Application of phospholipase in promoting phosphorus removal in water body

Abstract

The application discloses application of phospholipase in promoting phosphorus removal in water body, and belongs to the technical field of water treatment. The phospholipase is added to a wetland or a constructed wetland system, the phospholipase catalyzes degradation of organic phosphorus into intermediates, and then shortens the time for conversion of the organic phosphorus into absorbable phosphorus, accelerates the conversion of the phosphorus into the absorbable phosphorus, improves the absorption efficiency of plants on the phosphorus, and thus improves the removal efficiency of the wetland or the constructed wetland system on the phosphorus in the water body.

Description

Technical Field

[0001] This application belongs to the field of water treatment technology, specifically relating to the application of phospholipase in promoting phosphorus removal from water. Background Technology

[0002] Phosphorus loads generated by human production and daily life have led to eutrophication of lakes. Therefore, phosphorus removal is one of the key aspects of water body management in advanced wastewater treatment.

[0003] Wetlands, or constructed wetlands, encompassing unique soil (filler)-plant-microbe ecosystems, are a cost-effective and efficient technology for controlling phosphorus load. Their mechanisms for removing phosphorus from water primarily involve physical, chemical, and biological processes. Physical processes include sedimentation, mainly referring to the process by which soluble phosphates in the influent are physically absorbed and stored within the wetland. Chemical processes include adsorption and precipitation, primarily referring to the adsorption of phosphorus by the soil (filler) and its precipitation within the wetland or constructed wetland system. Biological processes include plant uptake and normal and excessive accumulation by microorganisms; plant uptake mainly refers to inorganic phosphates (PO42-). 3- HPO4 2- H2PO4 - Phosphorus is absorbed by plant roots and assimilated into the plant's organic components (such as ATP, phospholipids, coenzymes, DNA, and RNA), and then carried out of the system during harvesting. Normal absorption and excessive accumulation of phosphorus by microorganisms mainly refer to the absorption of phosphorus into cells and its transformation into components of microbial cells during microbial growth.

[0004] Chemical action is considered one of the most important phosphorus removal mechanisms, thus improving the adsorption capacity of soil (filler) has become a research hotspot. For example, Chinese invention patent CN105776552A discloses a phosphorus removal filler for constructed wetlands and its preparation method, providing a core-shell structure phosphorus removal filler with a core composed of Ca(OH)2 and ferric salts, and an outer layer covered with CaCO3. Chinese invention patent CN112979086A discloses an enhanced phosphorus removal filler for constructed wetlands, composed of a composite filler and polyphosphate-accumulating bacteria activation liquid. The composite filler is selected from zeolite, maifanite, crushed stone, magnetite, volcanic rock, dolomite, shale ceramsite, oyster shells, and marine materials. Any combination of clam shells, cinders, slag, peat moss, coarse sand, gravel, clay ceramsite, fly ash, and steel slag; Chinese invention patent publication number CN115180713A describes a high-efficiency phosphorus removal wetland filler and its preparation method. The high-efficiency phosphorus removal wetland filler includes: gravel, soil, shale, limestone, manganese sand, sludge, zeolite, and fly ash, wherein the gravel accounts for 40-60%, soil 10-15%, shale 5-8%, limestone 5-8%, manganese sand 3-5%, sludge 10-15%, zeolite 2-3%, and fly ash 4-6%. However, after the system has been running for a period of time, the phosphorus removal rate of the soil (filler) will decrease to a certain extent, and the adsorbed and precipitated phosphorus will still exist in the wetland or artificial wetland system, still posing a risk of recycling and release.

[0005] Correspondingly, plant absorption deserves more attention because it absorbs phosphorus and assimilates it into organic components, which are then removed from the system during harvesting. However, it also has certain limitations, mainly because phosphorus in wastewater often exists as inorganic phosphate (PO4). 3- HPO4 2- H2PO4 - Phosphorus exists in the form of polyphosphates and organic phosphorus. Among them, only inorganic phosphates can be absorbed by plants. Although organic phosphorus can be gradually converted into inorganic phosphate (i.e., absorbable phosphorus) that plants can absorb through various complex pathways under natural conditions, the conversion process is very slow and time-consuming. Often, the conversion process is not completed before the wastewater is discharged from the system, which affects the removal efficiency of phosphorus in the water, is not conducive to improving the quality of effluent, and increases the risk of eutrophication of the receiving water body. Summary of the Invention

