Porphyridium, aquaculture sewage treatment agent containing porphyridium and preparation method of aquaculture sewage treatment agent
By immobilizing *Porphyra yezoensis* QDHP-404 algal powder and chitosan sodium alginate, the problem of nitrogen and phosphorus pollution in aquaculture has been solved, achieving efficient and low-cost wastewater treatment and ecological balance improvement.
Patent Information
- Application Number
- CN202511684409.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-03-29
- Filing Date
- 2025-11-17
- Publication Date
- 2026-03-17
AI Technical Summary
In aquaculture, nitrogen and phosphorus pollution caused by feed and aquatic excrement is difficult to remove effectively. Traditional methods are costly or harmful to water quality, and existing microalgae treatment technologies are not widely used in aquaculture.
The aquatic wastewater treatment agent QDHP-404 of *Porphyra yezoensis* was used. By preparing *Porphyra yezoensis* powder and using chitosan and sodium alginate immobilization technology, combined with specific culture media and light conditions, the growth rate and biomass of microalgae were improved, and the wastewater treatment agent was prepared to remove pollutants such as nitrogen and phosphorus.
It effectively removes pollutants such as nitrogen and phosphorus from aquaculture wastewater, reduces costs, maintains the ecological balance of water bodies, reduces the use of chemical agents, and promotes water quality improvement and the health of aquatic organisms.
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Abstract
Description
[0001] This application claims priority to Chinese patent application (application number 2025103856943) with a filing date of 2025-03-29, the entire contents of which are hereby incorporated by reference herein. TECHNICAL FIELD
[0002] The present application relates to the technical field of microorganisms, more particularly, it relates to a Porphyridium cruentum, a water breeding sewage treatment agent containing the Porphyridium cruentum and a preparation method thereof. BACKGROUND
[0003] As an important part of agricultural production, aquaculture provides an important food source for people all over the world. Feeding is the most basic and key link in aquaculture. However, only about 9% and 17% of nitrogen and phosphorus in the feed are absorbed by the cultured animals, and the rest is discharged into the water. In addition, the excretion of cultured animals is also rich in nitrogen and phosphorus. Therefore, both the feeding and the excretion of cultured animals in the process of aquaculture will input a large amount of nitrogen and phosphorus into the aquaculture water, causing serious pollution to the water.
[0004] For the treatment of aquaculture wastewater, traditional methods include physical and chemical methods. The physical method has better removal effect on suspended particulate matter (such as leftover feed and excretion) in aquaculture wastewater, but it is difficult to remove dissolved pollutants such as nitrogen and phosphorus. The chemical method has good treatment effect, but the cost is high and it is easy to cause harm to water quality, so it is difficult to be widely applied in aquaculture.
[0005] As an important primary producer in aquatic ecosystems, algae play an important role in water environment governance. Using microalgae to treat wastewater can not only purify wastewater, but also obtain high-value products. Algae can efficiently remove nitrogen and phosphorus and other pollutants in wastewater, and the construction and operation cost of the treatment process is low, and the maintenance method is simple, so it has attracted widespread attention at home and abroad.
[0006] Porphyridium cruentum is a unicellular red alga belonging to the genus Porphyridium of the family Porphyridiaceae. Porphyridium cruentum can synthesize a large amount of phycobiliprotein and extracellular polysaccharide and other high-value active substances during growth, and has broad application prospects. At present, the research on Porphyridium cruentum mainly focuses on the optimization and improvement of the culture process and the development and utilization of various bioactive substances, and there are few reports on the use of Porphyridium cruentum to treat aquaculture wastewater. SUMMARY
[0007] In order to improve the treatment effect of microalgae on aquaculture wastewater, the present application provides a Porphyridium cruentum, a water breeding sewage treatment agent containing the Porphyridium cruentum and a preparation method thereof.
[0008] In the first aspect, this application provides a purple algae, which adopts the following technical solution: A species of purple algae, named *Porphyridium cruentum* QDHP-404, is deposited at the China Center for Type Culture Collection (CCTCC) on March 9, 2023, with accession number CCTCC NO: M2023273.
[0009] By adopting the above technical solutions, *Porphyrinus purpureus* can effectively accumulate high-value-added natural bioactive components such as phycobiliproteins, polysaccharides, and polyunsaturated fatty acids during its growth process, resulting in high economic value and broad application prospects. As a microalga with great development potential, it possesses excellent biological purification functions, and its application in water treatment can yield potentially high-value biomass and functional products.
[0010] Secondly, this application provides an aquaculture wastewater treatment agent containing *Porphyra yezoensis*, employing the following technical solution: A wastewater treatment agent for aquaculture containing *Porphyra yezoensis*, specifically algae powder containing *Porphyra yezoensis* QDHP-404.
[0011] By adopting the above technical solution, the purple algae QDHP-404 can be made into algae powder for wastewater treatment. Relying on the proliferation of microalgae cells and the accumulation of nutrients, the wastewater discharge for aquaculture can meet national standards, while also taking into account cost issues. At the same time, it can realize the resource utilization of wastewater and reduce the cost of microalgae cultivation.
