction solvent 2 is 0.05-0.1 M, and the concentration of sodium hypochlorite is 0.2%-0.4% (available chlorine).

[0023] In step A3) above, preferably, 300-600 mL of extraction solvent 3 is added to every 100 g of algal cell precipitate 2; the concentration of NaOH in the extraction solvent 3 is 0.05-0.1 M, and the concentration of sodium chloride is 0.2-0.5 M.

[0024] In step A4) above, the anion exchange column can be a Q-agarose gel FF column, a Q-agarose gel HP column, etc. When separating the supernatant using an anion exchange column, the supernatant to be separated is added to the anion exchange column, and then eluted with a gradient of NaCl solution. The eluent containing organic components with a high carbon-to-nitrogen ratio is collected.

[0025] In step B1) above, water is used as the extraction solvent to extract the cyanobacteria sample once or multiple times. Preferably, 50-100 mL of water is added for each 100 g of cyanobacteria sample during each extraction. This step mainly extracts the extracellular organic matter of cyanobacteria.

[0026] In step B2) above, preferably, 100-200 mL of extraction solvent 2 is added to every 100 g of algal cell precipitate 1; the concentration of NaOH in the extraction solvent 2 is 0.05-0.1 M.

[0027] In step B3) above, preferably, 160-320 mL of extraction solvent 3 is added to every 100 g of algal cell precipitate 2.

[0028] The main advantages of the method for extracting and separating high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria in this invention include:

[0029] (1) High extraction efficiency and good selectivity. This invention cleverly adopts a step-by-step extraction process using alkaline solution. When necessary, a low concentration of oxidant (sodium hypochlorite or hydrogen peroxide) is added to assist in gentle cell disruption. Sodium chloride is used to weaken the interaction between biomolecules. This not only effectively decomposes and extracts high-carbon organic components such as polysaccharides and lipids, but also largely prevents the outflow of high-nitrogen and high-phosphorus components such as proteins and nucleic acids, thus successfully obtaining high-carbon, low-nitrogen and low-phosphorus organic components from cyanobacteria.

[0030] (2) The reagents used are inexpensive, readily available, safe, and environmentally friendly, and do not involve organic solvents. The extraction process is simple, energy-efficient, requires no complex equipment, and is easy to implement on a large scale. The extracted aqueous solution is easy to store and use.

[0031] (3) The carbon, nitrogen, and phosphorus composition of the extract meets the requirements for targeted acid production or other transformation and utilization methods. The algal cell precipitate is rich in nitrogen and phosphorus and can be used for further extraction of nucleic acids, proteins, polypeptides or amino acids, etc., or it can be used directly as fertilizer.

[0032] (4) It has realized the resource transformation of cyanobacteria and made an important contribution to reducing carbon emissions. Attached Figure Description

[0033] Figure 1 A schematic diagram of the method for extracting and separating high-carbon, low-nitrogen, and low-phosphorus organic matter from cyanobacteria in this invention.

[0034] Figure 2 Microscopic images of cyanobacteria samples before and during treatment in Example 1 of this invention, wherein: (a) microscopic image of cyanobacteria samples before treatment; (b) microscopic image of cyanobacteria samples after treatment with NaClO solution in the second step. Scale bar in the figures: 1 μm. The cyanobacteria samples used were extracted directly after harvesting without storage.

[0035] Figure 3 The percentage distribution of organic carbon in the extract of each step in Example 1 of this invention, relative to the total organic carbon (TOC) in the original algae. The cyanobacteria samples used were extracted directly after being harvested without storage.

[0036] Figure 4 The percentage of total phosphorus in the extract of each step in Example 1 of this invention relative to the total phosphorus content in the original algae. The cyanobacteria samples used were extracted directly after being harvested without storage.

[0037] Figure 5 The carbon-nitrogen ratio in the extract of each step in Example 1 of this invention. The cyanobacteria samples used were extracted directly after being harvested without storage.

[0038] Figure 6 Microscopic images of cyanobacteria samples before and during treatment in Example 2 of this invention, wherein: (a) microscopic image of cyanobacteria samples before treatment; (b) microscopic image of cyanobacteria samples after NaOH treatment in the third step. Scale bar in the figures: 1 μm. The cyanobacteria samples used were collected and stored in a sealed container at 4°C for 5 months before extraction.

