Method for relieving microalgae high-temperature stress and improving sewage treatment performance by utilizing ferroptosis inhibitor

Abstract

The invention discloses a method for relieving microalgae high-temperature stress and improving sewage treatment performance by utilizing a ferroptosis inhibitor. According to the method, a ferroptosis inhibitor Fer-1 is added into a microalgae growth environment, so that Fer-1 fully enters microalgae cells, and then the ferptosis inhibitor Fer-1 is applied to sewage treatment. According to the method, the ferroptosis inhibitor Fer-1 is added to regulate and control the way of programmed cell death of the microalgae, so that the inhibition effect of high temperature on the growth of the microalgae is remarkably relieved, the balance of an intracellular oxidation-anti-oxidation system is effectively maintained, the influence of a lipid metabolism way of the chlorella is relieved, high-temperature stress resistance is realized, and the yield of the chlorella is improved. Meanwhile, the nitrogen and phosphorus removal performance of the sewage is improved, the removal rates of ammonia nitrogen and phosphorus are improved by 44.50% and 30.72% respectively, and the method has important application value for effectively protecting microalgae cells under the extreme temperature condition and promoting low-carbon resourceful treatment of the microalgae in the sewage.

Description

Technical Field

[0001] The present invention belongs to the field of sewage resource treatment technology under extreme temperature conditions, and relates to a method for utilizing ferroptosis inhibitors to alleviate high temperature stress of microalgae and improve sewage treatment performance. Background Art

[0002] The combination of algae cultivation and sewage treatment shows great potential in alleviating the energy crisis and reducing the environmental burden. In theory, it can achieve a win-win effect of sewage resource utilization and microalgae biomass harvesting. However, the environmental stress faced during the outdoor cultivation of microalgae is one of the key factors limiting the efficiency of this technology and its application. Among them, temperature pressure is an important environmental factor affecting the microalgae sewage biological treatment system. Seasonal temperature changes and rapid daily temperature fluctuations can significantly change the growth conditions of microalgae, which directly affects the efficiency of sewage treatment.

[0003] Previous studies on temperature stress have mostly focused on the effects of constant moderate temperatures (20-35°C) on the growth of microalgae and the ability to remove pollutants, and relatively few studies have been conducted on high temperatures (>40°C) (Zhu J, Cai Y, Wakisaka M, et al. Mitigation of oxidative stress damage caused by abiotic stress to improve biomass yield of microalgae: A review [J]. Science of the Total Environment, 2023: 165-200.). In addition, current methods for overcoming temperature stress mainly rely on long-term domestication to obtain microalgae species that can adapt to a specific temperature range. Studies have shown that Scenedesmus intermedius sampled from terrestrial waters can adapt to 30°C after 15 generations, 35°C after 30 generations, and up to 40°C after 135 generations (Huertas IE, Rouco M, Lopez-Rodas V, et al. Warming will affect phytoplankton differently: evidence through a mechanistic approach [J]. Proceedings of the Royal Society B: Biological Sciences, 2011, 278 (1724): 3534-3543.). Therefore, microalgae can gradually adapt to high temperatures through generations of reproduction.

[0004] The adaptability of microalgae at the genetic level provides a potential solution for outdoor cultivation under long-term high temperature conditions in the future. Intergenerational adaptation can ensure that microalgae achieve stable physiological activity, and seasonal temperature changes help microalgae obtain sufficient adaptation time. However, the rapid temperature fluctuations in outdoor photobioreactors may have an amplitude of up to 10°C in a day, which makes it difficult for microalgae to achieve intergenerational adaptation in such a short period of time. In addition to slow intergenerational adaptation, microalgae also have rapid physiological adaptation mechanisms. Studies have shown that both Microcystis aeruginosa and Scenedesmus acutus exhibit higher photosynthetic rates and lower respiration rates under high temperature conditions, and their cell volumes are reduced. This change may be to solve the imbalance between catabolism and biosynthesis caused by increased temperature, by reducing cell volume to increase nutrient absorption rate and reduce metabolic costs (Staehr PA, Birkeland MJ. Temperature acclimation of growth, photosynthesis and respiration in two mesophilic phytoplankton species [J]. Phycologia, 2006, 45 (6): 648-656.). However, the current methods for improving the high temperature resistance of microalgae are still insufficient. Therefore, it is necessary to further study the effects of high temperature stress on the physiological responses of microalgae and propose a new method to enhance the high temperature resistance of microalgae. Summary of the invention