[0012] Optionally, the algal powder of *Porphyra yezoensis* QDHP-404 is prepared as follows: *Porphyra yezoensis* QDHP-404 is placed in KOCK medium and heated at 23-26℃ with a light intensity of (150-180) μmol / m². 2 The culture was carried out under the condition of / s, and the culture was centrifuged and dried to obtain the algal powder of *Porphyra yezoensis* QDHP-404.
[0013] By adopting the above technical solution, the KOCK medium is used to cultivate the purple algae QDHP-404, providing the necessary nutrients for its growth and reproduction. Moreover, the specific temperature and light intensity enable the purple algae to maintain a high growth rate and biomass yield. After centrifugation and drying, algae powder with few impurities, high purple algae content, and high purity can be obtained. In addition, the low moisture content makes it less prone to deterioration, and the algae powder form is easy to package and transport, which helps to reduce storage and transportation costs.
[0014] Optionally, the centrifugation speed is 8000-8500 r / min and the centrifugation time is 8-10 min.
[0015] By adopting the above technical solution, the centrifugation speed and time can effectively separate the purple algae from the culture medium, improve the harvest rate, and increase the algae powder yield. If the speed is too low, the purple algae cells will not be effectively separated, while if the speed is too high, it may damage the cells, affecting the purity of the purple algae and the activity of the cells.
[0016] Optionally, the drying process can be carried out by natural air drying at 15-20℃ or freeze drying.
[0017] By adopting the above technical solutions, low-temperature natural air drying can avoid the damage of high temperature to the active ingredients in *Porphyra yezoensis*, retain as many nutrients and bioactive substances in the algal powder as possible, improve the quality and application effect of the algal powder, and at the same time, natural air drying can also prevent the algal powder from deteriorating and reduce the possibility of oxidation; while freeze drying can retain the nutrients and bioactive substances in the algal powder to the maximum extent at a lower temperature, and maintain the structural integrity of the algal powder organism, which is beneficial to its utilization effect in subsequent applications.
[0018] Optionally, the KOCK culture medium is formulated as follows: 10 ml / L potassium nitrate, 1 ml / L dipotassium hydrogen phosphate, 10 ml / L magnesium sulfate heptahydrate, 1 ml / L ferric ammonium citrate, 10 ml / L soil extract, 484 mL artificial seawater, and 484 mL distilled water.
[0019] By adopting the above technical solution, the above culture medium contains a variety of nutrients required for the growth of *Porphyra yezoensis*, such as inorganic sources, nitrogen sources and phosphorus sources, which can meet the nutritional needs of microalgae during the growth process, promote their rapid growth and reproduction. Moreover, when using KOCK medium for algal powder culture, the operation steps are relatively simple and easy to control, which can reduce the difficulty and cost of operation and improve production efficiency.
[0020] Optionally, the artificial seawater formulation is as follows: 27g sodium chloride, 6.6g magnesium sulfate heptahydrate, 5.6g magnesium chloride hexahydrate, 1.5g calcium chloride, 1g potassium nitrate, 0.07g potassium dihydrogen phosphate, 0.04g sodium bicarbonate, 20ml 1M Tris-HCl, 1ml trace element stock solution, and 1ml Fe-EDTA solution; wherein the trace element stock solution formulation is: 4mg / L ZnCl2, 60mg / L H3BO3, 4mg / L CoCl2·2H2O, 4mg / L CuCl2·2H2O, 40mg / L MnCl2·4H2O, and (NH4)6Mo7O 24 The Fe-EDTA solution contains 37 mg / L of 0.05 M Na2EDTA (pH 7.6) 100 ml and 240 mg of FeCl3·4H2O.
[0021] Optionally, the soil extract can be prepared as follows: Take 200g of garden soil, place it in a beaker, add 1000ml of distilled water, seal the mouth of the beaker with plastic wrap, make a vent, heat in a boiling water bath for 3 hours, cool and let stand for 24 hours, repeat the above steps 3 times, filter to obtain a clear liquid, autoclave at 121℃ for 20 minutes, and refrigerate for later use.
[0022] Thirdly, this application provides a method for preparing an aquaculture wastewater treatment agent containing *Porphyra yezoensis*, employing the following technical solution: A method for preparing an aquaculture wastewater treatment agent containing *Porphyra yezoensis* includes the following steps: Sodium alginate was added to distilled water, sodium chloride was added, and the mixture was stirred to dissolve, thus obtaining a gel solution. Chitosan was dissolved in dilute acetic acid solution to obtain chitosan solution, and calcium chloride was dissolved in distilled water to obtain calcium chloride solution. Chitosan solution was uniformly sprayed onto the algal powder of *Porphyra yezoensis* QDHP-404, then added to the adhesive solution. After mixing evenly, the mixture was dripped into a calcium chloride solution. The mixture was filtered, washed, and an aquaculture wastewater treatment agent containing *Porphyra yezoensis* was obtained. The mass ratio of chitosan solution to *Porphyra yezoensis* QDHP-404 algal powder was 0.2-0.3:1, and the mass of the adhesive solution was 4 times the total weight of the chitosan solution and the *Porphyra yezoensis* QDHP-404 algal powder.