[0039] Figure 7 The percentage distribution of organic carbon in the extract of each step in Example 2 of this invention, relative to the total organic carbon (TOC) in the original algae. The cyanobacterial samples used were collected and stored in a sealed container at 4°C for 5 months before extraction.

[0040] Figure 8 The percentage of total phosphorus in the extract of each step in Example 2 of this invention relative to the total phosphorus content in the original algae. The cyanobacterial samples used were collected and stored in a sealed container at 4°C for 5 months before extraction.

[0041] Figure 9 The carbon-nitrogen ratio in the extract of each step in Example 2 of this invention. The cyanobacteria samples used were collected and stored in a sealed container at 4°C for 5 months before extraction. Detailed Implementation

[0042] The present invention is further illustrated below with reference to specific embodiments. Those skilled in the art should understand that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] Example 1

[0044] Weigh 100g of pre-dehydrated cyanobacteria sample (88.0% water content, C / N ratio 4.9) collected from Taihu Lake, and add 50mL of NaOH solution to bring the final NaOH concentration to 0.1M. After extraction with stirring (magnetic stirring, 200-300 rpm) for two hours, centrifuge (8000-9000 rpm), collecting the supernatant 1 and algal cell precipitate 1 separately. Add 160mL of 0.4% (mass fraction) NaClO solution (containing approximately 0.05M NaOH) to algal cell precipitate 1, stir for two hours, and centrifuge, collecting the supernatant 2 and algal cell precipitate 2 separately. The algal cells can be effectively disrupted (…). Figure 2 ).

[0045] Add 300 mL of 0.2 M NaCl solution containing 0.05 M NaOH to algal cell precipitate 2, stir in a 70 °C water bath for two hours, and then centrifuge to separate the supernatant 3 and algal cell precipitate 3. Quantitatively determine the total organic carbon (TOC) content in the original wet algal sample, supernatants 1 to 3, and algal cell precipitate 3. The results are as follows: Figure 3 As shown.

[0046] from Figure 3 As can be seen, the three extraction steps effectively extracted 15-20% of the organic carbon from the original algae, with a total extraction efficiency exceeding 50%. We further analyzed the percentage of total phosphorus in the extracts from each step relative to the total phosphorus content in the original algae, and the results are as follows: Figure 4 As shown, the total phosphorus extracted in the first two steps is less than 0.5% of the total phosphorus in the original algae, indicating that under the extraction conditions used, sodium hypochlorite only disrupted the cell wall and did not cause severe damage to the cell membrane or a large outflow of intracellular material, which is crucial for the selectivity of the extraction. A small amount of phosphorus is released in the third extraction step, accounting for only about 6.5% of the total phosphorus in the original algae, indicating that the extract is still mainly composed of high-carbon, low-phosphorus organic matter.

[0047] Next, we further analyzed the carbon-to-nitrogen ratio in the extract at each step, and the results are as follows: Figure 5 As shown.

[0048] Compared to the original algal sample (C / N ratio 4.9), the C / N ratios of the first two extraction steps were relatively high, indicating that the main components were carbohydrates and lipids, with relatively lower levels of proteins or peptides. The C / N ratio of the third extraction step was close to that of the original algal sample, but still higher than that of proteins (C / N ratio approximately 3) and nucleic acids (C / N ratio 2–3), indicating that there were still a relatively large amount of carbohydrates and lipids, while a small amount of proteins and phosphorus-containing inorganic substances (polyphosphates) or organic substances (nucleic acids, nucleotides, and phospholipids, etc.) entered the extraction solution.

[0049] We mixed the supernatants 1 to 3 obtained from the three-step extraction and separated them using an anion exchange column (Q-agarose gel FF column): 5–15 mL of the supernatant was taken and filtered under reduced pressure using a 0.45 μm filter membrane. The filtrate was collected and added to a 5 mL Q-agarose gel FF column at a flow rate of 50–100 cm / h. The eluent was collected and eluted sequentially with 5 mL of water, 5 mL of 0.1 M NaCl, 5 mL of 0.5 M NaCl, 5 mL of 1 M NaCl, and 5 mL of 2 M NaCl. The eluents were collected separately, and the carbon-nitrogen content was determined. The high carbon-nitrogen ratio fraction was collected, and the organic carbon content was found to be 40% of the total organic carbon in the original algal sample, with a carbon-nitrogen ratio of 8.1 and a carbon-phosphorus ratio greater than 120. The above experimental results indicate that the extraction and separation method established in this invention can extract high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria that have not been stored after harvesting. The extracted and separated solution fully meets the requirements for further targeted acid production reactions.

