A method for treating high-salinity wastewater using stephanodiscus muelleri

Abstract

This invention provides a method for treating high-salinity wastewater using *Chaetoceros muelleri*, belonging to the field of water treatment: *Chaetoceros muelleri* strains in a preservation state are transferred to a nutritionally complete and suitable standard f / 2 culture medium for activation culture under standard f / 2 conditions; actual marine aquaculture wastewater is used as the treatment target, and after filtering out impurities, the salinity of the wastewater is adjusted to 30-35‰; the *Chaetoceros muelleri* treated in step S1 is inoculated into the high-salinity wastewater at 0.05–0.2 g / L (dry weight), and the lighting system is activated after inoculation; during system operation, the algal biomass and changes in ammonia nitrogen, total nitrogen, and phosphate in the water are monitored regularly; when the pollutant removal rate remains stable at a high level and the algal biomass continues to increase, the system can be considered to be operating stably. This invention efficiently removes nitrogen, phosphorus, and other pollutants from wastewater using *Chaetoceros muelleri* technology, while maintaining stable algal growth, thus improving wastewater treatment efficiency and resource utilization rate.

Description

Technical Field

[0001] This invention belongs to the field of water treatment, and more specifically, relates to a method for treating high-salinity wastewater using Chaetoceros muelleri. Background Technology

[0002] With the continuous advancement of industrialization, urbanization, and resource development activities, the generation of high-salinity wastewater is increasing year by year. High-salinity wastewater originates widely from various sectors, including mariculture and processing, seafood refrigeration and cleaning, chemical and pharmaceutical industries, oil extraction, food processing, and municipal mixed wastewater in coastal areas. This wastewater typically contains high levels of dissolved inorganic salts, primarily sodium chloride, sulfates, and carbonates, with salinity significantly higher than conventional freshwater wastewater; some high-salinity wastewater can reach salinity of 3%–5% or even higher. In addition to high salinity, this type of wastewater is often accompanied by the enrichment of nutrients such as nitrogen and phosphorus, a low carbon-to-nitrogen ratio, and significant fluctuations in water quality. High-salinity environments exert a significant stress on biological systems, easily leading to osmotic pressure imbalances in cells, metabolic disorders, and alterations in microbial community structure, making high-salinity wastewater one of the most difficult types of wastewater to treat in the current field of water pollution control.

[0003] High-salinity wastewater, if discharged directly into receiving water bodies without effective treatment, will have a significant adverse impact on aquatic ecosystems. On the one hand, high salinity alters the ionic composition and osmotic pressure conditions of the water, inhibiting or disrupting the normal physiological activities of organisms in existing freshwater or nearshore ecosystems, leading to a decline in biodiversity. On the other hand, nutrients such as nitrogen and phosphorus accumulated in the wastewater will continuously enter the environment, inducing eutrophication, promoting abnormal phytoplankton reproduction, and consequently causing a decrease in dissolved oxygen, hypoxia or even anaerobic conditions in the bottom water, thus disrupting ecosystem stability. Therefore, achieving efficient removal of pollutants from high-salinity wastewater under salinity-adapted conditions is a crucial technical challenge in current water environment management.

[0004] Currently, high-salinity wastewater treatment technologies mainly include physical, chemical, and biological methods. Physical methods, such as evaporation crystallization, membrane separation, and adsorption, can achieve separation of salts or pollutants to a certain extent, but they typically suffer from high energy consumption, large equipment investment, complex operation and maintenance, and difficulties in treating concentrated brine, making long-term stable operation difficult in large-volume scenarios. Chemical methods remove pollutants by adding oxidants, flocculants, or using electrochemical technologies. Although the treatment efficiency is relatively fast, they are prone to secondary pollution, have high reagent costs, and fail to achieve resource recovery of pollutants. Biological treatment technologies have attracted attention due to their relatively low operating costs and environmental friendliness. However, in high-salinity environments, traditional bacterial-based biological treatment systems are susceptible to salt stress, resulting in reduced microbial activity, significantly decreased nitrification, denitrification, and organic matter degradation efficiencies, long system start-up cycles, and poor stability. Furthermore, high-salinity wastewater often has a low carbon-to-nitrogen ratio, requiring additional carbon sources to maintain the denitrification process, further increasing operating costs and management difficulty.

[0005] In recent years, microalgae treatment technology has been recognized as a green treatment technology that utilizes microalgae to absorb inorganic nitrogen, phosphorus, and carbon dioxide from water through photosynthesis under light conditions, converting pollutants into algal biomass. This technology combines pollutant removal with resource recovery potential and is considered a green treatment technology that aligns with the concept of low-carbon development. Existing research indicates that microalgae can achieve high nitrogen and phosphorus removal efficiencies in freshwater or low-salinity wastewater treatment, while simultaneously generating economically valuable biomass.