[0023] By employing the above technical solution, the algal powder of *Porphyra yezoensis* QDHP-404 is first sprayed with a chitosan solution, and then added to a colloid containing sodium alginate. The positively charged chitosan binds tightly to the negatively charged sodium alginate. Subsequently, sodium alginate undergoes ion exchange with calcium chloride, with calcium ions replacing sodium ions in the sodium alginate. Furthermore, chitosan has the ability to complex metal ions and can also adsorb a certain amount of calcium ions. This allows calcium chloride, sodium alginate, and chitosan to react fully, thereby encapsulating the *Porphyra yezoensis* QDHP-404 algal powder and achieving microalgae immobilization technology. This enhances the anabolic activity of microalgae and reduces their catabolic activity to some extent, allowing the microalgae activity in the wastewater treatment agent to remain stable for a longer period. This ensures the population dominance of *Porphyra yezoensis* to a certain extent, enabling it to more effectively exert its biological characteristics in aquaculture environments. It can also control the species composition of *Porphyra yezoensis* to a certain extent, preventing excessive numbers of *Porphyra yezoensis* in the aquaculture pond. Therefore, it is easier to maintain the dynamic balance of the entire aquaculture system. Furthermore, sodium alginate and chitosan themselves have a certain absorption effect on nitrogen and phosphorus, which can effectively remove nitrogen and phosphorus from the aquatic environment.
[0024] Optionally, the mass ratio of sodium alginate to sodium chloride is 42-48:2, the concentration of the adhesive solution is 3.5-4 wt%, the concentration of the chitosan solution is 3-3.5 wt%, and the concentration of the calcium chloride solution is 3-3.5 wt%.
[0025] By adopting the above technical solution, the dosage and concentration of each raw material can enable chitosan and sodium alginate to deposit on the algal powder of *Porphyra yezoensis* QDHP-404 to form a film, thereby maintaining the viability of *Porphyra yezoensis* for a longer time and ensuring its viability.
[0026] Optionally, the inoculation concentration of the aquaculture wastewater treatment agent containing *Porphyra yezoensis* in the wastewater is 200-400 mg / ml.
[0027] By adopting the above technical solution and adding the wastewater treatment agent to the aquaculture wastewater in an appropriate amount, *Porphyra yezoensis* can effectively remove pollutants such as nitrogen, phosphorus, and COD from the wastewater, improve the quality of the effluent, and reduce costs by using wastewater treatment agents containing *Porphyra yezoensis*. It will not cause secondary pollution and will also reduce the use of chemical agents, thus reducing damage to the environment.
[0028] In summary, this application has the following beneficial effects: 1. Since this application uses the algal powder of *Porphyra yezoensis* as the raw material for the wastewater treatment agent, it can efficiently remove pollution, reduce costs, prevent secondary pollution, reduce the use of chemical agents, reduce damage to the environment, promote the ecological balance of the water body, improve the self-purification capacity of the water body, and the microalgae can also provide food sources for aquatic organisms, promoting the stable development of the aquatic ecosystem.
[0029] 2. In this application, the wastewater treatment agent is preferably prepared by combining chitosan with algae powder such as sodium alginate and calcium chloride (e.g., *Plasmodium purpureus* QDHP-404). This can improve the aquaculture environment, enhance the disease resistance of aquatic organisms, regulate the balance of the ecosystem, maintain good water quality, and reduce the outbreak of diseases while maintaining high purification efficiency of ammonia nitrogen and total phosphorus. Detailed Implementation
[0030] The following embodiments provide a further detailed description of this application.
[0031] Isolation and identification of *Porphyra yezoensis* QDHP-404 The solid culture medium was selected from Qingdao Haibo Biotechnology, catalog number HB8723, and the BG11 culture medium was selected from Qingdao Haibo Biotechnology, catalog number HB8793.
[0032] (1) Separation: Natural water samples were collected, impurities were removed, and the algae were streaked onto a solid culture medium using the streak plate method. The culture was then kept at 27°C and a light intensity of 108 μmol / m². -2 s -1After culturing for 20 days under a 12:12h light cycle, single algal colonies were picked out using an inoculation loop and transferred to sterile test tubes containing 10mL of culture medium. These were then cultured under the same conditions for 15 days. BG11 medium was then aliquoted into 500mL Erlenmeyer flasks (250mL per flask) and sterilized at 121℃ for 30min. After cooling, 5mL of well-grown algal solution was inoculated into each Erlenmeyer flask and cultured for 15 days in a light shaker set at 120rpm, 27℃, and a light intensity of 108μmol / m². -2 s -1 .
