Method for improving wastewater treatment performance of microalgae under high temperature stress by using ferroptosis inhibitor

Abstract

The application discloses a method for relieving high-temperature stress of microalgae by using ferroptosis inhibitors to improve wastewater treatment performance. The method adds ferroptosis inhibitor Fer-1 into the microalgae growth environment, so that Fer-1 fully enters the algal cells, and is then applied to wastewater treatment. By adding ferroptosis inhibitor Fer-1 to regulate the programmed cell death pathway of microalgae, the application significantly relieves the inhibitory effect of high temperature on the growth of microalgae, effectively maintains the balance of the intracellular oxidation-antioxidation system, reduces the influence on the lipid metabolism pathway of chlorella, realizes the resistance to high-temperature stress, improves the nitrogen and phosphorus removal performance of wastewater, and increases the removal rates of ammonia nitrogen and phosphorus by 44.50% and 30.72% respectively. The application has important application value for effectively protecting microalgae cells under extreme temperature conditions and promoting the low-carbon resource treatment of wastewater microalgae.

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 integration of algae cultivation and wastewater treatment shows great potential for alleviating energy crises and reducing environmental burdens. Theoretically, it can achieve a win-win situation by both wastewater resource utilization and microalgae biomass harvesting. However, environmental stress during outdoor microalgae cultivation is a key factor limiting the efficiency and application of this technology. Among these, temperature stress is a significant environmental factor affecting microalgae wastewater biological treatment systems. Seasonal temperature changes and rapid daily temperature fluctuations can significantly alter microalgae growth conditions, directly impacting wastewater treatment efficiency.

[0003] Previous studies on temperature stress have mostly focused on the effects of constant moderate temperatures (20-35°C) on microalgae growth and pollutant removal capabilities, with relatively little research 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.). Furthermore, current methods for overcoming temperature stress rely primarily on long-term acclimation 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 future outdoor cultivation under long-term high temperature conditions. 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 within a day, making 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 rising temperatures, 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 stress resistance of microalgae. Summary of the Invention

[0005] To address the problems of low microalgae survival and nitrogen and phosphorus removal efficiency caused by extreme temperature fluctuations, this paper provides a method for alleviating high-temperature stress in microalgae using ferroptosis inhibitors to improve wastewater treatment performance. This method uses ferroptosis inhibitors to specifically block high-temperature-induced ferroptosis in microalgae cells, thereby rapidly restoring microalgae activity and efficiently removing nitrogen and phosphorus.

[0006] The technical solutions of the present invention are as follows:

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

[0008] 10 μM of the ferroptosis inhibitor Ferrostatin-1 (Fer-1) was added to the microalgae culture medium and pre-incubated in a plant growth chamber to allow Fer-1 to fully enter the algae cells before the microalgae were used for wastewater 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, Phaeodactylum tricornutum, etc.

[0011] Preferably, the pre-incubation conditions are: temperature 20-30°C, light intensity 26-400 μmol / m 2 / s, daily light duration 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 to 0.6.

[0013] The sewage to be treated described in the present invention is 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 microalgae in the early logarithmic growth phase with the ferroptosis inhibitor Fer-1. Compared with 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 has greatly enhanced the microalgae's ability to resist high temperature stress and effectively alleviated the growth-inhibiting effects of high temperature on microalgae. In the subsequent recovery phase, the pre-incubated microalgae were able to recover their activity relatively quickly, with an average increase of 349.02% in biomass and an increase of 44.50% and 30.72% in the average removal rates of ammonia nitrogen and phosphorus, respectively.

[0016] In summary, the present invention utilizes the ferroptosis inhibitor Fer-1 to significantly enhance microalgae's resistance to high-temperature stress, accelerate their recovery from heat stress, promote microalgal biomass accumulation, and improve nitrogen and phosphorus removal efficiency in microalgal wastewater. This invention provides a new regulatory approach to mitigate the inhibitory effects of extreme temperature fluctuations in tropical summers on wastewater treatment coupled with microalgae cultivation technologies, and has broad application prospects in the low-carbon resource recovery of wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 The growth, 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 vulgaris.

