Method for removing nutrient salts from aquaculture tail water by reinforcing chlorella

Abstract

The application discloses a method for removing nutrient salts in aquaculture tail water by using reinforced chlorella, and belongs to the technical field of aquaculture wastewater treatment. The method comprises the following steps: inoculating chlorella into aquaculture tail water for culture; adding agricultural composite plant hormones into the aquaculture tail water inoculated with chlorella, and uniformly mixing and then culturing; the agricultural composite plant hormones comprise gibberellin acid, indole-3-acetic acid and brassinolide. By adding the agricultural composite plant hormones with a suitable concentration, the chlorella cell proliferation and biomass accumulation can be effectively promoted, the content of photosynthetic pigments and the PSII photochemical performance can be improved, and the electron transfer efficiency can be enhanced; meanwhile, the activities of superoxide dismutase and catalase are improved, the oxidative damage is relieved, and the activities of key nitrogen metabolism enzymes such as nitrate reductase and glutamine synthetase are activated, so that the removal efficiency of nitrogen and phosphorus nutrient salts in aquaculture wastewater can be significantly improved.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a method for enhancing the removal of nutrients from aquaculture tailwater by Chlorella. Background Technology

[0002] In recent decades, the rapid expansion of aquaculture has played a significant role in increasing the supply of aquatic products and boosting economic benefits. However, intensive farming practices have also led to increasingly prominent wastewater discharge problems. During the farming process, residual feed, animal excrement, and organic matter decomposition products continuously release nutrients such as nitrogen and phosphorus into the water, resulting in increased NH4+ levels. + -N (ammonium nitrogen), NO3 - Pollutants such as nitrogen (nitrate), total nitrogen (TN), and total phosphorus (TP) accumulate in aquaculture wastewater. If this nutrient-rich wastewater is discharged directly into rivers, lakes, or nearshore waters without effective treatment, it will significantly increase the exogenous nitrogen and phosphorus load on the receiving water bodies, posing a potential threat to water quality safety and the stability of aquatic ecosystems. In particular, excessive nitrogen and phosphorus input can accelerate eutrophication, induce abnormal algal proliferation, and further lead to decreased water transparency, dissolved oxygen depletion, and deterioration of aquatic habitats. Therefore, developing wastewater treatment technologies that can efficiently reduce nitrogen and phosphorus nutrients while being energy-efficient and environmentally friendly is of great significance for reducing environmental risks in aquaculture and promoting the development of green aquaculture.

[0003] In recent years, microalgae technology has gradually become a research hotspot in the field of aquaculture wastewater treatment due to its ability to simultaneously achieve nutrient removal, CO2 fixation, and biomass accumulation. As a typical green alga, Chlorella has the characteristics of rapid growth, strong environmental adaptability, and high nitrogen and phosphorus absorption capacity, and has been widely used in wastewater treatment research. Microalgae can absorb and utilize nitrogen and phosphorus nutrients in water through their own growth and metabolic activities, converting them into cellular biomass, thereby achieving nutrient reduction in wastewater. However, the efficiency of microalgae wastewater treatment is still limited by factors such as cell proliferation rate, photosynthetic activity, antioxidant capacity, and nutrient assimilation efficiency. Natural algal growth alone often fails to achieve stable and efficient nitrogen and phosphorus reduction effects in a short period of time, and its tolerance to water quality fluctuations is limited.

[0004] Therefore, existing microalgae wastewater treatment technologies still suffer from unstable efficiency, and improving the treatment efficiency of microalgae is a technical problem that needs to be solved. Summary of the Invention

[0005] In view of this, the main objective of the present invention is to provide a method for enhancing the removal of nutrients from aquaculture tailwater by Chlorella, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for enhancing the removal of nutrients from aquaculture wastewater by Chlorella, comprising the following steps: Chlorella was inoculated into the wastewater of aquaculture and cultured. Add agricultural compound plant hormones to the aquaculture tail water inoculated with Chlorella, mix well and then culture. The agricultural compound plant hormone comprises gibberellinic acid, indole-3-acetic acid, and brassinolide; by mass percentage, the content of gibberellinic acid is 0.1-0.2%, the content of indole-3-acetic acid is 0.0003-0.0007%, and the content of brassinolide is 0.0002-0.0004%. The final concentration of the agricultural compound plant hormone in the aquaculture wastewater system is 10~30 mg / L.

[0007] Furthermore, the final concentration of the agricultural compound plant hormone in the wastewater system is 20 mg / L.

[0008] Furthermore, by mass percentage, the agricultural compound plant hormone contains 0.135% gibberellinic acid, 0.00052% indole-3-acetic acid, and 0.00031% brassinolide.

[0009] Furthermore, the initial inoculation density of the Chlorella is 10. 5 ~10 7 The cells / mL indicates that the Chlorella used for inoculation was in the logarithmic growth phase.

[0010] Furthermore, the culture includes: a culture temperature of 23–27°C, a light-dark cycle of alternating 12 h light and 12 h darkness, a light intensity of 1500–2500 lx, and a culture time of not less than 8 days.

[0011] Furthermore, the aquaculture wastewater contains phosphorus, ammonium nitrogen, and nitrate nitrogen simultaneously.

[0012] In a second aspect, this invention provides the application of an agricultural compound plant hormone in the preparation of a biological agent for enhancing the removal of nutrients from aquaculture wastewater by Chlorella, wherein the agricultural compound plant hormone comprises gibberellinic acid, indole-3-acetic acid, and brassinolide, and the concentration of the agricultural compound plant hormone used is 10–30 mg / L; by mass percentage, the content of gibberellinic acid in the agricultural compound plant hormone is 0.1–0.2%, the content of indole-3-acetic acid is 0.0003–0.0007%, and the content of brassinolide is 0.0002–0.0004%.