[0033] (2) Extraction: Take the logarithmic growth phase of Porphyra yezoensis, centrifuge at 8000 r / min for 10 min, collect Porphyra yezoensis in a 1.5 ml sterile centrifuge tube, freeze and thaw three times in liquid nitrogen, and grind rapidly with a sterile glass grinding rod. (3) Identification: The algal strain is spherical, with one cup-shaped chloroplast in each cell, one nucleus, and a relatively thin cell wall. A search of "Chinese Freshwater Algae - Systematics, Classification and Ecology" revealed that this algae is morphologically classified as belonging to the phylum Porphyra, order Porphyridiales, family Porphyridaceae, and genus Porphyrdium. The algal strain was named Porphyrdium QDHP-404 and deposited on March 9, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China. The deposit date is March 9, 2023, and the accession number is CCTCC NO: M2023273. Example
[0034] Example 1: An aquaculture wastewater treatment agent containing *Porphyra yezoensis*, comprising the following raw materials in parts by weight: 250g of *Porphyra yezoensis* QDHP-404 algal powder, 42g of sodium alginate, 2g of sodium chloride, 1.5g of chitosan, and 3.5g of calcium chloride; the *Porphyra yezoensis* QDHP-404 algal powder is prepared by the following method: ① Seed culture: Single colonies were obtained by streaking on a medium plate and incubated statically for 3 days at 23℃ and light intensity of 35000lx. The medium formula was: CaCl2·2H2O 0.3g / L, FeSO4·7H2O 15mg / L, MgSO4·7H2O 0.1g / L, NaNO3 2g / L, K2HPO4 0.3g / L, yeast extract 1g / L, glucose 8g / L, agar 1.5%, pH=6.0~8.0, and purified water was added to make up to 1L.
[0035] ② Seed activation: Pick a single algal colony from the culture medium plate and inoculate it onto KOCK medium. Incubate at 26℃ and a light intensity of 180 μmol / m². 2 Under static conditions for 5 days, the KOCK medium formula was as follows: 10 ml / L potassium nitrate, 1 ml / L dipotassium hydrogen phosphate, 10 ml / L magnesium sulfate heptahydrate, 1 ml / L ferric ammonium citrate, 10 ml / L soil extract, 484 ml artificial seawater, and 484 ml distilled water. The artificial seawater formula was as follows: 27 g sodium chloride, 6.6 g magnesium sulfate heptahydrate, 5.6 g magnesium chloride hexahydrate, 1.5 g calcium chloride, 1 g potassium nitrate, 0.07 g potassium dihydrogen phosphate, 0.04 g sodium bicarbonate, 20 ml 1M Tris-HCl, 1 ml trace element stock solution, and 1 ml Fe-EDTA solution. The trace element stock solution formula was as follows: 4 mg / L ZnCl2, 60 mg / L H3BO3, 4 mg / L CoCl2·2H2O, 4 mg / L CuCl2·2H2O, 40 mg / L MnCl2·4H2O, and (NH4)6Mo7O 24 ·4H2O 37mg / L, Fe-EDTA solution includes: 0.05M Na2EDTA (pH7.6) 100ml, FeCl3·4H2O 240mg, soil extract preparation method: take 200g garden soil, place it in a beaker, add 1000ml distilled water, seal the mouth of the beaker with plastic wrap, make a vent, heat in a boiling water bath for 3h, cool and let stand for 24h, repeat the above steps 3 times, filter to obtain clear liquid, autoclave at 121℃ for 20min, and refrigerate for later use.
[0036] ③ Large-scale culture: The *Porphyra yezoensis* was cultured in batches of 100 ml, 5 L, and 100 L. The large-scale culture conditions were 26℃ and a light intensity of 180 μmol / m². 2 / s, cultured in a bioreactor at 120 rpm.
[0037] ④ Algal powder preparation: Centrifuge the culture medium of *Porphyra yezoensis* at 8000 r / min for 10 min, and then air-dry the centrifuged material at 20℃ to obtain algal powder of *Porphyra yezoensis* QDHP4-4.
[0038] The preparation method of the aquaculture wastewater treatment agent containing *Porphyra yezoensis* includes the following steps: Add 42g of sodium alginate to 1156g of distilled water, add 2g of sodium chloride, stir to dissolve, and obtain a 3.5wt% gel solution. 1.5 g of chitosan was dissolved in 0.01 mol / L dilute acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%. 3.5 g of calcium chloride was dissolved in distilled water to prepare a calcium chloride solution with a concentration of 3.5 wt%. Chitosan solution was evenly sprayed onto the algal powder of *Porphyra yezoensis* QDHP-404, then added to the adhesive solution. After mixing evenly, it was dripped into the calcium chloride solution, filtered, and washed with distilled water to obtain an aquaculture wastewater treatment agent. The amount of chitosan solution used was 0.2 times the mass of *Porphyra yezoensis* QDHP-404 algal powder, and the amount of adhesive solution used was 4 times the total weight of *Porphyra yezoensis* QDHP-404 algal powder and chitosan solution.
[0039] Example 2: An aquaculture wastewater treatment agent containing *Porphyra yezoensis*, comprising the following raw materials in parts by weight: 230.8g of *Porphyra yezoensis* QDHP-404 algal powder, 48g of sodium alginate, 2g of sodium chloride, 2.42g of chitosan, and 3g of calcium chloride; the *Porphyra yezoensis* QDHP-404 algal powder is prepared by the following method: ① Seed culture: Single colonies were obtained by streaking on a medium plate and incubated statically for 5 days at 23℃ and light intensity of 35000lx. The medium formula was: CaCl2·2H2O 0.3g / L, FeSO4·7H2O 15mg / L, MgSO4·7H2O 0.1g / L, NaNO3 2g / L, K2HPO4 0.3g / L, yeast extract 1g / L, glucose 8g / L, agar 1.5%, pH=6.0~8.0, and purified water was added to make up to 1L.