[0006] However, under high-salinity wastewater conditions, most commonly used freshwater microalgae species struggle to adapt to the high osmotic pressure environment, exhibiting problems such as inhibited growth, decreased photosynthetic efficiency, and even death, resulting in insufficient system stability. Although some marine or salt-tolerant microalgae possess a certain degree of salt adaptability, systematic research on their long-term stable operation in high-salinity wastewater, their synergistic removal mechanisms for multiple pollutants, and their feasibility for engineering applications remains limited, and related technical solutions have not yet formed a mature system. Therefore, there is an urgent need to develop a microalgae treatment technology that can stably grow in high-salinity environments, effectively absorb nutrients from high-salinity wastewater, and possess application potential. Summary of the Invention

[0007] This invention provides a method for treating high-salt wastewater using Chaetoceros muelleri, aiming to solve the technical problems of the suppression of activity of conventional activated sludge and bacterial systems under high-salt conditions in existing high-salt wastewater treatment; the limited ability of existing salt-tolerant biological methods to synergistically remove multiple pollutants (nitrogen and phosphorus); poor process stability; high energy consumption; and low resource utilization.

[0008] This invention is implemented as follows: This invention provides a method for treating high-salinity wastewater using Chaetoceros muelleri, wherein, S1: Transfer the Chaetoceros muelleri algal strain, which is in storage, to a standard f / 2 medium with complete nutrition and suitable conditions, and carry out activation culture under the standard f / 2 medium conditions; S2: Using actual marine aquaculture wastewater as the treatment target, after filtering out impurities, the salinity of the wastewater is adjusted to 30~35‰. S3: Inoculate the *Chaetoceros muelleri* treated in S1 into high-salt wastewater at a concentration of 0.05–0.2 g / L (dry weight). After inoculation, start the lighting system. S4: During system operation, regularly monitor algal biomass and changes in ammonia nitrogen, total nitrogen, and phosphate in the water. When the pollutant removal rate remains stable at a high level and the algal biomass continues to increase, the system can be considered to be operating stably.

[0009] Based on the above technical solution, the method of treating high-salinity wastewater using Chaetoceros muelleri of the present invention can be further improved as follows: Furthermore, the culture conditions for activation culture in S1 under standard f / 2 medium are as follows: The culture temperature should be controlled at 24–26℃; The light-dark cycle is 14 h: 10 h; Light intensity is controlled at 10,000–15,000 lux; Shake well daily to maintain a uniform algae solution; When the algae enter the logarithmic growth phase, and the cells are observed to be intact and without obvious aggregation under a microscope, it is used as the inoculum for subsequent treatment systems.

[0010] Furthermore, the seawater treatment in S2 also includes the following conditions: The initial ammonia nitrogen concentration was controlled at 5–35 mg / L, the phosphate concentration at 0.5–6 mg / L, and the carbon source concentration at 0.5–2.5 g / L. Under system operating conditions, except for the factor to be investigated, the other conditions were fixed as follows: inoculum size 0.1 g / L, ammonia nitrogen concentration 15 mg / L, phosphate concentration 2.5 mg / L, and sodium bicarbonate concentration 1.5 g / L.

[0011] Furthermore, during system operation, S3 performs the following coordinated control on key parameters: S31: The light intensity is maintained in the range of 10,000-15,000 lux, rather than a fixed optimal value, in order to adapt to actual light fluctuations; S32: By adding sodium bicarbonate, the inorganic carbon supply is actively regulated, which directly addresses the "low carbon-to-nitrogen ratio" limitation commonly found in high-salt wastewater. At the same time, the pH of the system is dynamically controlled within a buffer zone of 7.5-8.5 to avoid the inhibition of algal cells by excessively high or low pH, thus ensuring that the algae maintain efficient metabolic activity and pollutant removal capacity. S33: Based on changes in influent water quality, maintain the concentrations of ammonia nitrogen and phosphate within the tolerance window of the algae's efficient metabolism, rather than pursuing a fixed concentration, in order to ensure the long-term stability of algal metabolic activity and pollutant removal efficiency. S34: When the influent water quality fluctuates or the light conditions are unstable, a mixed nutrient culture mode can be adopted as an operation control strategy. By adding an appropriate amount of organic carbon source to the water, Chaetoceros muelleri can simultaneously utilize light energy and organic substrates for metabolic growth, thereby maintaining the metabolic activity of the algae and improving the system's adaptability to complex conditions.