[0013] Furthermore, the biological agent enhances the removal efficiency of nitrogen and phosphorus nutrients in aquaculture tailwater by Chlorella through synergistic regulation of Chlorella's photosynthetic function, redox homeostasis, and nitrogen assimilation process.

[0014] Furthermore, the biological agent synergistically regulates the physiological metabolism of Chlorella in the following ways: (1) Enhance the photochemical efficiency and electron transport capacity of the PSII reaction center in Chlorella vulgaris; (2) Increase the activity of superoxide dismutase and catalase to maintain cellular redox homeostasis; (3) Upregulate the activity of glutamine synthase and nitrate reductase to enhance nitrogen assimilation capacity.

[0015] Furthermore, the biological agent also regulates the coupling process of carbon and nitrogen metabolism in Chlorella at the transcriptional level, including upregulating the expression of light-harvesting complex gene LHCB7, carbonic anhydrase gene CAH, glycolysis-related genes GAPDH / gapA, PEP and gpmI, nitrate reductase gene NR and aminotransfer-related genes ALT and AGT, and downregulating the expression of TCA cycle-related gene DLST / sucB.

[0016] The beneficial effects of this invention include at least the following: (1) This invention can effectively promote the proliferation of Chlorella cells, biomass and photosynthetic pigment accumulation by inducing with appropriate concentrations of GIB, significantly optimize the photochemical performance and electron transfer efficiency of PSII, improve the light energy conversion and utilization rate, improve the problem of limited growth of microalgae and insufficient photosynthetic efficiency in the tailwater environment, and provide sufficient biomass and energy support for the continuous purification of nitrogen and phosphorus pollutants.

[0017] (2) The present invention utilizes the regulatory effect of GIB to significantly enhance the activity of SOD and CAT antioxidant enzymes in Chlorella, alleviate the oxidative damage caused by tailwater stress, and maintain the metabolic homeostasis of algal cells; at the same time, it effectively activates the activity of key nitrogen metabolism enzymes NR and GS, opens up the assimilation metabolic pathway of nitrate nitrogen and ammonium nitrogen, and greatly enhances Chlorella's ability to degrade and assimilate different forms of nitrogen.

[0018] (3) This invention effectively solves the problems of uneven nitrogen and phosphorus removal and low efficiency in traditional microalgae water purification by strengthening the regulation of GIB, and improves the NH4 in aquaculture wastewater. + -N, NO3 - The removal rates of -N, TN, and TP can reach 89.4%, 82.4%, 71.3%, and 96.9%, respectively, which can effectively reduce the core pollutants of eutrophication in water bodies and significantly improve the water quality of aquaculture tailwater.

[0019] (4) The present invention has verified through transcriptome analysis that appropriate concentrations of GIB can target and activate the energy metabolism, carbon and nitrogen metabolism and cell component renewal pathways of Chlorella, achieve efficient coupling of carbon and nitrogen metabolism, precisely enhance the nutrient assimilation capacity, and will not cause algal transcriptome disorder, effectively avoid the metabolic stress caused by high concentrations of hormones, and ensure that the tailwater purification process is efficient, stable and controllable.

[0020] (5) This invention uses commercially available agricultural GIB preparations, which are readily available, inexpensive, and produce no secondary pollution. The operation process is simple and highly repeatable. This technology is suitable for the treatment of wastewater from various types of aquaculture, and relies on the native metabolism of microalgae to realize the resource utilization of pollutants. It is environmentally friendly and inexpensive, and has good prospects for industrial application. Attached Figure Description

[0021] Figure 1 Effects of GIB treatment on the growth and photosynthetic pigment accumulation of Chlorella. (a) Cell density; (b) Biomass; (c) Chlorophyll a (Chl-a) content; (d) Chlorophyll b (Chl-b) content.

[0022] Figure 2 Effects of GIB treatment on the rapid chlorophyll fluorescence induction curve and JIP-test parameters of Chlorella vulgaris via OJIP. (a) OJIP curve on day 2 of culture; (b) OJIP curve on day 10 of culture; (c) JIP-test parameters.

[0023] Figure 3 Effects of GIB treatment on the activities of antioxidant enzymes and key nitrogen metabolism enzymes in Chlorella. (a) Superoxide dismutase (SOD) activity; (b) Catalase (CAT) activity; (c) Glutamine synthase (GS) activity; (d) Nitrate reductase (NR) activity.

[0024] Figure 4 The effect of GIB treatment on the removal of nitrogen and phosphorus nutrients from aquaculture wastewater by Chlorella. (a) NH4 + -N concentration and removal rate; (b) NO3 - (c) TP concentration and removal rate; (d) TN concentration and removal rate. The bar chart shows the concentrations of each nutrient on day 6 and day 10 of culture, and the red dots represent the removal rate at the end of the culture period.

[0025] Figure 5 Differential expression and functional enrichment analysis of the Chlorella transcriptome under GIB-M treatment. (a) Volcano plot of differentially expressed genes; (b) Bubble plot of KEGG pathway enrichment analysis; (c) GO functional enrichment analysis.

[0026] Figure 6To explore the potential mechanism by which GIB-induced enhanced removal of nutrients from aquaculture wastewater by Chlorella. Red arrows indicate upward adjustment, and blue arrows indicate downward adjustment. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0029] The following specific embodiments illustrate the solution proposed in this invention: Example 1 1. Materials and Methods 1.1. Preparation of microalgae, plant hormones, and aquaculture wastewater The *Chlorella* sp. used in this study was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Before the experiments, the *Chlorella* was inoculated into BG11 medium for pre-culture under the following conditions: 25 ± 1 ℃, 12 h:12 h light-dark cycle, and 2000 lx light intensity. The cells were shaken daily during the culture period, and were used in subsequent experiments after reaching the logarithmic growth phase.