[0005] In view of the problem that extreme temperature fluctuations lead to low survival rate of microalgae and low nitrogen and phosphorus removal efficiency, the present invention provides a method for using ferroptosis inhibitors to alleviate microalgae high temperature stress and improve sewage treatment performance. The method uses ferroptosis inhibitors to specifically block the ferroptosis of microalgae cells caused by high temperature, thereby achieving rapid recovery of microalgae activity and efficient removal of nitrogen and phosphorus.

[0006] The technical solution of the present invention is as follows:

[0007] The method of using ferroptosis inhibitors to alleviate microalgae high temperature stress and improve sewage treatment performance is specifically as follows:

[0008] Add 10 μM of ferroptosis inhibitor Ferrostatin-1 (Fer-1) to the microalgae culture medium, place it in a plant growth chamber for pre-incubation to allow Fer-1 to fully enter the algae cells before using the microalgae for sewage treatment;

[0009] Alternatively, microalgae and 10 μM ferroptosis inhibitor Fer-1 are directly added to the wastewater to be treated for wastewater treatment.

[0010] Preferably, the microalgae species are Chlorella, Chlamydomonas, Cyanobacteria, Phaeophyta, etc.

[0011] Preferably, the pre-incubation conditions are: temperature 20-30°C, light intensity 26-400 μmol / m 2 / s, daily light time 12 to 18 hours.

[0012] Preferably, the initial optical density (OD 680 ) is 0.2-0.3, and pre-incubated until the microalgae grow to OD 680 It is 0.5~0.6.

[0013] The sewage to be treated described in the present invention is the sewage commonly used in the art that can be biologically treated using microalgae, including but not limited to municipal sewage, industrial wastewater, agricultural wastewater, etc.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] The method of the present invention first pre-incubates the microalgae in the early logarithmic growth phase with the ferroptosis inhibitor Fer-1. Compared with the microalgae that have not been pre-incubated with the inhibitor, the pre-incubated microalgae show a significant increase in chlorophyll levels and photosynthetic activity, indicating that the ferroptosis inhibitor greatly enhances the microalgae's ability to resist high temperature stress and effectively alleviates the growth inhibitory effect of high temperature on the microalgae. In the subsequent recovery stage, the pre-incubated microalgae can quickly recover its activity, its biomass increased by an average of 349.02%, and the average removal rates of ammonia nitrogen and phosphorus increased by 44.50% and 30.72%, respectively.

[0016] In summary, the present invention utilizes the ferroptosis inhibitor Fer-1 to significantly improve the resistance of microalgae to high temperature stress, accelerate the recovery process of microalgae in response to high temperature stress, promote the accumulation of microalgae biomass, and improve the nitrogen and phosphorus removal efficiency of microalgae wastewater. The present invention provides a new control method to weaken the inhibitory effect of extreme temperature fluctuations in hot summer in tropical areas on wastewater treatment coupled with microalgae cultivation technology, and has broad application prospects in low-carbon resource treatment of wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To compare the growth performance of Chlorella under the conditions of adding different concentrations of ferroptosis inhibitors.

[0018] Figure 2 The growth conditions, intracellular iron ion content and oxidative stress indicators of Chlorella with or without the addition of ferroptosis inhibitors.

[0019] Figure 3 Comparison of the photobioreactors of the control group and the two experimental groups.

[0020] Figure 4 (A) Denitrification, (B) Phosphorus removal efficiency and changes in the contents of nitrogen metabolism-related enzymes (C) glutamine synthetase (GS) and (D) glutamate synthetase (GOGAT) in Chlorella.

[0021] Figure 5 Changes in iron death-related indicators of Chlorella in the control group and two experimental groups.

[0022] Figure 6 Changes in lipid content of Chlorella in the control group and two experimental groups.

[0023] Figure 7 Changes in the expression of specific genes in Chlorella ferroptosis, fatty acid synthesis, glycerophospholipid metabolism, peroxisomes, energy metabolism, and photosynthetic activity.