[0012] Furthermore, S4 includes: S41: During system operation, samples are taken daily from different locations at the top, middle and bottom of the reactor and thoroughly mixed to obtain a microalgae suspension. S42: Take a portion of the suspension and, using a blank culture medium as a reference, measure the OD at a wavelength of 675 nm using a spectrophotometer. 675 Absorbance was used to calculate the growth of Chaetoceros muelleri based on the change in absorbance before and after the reaction, and OD was measured separately. 675 A linear relationship between absorbance and corresponding algal cell dry weight was established, based on OD... 675 Rapid calculation of algal biomass; algal biomass is determined by spectrophotometry using OD. 675 Absorbance is used to characterize algal cell density and growth. S42: The remaining microalgal suspension was filtered through a 0.45 μm microporous membrane, and the clear supernatant was collected. The supernatant after filtration was used to determine the concentrations of chemical oxygen demand, nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, and total phosphorus. The initial concentration was measured before inoculation, and the final concentration was measured after treatment. The nitrogen and phosphorus removal rates were obtained by calculating the changes in pollutant concentrations, and the assimilation capacity of Chaetoceros muelleri to nitrogen and phosphorus pollutants was analyzed in combination with changes in biomass.

[0013] Compared with existing technologies, the beneficial effects of the method for treating high-salinity wastewater using Chaetoceros muelleri provided by this invention are: The present invention aims to provide a technical solution for treating high-salinity wastewater using Chaetoceros muelleri. This technology can efficiently remove pollutants such as nitrogen and phosphorus from wastewater while maintaining stable algal growth, thereby improving wastewater treatment efficiency and resource utilization rate.

[0014] Chaetoceros muelleri was used as a functional microalgae and introduced directly or after domestication into a high-salt wastewater system under specific environmental conditions. Through the photosynthetic growth, nutrient absorption and co-metabolism of Chaetoceros muelleri, the simultaneous removal of ammonia nitrogen, total nitrogen, phosphate and organic matter in the high-salt wastewater was achieved.

[0015] This invention utilizes *Chaetoceros muelleri* as the core functional microalga. By synergistically regulating key parameters such as inoculum size, inorganic carbon supply, and ammonia nitrogen and phosphate concentrations, a dynamic matching relationship is established between the algal physiological metabolic processes and the characteristics of high-salinity wastewater, thereby achieving a stable conversion of pollutants into algal biomass. This constructs a self-sustaining biological treatment system that dynamically adapts to the complex conditions of high-salinity wastewater. By systematically integrating the physiological characteristics of the algae with controllable operating parameters, the system possesses inherent stability. This technical solution does not rely on complex physicochemical treatment units, has clearly defined operating conditions, and is suitable for treating high-salinity aquaculture effluent and similar wastewater.

[0016] To address the limitations of existing technologies that rely primarily on salt-tolerant or domesticated green algae and have limited adaptability to changes in operating conditions, this invention selects *Chaetoceros muelleri* (a diatom naturally adapted to seawater environments) as the core treatment unit. *Chaetoceros muelleri* possesses a siliceous cell wall structure, which effectively buffers osmotic pressure changes caused by high-salt environments, reducing damage to algal cells and making it more tolerant of salinity and water quality fluctuations than green algae. By systematically regulating key factors such as light, nutrients, inorganic carbon, organic load, and pH, the alkali's physiological metabolism dynamically adapts to the high-salt wastewater environment. This fundamentally overcomes the limitations of traditional microalgae technology, which is only applicable to "simulated seawater" or "low-complexity wastewater," making it easier to maintain normal algal growth within short-term operating timeframes and reducing the risk of system instability.