[0030] The agricultural mixed plant hormone GIB was purchased from Agfle Agro-Forestry-Environment Biotechnology AG, Germany. Its main components include 0.135% gibberellic acid (GA3), 0.00052% indole-3-acetic acid (IAA), 0.00031% brassinolide (BL), and some solvent. In the experiment, GIB was added to aquaculture wastewater according to treatment requirements, resulting in final concentrations of 10, 20, and 30 mg / L.

[0031] The aquaculture wastewater used in the experiment was artificially prepared based on the main water quality characteristics of high-density aquaculture wastewater, with a focus on simulating NH4. + -N, NO3 - The water consists of nitrogen and phosphorus nutrients such as nitrogen (N), total nitrogen (TN), and total phosphorus (TP). Initial water quality was measured before the experiment to ensure consistent initial conditions across all treatment groups.

[0032] Table 1 Composition of simulated aquaculture wastewater

[0033] 1.2 Experimental Design To investigate the effect of the agricultural plant hormone GIB on the removal of nutrients from aquaculture wastewater by Chlorella, this study set up one blank control group (Group Z) and three hormone treatment groups, with three replicates for each treatment (see Table 2). Each reaction system was supplemented with 500 mL of aquaculture wastewater and cultured in 1 L Erlenmeyer flasks. Except for the blank control group, all other treatment groups were inoculated with Chlorella to achieve an initial algal cell density of 102. 6 The cells / mL. The experiment was conducted in an artificial climate incubator under the following conditions: temperature 25 ± 1 ℃, light-dark cycle 12 h:12 h, and light intensity 2000 lx. The experimental period was 10 days.

[0034] Table 2 Experimental treatment group settings

[0035] 1.3. Measurement of physiological and biochemical parameters 1.3.1. Algae density To determine algal density, samples were collected every two days from the day of inoculation. After treating the samples with Luger's reagent, algal cells were observed and measured using an automated cell counter (Countstar BioMarine, ALIT Life Sciences, China).

[0036] 1.3.2. Measurement of photosynthetic pigments Algal solution (5 mL) was collected. Centrifuged at 8000 rpm for 10 minutes, and the supernatant was discarded. Then, 90% acetone (5 mL) was added to the precipitate and thoroughly mixed with a vortex mixer until the precipitate was completely dissolved in the acetone solution. After standing in the dark at 4°C for 24 hours, algal cells were separated again by centrifugation at 8000 rpm. The supernatant was collected, and its absorbance at wavelengths of 630, 645, 663, and 750 nm was measured using a UV-Vis spectrophotometer. 90% acetone was used as a blank control in this measurement. The concentrations of Chl-a and Chl-b in the microalgae were calculated using formulas.

[0037] 1.3.3. Measurement of photosynthetic activity The photosynthetic activity of Chlorella was measured using a portable pulse-modulated chlorophyll fluorescence spectrometer (AquaPen, PhotonSystems Instruments, Czech Republic). Before measurement, algal samples were acclimatized to the dark for 20 min to ensure full opening of the PSII reaction centers. Subsequently, rapid chlorophyll fluorescence induction curves (OJIP transient) were recorded, and fluorescence parameters such as FO, FJ, FI, and FM were obtained. To evaluate the effects of GIB treatment on the PSII photochemical performance and electron transport processes of Chlorella, JIP-test parameters including Fv / Fm, PIABS, VJ, ψEo, ETo / RC, and DIo / RC were analyzed. Here, Fv / Fm represents the maximum photochemical efficiency of PSII, PIABS represents the overall PSII performance index, VJ reflects the degree of electron accumulation on the PSII acceptor side, and ψEo represents the electron entry into the QA region. - The probability of the downstream electron transport chain, ETo / RC represents the electron transport flux per unit reaction center, and DIo / RC represents the energy dissipation flux per unit reaction center.

[0038] 1.3.4. Determination of nitrogen metabolism-related enzymes and antioxidant enzymes In this study, algal cells were collected during four consecutive days of batch operation under plant hormone stimulation and at the end of the experiment. The activities of nitrate reductase (NR), glutamine synthase (GS), superoxide dismutase (SOD), and catalase (CAT) were measured using detection kits.

[0039] 1.3 Measurement of Nitrogen and Phosphorus During the experiment, the NH4 content in the water samples was measured. + -N, NO3 - -N, TN, and TP concentrations. After water sample collection, the samples were centrifuged at 8000 rpm for 10 min, and the supernatant was used for water quality analysis. All indicators were analyzed according to the corresponding standard methods.

[0040] 1.4. Transcriptome Analysis After the experiment, samples were collected, and algal cells were collected by centrifugation at 4000 rpm and washed with PBS buffer. The washed algal cells were centrifuged again, and the resulting precipitate was flash-frozen in liquid nitrogen and stored at -80 °C. The precipitate was then sent to Shanghai Majorbio Biotechnology Co., Ltd. (Shanghai, China) for eukaryotic transcriptome sequencing. The sequencing data were analyzed on the Majorbio Cloud Platform online platform.

[0041] 1.5 Statistical Analysis Data were processed using SPSS 27.0 and Origin 2022. Differences between treatments were measured using analysis of variance (n=3). All experimental data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used to test differences between treatment groups, and Tukey multiple comparisons were used for significance analysis, with a significance level set at P < 0.05.

[0042] 2. Results and Discussion 2.1 Effects of GIB induction on the growth and photosynthetic pigment accumulation of Chlorella Different GIB treatments significantly affected the growth performance of Chlorella in aquaculture wastewater. Figure 1 a). During the 10-day culture period, the cell density in all treatment groups continuously increased, indicating that the artificially prepared nutrient conditions in the effluent could support the proliferation of Chlorella. Compared with group Z (control group), the addition of GIB further promoted the increase in cell number, with the 20 mg / L treatment showing the highest cell density in the later stage of culture. The cell density of the 30 mg / L treatment was lower than that of the 20 mg / L treatment, but still higher than that of group Z, indicating that the promoting effect of GIB on Chlorella proliferation is significantly concentration-dependent and does not continuously increase with increasing concentration. The changes in biomass were basically consistent with the trend of cell density. Figure 1 (b) In the early stages of cultivation, the biomass differences among the treatment groups were small; however, as the cultivation time increased, the biomass of the GIB-treated group gradually exceeded that of the Z group, reaching its highest level in the 20 mg / L treatment, indicating that an appropriate concentration of GIB is more conducive to the proliferation of Chlorella cells and the accumulation of dry matter. Similar dose-response effects have also been observed in studies on the regulation of microalgal growth by plant hormones, where appropriate concentrations of hormones can promote cell division, carbon assimilation, and biomass accumulation, while excessively high concentrations may weaken their promoting effect.