[0006] 1. The problem to be solved

[0007] This application addresses the problem that plants in wetland or constructed wetland systems cannot absorb organic phosphorus, resulting in low phosphorus absorption efficiency. It provides the application of phospholipase in promoting phosphorus removal from water bodies. Through the catalytic action of phospholipase, organic phosphorus is converted into intermediates, thereby shortening the time for organic phosphorus to be converted into absorbable phosphorus, accelerating the conversion of phosphorus to absorbable phosphorus, improving the efficiency of phosphorus absorption by plants, and thus promoting the efficiency of phosphorus removal from water bodies.

[0008] 2. Technical Solution

[0009] To solve the above problems, the technical solution adopted in this application is as follows:

[0010] This application provides the application of phospholipases in promoting phosphorus removal from water bodies. This application involves adding phospholipases to wetland or constructed wetland systems. The phospholipases catalyze the degradation of organic phosphorus into intermediates, thereby shortening the time required for organic phosphorus to convert to absorbable phosphorus, accelerating the conversion of phosphorus to absorbable phosphorus, and improving the efficiency of phosphorus absorption by plants. This, in turn, enhances the phosphorus removal efficiency of wetland or constructed wetland systems. Phospholipases are a class of enzymes responsible for phospholipid metabolism and biosynthesis in organisms, catalyzing the hydrolysis of glycerophospholipids. Phospholipases are generally widely used in the food processing industry, where the purpose of hydrolyzing phospholipids is to improve food texture, such as enhancing the flavor of pasta, or to focus on their role in the phosphorus cycle in plants, such as in the agricultural sector. However, their application in environmental protection and water treatment has been largely unexplored.

[0011] Furthermore, the aforementioned phospholipases include any one or more of phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase B (PLB), phospholipase C (PLC), and phospholipase D (PLD); these phospholipases are classified according to their different phospholipid hydrolysis sites, which are as follows: Figure 1As shown, phospholipase A1 (PLA1) and phospholipase A2 (PLA2) mainly act on specific ester bonds in phospholipid molecules (such as the 1- or 2-position ester bond in glycerophospholipids), hydrolyzing them to produce fatty acids (such as arachidonic acid) and lysophospholipids, but do not directly hydrolyze them to produce phosphate. Phospholipase B (PLB) has broader substrate specificity, capable of hydrolyzing two ester bonds in phospholipids, including those containing phosphate groups, but its main product is not phosphate alone, but rather free fatty acids and glycerophosphatidylcholine, etc. Phospholipase C (PLC) mainly acts on... Phospholipids contain phosphate ester bonds, but their main products are diacylglycerols and phosphorylated head groups, not just phosphate groups. Phospholipase D (PLD) mainly acts on the head groups of phospholipids to produce phosphatidic acids and alcohols, but it does not directly produce phosphate groups. However, although the products of the above-mentioned phospholipase hydrolysis of phospholipids are not inorganic phosphates (i.e., absorbable phosphorus) that can be directly absorbed by plants, this invention has found that phospholipase treatment can promote the absorption efficiency of phosphorus by plants. It is speculated that the conversion of organic phosphorus into intermediates accelerates the conversion of phosphorus into absorbable phosphorus.

[0012] Furthermore, the aforementioned phospholipases include phospholipase A1 and / or phospholipase B.

[0013] Furthermore, the above applications include: using nanospheres as a filler matrix, loading the above-mentioned phospholipase, and then encapsulating it with sodium alginate for use as a filler in artificial wetlands; loading and encapsulating the phospholipase can fix and encapsulate the phospholipase, allowing it to be released slowly, prolonging the effective time of phospholipase action, and achieving the long-lasting effectiveness of phospholipase in flowing water.

[0014] Furthermore, the aforementioned constructed wetland filler material also includes CaCl2, Ca... 2+ It promotes the hydrolysis of phospholipase. Metal ions, such as calcium ions, can promote the hydrolysis reaction by interacting with specific groups in organophosphorus molecules.