[0024] Figure 8 The comprehensive mechanism of high temperature stress-induced ferroptosis in Chlorella. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0026] The reagents used in the following examples are all available from commercial sources.

[0027] Example 1

[0028] Enrichment culture of microalgae:

[0029] The algae used in the experiment was Chlorella sp. (No.: MN906179) isolated from a laboratory batch reactor used to treat municipal sewage. The culture was expanded with sterilized (121°C, 30 min) simulated municipal sewage and subcultured at 25±2°C with a light:dark cycle of 14:10 (the light intensity of the light cycle was 3000 lux) until stable cell activity was obtained, which was suitable for subsequent experiments.

[0030] The composition of simulated municipal sewage is as follows: NaAc (230.77 mg / L), NH4Cl (114.64 mg / L), K2HPO4 (21.95 mg / L), trace element solution (1 mL / L). The trace element solution consists of: 1.5 g / L FeCl3·6H2O, 0.38 g / L MgSO4·7H2O, 0.01 g / L CaCl2, 0.18 g / L KI, 0.15 g / L H3BO3, 0.15 g / L CoCl2·6H2O, 0.12 g / L MnCl2·4H2O, 0.12 g / L ZnSO4·7H2O, 0.06 g / L Na2MoO4·2H2O, 0.03 g / L CuSO4·5H2O, 10 g / L EDTA.

[0031] Example 2

[0032] Determination of the optimal concentration of ferroptosis inhibitor:

[0033] The experiment was divided into four groups: (i) control group; (ii) high temperature group (50℃ group), in which the microalgae culture was subjected to instantaneous high temperature treatment on the third day, exposed to a high temperature environment of 50℃ for 30 minutes, and then restored to normal suitable conditions for continued cultivation, in order to explore the inhibitory effect of high temperature on the growth of microalgae; (iii) control group (Fer group) with the addition of ferroptosis inhibitor Fer-1, in which Fer-1 was set at three concentrations: 5μM, 10μM, and 20μM, in order to explore whether the addition of Fer-1 had any adverse effects on the growth of microalgae; (iiii) high temperature group (Fer-50℃ group) with the addition of ferroptosis inhibitor Fer-1, correspondingly, the Fer-1 concentrations were 5μM, 10μM, and 20μM, respectively. Different concentrations of Fer-1 were added to the microalgae culture on the first day, and subjected to instantaneous high temperature treatment on the third day, exposed to a high temperature environment of 50℃ for 30 minutes, and then restored to normal suitable conditions for continued cultivation, in order to explore whether ferroptosis was the main inhibitory mechanism.

[0034] All experimental groups were placed in a plant growth chamber to maintain a constant temperature (25°C), light intensity (3000 lux) and light-dark cycle (14:10). In the initial stage, the same operation steps were used in each group, but after one day of cultivation, different growth performances were observed. Based on this, on the second day, the cultures of all groups were diluted to the same biomass concentration (OD 680 Diluted to 0.20) to eliminate the influence of different microalgae biomass concentrations on the experimental results. Subsequently, on the third day, the 50℃ group and the Fer-50℃ group were subjected to instantaneous high temperature treatment in a 50℃ water bath under dark conditions for 30 minutes. Thereafter, the two groups returned to the plant growth chamber for subsequent experiments.

[0035] Figure 1 The effects of adding different concentrations of ferroptosis inhibitors on the growth performance of Chlorella were compared. Compared with the control group, there was no significant difference in the growth of the Fer group, proving that the addition of Fer-1 had no adverse effects on the growth of microalgae. The growth of microalgae in the 50℃ group and the Fer-50℃ group, which were subjected to instantaneous high temperature treatment, was significantly inhibited, but in comparison, the degree of inhibition in the Fer-50℃ group was significantly reduced. Among them, the experimental group with 10μM Fer-1 was the least inhibited, and the inhibitory effect on the growth of microalgae on the 7th day was significantly reduced by 125.24% compared with the 50℃ group, indicating that the optimal concentration of adding Fer-1 to alleviate the high temperature stress of microalgae is 10μM.