[0043] Changes in photosynthetic pigment content further reflect the regulatory role of GIB on the photosynthetic metabolism of Chlorella. Chl-a is an important component of the light reaction center and light-harvesting complex, and its content is closely related to light energy absorption and photochemical conversion capacity. The 20 mg / L treatment showed the highest Chl-a content on day 6 and maintained a high level in the later stages of cultivation. Figure 1 c) indicates that appropriate concentrations of GIB may promote chlorophyll synthesis and photosynthetic system construction. The response trend of Chl-b is basically consistent with that of Chl-a. Figure 1 d). Treatment with 20 mg / L significantly increased Chl-b content, suggesting that this treatment may enhance the formation of the light-harvesting pigment system. Since Chl-b is mainly involved in expanding the spectral absorption range and transferring captured energy to Chl-a, its accumulation helps to improve the light energy utilization potential of Chlorella.

[0044] It is noteworthy that while the 30 mg / L treatment promoted Chlorella growth and pigment accumulation, its effect was weaker than that of the 20 mg / L treatment. This suggests that high concentrations of GIB may alter intracellular metabolic distribution or induce certain physiological regulatory stresses, thereby limiting the continued accumulation of chlorophyll and further increases in biomass. These results indicate that 20 mg / L GIB is more beneficial for the growth of Chlorella and the accumulation of photosynthetic pigments in the aquaculture effluent, providing a physiological basis for improved nutrient assimilation and removal efficiency.

[0045] 2.2 Effects of GIB induction on the photochemical properties of Chlorella PSII Photosynthesis is the energy basis for microalgal growth and nutrient assimilation. The activity of the PSII reaction center and the efficiency of electron transport directly affect the cell's ability to utilize light energy. To assess the effect of GIB treatment on the photosynthetic system function of Chlorella, this study measured the OJIP rapid chlorophyll fluorescence induction curves on days 2 and 10 of culture, and further calculated the JIP-test parameters (…). Figure 2 ).

[0046] On day 2, all treatment groups showed a typical O–J–I–P fluorescence rise process, indicating that the Chlorella PSII reaction center maintained normal rapid fluorescence induction characteristics. Figure 2 a) Compared to group Z, the OJIP curves of the GIB-treated group shifted upwards overall. The 20 mg / L and 30 mg / L treatments showed higher fluorescence responses in the J, I, and P phases, indicating that GIB had already modulated the PSII photochemical process in the early stages of culture. The O–J phase of the OJIP curve mainly reflects the QA reduction process on the PSII acceptor side, while the J–I and I–P phases are related to electron transfer to the quinone library and further downstream processes. Therefore, the fluorescence changes in these phases in the GIB-treated group suggest that the activity of its PSII reaction center and electron transfer processes may be improved.

[0047] By day 10 of cultivation, the OJIP fluorescence intensity in all treatment groups significantly increased, reflecting that as cultivation progressed, the accumulation of photosynthetic pigments and the activity of the photosystem in Chlorella were further enhanced. Figure 2 (b) The 20 mg / L treatment maintained a high fluorescence response during the J–I–P phase, indicating that the appropriate concentration of GIB has a certain degree of persistence in promoting the photochemical function of PSII. The improvement of the 10 mg / L treatment was relatively limited, while the 30 mg / L treatment, although higher than group Z, did not further exceed the 20 mg / L treatment, indicating that the regulation of photosynthetic performance by GIB is concentration-dependent, and excessively high concentrations did not bring a stronger promoting effect.

[0048] The JIP-test parameters further illustrate the impact of GIB treatment on PSII performance and electron transport processes. Figure 2c). Fv / Fm reflects the maximum photochemical efficiency of PSII. The 20 mg / L treatment was significantly higher than group Z, indicating that the appropriate concentration of GIB improved the potential photochemical activity of the PSII reaction center. PIABS also showed a high level in the 20 mg / L treatment, indicating that its overall PSII performance was improved. Meanwhile, the 20 mg / L treatment significantly decreased VJ and increased ψEo, indicating that QA... - The reduced accumulation at the J point allows the captured excitation energy to more effectively enter the downstream electron transport chain of QA. The increase in ETo / RC further indicates an enhanced electron transport flux per unit reaction center, suggesting that GIB-M treatment promotes the downstream electron transport process of PSII.

[0049] The simultaneous increase in DIo / RC under 20 mg / L treatment indicates an increased level of energy dissipation per unit reaction center. Given the simultaneous high Fv / Fm, PIABS, ψEo, and ETo / RC levels associated with this treatment, the increase in DIo / RC is more likely to reflect photoprotective regulation under higher photochemical activity rather than PSII functional impairment. This suggests that GIB-M treatment, while enhancing electron transport capacity, may maintain photosystem stability by increasing non-photochemical energy dissipation. The improved PSII photochemical performance provides a functional explanation for the higher biomass accumulation and nutrient assimilation under GIB-M treatment. The increases in Fv / Fm, PIABS, ψEo, and ETo / RC indicate that Chlorella can more effectively convert captured light energy into electron transport, thus providing the energy and reducing power basis for carbon fixation and nitrogen reduction. This result also provides functional support for subsequent transcriptome-related responses related to light capture, electron transport, and energy metabolism.