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

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

[0023] Figure 7 The results were for changes in the expression of specific genes involved in ferroptosis, fatty acid synthesis, glycerophospholipid metabolism, peroxisomes, energy metabolism, and photosynthetic activity in Chlorella vulgaris.

[0024] Figure 8 This is a comprehensive mechanism of high temperature stress-induced iron death in Chlorella. DETAILED DESCRIPTION

[0025] The present invention will be described in further detail below with reference to 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 this experiment was Chlorella sp. (Cat. No. MN906179), isolated from a laboratory sequencing batch reactor used to treat municipal wastewater. The culture was expanded using sterilized (121°C, 30 minutes) simulated municipal wastewater and subcultured at 25±2°C with a 14:10 light:dark cycle (with a light intensity of 3000 lux during the photoperiod) until stable cell viability was achieved, suitable for subsequent experiments.

[0030] The composition of simulated municipal wastewater was as follows: NaAc (230.77 mg / L), NH4Cl (114.64 mg / L), K2HPO4 (21.95 mg / L), and a trace element solution (1 mL / L). The trace element solution consisted 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, and 10 g / L EDTA.

[0031] Example 2

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

[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, Fer-1 concentrations of 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 operating procedures were used for 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 The 50°C and Fer-50°C groups were then exposed to a 50°C water bath for 30 minutes in the dark. The two groups were then 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 vulgaris were compared. There was no significant difference in growth in the Fer group compared to the control group, demonstrating that the addition of Fer-1 had no adverse effects on microalgal growth. Microalgal growth in the 50°C and Fer-50°C groups, which were subjected to transient high temperature treatment, was significantly inhibited. However, the degree of inhibition in the Fer-50°C group was significantly reduced. The group with 10 μM Fer-1 experienced the least inhibition, significantly reducing the inhibitory effect on microalgal growth by 125.24% on day 7 compared to the 50°C group. This indicates that the optimal concentration of Fer-1 to alleviate high temperature stress in microalgae is 10 μM.

[0036] Example 3

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

[0038] The experiment was divided into three groups, each with three replicates: (i) a control group; (ii) a high-temperature group (50°C group), in which the microalgae culture was subjected to transient high-temperature treatment on day 3 to investigate the inhibitory effect of high temperature on microalgae growth; and (iii) a high-temperature group (Fer-50°C group) supplemented with the ferroptosis inhibitor Fer-1. 10 μM Fer-1 was added to the microalgae culture on day 1 and subjected to transient high-temperature treatment on day 3 to investigate whether ferroptosis was the primary inhibitory mechanism and to examine the feasibility of Fer-1 in improving the resistance of Chlorella to high-temperature stress and the efficiency of nitrogen and phosphorus removal. The specific operating procedures for each group are compared 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℃ 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℃ 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 to the 16th day. By the 16th day, the biomass reached 71.90% of the control group.

[0042] Figure 2 The growth, intracellular iron content, and oxidative stress indicators of Chlorella vulgaris were compared under conditions with or without the addition of Fer-1. Compared with the control group, both groups subjected to transient high temperature treatment showed a decrease in growth, but to varying degrees. The OD value of the group subjected to transient high temperature treatment at 50°C on the 8th day was 0.0447 W / m. 680Significantly decreased by 76.05% (p < 0.0001, see Figure 2 A), compared with the 50℃ group, the inhibitory effect on microalgae growth 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 comparison of the culture medium visual photos further confirmed that the biomass accumulation of Chlorella in the Fer-50℃ group was greater, 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. This indicates that Fer-1 effectively reduces 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°C group increased significantly by 1.67 times (p < 0.0001), while the MDA level in the Fer-50°C group was similar to that in 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 concentrations of different groups on the 8th day. Compared with the control group, the Fe 2 The content of Fe 3 The level increased significantly by 7.75 times (p<0.0001), indicating that high temperature stress promoted the 2 Fe 3 However, the addition of Fer-1 significantly slowed down this conversion. 3 The content was reduced by 45.58% compared with the 50℃ group (p<0.001, Figure 2 E).