[0036] Example 3

[0037] Effects of transient high temperature stress on physiological activity and nitrogen and phosphorus removal efficiency of microalgae:

[0038] The experiment was divided into three groups, each with three parallel samples: (i) control group; (ii) high temperature group (50℃ group), in which the microalgae culture was subjected to instantaneous high temperature treatment on the 3rd day to explore the inhibitory effect of high temperature on the growth of microalgae; (iii) high temperature group (Fer-50℃ group) with the addition of ferroptosis inhibitor Fer-1, in which 10μM Fer-1 was added to the microalgae culture on the 1st day and subjected to instantaneous high temperature treatment on the 3rd day to explore whether ferroptosis is the main inhibitory mechanism and to investigate the feasibility of Fer-1 in improving the resistance of Chlorella to high temperature stress and the removal efficiency of nitrogen and phosphorus. The specific operation procedures of each group are shown in Table 1.

[0039] Table 1 Comparison of operating procedures of the experimental groups

[0040]

[0041] According to the physiological response process of the microalgae in the 50°C group, the experiment was divided into two stages: (1) inhibition period (3 to 7 days): the high temperature stress on the 3rd day initially had a significant inhibitory effect on the growth of Chlorella. The biomass of the 50°C group decreased day by day and reached the lowest level on the 8th day, showing a significant inhibition of 76.05% compared with the control group; (2) recovery period (8 to 16 days): during this stage, the stress caused by high temperature on Chlorella was relieved, and the biomass gradually recovered and accumulated from the 8th day to the 16th day. On the 16th day, the biomass reached 71.90% of the control group.

[0042] Figure 2 The growth, intracellular iron content and oxidative stress indexes of Chlorella vulgaris with or without the addition of Fer-1 were compared. Compared with the control group, both groups subjected to instantaneous high temperature treatment showed a decrease in growth, but to different degrees. The OD of the 50℃ group subjected to only instantaneous high temperature treatment on the 8th day was 0.1477 W·m-1. 680Significantly decreased by 76.05% (p < 0.0001, see Figure 2 A), compared with the 50℃ group, the inhibitory effect on the growth of microalgae in the Fer-50℃ group was significantly reduced by 135.82% on the 8th day. In addition, there was no significant difference in the growth rate between the Fer-50℃ group and the control group on the 8th day (p>0.05, see Figure 2 B) Figure 3 As shown, the color contrast of the intuitive photos of the culture medium further confirmed that the biomass accumulation of Chlorella in the Fer-50℃ group was higher, indicating that the ferroptosis inhibitor Fer-1 played an important role in Chlorella's response to high temperature stress. High temperature stress caused the reactive oxygen species (ROS) levels in the 50℃ group and the Fer-50℃ group to be 159.97% (p<0.0001) and 43.20% (p<0.01) higher than those in the control group, respectively, indicating that Fer-1 effectively reduced the ROS production and subsequent oxidative damage caused by high temperature stress ( Figure 2 C) In addition, given the key role of ROS in lipid peroxidation, Figure 2 D shows the comparison of malondialdehyde (MDA) content. The MDA level in the 50℃ group increased significantly by 1.67 times (p<0.0001), while the Fer-50℃ group was similar to the control group, indicating the contribution of Fer-1 in protecting microalgae cells from membrane lipid peroxidation. Figure 2 E further compared the intracellular iron ion concentrations of different groups on the 8th day. Compared with the control group, the Fe2+ content in the 50℃ group decreased by 41.10% (p<0.0001), while the Fe3+ level increased significantly by 7.75 times (p<0.0001), indicating that high temperature stress promoted the conversion of Fe2+ to Fe3+. However, the addition of Fer-1 significantly slowed down this conversion, and the Fe3+ content in the Fer-50℃ group decreased by 45.58% compared with the 50℃ group (p<0.001, Figure 2 E).

[0043] Table 2 compares the nitrogen and phosphorus removal of each group. The results show that the nitrogen and phosphorus removal rate of the Fer-50℃ group is significantly higher than that of the 50℃ group, among which the average removal rates of ammonia nitrogen and phosphorus are increased by 44.50% and 30.72%, respectively. Figure 4 A and B further showed that the time required for the Fer-50℃ group to relieve the inhibitory effect of high temperature stress was significantly shorter than that of the 50℃ group, and the absorption of nitrogen and phosphorus nutrients could be quickly restored. In order to further explore the significant changes in ammonia nitrogen absorption under high temperature stress, the nitrogen metabolism response was evaluated. Figure 4As shown in C and D, the activity levels of glutamine synthetase (GS) and glutamate synthetase (GOGAT) in the 50℃ group were 56.77% and 52.04% lower than those in the control group, and 52.66% and 50.02% lower than those in the Fer-50℃ group, respectively. This indicates that nitrogen metabolism is significantly inhibited under high temperature stress conditions, and highlights the important role of Fer-1 in alleviating this stress.