[0050] 2.3 Effects of GIB induction on the antioxidant defense and key enzyme activities of nitrogen metabolism in Chlorella The complex nutrient composition, organic background, and exogenous hormone stimulation in aquaculture wastewater can all alter the redox state within Chlorella cells. Superoxide dismutase (SOD) and catalase (CAT), as key enzymes in the algal cell's antioxidant defense system, participate in the disproportionation of superoxide anion radicals and the scavenging of H₂O₂, respectively. Changes in their activity can be used to characterize the intensity of cellular responses to environmental stress and metabolic stimuli. Figure 3 As can be seen, GIB treatment significantly increased SOD activity in Chlorella on day 6, with the 20 mg / L treatment reaching the highest level, followed by the 30 mg / L treatment, and the 10 mg / L treatment showing a relatively low level. This result indicates that an appropriate concentration of GIB may enhance Chlorella's ability to scavenge reactive oxygen species, thereby reducing the impact of oxidative damage on cell proliferation and photosynthetic structural stability. CAT activity showed a similar response to GIB treatment as SOD. Figure 3(b) On day 6, the CAT activity under the 20 mg / L treatment was significantly higher than that of group Z and other treatment groups, indicating that 20 mg / L GIB not only promoted the primary scavenging of superoxide anions but also enhanced the further decomposition of H2O2. On day 10, the CAT activity of each treatment group changed compared to day 6, but the 20 mg / L treatment still maintained a high level, indicating that the antioxidant defense capacity of Chlorella under medium concentration GIB induction has a certain degree of sustainability. The phenomenon of plant hormones regulating the activity of antioxidant enzymes has been reported in many types of photosynthetic organisms, especially brassinolide, which is believed to improve the adaptability of photosynthetic organisms to abiotic stress by regulating the antioxidant defense system. The simultaneous increase in SOD and CAT activities in this study may help alleviate the oxidative stress in the tailwater environment and during the exogenous hormone induction process, enabling Chlorella to maintain high photosynthetic and growth activities.

[0051] Compared to antioxidant enzymes, GS and NR more directly reflect the nitrogen uptake and assimilation process in Chlorella. GS is a key enzyme in ammonium nitrogen assimilation, capable of catalyzing NH4+. + The reaction of glutamate with glutamate to form glutamine is a crucial step in the pathway of inorganic nitrogen entering amino acids and protein synthesis. Figure 3 As can be seen from c, GIB treatment significantly increased GS activity, and GIB-M maintained its highest level on both day 6 and day 10. On day 6, the GS activities of the 10 mg / L, 20 mg / L, and 30 mg / L treatments were significantly higher than those of group Z, indicating that GIB induction enhanced the inorganic nitrogen assimilation potential of Chlorella. The effects of exogenous plant hormones on microalgal nitrogen metabolism have gradually attracted attention. Recent studies have shown that plant hormones can jointly promote microalgal growth by regulating carbon and nitrogen metabolism networks and antioxidant systems, suggesting that hormone-induced growth promotion is not solely due to enhanced cell division, but may also involve the re-regulation of nitrogen assimilation processes.

[0052] NR is the rate-limiting enzyme in the reduction of nitrate nitrogen, and its activity determines the reduction of NO3. - -N to NO2 - The ability to convert -N. Figure 3The results showed that the 20 mg / L treatment significantly increased nitrate reduction (NR) activity, especially on day 6. While the 30 mg / L treatment was higher than the Z group and the 10 mg / L treatment, it was significantly lower than the 20 mg / L treatment. This indicates that 20 mg / L GIB is more beneficial for activating nitrate reduction in Chlorella. Both nitrate reduction and ammonia assimilation require photosynthesis to provide reducing power, ATP, and the carbon skeleton; therefore, the enhanced NR and GS activities may be related to the higher chlorophyll accumulation and biomass growth under the aforementioned 20 mg / L treatment. Previous studies on plant hormones alleviating nitrogen stress in microalgae have indicated that exogenous hormones can improve the growth performance of microalgae under different nitrogen conditions and may affect the interaction between nitrogen signaling and hormone signaling. Notably, the 30 mg / L treatment did not show the highest levels of antioxidant enzyme or nitrogen metabolism enzyme activity, indicating that there is a concentration threshold for GIB's metabolic regulation in Chlorella. Excessively high concentrations of exogenous hormones may cause intracellular signal regulation imbalance or cause cells to use more energy for maintaining homeostasis rather than growth and assimilation metabolism. Similar dose-response effects are common in microalgal plant hormone studies. Exogenous hormones can promote growth and metabolism at appropriate concentrations, but their promoting effect weakens or even turns into a stress effect when the optimal range is exceeded. Therefore, the simultaneous increase in the activities of SOD, CAT, GS, and NR under 20 mg / L treatment reflects that 20 mg / L GIB can form a relatively coordinated physiological response between antioxidant defense and nitrogen assimilation metabolism.

[0053] 2.4 Effects of GIB induction on nitrogen and phosphorus removal in aquaculture tailwater GIB induction significantly affected the removal efficiency of different forms of nutrients in aquaculture wastewater by Chlorella. Figure 4 For NH4 + -N concentrations in all treatment groups decreased with increasing culture time, indicating that Chlorella can effectively utilize ammonium nitrogen in the effluent. Compared with group Z, the GIB treatment further reduced NH4+. + -N residual concentration, with the 20 mg / L GIB treatment showing the best performance at the end of the culture period, NH4 + -N removal rate reached 89.4%. NH4 + -N is an inorganic nitrogen form that microalgae readily absorb and utilize, and it can enter the amino acid and protein synthesis processes via the GS / GOGAT pathway. Combined with the aforementioned enzyme activity results, treatment with 20 mg / L significantly increased GS activity, indicating that an appropriate concentration of GIB may promote NH4+ phosphate absorption by enhancing ammonium nitrogen assimilation. + -N is removed.