[0015] Furthermore, the preparation method of the above-mentioned artificial wetland filler includes: adding nanospheres to a phospholipase solution, shaking and then allowing it to stand to obtain catalytic nanospheres; adding the catalytic nanospheres to a sodium alginate solution and stirring until the surface of the nanospheres is covered with sodium alginate; then immersing them in a calcium chloride solution, embedding them, and then removing them to obtain the above-mentioned artificial wetland filler.

[0016] Furthermore, the aforementioned artificial wetland filler also includes indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc, magnesium, etc., which can promote plant root growth and thus improve phosphorus absorption efficiency.

[0017] Furthermore, the preparation method of the above-mentioned artificial wetland filler includes: adding nanospheres to a mixed auxiliary solution containing indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc and magnesium, shaking and then letting it stand to obtain auxiliary nanospheres; adding catalytic nanospheres and auxiliary nanospheres to a sodium alginate solution and stirring until the surface is covered with sodium alginate; then immersing it in a calcium chloride solution, embedding it and taking it out to obtain the above-mentioned artificial wetland filler.

[0018] Furthermore, the calcium mentioned above is calcium chloride.

[0019] Furthermore, the iron mentioned above is ferrous sulfate.

[0020] Furthermore, the zinc mentioned above is zinc sulfate.

[0021] Further, the magnesium mentioned above is magnesium sulfate. Further, the concentration of indoleacetic acid in the above mixed auxiliary solution is 10–1000 mg / L. Even further, the concentration of indoleacetic acid in the above mixed auxiliary solution is 1000 mg / L.

[0022] Furthermore, the concentration of vitamin B1 in the above-mentioned mixed auxiliary solution is 1–100 mg / L. Even more specifically, the concentration of vitamin B1 in the above-mentioned mixed auxiliary solution is 100 mg / L.

[0023] Furthermore, the concentration of vitamin B6 in the above-mentioned mixed auxiliary solution is 1–100 mg / L. Even more specifically, the concentration of vitamin B6 in the above-mentioned mixed auxiliary solution is 100 mg / L.

[0024] Furthermore, in the above-mentioned mixed auxiliary solution, the concentration of calcium chloride is 10–10000 mg / L. Even further, in the above-mentioned mixed auxiliary solution, the concentration of calcium chloride is 10000 mg / L.

[0025] Furthermore, in the above-mentioned mixed auxiliary solution, the concentration of ferrous sulfate is 10–10000 mg / L. Even further, in the above-mentioned mixed auxiliary solution, the concentration of ferrous sulfate is 10000 mg / L.

[0026] Furthermore, in the above-mentioned mixed auxiliary solution, the concentration of zinc sulfate is 1–1000 mg / L. Even more specifically, in the above-mentioned mixed auxiliary solution, the concentration of zinc sulfate is 1000 mg / L.

[0027] Furthermore, in the above-mentioned mixed auxiliary solution, the concentration of magnesium sulfate is 0.5–500 mg / L. Even more specifically, in the above-mentioned mixed auxiliary solution, the concentration of magnesium sulfate is 500 mg / L.

[0028] Furthermore, the mass ratio of the nanospheres to the phospholipase solution is 1:2 or higher, at which point the phospholipase solution can completely soak the nanospheres.

[0029] Furthermore, the mass ratio of the nanospheres to the mixed auxiliary liquid is 1:2 or higher, at which point the mixed auxiliary liquid can completely soak the nanospheres.

[0030] Furthermore, the mass ratio of the catalytic nanospheres to the auxiliary nanospheres is 9:1 or higher.

[0031] Furthermore, the above-mentioned sodium alginate solution is 4% sodium alginate, which is prepared by adding 4 grams of sodium alginate powder to every 100 ml of distilled water, stirring thoroughly until uniform and transparent, and then letting it stand for 2-3 hours.

[0032] Furthermore, the above-mentioned calcium chloride solution is 4% calcium chloride, which is prepared by adding 4 grams of calcium chloride to every 100 ml of distilled water and stirring thoroughly until it is uniform and transparent.

[0033] This application also provides an artificial wetland filler that promotes phosphorus removal from water, the filler comprising:

[0034] Nanospheres

[0035] Phospholipases include any one or more of phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase B (PLB), phospholipase C (PLC), and phospholipase D (PLD).