[0017] To address the issues of existing technologies neglecting the synergistic effects of key operating parameters and the imbalance in removal processes under conditions of multiple pollutant coexistence, this invention moves beyond isolated examination of the influence of single parameters. Instead, it systematically studies the synergistic relationships between light intensity, ammonia nitrogen concentration, phosphate concentration, sodium bicarbonate dosage, organic load, and culture mode through a single-factor experimental system. The suitable operating ranges for each parameter (e.g., inoculum size 0.05–0.2 g / L, ammonia nitrogen 10–25 mg / L, phosphate 2.5 mg / L, sodium bicarbonate 1.5 g / L) have been clarified, shifting the treatment process from empirical operation to parameterized control. This has led to the construction of an operating parameter system suitable for real high-salinity wastewater conditions, achieving simultaneous and efficient removal of multiple pollutants such as nitrogen and phosphorus. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The changes of different microalgae under high-salt wastewater conditions; Figure 2 Growth of Chaetoceros muelleri and removal of pollutants under different initial inoculation concentrations; Figure 3 Growth of Chaetoceros muelleri and removal of pollutants under different ammonia nitrogen concentrations; Figure 4 Growth of Chaetoceros muelleri and removal of pollutants under different phosphate concentrations; Figure 5 Growth of Chaetoceros muelleri and removal of pollutants under different inorganic carbon concentrations; Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0022] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0023] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0024] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0025] Example 1 like Figure 1-5 As shown, this invention provides a method for treating high-salinity wastewater using Chaetoceros muelleri, wherein... S1: Transfer the Chaetoceros muelleri algal strain, which is in storage, to a standard f / 2 medium with complete nutrition and suitable conditions, and carry out activation culture under the standard f / 2 medium conditions; S2: Using actual marine aquaculture wastewater as the treatment target, after filtering out impurities, the salinity of the wastewater is adjusted to 30~35‰. S3: Inoculate the treated Chaetoceros muelleri at a concentration of 0.05–0.2 g / L (dry weight) into the high-salt wastewater. After inoculation, start the lighting system. S4: During system operation, regularly monitor algal biomass and changes in ammonia nitrogen, total nitrogen, and phosphate in the water. To ensure the formation of diatom cell walls, a silicon source can be added to the system, preferably Na2SiO3·9H2O, with a concentration of 5–20 mg / L (calculated as Si). When the pollutant removal rate remains stable at a high level and the algal biomass continues to increase, the system can be considered to be operating stably.

[0026] Based on the above technical solution, the method of treating high-salinity wastewater using Chaetoceros muelleri of the present invention can be further improved as follows: Optionally, in the above technical solution, the culture conditions for activation culture in S1 under standard f / 2 medium are as follows: The culture temperature should be controlled at 24–26℃; The light-dark cycle is 14 h: 10 h; Light intensity is controlled at 10,000–15,000 lux; Shake well daily to maintain a uniform algae solution; The experiment lasted for 7 days, with samples taken every 24 hours.

[0027] When the algae enter the logarithmic growth phase, and the cells are observed to be intact and without obvious aggregation under a microscope, it is used as the inoculum for subsequent treatment systems.

[0028] Optionally, in the above technical solution, the seawater treatment of S2 also includes the following conditions: The initial ammonia nitrogen concentration was controlled at 5–35 mg / L, the phosphate concentration at 0.5–6 mg / L, and the carbon source concentration at 0.5–2.5 g / L. Under the system operating conditions, except for the factor to be investigated, the other conditions were fixed as follows: inoculum size 0.1 g / L, ammonia nitrogen concentration 15 mg / L, phosphate concentration 2.5 mg / L, and sodium bicarbonate concentration 1.5 g / L; The aforementioned fixed conditions can also be used in single-factor experiments.

[0029] Optionally, in the above technical solution, S3 performs the following coordinated control on key parameters during system operation: S31: The light intensity is maintained in the range of 10,000-15,000 lux, rather than a fixed optimal value, in order to adapt to actual light fluctuations; S32: By adding sodium bicarbonate, the inorganic carbon supply is actively regulated, which directly addresses the "low carbon-to-nitrogen ratio" limitation commonly found in high-salt wastewater. At the same time, the pH of the system is dynamically controlled within a buffer zone of 7.5-8.5 to avoid the inhibition of algal cells by excessively high or low pH, thus ensuring that the algae maintain efficient metabolic activity and pollutant removal capacity. S33: Based on changes in influent water quality, maintain the concentrations of ammonia nitrogen and phosphate within the tolerance window of the algae's efficient metabolism, rather than pursuing a fixed concentration, in order to ensure the long-term stability of algal metabolic activity and pollutant removal efficiency. S34: When the influent water quality fluctuates or the light conditions are unstable, a mixed nutrient culture mode can be adopted as an operation control strategy. By adding an appropriate amount of organic carbon source to the water, Chaetoceros muelleri can simultaneously utilize light energy and organic substrates for metabolic growth, thereby maintaining the metabolic activity of the algae and improving the system's adaptability to complex conditions.