[0054] NO3 - -N concentration also continued to decrease during the culture process, and the 20 mg / L treatment showed a stronger nitrate nitrogen removal capacity. At the end of the culture period, NO3- under the 20 mg / L GIB treatment...- -N removal rate reached 82.4%, higher than other treatments. (Compared to NH4) + -N is different, NO3 - -N usually needs to be reduced to NO2 by NR catalysis first. - -N, and must undergo further transformation before entering the nitrogen assimilation pathway. Therefore, NO3- - -N removal efficiency depends not only on cell growth requirements but also on nitrate reduction capacity. In this study, treatment with 20 mg / L significantly increased NR activity, compared with NO3-. - The increased -N removal rate suggests that 20 mg / L GIB may have enhanced Chlorella's ability to reduce and subsequently assimilate nitrate nitrogen.

[0055] The TP removal rate was relatively high among all nutrient indicators. At the end of the culture period, the TP removal rate reached 96.9% under the 20 mg / L GIB treatment, indicating that the 20 mg / L treatment significantly enhanced phosphorus removal by *Chlorella vulgaris*. Phosphorus is an important component of ATP, nucleic acids, phospholipids, and various phosphorylation intermediates, and its removal is usually closely related to algal cell proliferation, biomass accumulation, and enhanced metabolic activity. The aforementioned results showed that the cell density, biomass, and Chl-a and Chl-b contents of *Chlorella vulgaris* were all at high levels under the 20 mg / L treatment, indicating that an appropriate concentration of GIB promoted cell proliferation and photosynthetic metabolism, thereby increasing the cells' structural and metabolic demand for phosphorus. Furthermore, adsorption on the microalgal cell surface, binding to extracellular polymers, and phosphorus precipitation processes that may occur during culture may also contribute to TP removal.

[0056] TN removal reflects the combined effect of reduction in different nitrogen forms. During the cultivation process, the TN concentration in all Chlorella treatment groups showed a decreasing trend, indicating that Chlorella can effectively reduce the total nitrogen load in the effluent. Compared with group Z, the GIB treatment further improved the TN removal effect, with the 20 mg / L GIB treatment showing the best performance, achieving a TN removal rate of 71.3% at the end of the cultivation period. This result is consistent with NH4+. + -N and NO3 - The -N removal trend is consistent with the enhanced GS and NR activities under 20 mg / L treatment, indicating that an appropriate concentration of GIB may improve Chlorella's overall nitrogen reduction capacity by promoting inorganic nitrogen absorption, reduction and assimilation processes.

[0057] Overall, the 20 mg / L GIB treatment in NH4 + -N, NO3 -The treatment showed superior performance in the removal of nitrogen (N), total nitrogen (TN), and total phosphorus (TP), with final removal rates of 89.4%, 82.4%, 71.3%, and 96.9%, respectively. This result is consistent with the aforementioned changes in growth, photosynthetic pigments, and nitrogen metabolism enzyme activity, indicating that the higher biomass accumulation, photosynthetic pigment levels, and GS and NR activities of Chlorella under the 20 mg / L treatment jointly promoted nitrogen and phosphorus absorption and transformation. In contrast, the 30 mg / L GIB treatment did not further improve nutrient removal efficiency, suggesting that there is an optimal concentration range for GIB to enhance the purification capacity of Chlorella effluent; excessively high concentrations may weaken its promoting effect on growth and nutrient assimilation metabolism.

[0058] 2.5 Transcriptome response enhanced by nutrient removal in Chlorella induced by GIB To further elucidate the molecular basis of the enhanced growth and nutrient removal capacity of Chlorella under 20 mg / L treatment, a comparative transcriptome analysis was performed between group Z and group 20 mg / L. The results showed that 20 mg / L treatment induced a total of 311 differentially expressed genes, of which 112 genes were upregulated and 199 genes were downregulated. Figure 5 a) The relatively limited number of differentially expressed genes suggests that 20 mg / LGIB did not cause widespread transcriptional perturbation in Chlorella, but rather likely affected its physiological functions and nutrient assimilation capacity by regulating specific metabolic processes.

[0059] GO enrichment results showed that differentially expressed genes were mainly related to biological processes such as ATP synthesis coupled with proton transport, proton transmembrane transport, and ATP synthesis coupled with electron transport. Figure 5 c). These processes are closely related to transmembrane proton gradient formation, electron transport, and energy conversion, providing energy support for cell growth, substance transport, and nitrogen assimilation. At the cellular component level, differentially expressed genes were mainly enriched in the inner mitochondrial membrane and cytoplasm, suggesting that GIB-M treatment may affect cellular respiratory metabolism and intracellular energy balance. At the molecular function level, oxidoreductase activity, NADH dehydrogenase activity, cofactor binding, and transmembrane transport activity were particularly prominent, indicating that GIB-M treatment may enhance electron transport, redox regulation, and substance transport-related processes. This result corresponds to the aforementioned increase in SOD and CAT activities, suggesting that appropriate concentrations of GIB may improve Chlorella's adaptability to the tailwater environment by improving redox homeostasis and energy supply.