[0036] Sodium alginate,

[0037] Calcium chloride,

[0038] Phospholipase is loaded onto nanospheres, and sodium alginate is used to encapsulate the phospholipase-loaded nanospheres.

[0039] Furthermore, the aforementioned constructed wetland filler material also includes: indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc, and magnesium.

[0040] This application also provides an artificial wetland system in which the filler material includes the aforementioned artificial wetland filler material that promotes phosphorus removal from water.

[0041] This application also provides the above-mentioned constructed wetland filler material for promoting phosphorus removal in water and the application of the above-mentioned constructed wetland system in phosphorus removal in water.

[0042] 3. Beneficial effects

[0043] Compared with the prior art, the advantages of this application are as follows:

[0044] (1) The application of phospholipase in promoting phosphorus removal in water bodies provided in this application. Although phospholipase cannot directly convert organic phosphorus into absorbable phosphorus, the inventors accidentally discovered and verified through experiments that the intermediate products after phospholipase catalyzes hydrolysis can shorten the time for organic phosphorus to be converted into absorbable phosphorus and accelerate the conversion of phosphorus into absorbable phosphorus. In other words, the use of phospholipase promotes the conversion of organic phosphorus into absorbable phosphorus, improves the efficiency of phosphorus absorption by plants, and thus improves the phosphorus removal efficiency of wetland or artificial wetland systems.

[0045] (2) The application of phospholipase provided in this application in promoting phosphorus removal in water bodies uses nanospheres as the filler matrix, loads phospholipase and then encapsulates it with sodium alginate, and then uses it as a filler in artificial wetlands to fix and encapsulate phospholipase, so that phospholipase is released slowly, prolonging the effective time of phospholipase action and realizing the long-lasting effectiveness of phospholipase on flowing water bodies.

[0046] (3) The application of the phospholipase provided in this application in promoting phosphorus removal from water bodies, wherein the packing material containing the phospholipase also includes CaCl2, indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc, magnesium, etc.; wherein CaCl2... 2+ It promotes the hydrolysis of phospholipase. Indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc, magnesium and other substances can promote the growth of plant roots, thereby improving the adsorption efficiency of phosphorus. Attached Figure Description

[0047] Figure 1 This is a map showing the sites where different phospholipases hydrolyze phospholipids.

[0048] Figure 2 This is the simulated artificial wetland system of Example 2.

[0049] Figure 3 This refers to the concentration changes of organic phosphorus in the simulated constructed wetland system of Example 2.

[0050] Figure 4 This refers to the change in total phosphorus concentration in the simulated constructed wetland system of Example 2.

[0051] Figure 5 It is the cumulative removal rate of total phosphorus in the simulated constructed wetland system of Example 2.

[0052] Figure 6 This is the simulated artificial wetland system of Example 3.

[0053] Figure 7 This refers to the concentration change of organic phosphorus in the simulated constructed wetland system of Example 3.

[0054] Figure 8 This refers to the change in total phosphorus concentration in the simulated constructed wetland system of Example 3.

[0055] Figure 9 This is the daily removal rate of total phosphorus in the simulated constructed wetland system of Example 3.

[0056] Figure 10 It is the cumulative removal rate of total phosphorus in the simulated constructed wetland system of Example 3.

[0057] Figure 11 This refers to the height change of water hyacinth in the simulated artificial wetland system of Example 3.

[0058] Figure 12 This refers to the height variation of Acorus calamus in the simulated artificial wetland system of Example 3.

[0059] Figure 13 This refers to the height variation of *Acorus calamus* in the simulated artificial wetland system of Example 3. Detailed Implementation

[0060] The present application will be further described below with reference to specific embodiments.

[0061] It should be noted that terms such as "upper", "lower", "left", "right", and "middle" used in this specification are only for clarity of description and are not intended to limit the scope of implementation. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of this application.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0063] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0064] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0065] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0066] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0067] Those skilled in the art will understand that there are various types of plants used in constructed wetland systems, which can be selected based on factors such as location, climate, and water samples. In this application, the plants used to simulate the constructed wetland system include two or three of the following: water hyacinth (originating from Yongzhou, Hunan), sweet flag (originating from Suqian, Jiangsu), and red sweet flag (originating from Lishui, Zhejiang). All of these plants are commercially available. Before the experiment, the plants are pre-cultured according to their growth characteristics and experimental requirements for 5 to 7 days to allow them to adapt to the laboratory environment.