[0040] ② Seed activation: Pick a single algal colony from the culture medium plate and inoculate it onto KOCK medium. Incubate at 23℃ and a light intensity of 150 μmol / m². 2 Under static conditions for 5 days, the KOCK medium formula was as follows: 10 ml / L potassium nitrate, 1 ml / L dipotassium hydrogen phosphate, 10 ml / L magnesium sulfate heptahydrate, 1 ml / L ferric ammonium citrate, 10 ml / L soil extract, 484 ml artificial seawater, and 484 ml distilled water. The artificial seawater formula was as follows: 27 g sodium chloride, 6.6 g magnesium sulfate heptahydrate, 5.6 g magnesium chloride hexahydrate, 1.5 g calcium chloride, 1 g potassium nitrate, 0.07 g potassium dihydrogen phosphate, 0.04 g sodium bicarbonate, 20 ml 1M Tris-HCl, 1 ml trace element stock solution, and 1 ml Fe-EDTA solution. The trace element stock solution formula was as follows: 4 mg / L ZnCl2, 60 mg / L H3BO3, 4 mg / L CoCl2·2H2O, 4 mg / L CuCl2·2H2O, 40 mg / L MnCl2·4H2O, and (NH4)6Mo7O 24·4H2O 37mg / L, Fe-EDTA solution includes: 0.05M Na2EDTA (pH7.6) 100ml, FeCl3·4H2O 240mg, soil extract preparation method: take 200g garden soil, place it in a beaker, add 1000ml distilled water, seal the mouth of the beaker with plastic wrap, make a vent, heat in a boiling water bath for 3h, cool and let stand for 24h, repeat the above steps 3 times, filter to obtain clear liquid, autoclave at 121℃ for 20min, and refrigerate for later use.
[0041] ③ Scale-up culture: The *Porphyra yezoensis* was cultured in batches of 100 ml, 5 L, and 100 L. The scale-up culture conditions were 23℃ and a light intensity of 150 μmol / m². 2 / s, cultured in a bioreactor at 160 rpm.
[0042] ④ Algal powder preparation: Centrifuge the culture medium of *Porphyra yezoensis* at 8500 r / min for 8 min, and then air dry the centrifuged material at 15℃ to obtain algal powder of *Porphyra yezoensis* QDHP4-4.
[0043] The preparation method of the aquaculture wastewater treatment agent containing *Porphyra yezoensis* includes the following steps: Add 48g of sodium alginate to 1150g of distilled water, add 2g of sodium chloride, stir and dissolve to obtain a 4wt% gel solution; dissolve 2.42g of chitosan in 0.01mol / L dilute acetic acid solution to obtain a 3.5wt% chitosan solution; dissolve 3g of calcium chloride in distilled water to obtain a 3wt% calcium chloride solution. Chitosan solution was evenly sprayed onto the algal powder of *Porphyra yezoensis* QDHP-404, then added to the adhesive solution. After mixing evenly, it was dripped into calcium chloride solution, filtered, and washed with distilled water to obtain an aquaculture wastewater treatment agent. The amount of chitosan solution used was 0.3 times the mass of *Porphyra yezoensis* QDHP-404 algal powder, and the amount of adhesive solution used was 4 times the total weight of *Porphyra yezoensis* QDHP-404 algal powder and chitosan solution.
[0044] Example 3: An aquaculture wastewater treatment agent containing *Porphyra yezoensis*, comprising the following raw materials in parts by weight: 250g of *Porphyra yezoensis* QDHP-404 algal powder, 42g of sodium alginate, 2g of sodium chloride, 1.5g of chitosan, and 3.5g of calcium chloride; the *Porphyra yezoensis* QDHP-404 algal powder is prepared by the following method: ① Seed culture: Single colonies were obtained by streaking on a medium plate and incubated statically for 3 days at 23℃ and light intensity of 35000lx. The medium formula was: CaCl2·2H2O 0.3g / L, FeSO4·7H2O 15mg / L, MgSO4·7H2O 0.1g / L, NaNO3 2g / L, K2HPO4 0.3g / L, yeast extract 1g / L, glucose 8g / L, agar 1.5%, pH=6.0~8.0, and purified water was added to make up to 1L.