[0060] KEGG analysis further revealed that differentially expressed genes were mainly distributed in pathways such as endoplasmic reticulum protein processing, oxidative phosphorylation, nitrogen metabolism, arginine biosynthesis, fructose and mannose metabolism, and β-alanine metabolism. Figure 5(b) Among them, the oxidative phosphorylation pathway was consistent with the energy conversion-related items in the GO analysis, suggesting that 20 mg / L treatment may promote ATP generation and electron transport. The response of the endoplasmic reticulum protein processing pathway may be related to the synthesis, folding, and renewal of metabolic enzymes, transport proteins, and structural proteins. For Chlorella, which proliferates rapidly under 20 mg / L treatment, energy supply and protein renewal are essential for maintaining cell division, photosynthetic pigment accumulation, and nutrient assimilation. The responses of nitrogen metabolism and arginine biosynthesis pathways showed a good correlation with water quality and enzyme activity results. At the end of the culture period, NH4 under 20 mg / L treatment... + -N, NO3 - The removal rates of -N and TN reached 89.4%, 82.4%, and 71.3%, respectively, while the activities of GS and NR significantly increased. NR participates in NO3 removal. - -N restores, GS participates in NH4 + The enhanced activity of -N assimilation and its associated activity suggests that 20 mg / L treatment may promote the conversion of inorganic nitrogen into organic nitrogen components such as amino acids and proteins. The response of the arginine biosynthesis pathway further indicates that 20 mg / L treatment may promote the accumulation of nitrogen assimilation products into the organic nitrogen pool, thereby improving the overall nitrogen reduction capacity of *Chlorella* in the tailwater. Changes in carbon metabolism-related pathways also provide a metabolic explanation for the enhanced nitrogen assimilation. The responses of the fructose and mannose metabolic pathways suggest that 20 mg / L treatment may affect sugar metabolism and carbon flux allocation, providing carbon skeletons and energy support for cell proliferation and amino acid synthesis. Combined with the aforementioned higher cell density, biomass, and chlorophyll content, the increased nutrient removal capacity of *Chlorella* under 20 mg / L treatment may not be solely due to increased cell number, but rather related to a synergistic enhancement of energy metabolism, carbon metabolism, and nitrogen assimilation processes. Furthermore, phosphorus, as an important element for cellular components such as ATP, nucleic acids, and membrane lipids, may also see increased removal with cell proliferation, protein turnover, and enhanced metabolic activity, which is consistent with the result that TP removal reached 96.9% under 20 mg / L treatment.

[0061] These results indicate that 20 mg / L GIB primarily enhances the physiological activity and nutrient assimilation capacity of Chlorella in aquaculture wastewater by regulating pathways related to energy metabolism, redox processes, protein processing, nitrogen metabolism, and carbon metabolism. Transcriptome results corroborate those of growth, photosynthetic pigments, enzyme activity, and water quality removal, suggesting that the enhancing effect of 20 mg / L GIB is closely related to enhanced energy supply, carbon-nitrogen metabolism coupling, and improved cellular metabolic state.

[0062] 2.6 Potential mechanisms by which GIB induces enhanced nutrient removal from Chlorella Based on physiological responses, PSII photochemical performance, nutrient removal, and transcriptome results, this study further constructed a potential mechanism for enhanced nutrient removal in tailwater by Chlorella under GIB-induced stimulation. Figure 6 The results showed that 20 mg / L GIB treatment could improve Chlorella's ability to utilize nitrogen and phosphorus in aquaculture tailwater by improving light energy conversion, enhancing inorganic carbon utilization and central carbon metabolism, promoting nitrogen reduction and organic nitrogen synthesis, and accompanied by cell component renewal and increased phosphorus demand.

[0063] At the level of photosynthetic energy conversion and carbon metabolism, the 20 mg / L treatment enhanced metabolic processes related to light capture and inorganic carbon utilization. Upregulation of the light-capturing complex-related gene LHCB7 suggests a possible enhancement of the light-capturing capacity of the PSII antenna system; simultaneously, upregulation of the carbonic anhydrase gene CAH indicates a CO2 / HCO3 ratio... - The transformation process may be accelerated, which could improve the efficiency of inorganic carbon supply. Furthermore, the increased expression of glycolysis-related genes such as GAPDH / gapA, PEP, and gpmI suggests that central carbon metabolism and glycolysis flux may be enhanced. These changes can increase the supply of ATP, NAD(P)H, and the carbon skeleton, providing a metabolic basis for cell proliferation, nitrogen assimilation, and organic matter synthesis. This is consistent with the higher chlorophyll content, cell density, and biomass accumulation observed under the 20 mg / L treatment.

[0064] Enhanced carbon metabolism is further coupled with nitrogen transformation and organic nitrogen assimilation processes. Upregulation of NR expression under 20 mg / L treatment indicates that NO3- - -N to NO2 - The reduction of -N was promoted, which was related to the higher NR activity and 82.4% NO3 reduction in the 20 mg / L treatment. - The -N removal rate corresponds accordingly. Simultaneously, the upregulation of aminotransfer-related genes such as ALT and AGT indicates that the reduced inorganic nitrogen further enters the amino acid transformation network, including glutamic acid, alanine, aspartic acid, and glycine. The upregulation of arginine biosynthesis-related genes also suggests that 20 mg / L treatment may promote the conversion of inorganic nitrogen to the organic nitrogen pool. Therefore, the promotion of nitrogen removal by 20 mg / L GIB is not limited to inorganic nitrogen absorption, but involves a continuous process including nitrate nitrogen reduction, ammonium nitrogen assimilation, and amino acid synthesis. This can explain the final NH4+ ionization. + -N, NO3 - The removal rates of -N and TN reached 89.4%, 82.4%, and 71.3%, respectively.

[0065] Downregulation of the TCA cycle-related gene DLST / sucB further suggests a possible adjustment in carbon flux allocation. DLST / sucB is involved in the conversion of α-ketoglutarate to succinyl-CoA, and its downregulation may reduce the continued oxidative consumption of α-ketoglutarate in the TCA cycle, allowing it to be more retained for the synthesis of glutamate and other amino acids. α-ketoglutarate is a key carbon skeleton linking carbon metabolism and nitrogen assimilation; therefore, this change may contribute to enhancing the conversion of inorganic nitrogen to organic nitrogen components. In other words, the 20 mg / L treatment not only improves energy supply but may also enhance the carbon-nitrogen metabolism coupling efficiency by regulating carbon skeleton allocation.