[0043] Table 2 compares the nitrogen and phosphorus removal performance of each group. The results show that the nitrogen and phosphorus removal rates of the Fer-50℃ group were significantly improved compared with the 50℃ group, with the average removal rates of ammonia nitrogen and phosphorus increasing 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 effects of high temperature stress was significantly shorter than that of the 50℃ group, and that it was able to quickly recover the absorption of nitrogen and phosphorus nutrients. In order to further explore the significant changes in ammonia nitrogen absorption under high temperature stress, the nitrogen metabolic response was evaluated. Figure 4As shown in Figures C and D, the activity levels of glutamine synthetase (GS) and glutamate synthetase (GOGAT) in the 50°C 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°C group, respectively. This indicates that nitrogen metabolism is significantly inhibited under high temperature stress 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 ferroptosis-related indicators in the three groups of samples were summarized. The ATP level in the 50℃ group decreased by 42.22% compared with the control group (p < 0.0001, see Figure 5 A), indicating impaired mitochondrial function, which leads 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 SOD and CAT activities 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 peroxides on the cell membrane in the 50°C group changed the membrane permeability, leading to cell membrane rupture and a 192.53% increase in lactate dehydrogenase (LDH) levels (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%, respectively, p < 0.005 and p = 0.01, see Figure 5 Figures F, G, and H further demonstrate that high temperature induces ferroptosis in microalgae. In the Fer-50°C group, the changes in these indicators were as expected, demonstrating 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 addition of Fer-1 could significantly reverse the lipid remodeling of Chlorella caused by high temperature stress, 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. 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 regulatory factors such as phosphatidylglycerol (PG) and thioquinovose 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 supplementation 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 ). Although there was no significant change in carbon chain length, the addition of Fer-1 significantly increased the levels of a variety of 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 unsaturation level 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 exactly the 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 patterns of each group were analyzed, and the results showed that Fer-1 mainly responded to high temperature stress by regulating six metabolic pathways of Chlorella, including ferroptosis, fatty acid synthesis, glycerophospholipid metabolism, peroxisome, energy metabolism, and photosynthetic activity. Figure 7Figure A shows transcriptional changes in key genes associated with ferroptosis. Specifically, in the 50°C group, expression of the long-chain acyl-CoA synthetase gene ACSL was upregulated on day 4, indicating an increased cellular demand for long-chain fatty acids to cope with oxidative stress. However, this upregulation was successfully reversed in the Fer-1 group. Simultaneously, transient high-temperature treatment increased the transcription levels of the glutathione synthase gene GSS and the glutathione peroxidase gene GPX, while significantly downregulated the glutathione reductase gene GSR. However, the expression differences of these three genes were significantly reduced in the Fer-50°C group. Chlorella may protect against oxidative damage by upregulating GSS, but this still fails to maintain adequate antioxidant levels. Furthermore, a large amount of GSH may be oxidized to oxidized glutathione GSSG, which hinders the reduction pathway of GSSG, further disrupting intracellular glutathione levels and 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 promoting the Fenton reaction, ultimately increasing Fe 3 and lipid peroxide levels (see Figure 2 D and 2E). Figure 7 Figures B and 7C show changes in genes involved in fatty acid synthesis and glycerophospholipid metabolism. In the 50°C treatment, expression of genes involved in fatty acid synthesis (such as accC and ACACA) was downregulated on day 4, suggesting that Chlorella may rapidly respond to lipid metabolism disturbances by downregulating key enzymes. However, in the following days, expression of these genes recovered or exceeded control levels, indicating that synthesis capacity was restored after initial inhibition. Regarding glycerophospholipid synthesis, expression of genes involved in diacylglycerol (DAG) and triacylglycerol (TAG) synthesis (such as ATS1, plsC, DPP1, and DGAT1) decreased on day 4, while levels of TGL4, which is critical for TAG breakdown, increased. This suggests that the microalgae may promote TAG breakdown to release additional energy rather than enhancing DAG or TAG synthesis. Furthermore, decreased expression of genes involved in PE synthesis (such as ETNK, CHK, and EPT1) also suggests that transient high-temperature treatment impedes PE synthesis. However, the increased PE content observed in metabolomics may be due to a redistribution of lipid composition. Figure 7DF analyzed gene expression changes between groups related to oxidative stress, energy metabolism, and photosynthesis. Genes related to peroxisome function and biogenesis (e.g., CAT, SOD, PRDX5, etc.) were significantly upregulated in the 50°C group, indicating an enhanced antioxidant response to counteract the increased ROS caused by transient high-temperature treatment. Simultaneously, genes related to energy metabolism, such as the nitrate reductase gene NR and the nitrite reductase gene nirA, were significantly downregulated by transient high-temperature treatment. Furthermore, high temperature severely damaged the photosystem, with decreased expression of PSI and PSII subunits and genes involved in photosynthetic electron transfer (e.g., PetE and PetF). However, Fer-1 treatment significantly alleviated the expression changes of multiple genes (e.g., petH, psaE, and psbO), promoting linear electron transport (LET), enhancing ATP and NADPH production, helping to maintain the structural integrity of the oxygen-evolving complex (OEC), and mitigating excessive ROS production in PSII. Finally, on days 7 and 14, the expression levels of most genes in the 50°C group were comparable to those in the control group, with some genes even slightly upregulated. These results support the mechanism by which transient high temperature treatment induces ferroptosis in microalgae cells.