[0068] In this application, phospholipase A1 was purchased from Novozymes (Denmark, lecitase Ultra, 10 KLU / g, 50g), and phospholipase B was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. (catalog number: FDG-2238, 0.1kg, 2900U / g enzyme activity).

[0069] In this application, soy lecithin was used to simulate organic phosphorus in water. The soy lecithin was purchased from Henan Jiapai Biotechnology Co., Ltd., 100g / bag.

[0070] Example 1

[0071] This embodiment provides the application of phospholipase in promoting phosphorus removal in water. Specifically, nanospheres are used as the filler matrix, loaded with phospholipase, and then encapsulated with sodium alginate for use as a filler in artificial wetlands.

[0072] The artificial wetland filler material includes:

[0073] Nanospheres, used as a substrate for constructed wetland fillers, are used to load phospholipases;

[0074] Phospholipases, as catalytically active components, specifically break specific chemical bonds in organophosphates;

[0075] Indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc, and magnesium are used to promote plant root growth; Ca 2+ It can also promote the hydrolysis reaction of phospholipase;

[0076] Sodium alginate is used to immobilize and encapsulate phospholipase, indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc, and magnesium.

[0077] The preparation method of this artificial wetland filler includes:

[0078] (1) Solution preparation

[0079] Wash the nanospheres (nanobacterial nanospheres, Maibao) with distilled water and dry them at 103-105℃ for later use.

[0080] Preparation of the mixed auxiliary solution: Add 1g indoleacetic acid, 0.1g vitamin B1, 0.1g vitamin B6, 10g calcium chloride, 10g ferrous sulfate, 1g zinc sulfate, and 0.5g magnesium sulfate to 1 liter of water;

[0081] To prepare a 4% sodium alginate solution: add 4 grams of sodium alginate powder to every 100 ml of distilled water, stir thoroughly until uniform and transparent, and then let stand for 2-3 hours;

[0082] To prepare a 4% calcium chloride solution: add 4 grams of calcium chloride to every 100 ml of distilled water and stir thoroughly until homogeneous and transparent;

[0083] (2) Preparation

[0084] Add nanospheres to phospholipase solution at a mass ratio of 1:2, shake at 20-30°C for more than 30 minutes, and then let stand for 24 hours to obtain catalytic nanospheres.

[0085] Add nanospheres to a mixed auxiliary solution with a mass ratio of nanospheres to phospholipase solution of 1:2, shake at 20-30°C for 1 hour, and let stand for more than 2 hours to obtain auxiliary nanospheres.

[0086] Catalytic nanospheres and auxiliary nanospheres were added to a sodium alginate solution at a mass ratio of 9:1 and stirred until the surface was covered with sodium alginate. Then, the nanospheres were immersed in a calcium chloride solution, encapsulated, and removed to obtain the artificial wetland filler.

[0087] In this embodiment, the phospholipase solution is 10 KLU / g phospholipase A1, and an artificial wetland filler containing phospholipase A1 is prepared. The average adsorption capacity of the nanospheres on the solution is 10%. When the concentration of the phospholipase A1 solution is 10 KLU / g, 10g of nanospheres prepares catalytic nanospheres with a mass of approximately 11g, corresponding to 10 KLU phospholipase A1. The catalytic nanospheres and auxiliary nanospheres are prepared at a mass ratio of 9:1, and the auxiliary nanospheres have a mass of approximately 1.2g. Approximately 12.2g of filler corresponds to 10 KLU phospholipase A1.

[0088] In this embodiment, the phospholipase solution is 10 g / L phospholipase B, and an artificial wetland filler containing phospholipase B is prepared. The average adsorption capacity of the nanospheres on the solution is 10%. When the concentration of the phospholipase B solution is 10 g / L, 20 g of nanospheres prepares catalytic nanospheres with a mass of approximately 22 g. Based on an adsorption volume of approximately 2 mL (the solution density is calculated based on approximate water), this corresponds to approximately 20 mg of phospholipase B. Since the catalytic nanospheres and auxiliary nanospheres are in a mass ratio of 9:1, the auxiliary nanospheres have a mass of approximately 2.4 g. Approximately 24.4 g of filler corresponds to 20 mg of phospholipase B.