[0050] Example 2

[0051] Pre-dehydrated cyanobacteria samples (88.0% water content, C / N ratio 4.9) collected from Taihu Lake were stored in a sealed container at 4℃ for 5 months. 100g of the stored cyanobacteria sample (87.4% water content, C / N ratio 4.9) was weighed, 50mL of water was added, and the mixture was stirred at room temperature for one hour before centrifugation. The supernatant 1-1 was collected. Another 75mL of water was added to the algal cell precipitate 1-1, and the mixture was stirred at room temperature for one hour before centrifugation. Supernatant 1-2 and algal cell precipitate 1-2 were collected. Supernatant 1-1 and 1-2 were combined into Supernatant 1. 100mL of NaOH solution (0.075M, 3g / L) was added to the algal cell precipitate 1-2 obtained in the previous step, and the mixture was stirred at room temperature for one hour, followed by centrifugation. Supernatant 2 was collected. Another 160mL of 1.5g / L NaOH solution was added to algal cell precipitate 2, and the mixture was stirred at room temperature for one hour. Centrifugation was performed, and the supernatant (3) and algal cell precipitate (3) were collected separately. Microscopic observation showed that the algal cells could be effectively disrupted. Figure 6 )

[0052] The organic carbon content in the unextracted algal samples, supernatants 1 to 3, and algal cell precipitate 3 was quantitatively determined, and the results are as follows: Figure 7 As shown in the figure, the composition of the algae has changed significantly after storage. Adding water directly yields nearly 20% of the organic carbon from the original algae. Adding a low-concentration NaOH solution further extracts more organic carbon. In the third step, using a NaOH solution with a concentration halved, no sodium hypochlorite is needed as the cells have already been disrupted. The organic carbon obtained from these three extraction steps accounts for 37.4% of the total organic carbon in the original algae sample. The percentage of total phosphorus in the extract at each step relative to the total phosphorus content in the original algae is shown in the figure. Figure 8 As shown. Phosphorus was virtually undetectable in the first two extraction steps, and the phosphorus extracted in the third step was only about 1% of the total phosphorus in the original algae. This indicates that under the extraction conditions used, phosphorus remained in the algal cell precipitate, and the phosphorus in the extract was negligible. The carbon and nitrogen content of the extracts from each step is as follows: Figure 9 As shown.

[0053] Compared to the algal sample used (C / N ratio 4.9), the supernatant 2 had a higher C / N ratio, reaching 9.3. However, the C / N ratio of the supernatant 1 in this embodiment was lower than that of the supernatant 1 in Example 1, suggesting that the structure of components such as peptidoglycans or proteins in the algal cells changed during storage, allowing them to be directly dissolved in water and extracted. The C / N ratio in the third step was close to that of the original algal sample, but still higher than that of proteins (C / N ratio approximately 3) and nucleic acids (C / N ratio 2-3), indicating that there were still a certain amount of carbohydrates and lipids present. Since the C / N ratio of the supernatant 3 was already low, no further extraction was performed on the algal cell precipitate 3.

[0054] We attempted to separate the supernatants obtained from the above three extraction steps using an anion exchange column (Q-agarose gel FF column). However, the carbon-to-nitrogen ratio (C / N ratio) of the fractionated collected solutions was similar, around 6.5, which is significantly different from the results in Example 1. This further indicates that the algal cells underwent significant changes after storage, and the retention behavior of the extracted components on the anion exchange column was more similar, making it difficult to separate the components with higher C / N ratios. The above experimental results show that storing the harvested cyanobacteria in a sealed container at 4°C for 5 months before extraction further reduces the requirements for the extraction solvent. Only water and dilute sodium hydroxide solution are needed to extract high-carbon, low-nitrogen, and low-phosphorus organic components, with the phosphorus content being negligible. Although the carbon extraction amount is slightly less than in Example 1, the C / N ratio still meets the requirements for further targeted acid production reactions.