[0045] ② Seed activation: Pick a single algal colony from the culture medium plate and inoculate it onto KOCK medium. Incubate at 26℃ and a light intensity of 180 μmol / m². 2 Under static conditions for 5 days, the KOCK medium formula was as follows: 10 ml / L potassium nitrate, 1 ml / L dipotassium hydrogen phosphate, 10 ml / L magnesium sulfate heptahydrate, 1 ml / L ferric ammonium citrate, 10 ml / L soil extract, 484 ml artificial seawater, and 484 ml distilled water. The artificial seawater formula was as follows: 27 g sodium chloride, 6.6 g magnesium sulfate heptahydrate, 5.6 g magnesium chloride hexahydrate, 1.5 g calcium chloride, 1 g potassium nitrate, 0.07 g potassium dihydrogen phosphate, 0.04 g sodium bicarbonate, 20 ml 1M Tris-HCl, 1 ml trace element stock solution, and 1 ml Fe-EDTA solution. The trace element stock solution formula was as follows: 4 mg / L ZnCl2, 60 mg / L H3BO3, 4 mg / L CoCl2·2H2O, 4 mg / L CuCl2·2H2O, 40 mg / L MnCl2·4H2O, and (NH4)6Mo7O 24 ·4H2O 37mg / L, Fe-EDTA solution includes: 0.05M Na2EDTA (pH7.6) 100ml, FeCl3·4H2O 240mg, soil extract preparation method: take 200g garden soil, place it in a beaker, add 1000ml distilled water, seal the mouth of the beaker with plastic wrap, make a vent, heat in a boiling water bath for 3h, cool and let stand for 24h, repeat the above steps 3 times, filter to obtain clear liquid, autoclave at 121℃ for 20min, and refrigerate for later use.
[0046] ③ Large-scale culture: The *Porphyra yezoensis* was cultured in batches of 100 ml, 5 L, and 100 L. The large-scale culture conditions were 26℃ and a light intensity of 180 μmol / m². 2 / s, cultured in a bioreactor at 120 rpm.
[0047] ④ Algal powder preparation: Centrifuge the culture medium of *Porphyra yezoensis* at 8000 r / min for 10 min, and then air-dry the centrifuged material at 20℃ to obtain algal powder of *Porphyra yezoensis* QDHP4-4.
[0048] The preparation method of the aquaculture wastewater treatment agent containing *Porphyra yezoensis* includes the following steps: Add 42g of sodium alginate to 1156g of distilled water, add 2g of sodium chloride, stir to dissolve, and obtain a 3.5wt% gel solution. Dissolve 3.5g of calcium chloride in distilled water to prepare a calcium chloride solution with a concentration of 3.5wt%. The algae powder of *Porphyra yezoensis* QDHP-404 was added to the gel solution, mixed evenly, and then dripped into the calcium chloride solution. After filtration and washing with distilled water, an aquaculture wastewater treatment agent was obtained.
[0049] Example 4: An aquaculture wastewater treatment agent containing *Porphyra yezoensis*, comprising the following raw materials in parts by weight: 250g of *Porphyra yezoensis* QDHP-404 algal powder, 1.5g of chitosan; the *Porphyra yezoensis* QDHP-404 algal powder is prepared by the following method: ① Seed culture: Single colonies were obtained by streaking on a medium plate and incubated statically for 3 days at 23℃ and light intensity of 35000lx. The medium formula was: CaCl2·2H2O 0.3g / L, FeSO4·7H2O 15mg / L, MgSO4·7H2O 0.1g / L, NaNO3 2g / L, K2HPO4 0.3g / L, yeast extract 1g / L, glucose 8g / L, agar 1.5%, pH=6.0~8.0, and purified water was added to make up to 1L.
[0050] ② Seed activation: Pick a single algal colony from the culture medium plate and inoculate it onto KOCK medium. Incubate at 26℃ and a light intensity of 180 μmol / m². 2 Under static conditions for 5 days, the KOCK medium formula was as follows: 10 ml / L potassium nitrate, 1 ml / L dipotassium hydrogen phosphate, 10 ml / L magnesium sulfate heptahydrate, 1 ml / L ferric ammonium citrate, 10 ml / L soil extract, 484 ml artificial seawater, and 484 ml distilled water. The artificial seawater formula was as follows: 27 g sodium chloride, 6.6 g magnesium sulfate heptahydrate, 5.6 g magnesium chloride hexahydrate, 1.5 g calcium chloride, 1 g potassium nitrate, 0.07 g potassium dihydrogen phosphate, 0.04 g sodium bicarbonate, 20 ml 1M Tris-HCl, 1 ml trace element stock solution, and 1 ml Fe-EDTA solution. The trace element stock solution formula was as follows: 4 mg / L ZnCl2, 60 mg / L H3BO3, 4 mg / L CoCl2·2H2O, 4 mg / L CuCl2·2H2O, 40 mg / L MnCl2·4H2O, and (NH4)6Mo7O 24·4H2O 37mg / L, Fe-EDTA solution includes: 0.05M Na2EDTA (pH7.6) 100ml, FeCl3·4H2O 240mg, soil extract preparation method: take 200g garden soil, place it in a beaker, add 1000ml distilled water, seal the mouth of the beaker with plastic wrap, make a vent, heat in a boiling water bath for 3h, cool and let stand for 24h, repeat the above steps 3 times, filter to obtain clear liquid, autoclave at 121℃ for 20min, and refrigerate for later use.
[0051] ③ Large-scale culture: The *Porphyra yezoensis* was cultured in batches of 100 ml, 5 L, and 100 L. The large-scale culture conditions were 26℃ and a light intensity of 180 μmol / m². 2 / s, cultured in a bioreactor at 120 rpm.
[0052] ④ Algal powder preparation: Centrifuge the culture medium of *Porphyra yezoensis* at 8000 r / min for 10 min, and then air-dry the centrifuged material at 20℃ to obtain algal powder of *Porphyra yezoensis* QDHP4-4.