[0089] Example 2

[0090] This embodiment provides a study on the effects of different types of phospholipases on phosphorus removal from water.

[0091] In this embodiment, the plants used to simulate the artificial wetland system include water hyacinth and sweet flag, such as... Figure 2 As shown, there are 9-10 water hyacinths, about 5cm tall, and 3-4 water calamus plants, about 30cm tall.

[0092] In this embodiment, the experiment was divided into four groups (including a control group). Each group's simulated constructed wetland system was supplemented with 2L of natural lake water (water quality superior to Class III water in the "Surface Water Environmental Quality Standard"), and 10mL of soybean lecithin emulsion with a concentration of 25mg / mL was added to simulate wastewater containing organophosphorus compounds. The types and amounts of phospholipase-prepared constructed wetland fillers added in the four groups are shown below:

[0093] (1) Phospholipase B artificial wetland packing experimental group: 73.2g of phospholipase B-containing artificial wetland packing prepared in Example 1 was added (i.e., about 60mg of phospholipase B was added);

[0094] (2) Phospholipase A1 artificial wetland packing experimental group: 12.2g of phospholipase A1-containing artificial wetland packing prepared in Example 1 was added (i.e., about 10KLU of phospholipase A1 was added);

[0095] (3) Experimental group combining phospholipase A1 artificial wetland packing and phospholipase B artificial wetland packing: 73.2g of artificial wetland packing containing phospholipase B prepared in Example 1 and 12.2g of artificial wetland packing containing phospholipase A1 prepared in Example 1 were added;

[0096] (4) Experimental group without enzyme addition: No phospholipase was added;

[0097] The experiment lasted from April 14, 2024 to April 28, 2024, for a total of 15 days.

[0098] Results analysis:

[0099] (1) The effect of different types of phospholipases on the removal of organophosphates

[0100] Simulated changes in organic phosphorus concentration in constructed wetland systems, such as Figure 3 As shown in Table 1, the concentration and removal rate of organic phosphorus are as follows. Table 1 shows that within one day of adding phospholipase B, phospholipase A1, and a combination of phospholipase A1 and phospholipase B as filler, the concentration of organic phosphorus in the simulated constructed wetland system water sample decreased by 79.9%, 85.8%, and 89.6%, respectively, while the concentration of organic phosphorus in the blank sample decreased by 12.1%. This indicates that phospholipase A1's effect in promoting the conversion of lecithin to soluble orthophosphate is slightly stronger than that of phospholipase B within 24 hours, but within 48 hours and longer periods (e.g., 1-2 weeks), phospholipase B has a stronger removal effect. Furthermore, the combination of both has a better effect on the removal of organic phosphorus in water. The reason for this is that phospholipase B and phospholipase A1 hydrolyze lecithin into lysophospholipids and a free fatty acid, and further hydrolyze the phosphate bonds in lysophospholipids, thereby releasing phosphate ions, thus removing organic phosphorus. Figure 3 It was found that in the three simulated constructed wetland systems with added phospholipase, the concentration of organic phosphorus initially decreased rapidly, then stabilized and exhibited fluctuations; in the blank sample, the concentration of organic phosphorus initially decreased steadily, then stabilized and exhibited fluctuations. Comparing these three systems, the following conclusions can be drawn: both phospholipase A1 and phospholipase B have strong catalytic conversion effects on organic phosphorus, and the phosphorus-containing intermediates they generate are more easily converted into absorbable phosphorus or more easily absorbed by plants, which is beneficial for the removal of organic phosphorus from water.

[0101] Table 1

[0102]

[0103]

[0104] (2) The effect of different types of phospholipases on total phosphorus removal

[0105] Simulated changes in total phosphorus concentration in constructed wetland systems, such as Figure 4 As shown, the cumulative removal rate of total phosphorus changes as follows: Figure 5 As shown in the figure, the experimental group with the addition of phospholipase A1 and phospholipase B showed the highest total phosphorus removal rate. Furthermore, the removal rates of both the phospholipase A1 and phospholipase B groups were significantly higher than those of the experimental group without enzymes. This indicates that both phospholipase A1 and phospholipase B effectively promote the absorption and removal of total phosphorus by plants in the simulated artificial wetland, and their combined use yields even better results.