[0030] Optionally, in the above technical solution, S4 includes: S41: During system operation, samples are taken daily from different locations at the top, middle and bottom of the reactor and thoroughly mixed to obtain a microalgae suspension. S42: Take a portion of the suspension and, using a blank culture medium as a reference, measure the OD at a wavelength of 675 nm using a spectrophotometer. 675 Absorbance was used to calculate the growth of Chaetoceros muelleri based on the change in absorbance before and after the reaction, and OD was measured separately. 675 A linear relationship between absorbance and corresponding algal cell dry weight was established, based on OD... 675 Rapid calculation of algal biomass; algal biomass is determined by spectrophotometry using OD. 675 Absorbance is used to characterize algal cell density and growth. S42: The remaining microalgal suspension was filtered through a 0.45 μm microporous membrane, and the clear supernatant was collected. The supernatant after filtration was used to determine the concentrations of chemical oxygen demand, nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, and total phosphorus. The initial concentration was measured before inoculation, and the final concentration was measured after treatment. The nitrogen and phosphorus removal rates were obtained by calculating the changes in pollutant concentrations, and the assimilation capacity of Chaetoceros muelleri to nitrogen and phosphorus pollutants was analyzed in combination with changes in biomass.

[0031] The concentrations of pollutants were determined using standard water quality analysis methods: Chemical oxygen demand (COD) was determined using the potassium dichromate method (HJ 828-2017); ammonia nitrogen was determined using Nessler's reagent spectrophotometry (HJ 535-2009); nitrate nitrogen was determined using ultraviolet spectrophotometry (HJ / T 346); nitrite nitrogen was determined using the N-(1-naphthyl)-ethylenediamine spectrophotometry; and total phosphorus was determined using the ammonium molybdate spectrophotometry (GB 11893-89).

[0032] Optionally, S42 includes calculating the relationship between absorbance and dry weight using the following equation: DW = k × OD 675 + b; Where DW is the dry weight concentration of algae (g / L), and k and b are regression coefficients.

[0033] Optionally, S43 includes calculating the pollutant removal rate using the following formula: η = (C0 - Ct) / C0 × 100%; Where C0 is the initial concentration of pollutant (mg / L) and Ct is the concentration of pollutant at time t (mg / L).

[0034] Experiment 1: Comparison of the growth and pollutant removal efficiency of different algal species in high-salinity wastewater. Comparative analysis of the growth performance and pollutant removal efficiency of various marine and salt-tolerant microalgae under the same high-salinity wastewater conditions reveals significant differences in environmental adaptability and treatment efficiency among algal species. Previous studies have shown that diatoms, due to their cell wall structure and efficient inorganic nutrient absorption mechanisms, generally exhibit superior growth stability and nutrient assimilation capacity compared to green algae in seawater and high-salinity environments.

[0035] from Figure 1 The results showed that *Chaetoceros muelleri* maintained continuous biomass growth under high salinity conditions, and its growth stability was significantly better than that of other green algae such as *Chlorella vulgaris*, *Scenedesmus tetracauda*, and *Dunaliella salina*. Meanwhile, *Chaetoceros muelleri* exhibited rapid pollutant removal rates during ammonia nitrogen and phosphate removal processes, and demonstrated good tolerance to pollutants in high-salt wastewater.

[0036] In contrast, existing literature indicates that most green algae are susceptible to the toxicity of free ammonia under high salinity and high ammonium nitrogen conditions, leading to decreased photosynthetic efficiency and even algal death. The growth lag and decreased removal efficiency observed in some green algae in this experiment are consistent with existing research results. Therefore, it can be concluded that *Chaetoceros muelleri* has superior engineering application potential under high salinity wastewater conditions and is the preferred functional microalgae in this invention. The use of *Chaetoceros muelleri* (diatom), which is naturally adapted to high salinity environments, allows it to possess endogenous tolerance to high salinity and high ammonia nitrogen due to its siliceous cell wall structure and metabolic characteristics. This eliminates the need for complex acclimatization, resulting in faster system start-up and more stable operation.

[0037] Figure 1 In the figure, (a) changes in ammonia nitrogen concentration, (b) changes in phosphate concentration, and (c) changes in biomass.

[0038] Based on the identification of *Chaetoceros muelleri* as the dominant algal species, this invention further investigated the effects of key operating parameters on algal growth and pollutant removal performance through a single-factor experimental system. Related studies indicate that light intensity, nitrogen and phosphorus concentrations, and inorganic carbon supply are the core factors limiting the effectiveness of microalgal wastewater treatment, and their suitable ranges are prerequisites for stable operation.