[0053] The preparation method of the aquaculture wastewater treatment agent containing *Porphyra yezoensis* includes the following steps: 1.5 g of chitosan was dissolved in a 0.01 mol / L dilute acetic acid solution to prepare a chitosan solution with a concentration of 3 wt%. Chitosan solution was uniformly sprayed onto the algal powder of *Porphyra yezoensis* QDHP-404 and dried with hot air at 20°C to obtain an aquaculture wastewater treatment agent. The amount of chitosan solution used was 0.2 times the mass of the algal powder of *Porphyra yezoensis* QDHP-404.
[0054] Performance testing Wastewater treatment effect of eel farming: An eel farming pond in Qingdao was selected, and the farming water was sampled. The total nitrogen, ammonia nitrogen, total phosphorus, chemical oxygen demand and pH value in the farming water were tested. The test results are shown in Table 1.
[0055] Table 1 Water quality of wastewater from eel farming Item Water quality of aquaculture wastewater Discharge standard of aquaculture wastewater Total phosphorus (mg / L) 3.68 ≤5 Ammonia nitrogen (mg / L) 6.81 - Total nitrogen (mg / L) 24.65 ≤3 Chemical oxygen demand (mg / L) 21.38 ≤15 pH 6.7 6.0-9.0 Pretreatment of experimental water: After the eel farming wastewater was allowed to settle, it was filtered through gauze to remove larger particles; Experimental Methods: Pretreated aquaculture wastewater was placed in 500mL Erlenmeyer flasks. Wastewater treatment agents prepared in Examples 1-4 were added to each flask at a concentration of 400mg / ml. These were experimental groups. A blank group without wastewater treatment agent was also set up. Control groups 1 and 2 were also set up, with 100mg / ml of the wastewater treatment agent from Example 1 and 500mg / ml of the wastewater treatment agent from Example 1, respectively. Each experimental group, blank group, and control group had three parallel groups. The Erlenmeyer flasks were placed at a temperature of 25±1℃, a light-dark ratio of 12:12h, and a light intensity of 100μmol / m². 2 In an incubator with a rotation speed of 150 r / min, the water quality parameters of the wastewater and the biomass concentration of *Porphyra yezoensis* were measured on the 3rd and 5th days. The average value of each group was taken as the test results, and the test results are recorded in Table 2.
[0056] The detection methods are as follows: (1) Concentration of microalgae biomass: The concentration of microalgae biomass was determined by dry weight method; (2) Water quality parameters: Ammonia nitrogen content was determined by Nessler's reagent spectrophotometry (GB7479-87), nitrite nitrogen content was determined by N-(1-naphthyl)-ethylenediamine spectrophotometry (GB7593-87), nitrate nitrogen content was determined by ultraviolet spectrophotometry (HJ / T356-2007), phosphorus content was determined by molybdenum antimony spectrophotometry (GB11893-98), and COD was determined by dichromate method (GB11914-89).
[0057] Table 2. Treatment effect of wastewater treatment agents containing *Porphyra yezoensis* on eel farming wastewater. As can be seen from the data in Table 2, the wastewater treatment agents prepared in Examples 1 and 2 can effectively remove non-polluting substances such as COD, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total phosphorus from eel farming wastewater within 5 days. Controlling the amount of wastewater treatment agent added can improve the pollutant removal rate while shortening the wastewater treatment time, which is very beneficial for the future use of microalgae in the treatment of large-scale aquaculture wastewater.
[0058] Shrimp pond wastewater treatment effect: Healthy shrimp larvae were selected and placed in an indoor recirculating aquarium (150cm×70cm×60cm) for acclimatization for 2 weeks to adapt to the experimental conditions. During the temporary holding period, they were fed feed. The experimental water used was sand-filtered seawater, and a low-pressure blower was used to provide oxygen to increase the dissolved oxygen content of the aquaculture water.
[0059] At the start of the experiment, healthy shrimp larvae with an average initial weight of 0.71g and ethyl acetate were selected and stocked in 35 aquariums. The wastewater treatment agent prepared in Examples 1-4 was added to the aquariums at a concentration of 400mg / ml as experimental groups 1-4. No wastewater treatment agent was added; the larvae were fed only as a control group. The wastewater treatment agent prepared in Example 1 was added at a concentration of 100mg / ml as control group 1, and the wastewater treatment agent prepared in Example 1 was added at a concentration of 500ml / ml as control group 2, for a total of 7 treatment groups. Each group had 5 replicates. Each aquarium contained 30 shrimp larvae. During the experiment, the larvae were fed 4 times a day (at 8:00, 12:00, 16:00, and 20:00), with a feed amount of 7% of their body weight. Feeding and molting were observed during feeding, and uneaten food and feces were checked after feeding. The feeding amount was adjusted weekly based on the growth and feeding behavior of the larvae. The experiment lasted 21 days. Water quality and shrimp larvae were tested, and the results are recorded in Table 3.
[0060] After the experiment, water samples were taken from each group between 10:00 and 11:00 AM, and various water quality factors, including ammonia nitrogen (NH4), were immediately measured. + Nitrite nitrogen (NO2-N) was determined by the Nessler reagent method; nitrite nitrogen (NO2-N) was determined by the GR reagent method; dissolved oxygen (DO) was determined by iodometric titration.