[0044] Table 2 Nitrogen and phosphorus removal rates during the experimental period

[0045]

[0046] Figure 5 The changes in indicators related to ferroptosis in the three groups of samples were summarized. The ATP level in the 50℃ group was reduced by 42.22% compared with the control group (p<0.0001, see Figure 5 A), indicating impaired mitochondrial function, leading to increased ROS (see Figure 2 C). At the same time, the activities of superoxide dismutase (SOD) and catalase (CAT) in the 50℃ group increased significantly (from 12.89 and 1.80 U / mg protein to 16.09 and 10.85 U / mg protein, respectively, both p<0.0005, see Figure 5 B and 5C), indicating an enhanced antioxidant response. In contrast, in the Fer-50℃ group, the addition of Fer-1 effectively alleviated mitochondrial damage and even slightly increased adenosine triphosphate (ATP) levels (see Figure 5 A), while the activities of SOD and CAT decreased accordingly, indicating that Fer-1 may regulate the antioxidant balance in Chlorella. In addition, the synthesis of lipoxygenase (LOX) was significantly enhanced in the 50℃ group (p<0.0005, see Figure 5 D), activating the polyunsaturated fatty acid (PUFA) / LOX / LPO axis and inducing lipid peroxidation. In addition, the excessive accumulation of lipid peroxidation on the cell membrane in the 50°C group changed the membrane permeability, leading to cell membrane rupture and an increase in lactate dehydrogenase (LDH) level by 192.53% (p<0.0001, see Figure 5 E). In addition, several other ferroptosis-related indicators also showed abnormal changes, such as a 36.90% decrease in glutathione peroxidase (GPX4) content (p<0.001), and a significant depletion of antioxidants glutathione (GSH) and ascorbic acid (AsA) (reduced by 72.05% and 72.23%, p<0.005 and p=0.01, respectively, see Figure 5 F, G and H), further indicating that high temperature induced ferroptosis in microalgae. In the Fer-50℃ group, the changes in the above indicators were in line with expectations, showing the important role of Fer-1 in inhibiting ferroptosis and protecting microalgae.

[0047] Figure 6The lipid metabolites of each group were analyzed, and the results showed that the lipid remodeling of Chlorella caused by high temperature stress could be significantly reversed by adding Fer-1, which was mainly reflected in three key changes: changes in lipid abundance specific to chloroplast membranes or mitochondria, accumulation of low-unsaturated lipids, and formation of lipid peroxides. First, after instantaneous high temperature treatment, the abundance of various lipids closely related to photosynthesis and energy metabolism changed to varying degrees. Specifically, in the 50°C group, the abundance of digalactosyldiacylglycerol (DGDG) decreased, while the abundance of its substrate monogalactosyldiacylglycerol (MGDG) increased, and this ratio is crucial for chloroplast morphology. The increase in MGDG abundance may be due to the inhibition of the activity of the specific lipase PGD1, thereby promoting the accumulation of MGDG and changing the composition of the photosynthetic complex to inhibit photosynthesis. In addition, high temperature stress caused changes in the abundance of typical allosteric regulators such as phosphatidylglycerol (PG) and thioquinovosyl esters (SQDG), which damaged the activity of light-dependent protochlorophyllide oxidoreductase (POR), thereby affecting the efficiency of light energy utilization and the chlorophyll synthesis pathway. At the same time, high temperature stress also significantly downregulated cardiolipin (CL), but after supplementing with Fer-1, its level was restored to a level close to that of the control group. Secondly, lipids with different degrees of unsaturation responded differently to high temperature stress. The number of unsaturated components of most lipids increased, while the saturated components decreased relatively (see Figure 6 A). Although there was no significant change in carbon chain length, the addition of Fer-1 significantly increased the levels of various highly unsaturated lipids (such as phosphatidylserine PS, phosphatidylcholine PC, diacylglycerol DG and DGDG), while lower unsaturated lipids (such as phosphatidylethanolamine PE and MGDG) were downregulated in the Fer-50℃ group. This suggests that Fer-1 may alleviate high temperature-induced ferroptosis by regulating the degree of unsaturation of lipids. Finally, high temperature stress also induced the production of lipid peroxides. In the 50℃ group, PE levels increased significantly, and the accumulation of substrates provided conditions for peroxidation reactions and may induce the formation of lipid peroxides, which is a hallmark of the lethal mechanism of ferroptosis. In addition, most PC components in the 50℃ group increased compared with the control group, and PC peroxidation is also a key trigger for ferroptosis. It is worth noting that Fer-1 mainly inhibited the increase in PE levels caused by high temperature stress, while the changes in PC were smaller. This may be because PE phospholipids play a more significant role in ferroptosis, especially PE lipids rich in PUFA tails are more susceptible to oxidative stress and are therefore more likely to form lipid peroxides.