[0039] The effect of initial inoculum concentration on pollutant removal efficiency in Experiment Example 2 Previous studies have shown that the initial algal density directly affects the start-up speed and disturbance resistance of microalgal systems. Figure 2 The results showed that when the initial inoculum concentration was low, the algae entered the logarithmic growth phase more slowly, and the initial removal rate of ammonia nitrogen and phosphate by *Chaetoceros muelleri* was also slower. However, when the inoculum concentration was too high, the biomass growth rate did not increase proportionally, possibly due to a reduced growth efficiency per unit biomass caused by light shading. Figure 2 The growth curves (c) of *Chaetoceros muelleri* and its removal efficiency for ammonia nitrogen (a) and phosphate (b) at different initial inoculum concentrations (0.05, 0.1, 0.15, 0.20, 0.30 g / L) are shown. An inoculum concentration of 0.1-0.15 g / L exhibits the optimal balance between start-up rate and removal efficiency; excessively low or high inoculum concentrations are detrimental to the stable and continuous removal of pollutants. Within the suitable inoculum concentration range, *Chaetoceros muelleri* can rapidly enter the logarithmic growth phase, achieving a good coupling between biomass growth and pollutant removal, providing reliable conditions for the rapid start-up of high-salinity wastewater treatment systems.

[0040] Figure 2 In the figure, (a) changes in ammonia nitrogen concentration, (b) changes in phosphate concentration, and (c) changes in biomass.

[0041] Experiment Example 3: The effect of ammonia nitrogen concentration on algal growth and pollutant removal. Studies generally agree that ammonium nitrogen is the preferred nitrogen source for microalgae, but under high concentration conditions, the presence of free ammonia can cause toxic stress to algal cells. Figure 3 Comparing the growth (c) and pollutant removal (a, b) of *Chaetoceros muelleri* under different initial ammonia nitrogen concentrations (5, 10, 15, 25, 35 mg / L), the optimal algal growth and pollutant removal effects were observed within the 10-25 mg / L range. *Chaetoceros muelleri* exhibited high growth rate and ammonia nitrogen removal efficiency within the low to medium ammonia nitrogen concentration range. When the ammonia nitrogen concentration further increased, algal growth was inhibited, and biomass growth slowed significantly, indicating that ammonia nitrogen concentration has a clear threshold effect on algal growth and the removal process. This invention, by clarifying the suitable ammonia nitrogen concentration range, provides a basis for the stable operation of the denitrification process in high-salinity wastewater, thereby avoiding system performance fluctuations under high ammonia conditions.

[0042] Figure 3In the figure, (a) changes in ammonia nitrogen concentration, (b) changes in phosphate concentration, and (c) changes in biomass.

[0043] Experiment Example 4: The effect of phosphate concentration on algal growth and pollutant removal. Phosphorus is an essential element for energy metabolism and genetic material synthesis in microalgal cells. The intracellular nutrient limitation theory states that when phosphorus supply is insufficient, both microalgal growth and nitrogen assimilation processes are restricted. Figure 4 The effects of different phosphate concentrations (0.5, 1.5, 2.5, 4, and 6 mg / L) on the growth (c) and nitrogen and phosphorus removal (a and b) of *Chaetoceros muelleri* were analyzed. Phosphate concentrations of 1.5–4 mg / L showed the best promoting effect. Under low phosphorus conditions, insufficient phosphate supply limited the growth of *Chaetoceros muelleri*, and the ammonia nitrogen removal efficiency decreased significantly. With increasing phosphate concentration, algal growth and the simultaneous removal of nitrogen and phosphorus significantly improved. When the phosphorus concentration exceeded a certain level, its promoting effect tended to plateau. Therefore, it can be concluded that within a certain range, phosphate is an important limiting factor affecting system performance, and its promoting effect is no longer significant beyond this range. These results verify the importance of maintaining a reasonable nitrogen-to-phosphorus ratio in the treatment of high-salinity wastewater.

[0044] Figure 4 In the figure, (a) changes in ammonia nitrogen concentration, (b) changes in phosphate concentration, and (c) changes in biomass.

[0045] Experiment Example 5: Effect of Inorganic Carbon Supply on Algal Growth and Removal Performance Inorganic carbon is the direct substrate for microalgal photosynthesis. Previous studies have shown that insufficient inorganic carbon supply in high-salinity or alkaline water bodies significantly limits microalgal growth and nutrient uptake efficiency. This experiment investigated the effects of different sodium bicarbonate dosages (e.g., 0.5, 1.5, 2.5, 3.5 g / L) on the growth (c) and pollutant removal (a, b) of *Chaetoceros muelleri*. A dosage of 1.5–2.5 g / L achieved the optimal balance between promoting photosynthesis and avoiding high pH stress. Figure 5 The results showed that appropriate addition of sodium bicarbonate could effectively improve the growth rate and nitrogen and phosphorus assimilation capacity of *Chaetoceros muelleri*. However, when the amount of sodium bicarbonate added was too high, the pH of the system increased, which had an adverse effect on the algal cells. Therefore, this invention achieves a balance between promoting photosynthesis and avoiding environmental stress by limiting the range of inorganic carbon addition, so as to promote photosynthesis while avoiding the formation of adverse environmental conditions.