[0106] Example 3

[0107] This embodiment provides a study on the effect of different concentrations of phospholipase B on phosphorus removal from water.

[0108] In this embodiment, the plants used to simulate the artificial wetland system include water hyacinth, sweet flag, and rock sweet flag, such as... Figure 6 As shown, there are 3-5 water hyacinths, 6-7cm tall, 3 sweet flag plants, about 35cm tall, and 3 sweet flag plants, about 40cm tall.

[0109] In this embodiment, the experiment was divided into three groups (including a control group). Each simulated artificial wetland system contained 2L of natural lake water (water quality better than Class III water) and 3mL of plant nutrient solution (Miracle Grove general-purpose plant nutrient solution), and 10mL of soybean lecithin emulsion at a concentration of 25mg / mL was added to simulate wastewater containing organophosphorus compounds. The phospholipase addition amounts in the three groups are shown below:

[0110] (1) Low concentration enzyme experimental group: 24.4g of artificial wetland filler containing phospholipase B prepared in Example 1 (i.e., about 20mg of phospholipase B was added);

[0111] (2) High concentration enzyme experimental group: 73.2g of artificial wetland filler containing phospholipase B prepared in Example 1 (i.e., about 60mg of phospholipase B was added);

[0112] (3) Experimental group without enzyme addition: No phospholipase was added;

[0113] The experiment began on May 3, 2023, and lasted for a total of 2 weeks.

[0114] Results analysis:

[0115] (1) The effect of different concentrations of phospholipase B on the removal of organic phosphorus

[0116] Simulated changes in organic phosphorus concentration in constructed wetland systems, such as Figure 7 As shown in Table 2, the concentration and removal rate of organophosphorus compounds are as follows.

[0117] like Figure 7As shown, the organic phosphorus concentration in water samples from two simulated constructed wetland systems containing phospholipase B first decreased rapidly, then stabilized and exhibited fluctuations. In the untreated experimental group (blank sample), the organic phosphorus concentration first decreased steadily, then stabilized and exhibited fluctuations. Comparing these three results, the following conclusion can be drawn: the constructed wetland filler of this invention has a strong catalytic conversion effect on organic phosphorus.

[0118] Comparing the three groups of experiments, within one day of adding the artificial wetland filler, the concentration of organic phosphorus in the water sample of the high-concentration enzyme experimental group decreased by 89%, the concentration of organic phosphorus in the water sample of the low-concentration enzyme experimental group (with relatively less added enzyme) decreased by 64%, and the concentration of organic phosphorus in the blank sample (without enzyme) decreased by 15%. This indicates that the rate of conversion of lecithin into soluble orthophosphate is directly proportional to the amount of filler added in this invention within a certain range.

[0119] The organic phosphorus concentration in the blank sample began to approach that of the first two experimental samples by the fifth day. This is because plants themselves contain various microorganisms that participate in the decomposition of organic matter. Taking calamus as an example, the roots of calamus contain rhizosphere bacteria, rhizosphere fungi, degrading bacteria, nitrifying bacteria, and other microorganisms. These microorganisms can decompose organic matter and convert it into nutrients that plants can use. Organic phosphorus is also converted in this process and thus slowly absorbed.

[0120] Starting on the sixth day of the experiment, the concentration of organic phosphorus in the three water bodies tended to be low and stable, with slight fluctuations. It was inferred that this was because the metabolism of plants and microorganisms caused some cells to enter the water bodies, and the phosphorus in these cells would also affect the changes in the concentration of organic phosphorus in the water bodies.

[0121] In the experimental group with the artificial wetland filler of this invention, organophosphorus compounds reached low levels after 48 hours, while the blank control group without the filler only reached a relatively stable low level of organophosphorus compounds after one week. This demonstrates that adding the artificial wetland filler of this invention can significantly improve the conversion and removal rate of organophosphorus compounds in this type of plant-based water purification system.