[0055] Comparative Example 1

[0056] 100g of pre-dehydrated cyanobacteria sample (88.0% water content, C / N ratio 4.9) collected from Taihu Lake was weighed and added to 24mL of deionized water. After stirring and extraction for two hours, centrifugation was performed (magnetic stirring, speed 200-300rpm; centrifugation speed 8000-9000rpm). The supernatant and algal cell precipitate were collected separately. The TOC in the supernatant was found to be 2.9% of the original algal sample's TOC, with a C / N ratio of 10.2. Further extraction with water was performed on the algal cell precipitate, and almost no TOC was detected in the extract.

[0057] The TOC measurement results of Example 1 ( Figure 3 Compared with the above results, it can be seen that the TOC extracted by 50 mL of 0.1 M NaOH solution in Example 1 under the same conditions (accounting for 17.8% of the original algal sample TOC) is more than 6 times that extracted with pure water. The carbon-nitrogen ratio determination result of Example 1 (the carbon-nitrogen ratio of supernatant 1 is 6.7) is relatively low compared with the above results. These results indicate that using NaOH solution and increasing the volume of the extract helps to extract more organic carbon. Although the carbon-nitrogen ratio is lower, considering the extraction amount of organic carbon and the needs of subsequent applications, using 0.1 M NaOH solution is a better extraction method.

[0058] The TOC measurement results of Example 2 ( Figure 7Comparing the results with Comparative Example 1, it is evident that in Example 2, after storing the sample in a sealed container at 4°C for 5 months, a significant amount of organic carbon could be extracted by adding pure water. The TOC obtained through a two-step water extraction (50 mL + 75 mL) was 19.3% of the original algal sample's TOC, which was 6.7 times higher than that extracted directly with pure water without prior storage. We further determined the extraction efficiency of each of the two water extraction steps after storage. The TOC obtained from the first step of pure water extraction accounted for 13.3% of the original algal sample's TOC, and the TOC obtained from the second step accounted for 5.9%. These results indicate that the changes in algal cells after storing the sample in a sealed container at 4°C facilitate the direct extraction of a larger amount of organic carbon. It is particularly noteworthy that the phosphorus content in the supernatants 1 to 3 obtained in Examples 1 and 2 was very low, essentially negligible. Compared to the original algal sample, the carbon-to-nitrogen ratio was increased. The obtained high-carbon, low-nitrogen, and low-phosphorus extracts lay an important foundation for further development of biomass carbon sources.

[0059] Comparative Example 2

[0060] 100g of pre-dehydrated cyanobacteria sample (88.0% water content, C / N ratio 4.9) collected from Taihu Lake was weighed and 50mL of NaOH solution was added to a final concentration of 0.2M. After stirring in a 70℃ water bath for two hours, the sample was centrifuged (magnetic stirring, speed 200-300 rpm; centrifugation speed 8000-9000 rpm), and the supernatant and algal cell precipitate were collected separately. The TOC of the supernatant was found to be 13% of the original algal sample's TOC, and the C / N ratio was 6.4.

[0061] The TOC measurement results of Example 1 ( Figure 3 Comparing the results above, it is clear that using a higher concentration of NaOH solution and increasing the temperature does not increase the extracted TOC. Figure 5 Compared to the data for the carbon-to-nitrogen ratio in Example 1, Comparative Example 2 has a smaller carbon-to-nitrogen ratio. These results indicate that the extraction method using 0.1M NaOH solution at room temperature, as employed in Example 1, is a superior extraction protocol.

[0062] Comparative Example 3

[0063] The step in Example 2, "Add 160 mL of 1.5 g / L NaOH solution to algal cell precipitate 2, stir at room temperature for one hour. Centrifuge and collect the supernatant 3 and algal cell precipitate 3 separately," was replaced with "Add 160 mL of 0.23% NaClO solution (containing 1-1.5 g / L NaOH) to algal cell precipitate 2, stir at room temperature for one hour. Centrifuge and collect the supernatant and algal cell precipitate separately." The TOC of the supernatant was measured to be 14.2% of the original algal sample's TOC, which is consistent with... Figure 7 Compared to the corresponding 11.0%, the increase was limited. Further determination of the phosphorus content showed results consistent with... Figure 8 Similarly, these results indicate that stored algal samples can yield a significant amount of TOC using only NaOH, and the addition of NaClO has limited effect and does not significantly increase the extracted organic carbon content.