[0048] Figure 7 The gene expression profiles of each group were analyzed, and the results showed that Fer-1 responded to high temperature stress mainly by regulating six metabolic pathways of Chlorella, including ferroptosis, fatty acid synthesis, glycerophospholipid metabolism, peroxisome, energy metabolism, and photosynthetic activity. Figure 7A shows the transcriptional changes of key genes related to ferroptosis. Specifically, in the 50℃ group, the expression of the long-chain acyl-CoA synthetase gene ACSL was upregulated on the 4th day, indicating that the cell's demand for long-chain fatty acids increased to cope with oxidative stress, but the Fer-1 group successfully reversed this upregulation of expression. At the same time, the transcription levels of the glutathione synthetase gene GSS and the glutathione peroxidase gene GPX increased after instantaneous high temperature treatment, while the glutathione reductase gene GSR was significantly downregulated, but the expression difference of these three genes in the Fer-50℃ group was significantly reduced. Chlorella may resist oxidative damage by upregulating GSS, but it still cannot maintain sufficient antioxidant levels; at the same time, a large amount of GSH may be oxidized to oxidized glutathione GSSG, and the reduction pathway of GSSG is hindered, further destroying the intracellular glutathione level and the overall redox balance. In addition, the expression of most autophagy-related genes (such as ATG9, ATG11, and ATG13) increased after transient high temperature treatment, indicating that the autophagy process may promote ferroptosis, increase intracellular free iron by degrading ferritin and promote the Fenton reaction, and ultimately increase the levels of Fe3+ and lipid peroxides (see Figure 2 D and 2E). Figure 7 B and 7C show the changes in genes related to fatty acid synthesis and glycerophospholipid metabolism. In the 50℃ group, the expression of genes related to fatty acid synthesis (such as accC and ACACA) was downregulated on the 4th day, indicating that Chlorella may respond quickly to lipid metabolism disorders by downregulating key enzymes. However, in the following days, the expression of these genes recovered or exceeded the control level, indicating that the synthesis capacity was restored after the initial synthesis was inhibited. In terms of glycerophospholipid synthesis, the expression of genes related to diacylglycerol (DAG) and triacylglycerol (TAG) synthesis (such as ATS1, plsC, DPP1, DGAT1) decreased on the 4th day, while the level of TGL4, which is essential for TAG decomposition, increased, indicating that the microalgae may promote TAG decomposition to release additional energy rather than enhance DAG or TAG synthesis. In addition, the expression of genes related to PE synthesis (such as ETNK, CHK and EPT1) was reduced, which also indicated that transient high temperature treatment hindered PE synthesis, but the increase in PE content was observed in metabolomics, which may be due to the redistribution of lipid composition. Figure 7DF analyzed the gene expression changes among the groups in oxidative stress, energy metabolism, and photosynthesis. The peroxisome function and biogenesis-related genes (e.g., CAT, SOD, PRDX5, etc.) in the 50℃ group were significantly upregulated, indicating that the antioxidant response was enhanced to counteract the increase in ROS caused by transient high temperature treatment. At the same time, energy metabolism-related genes, such as the nitrate reductase gene NR and the nitrite reductase gene nirA, also showed significant downregulation due to transient high temperature treatment. Moreover, the photosystem was severely damaged under high temperature, and the expression of PSI and PSII subunits and photosynthetic electron transfer-related genes (e.g., PetE, PetF, etc.) decreased; however, Fer-1 treatment significantly alleviated the expression changes of multiple genes (e.g., petH, psaE, and psbO), promoted linear electron transfer (LET), strengthened ATP and NADPH production, helped maintain the structural integrity of the oxygen-evolving complex (OEC), and alleviated the excessive ROS produced in PSII. Finally, on the 7th and 14th days, the expression levels of most genes in the 50℃ group were comparable to those in the control group, and some genes were even slightly upregulated. The above results support the mechanism of transient high temperature treatment inducing ferroptosis of microalgae cells.