[0122] Table 2

[0123] Comparison of organophosphate removal rates Low concentration enzyme experimental group High concentration enzyme experimental group No enzyme experimental group Initial organic phosphorus (as phosphorus phosphorus) (mg / L) 3.72 4.18 3.98 24-hour organic phosphorus (as phosphorus phosphorus) (mg / L) 1.04 0.46 3.37 48h organic phosphorus (calculated as phosphorus P) (mg / L) 0.36 0.09 2.2 One week's organic phosphorus (calculated as phosphorus phosphorus) (mg / L) 0.23 0.1 0.54 24-hour removal rate 72.04％ 89.00％ 15.33％ 48-hour removal rate 90.32％ 97.85％ 44.72％ Weekly removal rate 93.82％ 97.61％ 86.43％ Average removal rate in the second week 95.65％ 97.94％ 95.98％

[0124] (2) Effect of different concentrations of phospholipase on total phosphorus removal

[0125] Simulated changes in total phosphorus concentration in constructed wetland systems, such as Figure 8 As shown, the daily total phosphorus removal rate changes as follows: Figure 9 As shown, the cumulative removal rate of total phosphorus changes as follows: Figure 10 As shown.

[0126] from Figure 8The results show that the constructed wetland with high concentrations of phospholipase exhibits the highest removal rate of total phosphorus by plants. Furthermore, within the same timeframe, the removal rate is significantly higher than that of the constructed wetland without added enzymes, indicating that phospholipase B accelerates phosphorus absorption by plants in constructed wetlands.

[0127] from Figure 9 The results show that the overall daily removal rate of the enzyme-added sample was higher than that of the untreated sample, demonstrating the promoting effect of phospholipase on phosphorus absorption by plants. The daily removal rate of total phosphorus remained stable in a low range, indicating that the removal of total phosphorus in constructed wetlands is a long-term process. This process mainly relies on phosphorus absorption by plants. Therefore, in terms of daily removal rate, there was no significant difference between adding and not adding enzymes.

[0128] from Figure 10 The results show that the cumulative total phosphorus removal rate over two weeks, i.e., the total removal rate, was significantly higher in the treatment group with both high and low concentrations of phospholipase than in the group without enzyme. This indicates that adding phospholipase not only facilitates the catalytic conversion of organic phosphorus but also promotes the absorption and removal of total phosphorus to a certain extent.

[0129] (3) Effects of different concentrations of phospholipase on plant height changes in a simulated constructed wetland system

[0130] Simulated plant height changes in constructed wetland systems, such as Figures 11-13 As shown, all three plant species increased in height, but the increase in height of the experimental groups with added phospholipase was significantly higher than that of the control group without phospholipase. Water hyacinth and sweet flag showed more significant height increases in the high-concentration phospholipase groups. This indicates that the addition of phospholipase facilitates the catalytic conversion of organic phosphorus, making it easier for plants to absorb and resulting in better plant growth. The promoting effect was particularly strong on sweet flag, with increases of 16.02 cm and 16.47 cm in the low-concentration and high-concentration groups, respectively, nearly twice the 8.43 cm increase in the control group.

Claims

1. An artificial wetland filler that promotes phosphorus removal from water, characterized in that, The artificial wetland filler material includes: Nanospheres Phospholipases, including phospholipase A1 and / or phospholipase B, Indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc, magnesium, Sodium alginate, Calcium chloride, Phospholipase is loaded onto nanospheres, and sodium alginate is used to encapsulate the phospholipase-loaded nanospheres. The method for preparing the artificial wetland filler includes: adding nanospheres to a phospholipase solution, shaking and then allowing it to stand to obtain catalytic nanospheres; adding nanospheres to a mixed auxiliary solution containing indoleacetic acid, vitamin B1, vitamin B6, calcium, iron, zinc and magnesium, shaking and then allowing it to stand to obtain auxiliary nanospheres; adding the catalytic nanospheres and auxiliary nanospheres to a sodium alginate solution, stirring until the surface is covered with sodium alginate; then immersing them in a calcium chloride solution, embedding them and then removing them.

2. An artificial wetland system, characterized in that, The filler material in the constructed wetland system includes the constructed wetland filler material for promoting phosphorus removal from water as described in claim 1.

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