[0066] Compared to nitrogen removal, transcriptomic evidence related to phosphorus removal leans more towards increased cellular component turnover and metabolic demands. BTA1-related glycerophospholipid metabolic responses suggest potential remodeling of cell membrane lipid composition, while upregulation of pyrimidine turnover / recycling-related genes such as UPP and pydC indicates enhanced nucleotide metabolic turnover. Phosphorus is a crucial component of ATP, nucleic acids, phospholipids, and phosphorylation intermediates; therefore, cell proliferation, membrane structure turnover, and enhanced nucleic acid metabolism may all increase the phosphorus requirement of Chlorella. Considering the 96.9% TP removal rate in the 20 mg / L treatment, phosphorus removal is more likely primarily contributed by biomass accumulation-driven assimilation and uptake, accompanied by phosphorus redistribution during membrane lipid remodeling and nucleotide cycling.

[0067] Therefore, the enhanced nutrient removal of Chlorella induced by 20 mg / L GIB can be attributed to an interconnected metabolic regulatory chain: GIB first enhances light capture, inorganic carbon conversion, and glycolysis, increasing energy, reducing power, and carbon skeleton supply; subsequently, these metabolic resources are used for nitrate reduction, ammonium nitrogen assimilation, and amino acid synthesis; at the same time, TCA cycle flux adjustment further promotes the allocation of carbon skeleton to nitrogen assimilation processes; finally, enhanced cell proliferation and component renewal jointly drive the improvement of nitrogen removal efficiency and synergistically promote phosphorus removal.

[0068] 3. Conclusion This study systematically evaluated the effect of Chlorella vulgaris on nutrient removal in aquaculture tailwater induced by the agricultural compound plant hormone GIB, and its physiological and molecular response mechanisms. The results showed that 20 mg / L GIB treatment most effectively promoted Chlorella vulgaris cell proliferation, biomass accumulation, and photosynthetic pigment synthesis, and improved PSII photochemical performance and electron transport capacity. This treatment also increased the activities of SOD, CAT, GS, and NR, and enhanced cellular antioxidant defense and NH4+. + -N assimilation and NO3 - -N reducing power makes NH4 + -N, NO3 -The removal rates of -N, TN, and TP reached 89.4%, 82.4%, 71.3%, and 96.9%, respectively. Transcriptome results further indicated that the 20 mg / L treatment likely drove Chlorella growth and nutrient assimilation by enhancing light capture, inorganic carbon utilization, glycolysis, nitrogen metabolism, and amino acid synthesis, accompanied by increased cell component renewal and phosphorus demand. Overall, appropriate concentrations of agricultural compound plant hormones can effectively improve the purification performance of Chlorella wastewater, providing a feasible regulatory strategy for enhanced treatment of aquaculture wastewater by microalgae.

[0069] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0070] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0071] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for enhancing the removal of nutrients from aquaculture tailwater by Chlorella, characterized in that, Includes the following steps: Chlorella was inoculated into the wastewater of aquaculture and cultured. Add agricultural compound plant hormones to the aquaculture tail water inoculated with Chlorella, mix well and then culture. The agricultural compound plant hormone comprises gibberellinic acid, indole-3-acetic acid, and brassinolide; by mass percentage, the content of gibberellinic acid is 0.1-0.2%, the content of indole-3-acetic acid is 0.0003-0.0007%, and the content of brassinolide is 0.0002-0.0004%. The final concentration of the agricultural compound plant hormone in the aquaculture wastewater system is 10~30 mg / L.

2. The method according to claim 1, characterized in that, The final concentration of the agricultural compound plant hormone in the wastewater system is 20 mg / L.

3. The method according to claim 1, characterized in that, By mass percentage, the agricultural compound plant hormone contains 0.135% gibberellinic acid, 0.00052% indole-3-acetic acid, and 0.00031% brassinolide.

4. The method according to claim 1, characterized in that, The initial inoculation density of the Chlorella was 10. 5 ~10 7 The cells / mL indicates that the Chlorella used for inoculation was in the logarithmic growth phase.

5. The method according to claim 1, characterized in that, The culture includes: a culture temperature of 23-27℃, a light-dark cycle of alternating 12 h light and 12 h darkness, a light intensity of 1500-2500 lx, and a culture time of not less than 8 days.

6. The method according to claim 1, characterized in that, The aquaculture wastewater contains phosphorus, ammonium nitrogen, and nitrate nitrogen.

7. The application of agricultural compound plant hormones in the preparation of biological agents for enhancing the removal of nutrients from aquaculture tailwater by Chlorella, characterized in that, The agricultural compound plant hormone comprises gibberellinic acid, indole-3-acetic acid, and brassinolide, and the concentration of the agricultural compound plant hormone used is 10-30 mg / L; by mass percentage, the content of gibberellinic acid in the agricultural compound plant hormone is 0.1-0.2%, the content of indole-3-acetic acid is 0.0003-0.0007%, and the content of brassinolide is 0.0002-0.0004%.

8. The application according to claim 7, characterized in that, The biological agent improves the removal efficiency of nitrogen and phosphorus nutrients in aquaculture tailwater by Chlorella through synergistic regulation of Chlorella's photosynthetic function, redox homeostasis and nitrogen assimilation process.

9. The application according to claim 8, characterized in that, The biological agent synergistically regulates the physiological metabolism of Chlorella in the following ways: (1) Enhance the photochemical efficiency and electron transport capacity of the PSII reaction center in Chlorella vulgaris; (2) Increase the activity of superoxide dismutase and catalase to maintain cellular redox homeostasis; (3) Upregulate the activity of glutamine synthase and nitrate reductase to enhance nitrogen assimilation capacity.

10. The application according to claim 8, characterized in that, The biopharmaceutical also regulates the coupling process of carbon and nitrogen metabolism in Chlorella at the transcriptional level, including upregulating the expression of light-harvesting complex gene LHCB7, carbonic anhydrase gene CAH, glycolysis-related genes GAPDH / gapA, PEP and gpmI, nitrate reductase gene NR and aminotransfer-related genes ALT and AGT, and downregulating the expression of TCA cycle-related gene DLST / sucB.

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