[0061] After the experiment, 10 shrimp were taken from each aquarium, and blood was drawn from their hearts using a syringe. The blood was placed in Eppendorf tubes and left to stand at 4°C for several hours. The blue serum was then collected and stored at -20°C for later use. Phenol oxidase (PO) activity was determined according to the colorimetric method using L-dopa as a substrate, as described by Ashida (1971). Lysozyme activity (LSZ) was determined according to the method described by Wang Lei, Li Guangyou, et al., "Determination and Characterization of Antibacterial, Lysozyme and Phenol Oxidase Activities in the Hemolymph of Chinese Shrimp." Micrococcus lysate, after secondary activation, was inoculated into beef extract peptone solid medium and cultured for 48 hours. After centrifugation, the bacterial cells were collected and then diluted with 0.1 mol / L potassium phosphate buffer (pH = 6.4) according to the improved method of Hultmark et al. 570nm =0.3, prepare a substrate suspension for testing, and under the conditions specified in this method, the lysozyme activity (U / mL) = (A0-A) / A0.
[0062] Table 3. Treatment effects of wastewater treatment agents containing *Porphyra yezoensis* on shrimp farming wastewater. As can be seen from the data in Table 3, compared with the blank control group and the control group, the wastewater treatment agent containing *Porphyra yezoensis* prepared in Examples 1-2 can effectively reduce nitrite nitrogen and ammonia nitrogen in aquaculture water, reduce the possibility of water and bottom sediment decay inducing the reproduction of pathogenic bacteria such as Vibrio, reduce the occurrence of shrimp diseases, and at the same time improve the disease resistance of shrimp, improve the activity of PO enzyme and LSZ, increase resistance to pathogenic bacteria, thereby regulating the balance of the ecosystem, maintaining good water quality, enhancing the disease resistance of shrimp, and reducing the outbreak of diseases.
[0063] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A Porphyridium sp. characterized in that, The Porphyridium cruentum QDHP-404 is deposited in the China Center for Type Culture Collection on March 9, 2023, and the deposit number is CCTCC NO: M2023273.
2. An aquaculture wastewater treatment agent containing Porphyridium, characterized in that, The algal powder containing the Porphyridium cruentum QDHP-404 of claim 1.
3. The Porphyridium-containing aquaculture effluent treatment agent of claim 2, characterized in that: The method for preparing the algal powder of the Porphyridium QDHP-404 is as follows: the Porphyridium QDHP-404 is placed in KOCK medium and cultured at 23-26°C under the condition of light intensity of (150-180) μmol / m 2 / s, and the culture is centrifuged and dried to obtain the algal powder of the Porphyridium QDHP-404.
4. The Porphyridium-containing aquaculture effluent treatment agent of claim 3, characterized in that: The centrifugal speed is 8000-8500 r / min, and the centrifugal time is 8-10 min.
5. The Porphyridium-containing aquaculture effluent treatment agent according to claim 3, characterized in that: The drying is natural air drying or freeze drying at 15-20 ℃.
6. The Porphyridium-containing aquaculture effluent treatment agent of claim 3, characterized in that: The formula of the KOCK culture medium is as follows: 10 ml / L potassium nitrate, 1 ml / L dipotassium hydrogen phosphate, 10 ml / L magnesium sulfate heptahydrate, 1 ml / L ferric ammonium citrate, 484 mL artificial seawater, and 484 mL distilled water.
7. The preparation method of the aquaculture wastewater treatment agent containing the Porphyridium cruentum according to any one of claims 2-6, characterized in that: Sodium alginate is added to distilled water, and sodium chloride is added, stirred and dissolved to prepare a glue solution; Chitosan is dissolved in a dilute acetic acid solution to prepare a chitosan solution, and calcium chloride is dissolved in distilled water to prepare a calcium chloride solution; The chitosan solution is uniformly sprayed on the algal powder of the Porphyridium cruentum QDHP-404, and then added to the glue solution, mixed uniformly, and then dropped into the calcium chloride solution, filtered, washed, and prepared into the aquaculture wastewater treatment agent containing the Porphyridium cruentum, the mass ratio of the chitosan solution and the algal powder of the Porphyridium cruentum QDHP-404 is 0.2-0.3:1, and the mass of the glue solution is 4 times the total weight of the chitosan solution and the algal powder of the Porphyridium cruentum QDHP-404.
8. The method of claim 7, wherein the preparation of the Porphyridium- containing aquaculture effluent treatment agent is characterized by: The mass ratio of the sodium alginate and the sodium chloride is 42-48:2, and the concentration of the glue solution is 3.5-4 wt%; The concentration of the chitosan solution is 3-3.5 wt%, and the concentration of the calcium chloride solution is 3-3.5 wt%.
9. The method of claim 7, wherein the preparation of the Porphyridium- containing aquaculture effluent treatment agent is characterized by: The inoculation concentration of the aquaculture wastewater treatment agent containing the Porphyridium cruentum in the wastewater is 200-400 mg / ml.