Claims

1. A method for extracting and separating high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria, wherein the cyanobacteria sample, after being pre-dehydrated following harvesting, is directly extracted or stored in a sealed container at 4°C. Different extraction solvents are used depending on the storage time. Specifically: A. For cyanobacterial samples that have not been stored or have been stored for no more than 3 months, the extraction and separation steps are as follows: A1) Using NaOH solution as extraction solvent 1, the sample was stirred and mixed at room temperature. After extraction for 1.5-3 h, the sample was centrifuged and the extract 1 and algal cell precipitate 1 were collected separately. A2) Add NaOH solution containing sodium hypochlorite or hydrogen peroxide to algal cell precipitate 1 as extraction solvent 2, stir and mix at room temperature for 1.5-3 h, then centrifuge to separate and collect extract 2 and algal cell precipitate 2 respectively. A3) Add NaOH solution containing sodium chloride as extraction solvent 3 to algal cell precipitate 2, stir and mix at 50~70℃ for 1.5~3 h, then centrifuge to separate and collect extract 3 and algal cell precipitate 3 respectively. A4) Combine extracts 1 to 3 and separate and purify them using an anion exchange chromatography column to obtain a water-soluble extract of high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria. B. For cyanobacteria samples stored for more than 3 months, the extraction and separation steps are as follows: B1) Using water as extraction solvent 1, the cyanobacteria sample was stirred and mixed at room temperature. After extraction for 0.5-2 h, the sample was centrifuged and the extract 1 and algal cell precipitate 1 were collected separately. B2) Add NaOH solution to algal cell precipitate 1 as extraction solvent 2, stir and mix at room temperature for 0.5-2 h, then centrifuge to separate and collect extract 2 and algal cell precipitate 2 respectively; B3) Add NaOH solution with a concentration of 1 / 3 to 2 / 3 of extraction solvent 2 to algal cell precipitate 2 as extraction solvent 3, stir and mix at room temperature for 0.5 to 2 h, then centrifuge to separate and collect extract 3 and algal cell precipitate 3 respectively. B4) Combine extracts 1 to 3 to obtain a water-soluble extract of high-carbon, low-nitrogen, and low-phosphorus organic components from cyanobacteria.

2. The extraction and separation method as described in claim 1, characterized in that, The pre-dehydration process involves removing water from the original water body and water adsorbed on the extracellular surface of the cyanobacteria after harvesting. The water content of the cyanobacteria sample after pre-dehydration is between 87% and 90%.

3. The extraction and separation method as described in claim 1, characterized in that, In step A1), every 100 g of cyanobacteria sample is mixed with 50-100 mL of extraction solvent 1, and the final concentration of NaOH in the mixed system is 0.1-0.2 M.

4. The extraction and separation method as described in claim 1, characterized in that, In step A2), 160-320 mL of extraction solvent 2 is added to every 100 g of algal cell precipitate 1. The extraction solvent 2 is a NaOH solution containing sodium hypochlorite, wherein the NaOH concentration is 0.05-0.1 M and the effective chlorine concentration is 0.2%-0.4%.

5. The extraction and separation method as described in claim 1, characterized in that, In step A3), 300-600 mL of extraction solvent 3 is added to every 100 g of algal cell precipitate 2. The concentration of NaOH in the extraction solvent 3 is 0.05-0.1 M, and the concentration of sodium chloride is 0.2-0.5 M.

6. The extraction and separation method as described in claim 1, characterized in that, The anion exchange column mentioned in step A4) is a Q-agarose gel FF column or a Q-agarose gel HP column.

7. The extraction and separation method as described in claim 1, characterized in that, Step A4) Add the supernatant to be separated to an anion exchange chromatography column, then elute with NaCl solution in a gradient, and collect the eluent containing organic components with a high carbon-to-nitrogen ratio.

8. The extraction and separation method as described in claim 1, characterized in that, In step B1), water is used as the extraction solvent to extract the cyanobacteria sample once or multiple times, with 50-100 mL of water added for each 100 g cyanobacteria sample for each extraction.

9. The extraction and separation method as described in claim 1, characterized in that, In step B2), 100-200 mL of extraction solvent 2 is added to every 100 g of algal cell precipitate 1, wherein the concentration of NaOH in extraction solvent 2 is 0.05-0.1 M.

10. The extraction and separation method as described in claim 1, characterized in that, In step B3), 160-320 mL of extraction solvent 3 is added to every 100 g of algal cell precipitate 2.

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