[0046] Figure 5 In the figure, (a) changes in ammonia nitrogen concentration, (b) changes in phosphate concentration, and (c) changes in biomass.

[0047] The combined data from the single-factor experiments show that light intensity and inorganic carbon supply are fundamental conditions for the stable growth of *Chaetoceros muelleri* in high-salinity wastewater, while ammonia nitrogen and phosphate concentrations directly determine pollutant removal efficiency and the safety margin of system operation. The data from the algal species screening analysis indicate that *Chaetoceros muelleri* exhibits significantly better ammonia nitrogen removal rate (93.6%), phosphate removal rate (94.2%), and biomass growth rate (6.4 times) than other green and golden algae species, providing high-quality functional microbial resources for high-salinity wastewater treatment.

[0048] Regarding the optimization of process parameters, under the combined conditions of initial inoculum amount of 0.1-0.15 g / L, ammonia nitrogen concentration of 10-25 mg / L, phosphate concentration of 1.5-2.5 mg / L, and sodium bicarbonate dosage of 1.5-4 g / L, the data in the figure show that *Chaetoceros muelleri* can achieve synergistic optimization of growth and pollutant removal. The removal rates of ammonia nitrogen and phosphate both reached over 90% after 7 days. Unlike existing technologies that mostly focus on performance evaluation under single nutrient conditions or ideal cultivation conditions, this invention constructs an operating parameter system suitable for real high-salinity wastewater conditions by systematically identifying and quantifying the data of key control factors in the figure, thus solving the problem of lack of engineering operation guidance in existing technologies.

[0049] Based on the shortcomings of the existing technologies mentioned above, namely the poor stability of biological treatment systems under high salinity conditions, the limited ability to synergistically remove multiple pollutants, and the lack of engineering guidance for operating parameters, the technical solution proposed in this invention, which uses Chaetoceros muelleri as the core and achieves high salinity wastewater purification through the synergistic regulation of multiple key factors, can provide an efficient, stable, and green technical path for high salinity wastewater treatment.

[0050] Among them, Chaetoceros muelleri of the present invention is a salt-tolerant diatom microalgae belonging to the genus Chaetoceros (Latin name: Chaetoceros muelleri). In the present invention, it is used for the treatment of high-salt wastewater and has strong salt tolerance and nutrient absorption capacity.

[0051] The high-salinity wastewater of this invention refers to wastewater with a salinity higher than that tolerated by ordinary freshwater biological treatment systems, typically with a salinity ≥25 g / L, such as effluent from marine aquaculture.

[0052] The nutrients in this invention refer to inorganic nitrogen and inorganic phosphorus in wastewater that can be absorbed and utilized by microalgae, including but not limited to ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and phosphate.

[0053] Among them, the f / 2 culture medium of the present invention is a standard artificial seawater culture medium commonly used for the cultivation of marine microalgae, proposed by Guillard and Ryther in 1962, which contains various inorganic salts, vitamins and trace elements required for the growth of microalgae.

[0054] The OD value (optical density value) of this invention is a measure of the degree of absorption of a sample by a spectrophotometer to a specific wavelength of light, used to indirectly reflect the density or concentration of microalgal cells.

[0055] Among them, the mixed nutrient culture mode of the present invention is a culture method in which microalgae obtain energy and carbon sources by simultaneously utilizing both photosynthesis (photoautotrophy) and organic matter (heterotrophy).

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for treating high-salinity wastewater using Chaetoceros muelleri, characterized in that, S1: The Chaetoceros muelleri algal strain, which is in storage, is transferred to a standard f / 2 medium with complete nutrition and suitable conditions, and activated under the conditions of standard f / 2 medium. S2: Using actual marine aquaculture wastewater as the treatment target, after filtering out impurities, the salinity of the wastewater is adjusted to 30~35‰. S3: Inoculate the *Chaetoceros muelleri* treated in S1 into high-salt wastewater at a concentration of 0.05–0.2 g / L (dry weight). After inoculation, start the lighting system. S4: During system operation, regularly monitor algal biomass and changes in ammonia nitrogen, total nitrogen, and phosphate in the water. When the pollutant removal rate remains stable at a high level and the algal biomass continues to increase, the system can be considered to be operating stably.