[0049] Figure 8 A comprehensive mechanism was proposed to clarify the specific pathway by which Fer-1 inhibits high temperature stress-induced ferroptosis in the microalga Chlorella sp. First, high temperature stress promotes the Fenton reaction by increasing oxidative stress, leading to Fe 2 Converted to Fe 3 , thereby increasing the intracellular Fe 3 accumulation. Secondly, under high temperature stress, the level of ROS in the cell increases, activating the antioxidant defense system, enhancing the activity of SOD and CAT, while reducing the abundance of GPX4, and consuming AsA and GSH early. Third, high temperature stress induces lipid remodeling, which is manifested as 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 LOX enzyme activation. 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 Fe 2 Fe 3 conversion, avoiding the intracellular Fe 3accumulation. Secondly, Fer-1 helps to reduce the level of ROS in the cell, 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, the lipid remodeling caused by high temperature stress is alleviated, which is manifested in that the changes in the abundance of key lipids (such as DGDG, MGDG, PG, SQDG and CL) in chloroplasts and mitochondria are controlled, the accumulation of lipids with lower unsaturation (such as PE) is inhibited, and the formation of lipid peroxides is restricted. 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 due to excessive toxic lipid peroxides, thereby avoiding triggering cell iron death. The present invention's in-depth understanding of the inhibition of microalgae activity under high temperature stress provides new insights for effectively regulating the growth of microalgae, such as by adding the iron death inhibitor Fer-1 to the system to formulate an environmental stress relief strategy 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, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the scope of protection 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 appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0052] In addition, the 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 and pre-incubate it in a plant growth chamber to allow Fer-1 to fully enter the algae cells before using the microalgae for wastewater 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 Phaeodactylum tricornutum.

3. The method according to claim 1, wherein The pre-incubation conditions are: temperature 20-30°C, light intensity 26-400 µmol / m² / s, and daily photoperiod of 12-18 hours.

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

6.

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

Images