[0049] Figure 8A comprehensive mechanism was proposed to clarify the specific pathway of using Fer-1 to inhibit ferroptosis induced by heat stress in the microalga Chlorella sp. First, heat stress promotes the Fenton reaction by increasing oxidative stress, resulting in the conversion of Fe2+ to Fe3+, thereby accumulating intracellular Fe3+. Second, under heat stress, intracellular ROS levels increase, activating the antioxidant defense system, enhancing the activities of SOD and CAT, while reducing the abundance of GPX4 and consuming AsA and GSH early. Third, heat stress induces lipid remodeling, which is manifested by changes in the abundance of key lipids (such as DGDG, MGDG, PG, SQDG, and CL) in chloroplasts and mitochondria, the accumulation of less unsaturated lipids (such as PE), and the formation of lipid peroxides, and the above changes are mediated by increased ROS levels and activation of LOX enzymes. Ultimately, excessive toxic lipid peroxides in the cell membrane lead to membrane rupture, thereby triggering ferroptosis of the cell. The typical ferroptosis inhibitor Fer-1 can effectively prevent the occurrence of this process. First, under the action of Fer-1, oxidative stress is inhibited, thereby curbing the conversion of Fe2+ to Fe3+ and avoiding the accumulation of Fe3+ in cells. Secondly, Fer-1 helps to reduce the level of intracellular ROS, thereby weakening the activation of the antioxidant defense system, maintaining the abundance of GPX4, and reducing the early consumption of AsA and GSH. In addition, under the influence of Fer-1, lipid remodeling caused by high temperature stress is alleviated, as shown by the controlled changes in the abundance of key lipids (such as DGDG, MGDG, PG, SQDG, and CL) in chloroplasts and mitochondria, the accumulation of lipids with low unsaturation (such as PE) is inhibited, and the formation of lipid peroxides is limited. At the same time, Fer-1 can directly scavenge lipid free radicals and inhibit the accumulation of lipid peroxides. These effects all help to protect the cell membrane and prevent membrane rupture caused by excessive toxic lipid peroxides, thereby avoiding triggering cell ferroptosis. The present invention's in-depth understanding of the inhibition of microalgae activity under high temperature stress provides new insights for effectively regulating microalgae growth, such as by adding the ferroptosis inhibitor Fer-1 to the system to develop environmental stress mitigation strategies to improve the efficiency of microalgae-based wastewater treatment systems.

[0050] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the specific details of the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical scheme of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0051] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0052] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for alleviating high temperature stress of microalgae and improving sewage treatment performance by using ferroptosis inhibitors, characterized in that: Specifically: Add 10 μM of the ferroptosis inhibitor Fer-1 to the microalgae culture medium, place it in a plant growth chamber for pre-incubation to allow Fer-1 to fully enter the algae cells before using the microalgae for sewage treatment; Alternatively, microalgae and 10 μM ferroptosis inhibitor Fer-1 can be directly added to the wastewater to be treated for wastewater treatment.

2. The method according to claim 1, characterized in that The microalgae species are Chlorella, Chlamydomonas, Cyanobacteria or Phaeophyta.

3. The method according to claim 1, characterized in that The pre-incubation conditions are: temperature 20-30 ℃, light intensity 26-400 µmol / m² / s, and daily light duration 12-18h.

4. The method according to claim 1, characterized in that: Initial OD of microalgae 680 The pH value is 0.2-0.3, and the microalgae are pre-incubated until they grow to OD 680 It is 0.5~0.

6.

5. The method according to claim 1, characterized in that The sewage to be treated is municipal sewage, industrial wastewater or agricultural wastewater.

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