2. The method for treating high-salinity wastewater using Chaetoceros muelleri according to claim 1, characterized in that, The culture conditions for activation culture in S1 under standard f / 2 medium are as follows: The culture temperature should be controlled at 24–26℃; The light-dark cycle is 14 h: 10 h; Light intensity is controlled at 10,000–15,000 lux; Shake well daily to maintain a uniform algae solution; When the algae enter the logarithmic growth phase, and the cells are observed to be intact and without obvious aggregation under a microscope, it is used as the inoculum for subsequent treatment systems.

3. The method for treating high-salinity wastewater using Chaetoceros muelleri according to claim 1, characterized in that, The seawater treatment in S2 also includes the following conditions: The initial ammonia nitrogen concentration was controlled at 5–35 mg / L, the phosphate concentration at 0.5–6 mg / L, and the carbon source concentration at 0.5–2.5 g / L. Under system operating conditions, except for the factor to be investigated, other conditions were fixed as follows: inoculum size 0.1 g / L, ammonia nitrogen concentration 15 mg / L, phosphate concentration 2.5 mg / L, and sodium bicarbonate concentration 1.5 g / L.

4. The method for treating high-salinity wastewater using Chaetoceros muelleri according to claim 1, characterized in that, During system operation, S3 performs the following coordinated control on key parameters: S31: The light intensity is maintained in the range of 10,000-15,000 lux, rather than a fixed optimal value, in order to adapt to actual light fluctuations; S32: By adding sodium bicarbonate, the inorganic carbon supply is actively regulated, which directly addresses the "low carbon-to-nitrogen ratio" limitation commonly found in high-salt wastewater. At the same time, the pH of the system is dynamically controlled within a buffer zone of 7.5-8.5 to avoid the inhibition of algal cells by excessively high or low pH, thus ensuring that the algae maintain efficient metabolic activity and pollutant removal capacity. S33: Based on changes in influent water quality, maintain the concentrations of ammonia nitrogen and phosphate within the tolerance window of the algae's efficient metabolism, rather than pursuing a fixed concentration, in order to ensure the long-term stability of algal metabolic activity and pollutant removal efficiency. S34: When the influent water quality fluctuates or the light conditions are unstable, a mixed nutrient culture mode can be adopted as an operation control strategy. By adding an appropriate amount of organic carbon source to the water, Chaetoceros muelleri can simultaneously utilize light energy and organic substrates for metabolic growth, thereby maintaining the metabolic activity of the algae and improving the system's adaptability to complex conditions.

5. A method for treating high-salinity wastewater using Chaetoceros muelleri according to claim 1, characterized in that, S4 includes: S41: During system operation, samples are taken daily from different locations at the top, middle and bottom of the reactor and thoroughly mixed to obtain a microalgae suspension. S42: Take part of the suspension, take blank medium as reference, use spectrophotometer to measure OD at 675 nm wavelength 675 Absorbance, according to the change of absorbance before and after reaction, calculate the growth of Mugilaria brevispina, respectively measure OD 675 Absorbance and corresponding algal cell dry weight, establish linear regression relationship between them, according to OD 675 Value, quickly calculate algal biomass; algal biomass uses spectrophotometry to measure OD 675 Absorbance, to characterize algal cell density and growth S43: Filter the remaining microalgae suspension through a 0.45 μm microporous membrane, take the clear supernatant, and use the supernatant after filtration for the determination of chemical oxygen demand, nitrate nitrogen, nitrite nitrogen, ammonia nitrogen and total phosphorus concentration; The initial concentration was measured before inoculation, and the final concentration was measured after treatment. The removal rates of nitrogen and phosphorus were obtained by calculating the changes in pollutant concentrations, and the assimilation capacity of Chaetoceros muelleri to nitrogen and phosphorus pollutants was analyzed in conjunction with changes in biomass.

6. A method for treating high-salinity wastewater using Chaetoceros muelleri according to claim 5, characterized in that, S42 includes calculating the relationship between absorbance and dry weight using the following equation: DW = k × OD 675 + b; Where DW is the dry weight concentration of algae (g / L), and k and b are regression coefficients.

7. A method for treating high-salinity wastewater using Chaetoceros muelleri according to claim 5, characterized in that, S43 includes calculating the pollutant removal rate using the following formula: η = (C0 - Ct) / C0 × 100%; Where C0 is the initial concentration of pollutant (mg / L) and Ct is the concentration of pollutant at time t (mg / L).

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